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镍柱纯化蛋白说明书

Instruction ManualProBond TM Purification SystemFor purification of polyhistidine-containing recombinant proteinsCatalog nos. K850-01, K851-01, K852-01, K853-01, K854-01,R801-01, R801-15Version K2 September200425-0006iiTable of ContentsKit Contents and Storage (iv)Accessory Products (vi)Introduction (1)Overview (1)Methods (2)Preparing Cell Lysates (2)Purification Procedure—Native Conditions (7)Purification Procedure—Denaturing Conditions (11)Purification Procedure—Hybrid Conditions (13)Troubleshooting (15)Appendix (17)Additional Protocols (17)Recipes (18)Frequently Asked Questions (21)References (22)Technical Service (23)iiiKit Contents and StorageTypes of Products This manual is supplied with the following products:Product CatalogNo.ProBond™ Purification System K850-01ProBond™ Purification System with Antibodywith Anti-Xpress™ Antibody K851-01with Anti-myc-HRP Antibody K852-01with Anti-His(C-term)-HRP Antibody K853-01with Anti-V5-HRP Antibody K854-01ProBond™ Nickel-Chelating Resin (50 ml) R801-01ProBond™ Nickel Chelating Resin (150 ml) R801-15ProBond™Purification System Components The ProBond™ Purification System includes enough resin, reagents, and columns for six purifications. The components are listed below. See next page for resin specifications.Component Composition Quantity ProBond™ Resin 50% slurry in 20% ethanol 12 ml5X NativePurification Buffer250 mM NaH2PO4, pH 8.02.5 M NaCl1 × 125 ml bottleGuanidinium LysisBuffer6 M Guanidine HCl20 mM sodium phosphate, pH 7.8500 mM NaCl1 × 60 ml bottleDenaturingBinding Buffer8 M Urea20 mM sodium phosphate, pH 7.8500 mM NaCl2 × 125 ml bottlesDenaturing WashBuffer8 M Urea20 mM sodium phosphate, pH 6.0500 mM NaCl2 × 125 ml bottlesDenaturing ElutionBuffer8 M Urea20 mM NaH2PO4, pH 4.0500 mM NaCl1 × 60 ml bottleImidazole 3 M Imidazole,20 mM sodium phosphate, pH 6.0500 mM NaCl1 × 8 ml bottlePurificationColumns10 ml columns 6Continued on next pageivKit Contents and Storage, ContinuedProBond™Purification System with Antibody The ProBond™ Purification System with Antibody includes resin, reagents, and columns as described for the ProBond™ Purification System (previous page) and 50 µl of the appropriate purified mouse monoclonal antibody. Sufficient reagents are included to perform six purifications and 25 Western blots with the antibody.For more details on the antibody specificity, subclass, and protocols for using the antibody, refer to the antibody manual supplied with the system.Storage Store ProBond™ resin at +4°C. Store buffer and columns at room temperature.Store the antibody at 4°C. Avoid repeated freezing and thawing of theantibody as it may result in loss of activity.The product is guaranteed for 6 months when stored properly.All native purification buffers are prepared from the 5X Native PurificationBuffer and the 3 M Imidazole, as described on page 7.The Denaturing Wash Buffer pH 5.3 is prepared from the Denaturing WashBuffer (pH 6.0), as described on page 11.Resin and ColumnSpecificationsProBond™ resin is precharged with Ni2+ ions and appears blue in color. It isprovided as a 50% slurry in 20% ethanol.ProBond™ resin and purification columns have the following specifications:• Binding capacity of ProBond™ resin: 1–5 mg of protein per ml of resin• Average bead size: 45–165 microns• Pore size of purification columns: 30–35 microns• Recommended flow rate: 0.5 ml/min• Maximum flow rate: 2 ml/min• Maximum linear flow rate: 700 cm/h• Column material: Polypropylene• pH stability (long term): pH 3–13• pH stability (short term): pH 2–14ProductQualificationThe ProBond™ Purification System is qualified by purifying 2 mg of myoglobinprotein on a column and performing a Bradford assay. Protein recovery mustbe 75% or higher.vAccessory ProductsAdditionalProductsThe following products are also available for order from Invitrogen:Product QuantityCatalogNo.ProBond™ Nickel-Chelating Resin 50 ml150 mlR801-01R801-15Polypropylene columns(empty)50 R640-50Ni-NTA Agarose 10 ml25 ml R901-01 R901-15Ni-NTA Purification System 6 purifications K950-01 Ni-NTA Purification Systemwith Antibodywith Anti-Xpress™ Antibody with Anti-myc-HRP Antibody with Anti-His(C-term)-HRP Antibodywith Anti-V5-HRP Antibody 1 kit1 kit1 kit1 kitK951-01K952-01K953-01K954-01Anti-myc Antibody 50 µl R950-25 Anti-V5 Antibody 50 µl R960-25 Anti-Xpress™ Antibody 50 µl R910-25 Anti-His(C-term) Antibody 50 µl R930-25 InVision™ His-tag In-gel Stain 500 ml LC6030 InVision™ His-tag In-gelStaining Kit1 kit LC6033Pre-Cast Gels and Pre-made Buffers A large variety of pre-cast gels for SDS-PAGE and pre-made buffers for your convenience are available from Invitrogen. For details, visit our web site at or contact Technical Service (page 23).viIntroductionOverviewIntroduction The ProBond™ Purification System is designed for purification of 6xHis-tagged recombinant proteins expressed in bacteria, insect, and mammalian cells. Thesystem is designed around the high affinity and selectivity of ProBond™Nickel-Chelating Resin for recombinant fusion proteins containing six tandemhistidine residues.The ProBond™ Purification System is a complete system that includespurification buffers and resin for purifying proteins under native, denaturing,or hybrid conditions. The resulting proteins are ready for use in many targetapplications.This manual is designed to provide generic protocols that can be adapted foryour particular proteins. The optimal purification parameters will vary witheach protein being purified.ProBond™ Nickel-Chelating Resin ProBond™ Nickel-Chelating Resin is used for purification of recombinant proteins expressed in bacteria, insect, and mammalian cells from any 6xHis-tagged vector. ProBond™ Nickel-Chelating Resin exhibits high affinity and selectivity for 6xHis-tagged recombinant fusion proteins.Proteins can be purified under native, denaturing, or hybrid conditions using the ProBond™ Nickel-Chelating Resin. Proteins bound to the resin are eluted with low pH buffer or by competition with imidazole or histidine. The resulting proteins are ready for use in target applications.Binding Characteristics ProBond™ Nickel-Chelating Resin uses the chelating ligand iminodiacetic acid (IDA) in a highly cross-linked agarose matrix. IDA binds Ni2+ ions by three coordination sites.The protocols provided in this manual are generic, and may not result in 100%pure protein. These protocols should be optimized based on the bindingcharacteristics of your particular proteins.Native VersusDenaturingConditionsThe decision to purify your 6xHis-tagged fusion proteins under native ordenaturing conditions depends on the solubility of the protein and the need toretain biological activity for downstream applications.• Use native conditions if your protein is soluble (in the supernatant afterlysis) and you want to preserve protein activity.• Use denaturing conditions if the protein is insoluble (in the pellet afterlysis) or if your downstream application does not depend on proteinactivity.• Use hybrid protocol if your protein is insoluble but you want to preserveprotein activity. Using this protocol, you prepare the lysate and columnsunder denaturing conditions and then use native buffers during the washand elution steps to refold the protein. Note that this protocol may notrestore activity for all proteins. See page 14.1MethodsPreparing Cell LysatesIntroduction Instructions for preparing lysates from bacteria, insect, and mammalian cellsusing native or denaturing conditions are described below.Materials Needed You will need the following items:• Native Binding Buffer (recipe is on page 8) for preparing lysates undernative conditions• Sonicator• 10 µg/ml RNase and 5 µg/ml DNase I (optional)• Guanidinium Lysis Buffer (supplied with the system) for preparing lysatesunder denaturing conditions• 18-gauge needle• Centrifuge• Sterile, distilled water• SDS-PAGE sample buffer• Lysozyme for preparing bacterial cell lysates• Bestatin or Leupeptin, for preparing mammalian cell lysatesProcessing Higher Amount of Starting Material Instructions for preparing lysates from specific amount of starting material (bacteria, insect, and mammalian cells) and purification with 2 ml resin under native or denaturing conditions are described in this manual.If you wish to purify your protein of interest from higher amounts of starting material, you may need to optimize the lysis protocol and purification conditions (amount of resin used for binding). The optimization depends on the expected yield of your protein and amount of resin to use for purification. Perform a pilot experiment to optimize the purification conditions and then based on the pilot experiment results, scale-up accordingly.Continued on next page2Preparing Bacterial Cell Lysate—Native Conditions Follow the procedure below to prepare bacterial cell lysate under native conditions. Scale up or down as necessary.1. Harvest cells from a 50 ml culture by centrifugation (e.g., 5000 rpm for5 minutes in a Sorvall SS-34 rotor). Resuspend the cells in 8 ml NativeBinding Buffer (recipe on page 8).2. Add 8 mg lysozyme and incubate on ice for 30 minutes.3. Using a sonicator equipped with a microtip, sonicate the solution on iceusing six 10-second bursts at high intensity with a 10-second coolingperiod between each burst.Alternatively, sonicate the solution on ice using two or three 10-secondbursts at medium intensity, then flash freeze the lysate in liquid nitrogen or a methanol dry ice slurry. Quickly thaw the lysate at 37°C andperform two more rapid sonicate-freeze-thaw cycles.4. Optional: If the lysate is very viscous, add RNase A (10 µg/ml) andDNase I (5 µg/ml) and incubate on ice for 10–15 minutes. Alternatively,draw the lysate through a 18-gauge syringe needle several times.5. Centrifuge the lysate at 3,000 ×g for 15 minutes to pellet the cellulardebris. Transfer the supernatant to a fresh tube.Note: Some 6xHis-tagged protein may remain insoluble in the pellet, and can be recovered by preparing a denatured lysate (page 4) followed bythe denaturing purification protocol (page 12). To recover this insolubleprotein while preserving its biological activity, you can prepare thedenatured lysate and then follow the hybrid protocol on page 14. Notethat the hybrid protocol may not restore activity in all cases, and should be tested with your particular protein.6. Remove 5 µl of the lysate for SDS-PAGE analysis. Store the remaininglysate on ice or freeze at -20°C. When ready to use, proceed to theprotocol on page 7.Continued on next page3Preparing Bacterial Cell Lysate—Denaturing Conditions Follow the procedure below to prepare bacterial cell lysate under denaturing conditions:1. Equilibrate the Guanidinium Lysis Buffer, pH 7.8 (supplied with thesystem or see page 19 for recipe) to 37°C.2. Harvest cells from a 50 ml culture by centrifugation (e.g., 5000 rpm for5 minutes in a Sorvall SS-34 rotor).3. Resuspend the cell pellet in 8 ml Guanidinium Lysis Buffer from Step 1.4. Slowly rock the cells for 5–10 minutes at room temperature to ensurethorough cell lysis.5. Sonicate the cell lysate on ice with three 5-second pulses at high intensity.6. Centrifuge the lysate at 3,000 ×g for 15 minutes to pellet the cellulardebris.Transfer the supernatant to a fresh tube.7. Remove 5 µl of the lysate for SDS-PAGE analysis. Store the remaininglysate on ice or at -20°C. When ready to use, proceed to the denaturingprotocol on page 11 or hybrid protocol on page 13.Note: To perform SDS-PAGE with samples in Guanidinium Lysis Buffer, you need to dilute the samples, dialyze the samples, or perform TCAprecipitation prior to SDS-PAGE to prevent the precipitation of SDS.Harvesting Insect Cells For detailed protocols dealing with insect cell expression, consult the manual for your particular system. The following lysate protocols are for baculovirus-infected cells and are intended to be highly generic. They should be optimized for your cell lines.For baculovirus-infected insect cells, when the time point of maximal expression has been determined, large scale protein expression can be carried out. Generally, the large-scale expression is performed in 1 liter flasks seeded with cells at a density of 2 × 106 cells/ml in a total volume of 500 ml and infected with high titer viral stock at an MOI of 10 pfu/cell. At the point of maximal expression, harvest cells in 50 ml aliquots. Pellet the cells by centrifugation and store at -70°C until needed. Proceed to preparing cell lysates using native or denaturing conditions as described on the next page.Continued on next page4Preparing Insect Cell Lysate—Native Condition 1. Prepare 8 ml Native Binding Buffer (recipe on page 8) containingLeupeptin (a protease inhibitor) at a concentration of 0.5 µg/ml.2. After harvesting the cells (previous page), resuspend the cell pellet in8 ml Native Binding Buffer containing 0.5 µg/ml Leupeptin.3. Lyse the cells by two freeze-thaw cycles using a liquid nitrogen or dryice/ethanol bath and a 42°C water bath.4. Shear DNA by passing the preparation through an 18-gauge needle fourtimes.5. Centrifuge the lysate at 3,000 ×g for 15 minutes to pellet the cellulardebris.Transfer the supernatant to a fresh tube.6. Remove 5 µl of the lysate for SDS-PAGE analysis. Store remaining lysateon ice or freeze at -20°C. When ready to use, proceed to the protocol on page 7.Preparing Insect Cell Lysate—Denaturing Condition 1. After harvesting insect cells (previous page), resuspend the cell pellet in8 ml Guanidinium Lysis Buffer (supplied with the system or see page 19for recipe).2. Pass the preparation through an 18-gauge needle four times.3. Centrifuge the lysate at 3,000 ×g for 15 minutes to pellet the cellulardebris. Transfer the supernatant to a fresh tube.4. Remove 5 µl of the lysate for SDS-PAGE analysis. Store remaining lysateon ice or freeze at -20° C. When ready to use, proceed to the denaturingprotocol on page 11 or hybrid protocol on page 13.Note: To perform SDS-PAGE with samples in Guanidinium Lysis Buffer, you need to dilute the samples, dialyze the samples, or perform TCAprecipitation prior to SDS-PAGE to prevent the precipitation of SDS.Continued on next pagePreparing Mammalian Cell Lysate—Native Conditions For detailed protocols dealing with mammalian expression, consult the manual for your particular system. The following protocols are intended to be highly generic, and should be optimized for your cell lines.To produce recombinant protein, you need between 5 x 106and 1 x 107 cells. Seed cells and grow in the appropriate medium until they are 80–90% confluent. Harvest cells by trypsinization. You can freeze the cell pellet in liquid nitrogen and store at -70°C until use.1. Resuspend the cell pellet in 8 ml of Native Binding Buffer (page 8). Theaddition of protease inhibitors such as bestatin and leupeptin may benecessary depending on the cell line and expressed protein.2. Lyse the cells by two freeze-thaw cycles using a liquid nitrogen or dryice/ethanol bath and a 42°C water bath.3. Shear the DNA by passing the preparation through an 18-gauge needlefour times.4. Centrifuge the lysate at 3,000 ×g for 15 minutes to pellet the cellulardebris. Transfer the supernatant to a fresh tube.5. Remove 5 µl of the lysate for SDS-PAGE analysis. Store the remaininglysate on ice or freeze at -20° C. When ready to use, proceed to theprotocol on page 7.Preparing Mammalian Cell Lysates—Denaturing Conditions For detailed protocols dealing with mammalian expression, consult the manual for your particular system. The following protocols are intended to be highly generic, and should be optimized for your cell lines.To produce recombinant protein, you need between 5 x 106and 1 x 107 cells. Seed cells and grow in the appropriate medium until they are 80–90% confluent. Harvest cells by trypsinization. You can freeze the cell pellet in liquid nitrogen and store at -70°C until use.1. Resuspend the cell pellet in 8 ml Guanidinium Lysis Buffer (suppliedwith the system or see page 19 for recipe).2. Shear the DNA by passing the preparation through an 18-gauge needlefour times.3. Centrifuge the lysate at 3,000 ×g for 15 minutes to pellet the cellulardebris. Transfer the supernatant to a fresh tube.4. Remove 5 µl of the lysate for SDS-PAGE analysis. Store the remaininglysate on ice or freeze at -20° C until use. When ready to use, proceed to the denaturing protocol on page 11 or hybrid protocol on page 13.Note: To perform SDS-PAGE with samples in Guanidinium Lysis Buffer, you need to dilute the samples, dialyze the samples, or perform TCAprecipitation prior to SDS-PAGE to prevent the precipitation of SDS.Purification Procedure—Native ConditionsIntroduction In the following procedure, use the prepared Native Binding Buffer, NativeWash Buffer, and Native Elution Buffer, columns, and cell lysate preparedunder native conditions. Be sure to check the pH of your buffers before starting.Buffers for Native Purification All buffers for purification under native conditions are prepared from the5X Native Purification Buffer supplied with the system. Dilute and adjust the pH of the 5X Native Purification Buffer to create 1X Native Purification Buffer (page 8). From this, you can create the following buffers:• Native Binding Buffer• Native Wash Buffer• Native Elution BufferThe recipes described in this section will create sufficient buffers to perform one native purification using one kit-supplied purification column. Scale up accordingly.If you are preparing your own buffers, see page 18 for recipe.Materials Needed You will need the following items:• 5X Native Purification Buffer (supplied with the system or see page 18 forrecipe)• 3 M Imidazole (supplied with the system or see page 18 for recipe)• NaOH• HCl• Sterile distilled water• Prepared ProBond™ columns with native buffers (next page)• Lysate prepared under native conditions (page 2)Imidazole Concentration in Native Buffers Imidazole is included in the Native Wash and Elution Buffers to minimize the binding of untagged, contaminating proteins and increase the purity of the target protein with fewer wash steps. Note that, if your level of contaminating proteins is high, you may add imidazole to the Native Binding Buffer.If your protein does not bind well under these conditions, you can experiment with lowering or eliminating the imidazole in the buffers and increasing the number of wash and elution steps.Continued on next page1X Native Purification Buffer To prepare 100 ml 1X Native Purification Buffer, combine:• 80 ml of sterile distilled water• 20 ml of 5X Native Purification Buffer (supplied with the system or see page 18 for recipe)Mix well and adjust pH to 8.0 with NaOH or HCl.Native Binding Buffer Without ImidazoleUse 30 ml of the 1X Native Purification Buffer (see above for recipe) for use as the Native Binding Buffer (used for column preparation, cell lysis, and binding).With Imidazole (Optional):You can prepare the Native Binding Buffer with imidazole to reduce the binding of contaminating proteins. (Note that some His-tagged proteins may not bind under these conditions.).To prepare 30 ml Native Binding Buffer with 10 mM imidazole, combine: • 30 ml of 1X Native Purification Buffer• 100 µl of 3 M Imidazole, pH 6.0Mix well and adjust pH to 8.0 with NaOH or HCl.Native Wash Buffer To prepare 50 ml Native Wash Buffer with 20 mM imidazole, combine:• 50 ml of 1X Native Purification Buffer• 335 µl of 3 M Imidazole, pH 6.0Mix well and adjust pH to 8.0 with NaOH or HCl.Native Elution Buffer To prepare 15 ml Native Elution Buffer with 250 mM imidazole, combine:• 13.75 ml of 1X Native Purification Buffer• 1.25 ml of 3 M Imidazole, pH 6.0Mix well and adjust pH to 8.0 with NaOH or HCl.Continued on next pageDo not use strong reducing agents such as DTT with ProBond™ columns. DTTreduces the nickel ions in the resin. In addition, do not use strong chelatingagents such as EDTA or EGTA in the loading buffers or wash buffers, as thesewill strip the nickel from the columns.Be sure to check the pH of your buffers before starting.PreparingProBond™ ColumnWhen preparing a column as described below, make sure that the snap-off capat the bottom of the column remains intact. To prepare a column:1. Resuspend the ProBond™ resin in its bottle by inverting and gentlytapping the bottle repeatedly.2. Pipet or pour 2 ml of the resin into a 10-ml Purification Columnsupplied with the kit. Allow the resin to settle completely by gravity(5-10 minutes) or gently pellet it by low-speed centrifugation (1 minuteat 800 ×g). Gently aspirate the supernatant.3. Add 6 ml of sterile, distilled water and resuspend the resin byalternately inverting and gently tapping the column.4. Allow the resin to settle using gravity or centrifugation as described inStep 2, and gently aspirate the supernatant.5. For purification under Native Conditions, add 6 ml Native BindingBuffer (recipe on page 8).6. Resuspend the resin by alternately inverting and gently tapping thecolumn.7. Allow the resin to settle using gravity or centrifugation as described inStep 2, and gently aspirate the supernatant.8. Repeat Steps 5 through 7.Storing PreparedColumnsTo store a column containing resin, add 0.02% azide or 20% ethanol as apreservative and cap or parafilm the column. Store at room temperature.Continued on next pagePurification Under Native Conditions Using the native buffers, columns and cell lysate, follow the procedure below to purify proteins under native conditions:1. Add 8 ml of lysate prepared under native conditions to a preparedPurification Column (page 9).2. Bind for 30–60 minutes using gentle agitation to keep the resinsuspended in the lysate solution.3. Settle the resin by gravity or low speed centrifugation (800 ×g), andcarefully aspirate the supernatant. Save supernatant at 4°C forSDS-PAGE analysis.4. Wash with 8 ml Native Wash Buffer (page 8). Settle the resin by gravityor low speed centrifugation (800 ×g), and carefully aspirate thesupernatant. Save supernatant at 4°C for SDS-PAGE analysis.5. Repeat Step 4 three more times.6. Clamp the column in a vertical position and snap off the cap on thelower end. Elute the protein with 8–12 ml Native Elution Buffer (seepage 2). Collect 1 ml fractions and analyze with SDS-PAGE.Note: Store the eluted fractions at 4°C. If -20°C storage is required, addglycerol to the fractions. For long term storage, add protease inhibitors to the fractions.If you wish to reuse the resin to purify the same recombinant protein, wash the resin with 0.5 M NaOH for 30 minutes and equilibrate the resin in a suitable binding buffer. If you need to recharge the resin, see page 17.Purification Procedure—Denaturing ConditionsIntroduction Instructions to perform purification using denaturing conditions with prepareddenaturing buffers, columns, and cell lysate are described below.Materials Needed You will need the following items:• Denaturing Binding Buffer (supplied with the system or see page 19 forrecipe)• Denaturing Wash Buffer, pH 6.0 (supplied with the system or see page 19 forrecipe) and Denaturing Wash Buffer, pH 5.3 (see recipe below)• Denaturing Elution Buffer (supplied with the system or see page 20 forrecipe)• Prepared ProBond™ columns with Denaturing buffers (see below)• Lysate prepared under denaturing conditions (page 11)Preparing the Denaturing Wash Buffer pH 5.3 Using a 10 ml aliquot of the kit-supplied Denaturing Wash Buffer (pH 6.0), mix well, and adjust the pH to 5.3 using HCl. Use this for the Denaturing Wash Buffer pH 5.3 in Step 5 next page.Be sure to check the pH of your buffers before starting. Note that thedenaturing buffers containing urea will become more basic over time. PreparingProBond™ ColumnWhen preparing a column as described below, make sure that the snap-off capat the bottom of the column remains intact.If you are reusing the ProBond™ resin, see page 17 for recharging protocol.To prepare a column:1. Resuspend the ProBond™ resin in its bottle by inverting and gentlytapping the bottle repeatedly.2. Pipet or pour 2 ml of the resin into a 10-ml Purification Columnsupplied with the kit. Allow the resin to settle completely by gravity(5-10 minutes) or gently pellet it by low-speed centrifugation (1 minuteat 800 ×g). Gently aspirate the supernatant.3. Add 6 ml of sterile, distilled water and resuspend the resin byalternately inverting and gently tapping the column.4. Allow the resin to settle using gravity or centrifugation as described inStep 2, and gently aspirate the supernatant.5. For purification under Denaturing Conditions, add 6 ml of DenaturingBinding Buffer.6. Resuspend the resin by alternately inverting and gently tapping thecolumn.7. Allow the resin to settle using gravity or centrifugation as described inStep 2, and gently aspirate the supernatant. Repeat Steps 5 through 7.Continued on next pagePurification Procedure—Denaturing Conditions, ContinuedPurification Under Denaturing Conditions Using the denaturing buffers, columns, and cell lysate, follow the procedure below to purify proteins under denaturing conditions:1. Add 8 ml lysate prepared under denaturing conditions to a preparedPurification Column (page 11).2. Bind for 15–30 minutes at room temperature using gentle agitation (e.g.,using a rotating wheel) to keep the resin suspended in the lysatesolution. Settle the resin by gravity or low speed centrifugation (800 ×g), and carefully aspirate the supernatant.3. Wash the column with 4 ml Denaturing Binding Buffer supplied with thekit by resuspending the resin and rocking for two minutes. Settle theresin by gravity or low speed centrifugation (800 ×g), and carefullyaspirate the supernatant. Save supernatant at 4°C for SDS-PAGEanalysis. Repeat this step one more time.4. Wash the column with 4 ml Denaturing Wash Buffer, pH 6.0 supplied inthe kit by resuspending the resin and rocking for two minutes. Settle the resin by gravity or low speed centrifugation (800 ×g), and carefullyaspirate the supernatant. Save supernatant at 4°C for SDS-PAGEanalysis. Repeat this step one more time.5. Wash the column with 4 ml Denaturing Wash Buffer pH 5.3 (see recipeon previous page) by resuspending the resin and rocking for 2 minutes.Settle the resin by gravity or low speed centrifugation (800 ×g), andcarefully aspirate the supernatant. Save supernatant at 4°C for SDS-PAGE analysis. Repeat this step once more for a total of two washes with Denaturing Wash Buffer pH 5.3.6. Clamp the column in a vertical position and snap off the cap on thelower end. Elute the protein by adding 5 ml Denaturing Elution Buffersupplied with the kit. Collect 1 ml fractions and monitor the elution bytaking OD280readings of the fractions. Pool the fractions that contain the peak absorbance and dialyze against 10 mM Tris, pH 8.0, 0.1% Triton X-100 overnight at 4°C to remove the urea. Concentrate the dialyzedmaterial by any standard method (i.e., using 10,000 MW cut-off, low-protein binding centrifugal instruments or vacuum concentrationinstruments).If you wish to reuse the resin to purify the same recombinant protein, wash the resin with 0.5 M NaOH for 30 minutes and equilibrate the resin in a suitable binding buffer. If you need to recharge the resin, see page 17.。

简述真核生物的转录后修饰过程和意义

英文回答：Eukaryotic transcriptional modification pertains to the post-transcriptional processing of the primary transcript, or pre-mRNA. This intricate process epasses a sequence of molecular events epassing 5' capping, splicing, and 3' polyadenylation. 5' capping involves the addition of a 7-methylguanosine cap to the 5' end of the pre-mRNA, conferring protection against degradation and facilitating translation initiation. Splicing denotes the removal of introns from the pre-mRNA and the subsequent ligation of exons, culminating in the formation of mature mRNA. This pivotal step significantly amplifies the coding potential of the genome and enables the generation of diverse protein isoforms from a single gene. Finally, 3' polyadenylation entails the addition of a poly(A) tail to the 3' end of the mRNA, which also governs mRNA stability and translation initiation.语录转录修改涉及语录后处理主语录，或mRNA前。

皱纹盘鲍(Haliotis discushannai Ino)胚胎RNA分离和cDNA文库构建

第37卷　第4期2006年7月海　洋　与　湖　沼OCEANOLOGI A ET L I M NOLOGI A SI N I CAVol 137,No 14July,2006皱纹盘鲍(H a liotis discus hanna i I no)胚胎RNA 分离和c D NA 文库构建3刘　晓　赵　敏　高其康　张国范(中国科学院海洋研究所　青岛　266071)(中国科学院海洋研究所　青岛　266071;中国科学院研究生院　北京　100039)(浙江大学分析测试中心　杭州　310029) 3国家自然科学基金项目,30271018号;国家863计划项目,2004AA626070号;青岛市自然科学基金项目,03222JZP 29号资助。

刘　晓,博士,研究员,E 2mail:liuxiao@m s .qdi o .ac .cn收稿日期:2005202216,收修改稿日期:2006201222提要从皱纹盘鲍胚胎样本中分离RNA 并构建了c DNA 文库。

分别收集孵化8、10和12h 的皱纹盘鲍胚胎,用抽滤除菌的海水反复悬浮胚胎,将这3个发育阶段的胚胎样本等量混合后用T R I Z OL 试剂提取总RNA,将提取物用酚2氯仿2异戊醇再抽提2次,分离获得高质量的总RNA 。

从总RNA 中分离mRNA,构建了皱纹盘鲍混合胚胎的c DNA 文库,未扩增胚胎文库的滴度为510×106pfu /m l,重组率为94112%,插入片段均大于400bp,7016%克隆的插入片段分布在1000—1500bp 之间,扩增后的文库滴度为3106×109pfu /m l 。

关键词皱纹盘鲍,胚胎,c DNA 文库,基因表达中图分类号 Q132 皱纹盘鲍(Haliotis discus hannai I no )自然分布于北太平洋海区。

从上世纪80年代中期实现人工育苗以来,我国的皱纹盘鲍养殖业发展较快,现已成为重要的养殖贝类。

Pichia Fermentation Process Guidelines

Version B Pichia Fermentation Process GuidelinesOverviewIntroduction Pichia pastoris, like Saccharomyces cerevisiae, is particularly well-suited forfermentative growth. Pichia has the ability to reach very high cell densities duringfermentation which may improve overall protein yields.We recommend that only those with fermentation experience or those who have accessto people with experience attempt fermentation. Since there are a wide variety offermenters available, it is difficult to provide exact procedures for your particular case.The guidelines given below are based on fermentations of both Mut+ and Mut S Pichiastrains in a 15 liter table-top glass fermenter. Please read the operator's manual for yourparticular fermenter before beginning. The table below provides an overview of thematerial covered in these guidelines.Step Topic Page1 Fermentation parameters 12 Equipment needed and preparation of medium 23 Measurement and use of dissolved oxygen (DO) in the culture 34 Growth of the inoculum 45 Generation of biomass on glycerol in batch and fed-batch phases 4-56 Induction of expression of Mut+ and Mut S recombinants in themethanol fed-batch phase6-77 Harvesting and lysis of cells 88 References 9-109 Recipes 11Fermentation Parameters It is important to monitor and control the following parameters throughout the fermentation process. The following table describes the parameters and the reasons for monitoring them.Parameter ReasonTemperature (30.0XC) Growth above 32XC is detrimental to protein expression Dissolved oxygen (>20%) Pichia needs oxygen to metabolize glycerol andmethanolpH (5.0-6.0 and 3.0) Important when secreting protein into the medium andfor optimal growthAgitation (500 to 1500 rpm) Maximizes oxygen concentration in the medium Aeration (0.1 to 1.0 vvm*for glass fermenters)Maximizes oxygen concentration in the medium whichdepends on the vesselAntifoam (the minimumneeded to eliminate foam)Excess foam may cause denaturation of your secretedprotein and it also reduces headspaceCarbon source (variablerate)Must be able to add different carbon sources at differentrates during the course of fermentationcontinued on next pageOverview, continuedRecommended Equipment Below is a checklist for equipment recommendations.ï A jacketed vessel is needed for cooling the yeast during fermentation, especially during methanol induction. You will need a constant source of cold water (5-10XC). This requirement may mean that you need a refrigeration unit to keep the water cold. ï A foam probe is highly recommended as antifoam is required.ï A source of O2--either air (stainless steel fermenters at 1-2 vvm) or pure O2(0.1-0.3 vvm for glass fermenters).ï Calibrated peristaltic pumps to feed the glycerol and methanol.ï Automatic control of pH.Medium Preparation You will need to prepare the appropriate amount of following solutions:ï Fermentation Basal Salts (page 11)ï PTM1Trace Salts (page 11)ï ~75 ml per liter initial fermentation volume of 50% glycerol containing 12 ml PTM1 Trace Salts per liter of glycerol.ï ~740 ml per liter initial fermentation volume of 100% methanol containing 12 mlPTM1Trace Salts per liter of methanol.Monitoring the Growth of Pichia pastoris Cell growth is monitored at various time points by using the absorbance at 600 nm (OD600) and the wet cell weight. The metabolic rate of the culture is monitored by observing changes in the concentration of dissolved oxygen in response to carbon availability (see next page).Dissolved Oxygen (DO) MeasurementIntroduction The dissolved oxygen concentration is the relative percent of oxygen in the mediumwhere 100% is O2-saturated medium. Pichia will consume oxygen as it grows, reducing the dissolved oxygen content. However, because oxygen is required for the first step ofmethanol catabolism, it is important to maintain the dissolved oxygen (DO) concentra-tion at a certain level (>20%) to ensure growth of Pichia on methanol. Accuratemeasurement and observation of the dissolved oxygen concentration of a culture willgive you important information about the state and health of the culture. Therefore, it isimportant to accurately calibrate your equipment. Please refer to your operator's manual.Maintaining the Dissolved Oxygen Concentration (DO) 1. Maintaining the dissolved oxygen above 20% may be difficult depending on theoxygen transfer rates (OTR) of the fermenter, especially in small-scale glassvessels. In a glass vessel, oxygen is needed to keep the DO above 20%, usually~0.1-0.3 vvm (liters of O2per liter of fermentation culture per minute). Oxygen consumption varies and depends on the amount of methanol added and the protein being expressed.2. Oxygen can be used at 0.1 to 0.3 vvm to achieve adequate levels. This can beaccomplished by mixing with the air feed and can be done in any glass fermenter.For stainless steel vessels, pressure can be used to increase the OTR. Be sure toread the operator's manual for your particular fermenter.3. If a fermenter cannot supply the necessary levels of oxygen, then the methanol feedshould be scaled back accordingly. Note that decreasing the amount of methanol may reduce the level of protein expression.4. To reach maximum expression levels, the fermentation time can be increased todeliver similar levels of methanol at the lower feed rate. For many recombinantproteins, a direct correlation between amount of methanol consumed and theamount of protein produced has been observed.Use of DO Measurements During growth, the culture consumes oxygen, keeping the DO concentration low. Note that oxygen is consumed whether the culture is grown on glycerol or methanol. The DO concentration can be manipulated to evaluate the metabolic rate of the culture and whether the carbon source is limiting. The metabolic rate indicates how healthy the culture is. Determining whether the carbon source is limiting is important if you wish to fully induce the AOX1 promoter. For example, changes in the DO concentrations (DO spikes) allow you to determine whether all the glycerol is consumed from the culture before adding methanol. Secondly, it ensures that your methanol feed does not exceed the rate of consumption. Excess methanol (> 1-2% v/v) may be toxic.Manipulation of DO If carbon is limiting, shutting off the carbon source should cause the culture to decrease its metabolic rate, and the DO to rise (spike). Terminate the carbon feed and time how long it takes for the DO to rise 10%, after which the carbon feed is turned back on. If the lag time is short (< 1 minute), the carbon source is limiting.Fermenter Preparation and Glycerol Batch PhaseInoculum Seed Flask Preparation Remember not to put too much medium in the baffled flasks. Volume should be 10-30% of the total flask volume.1. Baffled flasks containing a total of 5-10% of the initial fermentation volume ofMGY or BMGY are inoculated with a colony from a MD or MGY plate or from a frozen glycerol stock.2. Flasks are grown at 30XC, 250-300 rpm, 16-24 hours until OD600= 2-6. Toaccurately measure OD600> 1.0, dilute a sample of your culture 10-fold before reading.Glycerol Batch Phase 1. Sterilize the fermenter with the Fermentation Basal Salts medium containing 4%glycerol (see page 11).2. After sterilization and cooling, set temperature to 30XC, agitation and aeration tooperating conditions (usually maximum rpm and 0.1-1.0 vvm air), and adjust the pH of the Fermentation Basal Salts medium to 5.0 with 28% ammonium hydroxide(undiluted ammonium hydroxide). Add aseptically 4.35 ml PTM1trace salts/liter of Fermentation Basal Salts medium.3. Inoculate fermenter with approximately 5-10% initial fermentation volume from theculture generated in the inoculum shake flasks. Note that the DO will be close to 100% before the culture starts to grow. As the culture grows, it will consumeoxygen, causing the DO to decrease. Be sure to keep the DO above 20% by adding oxygen as needed.4. Grow the batch culture until the glycerol is completely consumed (18 to 24 hours).This is indicated by an increase in the DO to 100%. Note that the length of timeneeded to consume all the glycerol will vary with the density of the initial inoculum.5. Sampling is performed at the end of each fermentation stage and at least twice daily.We take 10 ml samples for each time point, then take 1 ml aliquots from this 10 mlsample. Samples are analyzed for cell growth (OD600and wet cell weight), pH, microscopic purity, and protein concentrations or activity. Freeze the cell pellets and supernatants at -80XC for later analysis. Proceed to Glycerol Fed-Batch Phase,page 5.Yield A cellular yield of 90 to 150 g/liter wet cells is expected for this stage. Recombinant protein will not yet be produced due to the absence of methanol.Introduction Once all the glycerol is consumed from the batch growth phase, a glycerol feed isinitiated to increase the cell biomass under limiting conditions. When you are ready toinduce with methanol, you can use DO spikes to make sure the glycerol is limited.Glycerol Fed-Batch Phase 1. Initiate a 50% w/v glycerol feed containing 12 ml PTM1trace salts per liter of glycerol feed. Set the feed rate to 18.15 ml/hr /liter initial fermentation volume.2. Glycerol feeding is carried out for about four hours or longer (see below). A cellularyield of 180 to 220 g/liter wet cells should be achieved at the end of this stage while no appreciable recombinant protein is produced.Note The level of expressed protein depends on the cell mass generated during the glycerolfed-batch phase. The length of this feed can be varied to optimize protein yield. A rangeof 50 to 300 g/liter wet cells is recommended for study. A maximum level of 4%glycerol is recommended in the batch phase due to toxicity problems with higher levelsof glycerol.Important If dissolved oxygen falls below 20%, the glycerol or methanol feed should bestopped and nothing should be done to increase oxygen rates until the dissolvedoxygen spikes. At this point, adjustments can be made to agitation, aeration, pressure oroxygen feeding.Proteases In the literature, it has been reported that if the pH of the fermentation medium islowered to 3.0, neutral proteases are inhibited. If you think neutral proteases aredecreasing your protein yield, change the pH control set point to 3.0 during the glycerolfed-batch phase (above) or at the beginning of the methanol induction (next page) andallow the metabolic activity of the culture to slowly lower the pH to 3.0 over 4 to 5 hours(Brierley, et al., 1994; Siegel, et al., 1990).Alternatively, if your protein is sensitive to low pH, it has been reported that inclusion ofcasamino acids also decreases protease activity (Clare, et al., 1991).Introduction All of the glycerol needs to be consumed before starting the methanol feed to fullyinduce the AOX1 promoter on methanol. However, it has been reported that a "mixedfeed" of glycerol and methanol has been successful to express recombinant proteins(Brierley, et al., 1990; Sreekrishna, et al., 1989). It is important to introduce methanolslowly to adapt the culture to growth on methanol. If methanol is added too fast, it willkill the cells. Once the culture is adapted to methanol, it is very important to use DOspikes to analyze the state of the culture and to take time points over the course ofmethanol induction to optimize protein expression. Growth on methanol also generates alot of heat, so temperature control at this stage is very important.Mut+ Methanol Fed-Batch Phase 1. Terminate glycerol feed and initiate induction by starting a 100% methanol feedcontaining 12 ml PTM1trace salts per liter of methanol. Set the feed rate to 3.6 ml/hr per liter initial fermentation volume.2. During the first 2-3 hours, methanol will accumulate in the fermenter and thedissolved oxygen values will be erratic while the culture adapts to methanol.Eventually the DO reading will stabilize and remain constant.3. If the DO cannot be maintained above 20%, stop the methanol feed, wait for theDO to spike and continue on with the current methanol feed rate. Increaseagitation, aeration, pressure or oxygen feeding to maintain the DO above 20%. 4. When the culture is fully adapted to methanol utilization (2-4 hours), and is limitedon methanol, it will have a steady DO reading and a fast DO spike time (generally under 1 minute). Maintain the lower methanol feed rate under limited conditions for at least 1 hour after adaptation before doubling the feed. The feed rate is then doubled to ~7.3 ml/hr/liter initial fermentation volume.5 After 2 hours at the 7.3 ml/hr/liter feed rate, increase the methanol feed rate to~10.9 ml/hr per liter initial fermentation volume. This feed rate is maintainedthroughout the remainder of the fermentation.6. The entire methanol fed-batch phase lasts approximately 70 hours with a total ofapproximately 740 ml methanol fed per liter of initial volume. However, this may vary for different proteins.Note: The supernatant may appear greenish. This is normal.Yield The cell density can increase during the methanol fed-batch phase to a final level of 350 to 450 g/liter wet cells. Remember that because most of the fermentation is carried out ina fed-batch mode, the final fermentation volume will be approximately double the initialfermentation volume.Fermentation of Mut S Pichia Strains Since Mut S cultures metabolize methanol poorly, their oxygen consumption is very low. Therefore, you cannot use DO spikes to evaluate the culture. In standard fermentations of a Mut S strain, the methanol feed rate is adjusted to maintain an excess of methanol in the medium which does not exceed 0.3% (may be determined by gas chromatography). While analysis by gas chromatography will insure that nontoxic levels of methanol are maintained, we have used the empirical guidelines below to express protein in Mut S strains. A gas chromatograph is useful for analyzing and optimizing growth of Mut S recombinants.continued on next pageMethanol Fed-Batch Phase, continuedMut S Methanol Fed- Batch Phase The first two phases of the glycerol batch and fed-batch fermentations of the Mut S strains are conducted as described for the Mut+ strain fermentations. The methanol induction phases of the Mut+ and Mut S differ in terms of the manner and amount in which the methanol feed is added to the cultures.1. The methanol feed containing 12 ml PTM1trace salts per liter of methanol is initiated at 1 ml/hr/liter initial fermentation volume for the first two hours. It is then increased in 10% increments every 30 minutes to a rate of 3 ml/hr which ismaintained for the duration of the fermentation.2.. The vessel is then harvested after ~100 hours on methanol. This time may be variedto optimize protein expression.Harvesting and Lysis of CellsIntroduction The methods and equipment listed below are by no means complete. The amount of cells or the volume of supernatant will determine what sort of equipment you need.Harvesting Cells and Supernatant For small fermentations (1-10 liters), the culture can be collected into centrifuge bottles (500-1000 ml) and centrifuged to separate the cells from the supernatant.For large fermentations, large membrane filtration units (Millipore) or a Sharples centrifuge can be used to separate cells from the supernatant. The optimal method will depend on whether you need the cells or the supernatant as the source of your protein and what you have available.Supernatants can be loaded directly onto certain purification columns or concentrated using ultrafiltration.Cell Lysis We recommend cell disruption using glass beads as described in Current Protocols inMolecular Biology, page 13.13.4. (Ausubel, et al., 1990) or Guide to ProteinPurification (Deutscher, 1990). This method may be tedious for large amounts of cells.For larger amounts, we have found that a microfluidizer works very well. Frenchpressing the cells does not seem to work as well as the glass beads or the microfluidizer.ReferencesIntroduction Most of the references below refer to papers where fermentation of Pichia wasperformed. Note that some of these are patent papers. You can obtain copies of patentsusing any of the following methods.ï Patent Depository Libraries. U. S. patents and international patents granted underthe Patent Cooperation Treaty (PCT) are available on microfilm. These can be copiedand mailed or faxed depending on length. There is a fee for this service. The referencelibrarian at your local library can tell you the location of the nearest Patent DepositoryLibrary.ï Interlibrary Loan. If you are not near a Patent Depository Library, you may request acopy of the patent through interlibrary loan. There will be a fee for this service.ï U. S. Patent Office. Requests may be made directly to the Patent Office, Arlington,VA. Please call 703-557-4636 for more information on cost and delivery.ï Private Library Services. There are private companies who will retrieve and sendyou patents for a fee. Two are listed below:Library Connection: 804-758-3311Rapid Patent Services: 800-336-5010Citations Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A.,Struhl, K., eds (1990) Current Protocols in Molecular Biology. GreenePublishing Associates and Wiley-Interscience, New York.Brierley, R. A., Siegel, R. S., Bussineau, C. M. Craig, W. S., Holtz, G. C., Davis, G. R.,Buckholz, R. G., Thill, G. P., Wondrack, L. M., Digan, M. E., Harpold, M. M.,Lair, S. V., Ellis, S. B., and William, M. E. (1989) Mixed Feed RecombinantYeast Fermentation. International Patent (PCT) Application. Publication No.WO 90/03431.Brierley, R. A., Bussineau, C., Kosson, R., Melton, A., and Siegel, R. S. (1990)Fermentation Development of Recombinant Pichia pastoris Expressing theHeterologous Gene: Bovine Lysozyme. Ann. New York Acad. Sci.589: 350-362.Brierley, R. A., Davis, G. R. and Holtz, G. C. (1994) Production of Insulin-Like GrowthFactor-1 in Methylotrophic Yeast Cells. United States Patent5,324,639.Clare, J. J., Romanos, M. A., Rayment, F. B., Rowedder, J. E., Smith, M. A., Payne, M.M., Sreekrishna, K. and Henwood, C. A. (1991) Production of EpidermalGrowth Factor in Yeast: High-level Secretion Using Pichia pastoris StrainsContaining Multiple Gene Copies. Gene105: 205-212.Cregg, J. M., Tschopp, J. F., Stillman, C., Siegel, R., Akong, M., Craig, W. S.,Buckholz, R. G., Madden, K. R., Kellaris, P. A., Davis, G. R., Smiley, B. L.,Cruze, J., Torregrossa, R., VeliÁelebi, G. and Thill, G. P. (1987) High-levelExpression and Efficient Assembly of Hepatitis B Surface Antigen in theMethylotrophic Yeast Pichia pastoris. Bio/Technology5: 479-485.Cregg, J. M., Vedvick, T. S. and Raschke, W. C. (1993) Recent Advances in theExpression of Foreign Genes in Pichia pastoris. Bio/Technology11: 905-910.Deutscher, M. P. (1990) Guide to Protein Purification. In: Methods in Enzymology (J.N. Abelson and M. I. Simon, eds.) Academic Press, San Diego, CA.continued on next pageReferences, continuedCitations, continuedDigan, M. E., Lair, S. V., Brierley, R. A., Siegel, R. S., Williams, M. E., Ellis, S. B., Kellaris, P. A., Provow, S. A., Craig, W. S., VeliÁelebi, G., Harpold, M. M. andThill, G. P. (1989) Continuous Production of a Novel Lysozyme via Secretionfrom the Yeast Pichia pastoris. Bio/Technology7: 160-164.Hagenson, M. J., Holden, K. A., Parker, K. A., Wood, P. J., Cruze, J. A., Fuke, M., Hopkins, T. R. and Stroman, D. W. (1989) Expression of Streptokinase inPichia pastoris Yeast. Enzyme Microbiol. Technol.11: 650-656.Laroche, Y., Storme, V., Meutter, J. D., Messens, J. and Lauwereys, M. (1994) High-Level Secretion and Very Efficient Isotopic Labeling of Tick AnticoagulantPeptide (TAP) Expressed in the Methylotrophic Yeast, Pichia pastoris.Bio/Technology12: 1119-1124.Romanos, M. A., Clare, J. J., Beesley, K. M., Rayment, F. B., Ballantine, S. P., Makoff,A. J., Dougan, G., Fairweather, N. F. and Charles, I. G. (1991) RecombinantBordetella pertussis Pertactin p69 from the Yeast Pichia pastoris High LevelProduction and Immunological Properties. Vaccine9: 901-906.Siegel, R. S. and Brierley, R. A. (1989) Methylotrophic Yeast Pichia pastoris Produced in High-cell-density Fermentations With High Cell Yields as Vehicle forRecombinant Protein Production. Biotechnol. Bioeng.34: 403-404.Siegel, R. S., Buckholz, R. G., Thill, G. P., and Wondrack, L. M. (1990) Production of Epidermal Growth Factor in Methylotrophic Yeast Cells. International Patent(PCT) Application. Publication No. WO 90/10697.Sreekrishna, K., Nelles, L., Potenz, R., Cruse, J., Mazzaferro, P., Fish, W., Fuke, M., Holden, K., Phelps, D., Wood, P. and Parker, K. (1989) High LevelExpression, Purification, and Characterization of Recombinant Human TumorNecrosis Factor Synthesized in the Methylotrophic Yeast Pichia pastoris.Biochemistry28(9): 4117-4125.©2002 Invitrogen Corporation. All rights reservedRecipesFermentation Basal Salts Medium For 1 liter, mix together the following ingredients:Phosphoric acid, 85% (26.7 ml)Calcium sulfate 0.93 gPotassium sulfate 18.2 gMagnesium sulfate-7H2O 14.9 gPotassium hydroxide 4.13 gGlycerol 40.0 gWater to 1 literAdd to fermenter with water to the appropriate volume and sterilize.PTM1 Trace Salts Mix together the following ingredients:Cupric sulfate-5H2O 6.0 gSodium iodide 0.08 gManganese sulfate-H2O 3.0 gSodium molybdate-2H2O 0.2 gBoric Acid 0.02 gCobalt chloride 0.5 gZinc chloride 20.0 gFerrous sulfate-7H2O 65.0 gBiotin 0.2 gSulfuric Acid 5.0 mlWater to a final volume of 1 literFilter sterilize and store at room temperature.Note: There may be a cloudy precipitate upon mixing of these ingredients. Filter-sterilize as above and use.11。

2008 Many species in one DNA barcoding overestimates the number of species PNAS-2008-Song-13486-91

Many species in one:DNA barcoding overestimates the number of species when nuclear mitochondrial pseudogenes are coamplifiedHojun Song*†,Jennifer E.Buhay*‡,Michael F.Whiting*,and Keith A.Crandall**Department of Biology,Brigham Young University,Provo,UT 84602;and ‡Belle W.Baruch Institute for Marine Sciences,University of South Carolina,Columbia,SC 29208Edited by W.Ford Doolittle,Dalhousie University,Halifax,NS,Canada,and approved July 14,2008(received for review March 28,2008)Nuclear mitochondrial pseudogenes (numts)are nonfunctional copies of mtDNA in the nucleus that have been found in major clades of eukaryotic organisms.They can be easily coampliﬁed with orthologous mtDNA by using conserved universal primers;however,this is especially problematic for DNA barcoding,which attempts to characterize all living organisms by using a short fragment of the mitochondrial cytochrome c oxidase I (COI)gene.Here,we study the effect of numts on DNA barcoding based on phylogenetic and barcoding analyses of numt and mtDNA se-quences in two divergent lineages of arthropods:grasshoppers and crayﬁsh.Single individuals from both organisms have numts of the COI gene,many of which are highly divergent from ortholo-gous mtDNA sequences,and DNA barcoding analysis incorrectly overestimates the number of unique species based on the standard metric of 3%sequence divergence.Removal of numts based on a careful examination of sequence characteristics,including indels,in-frame stop codons,and nucleotide composition,drastically re-duces the incorrect inferences of the number of unique species,but even such rigorous quality control measures fail to identify certain numts.We also show that the distribution of numts is lineage-speciﬁc and the presence of numts cannot be known a priori .Whereas DNA barcoding strives for rapid and inexpensive gener-ation of molecular species tags,we demonstrate that the presence of COI numts makes this goal difﬁcult to achieve when numts are prevalent and can introduce serious ambiguity into DNA barcoding.cytochrome c oxidase I ͉Decapoda ͉OrthopteraThe orthology of characters is one of the fundamental and implicit assumptions in the use of DNA sequence data to reconstruct phylogeny or to establish ‘‘barcodes’’for species.If the orthology assumption is violated,that is,whether paralogous sequences are unknowingly treated as orthologs,incorrect in-ferences are inevitable (1).This is especially true for the DNA barcoding initiative,which relies on the premise that all organ-isms have a unique and identifiable molecular tag,namely,a short region of mitochondrial cytochrome c oxidase subunit 1(COI)amplified by universal primers,and that one is comparing only orthologs among species when formulating barcodes (2).As such,DNA barcoding relies on the assumption that the COI fragments generated by PCR from genomic DNA represent orthologous copies of mitochondrial DNA (mtDNA).Increasing empirical evidence suggests;however,that this assumption does not always hold true and that there are a number of molecular evolutionary processes that can hinder correct amplification and identification of the orthologs (3),including (i)duplication of the gene of interest within the mitochondrial genome (4),(ii)heteroplasmy (5),(iii)bacterial infection biasing mtDNA vari-ation (6),and (iv)nuclear integration of mtDNA (7,8).If a portion of COI was duplicated in a given species,conventional PCR might amplify both the correct and duplicated COI frag-ments,thus introducing ambiguity into the barcoding whether the paralogous copy had diverged since duplication.Hetero-plasmy is the presence of a mixture of more than one type of mitochondrial genome within a single individual,and the coam-plification of divergent heteroplasmic copies of mtDNA would lead to an overestimation of the number of unique species under barcoding (3).Maternally inherited symbionts,such as Wolba-chia ,can cause linkage disequilibrium with mtDNA and,whether a population becomes infected with such symbionts,the mtDNA associated with the initial infection will spread through-out the population and result in the homogenization of mtDNA haplotypes (6).Among closely related species,these symbionts can break through the species barrier by hybridization followed by selective sweep,resulting in identical mtDNA sequences among different species,which would cause the underestimation of the number of unique species under barcoding (9).Whereas these three processes may be relatively uncommon and limited to a small number of organisms,a fourth process,the nuclear integration of mtDNA that gives rise to nuclear mitochondrial pseudogenes (numts),is a widespread phenomenon that has been reported in many eukaryotic clades (8,10).The effect of numts on DNA barcoding,however,has not been systematically studied to date.The first case of numts in Metazoa was reported in the grasshopper Locusta migratoria (11),in which a copy of a mitochondrial ribosomal RNA gene was found in the nuclear genome.Lopez et al.(12)found that nearly half of the mito-chondrial genome (7.9kb)was transferred to the nuclear ge-nome in the domestic cat and coined the term ‘‘numts.’’Since then,Ͼ82eukaryotes have been reported to have numts (8).A BLAST search of mitochondrial sequences in the published nuclear genomes suggests that nearly 99%of the mitochondrial sequences were transferred to different parts of the nucleus in both human and mouse (10).Pamilo et al.(13)reported Ͼ2,000possible numts in the honey bee genome and found a similarly large number of numt copies in the flour beetle genome.These findings collectively indicate that numts are extremely pervasive in nature and that there may be a large number of species with unrealized numts of the COI gene in the nucleus.The possible existence of COI numts poses a serious challenge to DNA barcoding.The fact that the COI gene can be amplifiedAuthor contributions:H.S.,J.E.B.,M.F.W.,and K.A.C.designed research;H.S.and J.E.B.performed research;H.S.and J.E.B.analyzed data;and H.S.,J.E.B.,M.F.W.,and K.A.C.wrote the paper.The authors declare no conﬂict of interest.This article is a PNAS Direct Submission.Data deposition:The sequences reported in this paper have been deposited in the GenBank database (accession nos.EU589049–EU589148,EU583504–EU583573,EU583577–EU583678,EF207161–EF207162,and EF207165–EF207168).Freely available online through the PNAS open access option.†Towhom correspondence may be addressed:Department of Biology,692Widtsoe Build-ing,Brigham Young University,Provo,UT 84602.E-mail:hojun ࿝song@.This article contains supporting information online at /cgi/content/full/0803076105/DCSupplemental .©2008by The National Academy of Sciences of the USA13486–13491͉PNAS ͉September 9,2008͉vol.105͉no.36 ͞cgi ͞doi ͞10.1073͞pnas.0803076105from diverse taxa by using a limited set of primers is heralded as one of the attractive features of this marker(14).It is true that relatively conserved regions within mtDNA allow the design of ‘‘universal’’primers,which can amplify mitochondrial fragments from an unknown species(15).However,conserved primers can be a double-edged sword when numts are present because they can coamplify numts in addition to the target mtDNA(7,8).If the nuclear integration of numts was an ancient and sufficient sequence divergence accumulated in the orthologous mtDNA, the conserved primers would be more likely to amplify numts in preference to mtDNA,which could possibly result in unambig-uous,paralogous sequences(8).Despite this serious problem, numts have been dismissed as a minor concern for DNA barcoding(16)and the issue of numts has not been adequately addressed.In this study,we investigate the effect of including numts in DNA barcoding in two divergent lineages of arthropods,insects, and crustaceans,which are known to have especially large numbers of numts(8,17–19).We also examine the effect of numts at different levels of divergence:subfamily-level(grass-hoppers)and species-and population-level(crayfish).Herein, we show that both grasshopper and crayfish species included in the study have numts of the COI gene and barcoding methods would incorrectly infer that single individuals belong to multiple, unique species.The prevalence of numts appears to be both species-specific and population-specific and the pattern of numt distribution is considerably different between lower-level and higher-level divergence among taxa.Finally,we demonstrate the importance of data exploration in DNA barcoding practice by examining sequence characteristics of numts.Results and DiscussionCoamplification of Numts with Orthologous mtDNA.Our results strongly suggest that a large number of paralogous haplotypes of various divergences are coamplified with the orthologous mtDNA sequences when conserved primers are used in both grasshoppers and crayfish,which can be identified by the pres-ence of indels,point mutations,and in-frame stop codons [supporting information(SI)Table S1].The majority of the coamplified paralogs can be easily considered nonfunctional numt haplotypes because of the presence of in-frame stop codons,which is especially evident in our crayfish data in which 97.3%of paralogs have stop codons.A large number of these numts have unusually high numbers of point mutations(meanϭ65.52,nϭ110),suggesting that nuclear integration of mtDNA would result in random accumulation of nucleotide changes.In the grasshopper data,however,there are many paralogs that cannot readily be categorized as numts because they lack in-frame stop codons and differ from the orthologous mtDNA sequences by one or two nucleotides.If the same haplotype that appears to be functional other than the ortholog is repeatedly found from a single individual,one can suspect heteroplasmy(5). Indeed,heteroplasmy seems to explain the presence of certain paralogous haplotypes in Schistocerca americana.However, there are many paralogs represented by single haplotypes with small nucleotide differences in all four grasshopper species. Although,it is unlikely that these are Taq polymerase errors during PCR because of the high fidelity polymerase we used (0.015%error rate or0.0732bp per reaction);we cannot rule out the possibility of PCR error amplified by additional cloning(5, 18).Also,heteroplasmy might in fact be a plausible explanation for these haplotypes because we limited our study to only30 clones per grasshopper species,thus not exploring the full extent of heteroplasmic diversity.If the proportion of numts is high compared with the ortholo-gous mtDNA fragments in a given PCR product,it is possible to generate unambiguous paralogous sequences(8).This is exac-erbated when the conserved primers preferentially amplify numts because of relatively ancestral sequence similarity of numts or divergence within the primer regions of the ortholo-gous mtDNA.In this case,typical indicators of different PCR products,such as multiple bands on gels and double peaks, background noise,and ambiguity in sequence chromatograms, will not be present;hence,paralogous sequences can be mistaken as orthologous mtDNA.In fact,this exact phenomenon was observed in18crayfish individuals of Orconectes barri and Orconectes australis from which numts were amplified and cleanly sequenced without cloning.Phylogenetic Analyses and Distribution of Numts.For the grasshop-per data,the parsimony and the Bayesian analyses both recov-ered the monophyly of the orthologous mtDNA and haplotypes for three of four species(Fig.1A);although,the topology was different in the placement of S.americana and Calliptamus italicus clades between the two methods.In each species,the largest clade was the polytomous clade consisting of the mtDNA ortholog and several similar haplotypes.The remaining haplo-types formed highly structured clades within each species,and this pattern was especially evident in Acrida willemsei and S. americana.For crayfish data,the Bayesian analysis recovered a topology mostly congruent with the parsimony analysis(Fig.1B) and both analyses found a large clade of numt haplotypes(84in parsimony and82in Bayesian)and a small clade of numts(18in both analyses),distinctly divergent from the clearly defined clades of the orthologous mtDNA of four species.These numt clades consisted of the haplotypes from O.australis,O.barri,and Orconectes packardi,which were not necessarily grouped either by the species or the populations.Only four numt haplotypes were placed among orthologs(one in Orconectes incomptus,one in O.australis,and two in O.barri).A clade consisting of three numt haplotypes of O.barri was robustly placed near the root of trees in both analyses.Among the orthologous mtDNA clades, three of four species formed monophyletic clades,with the exception of O.australis that had one large clade sister to O.barri and a small clade basal to the australisϩbarri clade.Based on the phylogenetic analyses,number of indels,point mutations, in-frame stop codons,and sequence divergence,it is possible to conclude:among grasshoppers,Locusta migratoria has three numts,A.willemsei has six,C.italicus has two,and S.americana has at least11numts;among crayfish,O.australis has60,O.barri has46,O.incomptus has one,and O.packardi has four numts. On average,32.54%and41.88%of haplotypes generated from grasshoppers and crayfish,respectively,were numts(Table S1). It is important to note that this is a conservative estimate of the number of numts per species because it is limited by the number of individuals and clones we generated.Both the grasshopper and crayfish data suggest that there can be multiple types of numts present within single individuals that vary considerably in nucleotide composition,suggesting multiple independent transfer events from the mitochondrial genome to the nucleus(8,17).Moreover,our data suggest that these independent nuclear integration events can give rise to a family of numts that can diverge at different substitution rates.For example,we found that the relationships among the numt haplotypes of S.americana are highly structured and a similar pattern was observed in other grasshopper and crayfish species. Not only can independent transfer occur multiple times,but it can occur at very different phylogenetic levels.Whereas many numts of S.americana are closely related to the orthologous mtDNA,two numt haplotypes form a strong clade with the mtDNA ortholog of Anabrus simplex,which belongs to a differ-ent suborder within Orthoptera.Similarly,three numt haplo-types of O.barri were placed near outgroups belonging to different crayfish genera.These findings collectively suggest that there could have been an ancient nuclear integration event and that enough time has passed for these numts to have accumu-Song et al.PNAS͉September9,2008͉vol.105͉no.36͉13487E V O L U T I ONlated substantial sequence divergence from the orthologous mtDNA.Our two datasets differ at the level of divergence among the ingroup species along with the distribution of numts deduced from the phylogenetic analyses.Except for two numt haplotypes of S.americana that grouped within an outgroup,all grasshopper numt haplotypes strongly grouped with their orthologous mtDNA.However,this pattern is not observed in the four closely related crayfish species.Only a small portion of numt haplotypes grouped with their orthologs,whereas the majority formed clades among themselves with no apparent population or spe-cies-specific groupings.In other words,numt haplotypes se-quenced from different crayfish individuals from different pop-ulations and different species form monophyletic groups.This finding implies that the closely related crayfish species share similar types of numts that must predate the speciation events.Both patterns have been reported from other studies looking at various mitochondrial genes in diverse metazoan lineages at different phylogenetic levels (18,20).We conclude that the distribution pattern of numts within a given group of organisms cannot be predicted a priori ,but depends on the timing and the frequency of nuclear integration,which can clearly predate and postdate speciation events.The distribution of numts in both datasets suggests that their prevalence may be lineage-specific.For example,we sequenced numts from individuals collected from 11of 56cave crayfish populations (Tables S2and S3).Among the four species,numtswere sequenced for 7of 34localities in O.australis (southern-most region of range,primarily caves in Alabama),2of 7in O.barri (southernmost region of range,caves in Tennessee),1of 3in O.incomptus ,and 1of 12in O.packardi .This observed pattern does not necessarily mean that the remaining 45populations are free of numts but it does mean that there is a nonrandom population-specific variation in the level of numt prevalence.Nuclear integration of mtDNA happens at the level of the individual (8)and a large population size can effectively dilute the amount of numts in a given population.In this case,the proportion of numts is much smaller than that of the orthologous mtDNA in a given individual,rendering the numt coamplifica-tion less likely,even with a possibility of the presence of plesiomorphic numts in the nucleus.However,whether a pop-ulation experiences genetic drift because of an extreme bottle-neck,numts can be fixed in a few founders,resulting in a disproportionately high level of numts.Another intrinsic factor,nuclear genome size,might also play a role that results in uneven distribution of numts.Whereas all grasshopper species have numts, A.willemsei and S.americana have especially high numbers that are divergent from each other.Bensasson et al.(21)suggested that a positive correlation between the number of numts and nuclear genome size might exist and it is possible that these two species might have larger nuclear genomes than the others.Alternatively,inherent species-specific differences in the frequency of DNA transfer from mitochondria to the nucleus and in the rate of loss of numts in the nucleus have also beenMT h12 (1) h3 (2)3%3%O. packardiO. incomptus O. australisO. australis O. barrinumts numtsOutgroupsOutgroups 3%3%A GrasshoppersB CrayfishBarcoding with numtsBarcoding with numts Barcoding after Barcoding after quality controlquality control8311414119211221121Fig.1.Phylogenetic and barcoding analyses based on orthologous mtDNA COI and paralogous numt haplotypes from grasshoppers and crayﬁsh.(A )Grasshoppers:the cladogram on the left is a strict consensus of 41MPTs (L ϭ1002;CI ϭ0.54;RI ϭ0.85).Dots above branch indicate the nodes with the bootstrap value of Ͼ75and posterior probability of Ͼ95%.Orthologous mtDNA is indicated in bold and putative numts are indicated as red terminals.Number in parenthesis represents the number of identical copies for a particular haplotypes (h)and asterisk indicates ones with in-frame stop codons.When DNA barcoding analysis (NJ analysis based on K2P distances)is performed on the complete dataset,the number of unique species inferred based on 3%sequence divergence (colored numbers nextto the vertical bars)is overestimated (barcoding with numts).After the removal of the haplotypes with indels and in-frame stop codons (barcoding after quality control),the number of unique species inferred under DNA barcoding is drastically reduced.Purple,Schistocerca americana (Sa );blue,Calliptamus italicus (Ci );green,Acrida willemsei (Aw );orange,Locusta migratoria (Lm );and gray,outgroups.(B )Crayﬁsh:the circular cladogram on top is the strict consensus of 94MPTs (L ϭ1064;CI ϭ0.39;RI ϭ0.91).Terminals are colored to indicate species.Purple,Orconectes australis ;orange,O.barri ;green,O.incomptus ;blue,O.packardi ;and gray,outgroups.All numt haplotypes are indicated as red terminals.Similarly,DNA barcoding overestimates the number of unique species when numts are included,but the removal of numts reduces the inferred number of species.Notice that even after rigorous quality control,the inferred number of unique species is actually higher than the actual number of species,suggesting that some numts are difﬁcult to identify.13488͉ ͞cgi ͞doi ͞10.1073͞pnas.0803076105Song et al.suggested as possible explanations for lineage-specific numt variation(10),which may explain our observed pattern.It is also possible that the conserved primers we used were simply more efficient in amplifying numts of some individuals than others. DNA Barcoding Overestimates the Number of Species with Coampli-fication of Numts.If numts of the COI region are unknowinglycoamplified with the orthologous mtDNA and used in DNA barcoding without careful exploration of sequence character-istics,the number of unique species inferred from the analysis would certainly be overestimated.For the grasshopper data, the barcoding analysis finds that the haplotypes generated from individuals from each of four species form several distinct clusters,which can be considered unique species based on the 3%sequence divergence threshold typically used in the bar-coding studies.Based on the clustering pattern,one would conclude that there are a total of17unique species,suggesting the discovery of13additional cryptic species(Fig.1A).For the crayfish data,our analysis finds that some numt haplotypes nested among the orthologous mtDNA are not divergent enough to consider them unique species under DNA barcod-ing.However,among the highly divergent numts,we find numerous distinct clusters that hadϾ3%sequence divergence among each other.O.australis and O.barri each had two ortholog clusters that could be considered unique species.The barcoding analysis would thus infer a total of25unique species,suggesting the discovery of21additional cryptic species(Fig.1B).A careful examination of sequence characteristics before barcoding analyses drastically reduces the possibility of incor-rect inferences.It is possible to identify numts on the basis of in-frame stop codons and indels and to remove these haplo-types from the original datasets.For grasshopper data,for example,the barcoding analysis recovers a total of six clusters that can be considered unique species after the removal of such obvious numt haplotypes,implying two additional cryptic species(Fig.1A).These two clusters are formed by three haplotypes of S.americana,which have neither indels nor stop codons.When we remove a total of108obvious numt haplo-types from the crayfish data,the barcoding analysis finds7 unique clusters consisting of three crayfish species,two dif-ferently sized clusters of O.australis,and two clusters formed by the numts with no stop codons(Fig.1B).In other words,the removal of numts considerably reduces the inferred number of unique species in both grasshoppers(17before,6after)and crayfish(25before,7after).However,even the most careful sequence examination does not eliminate all incorrect infer-ences because some numts can be of the expected length without any in-frame stop codons and are not readily distin-guishable from the orthologous mtDNA.An examination of nucleotide composition may serve as an effective filter for identifying highly divergent numts because of different com-positional bias between mtDNA and nuclear DNA(8).In fact, two paralogous haplotypes of S.americana with no stop codons have a significantly lower AT%compared with the ortholo-gous mtDNA,suggesting that they are numts and the clade formed by these haplotypes does not represent a cryptic species.However,even this approach cannot totally eliminate incorrect inferences because some numts(1haplotype of S. americana,1haplotype of O.australis,and2haplotypes of O. barri)have no indels,no in-frame stop codons,and a highly similar AT%to the orthologs.It is possible that in-frame stop codons and indels lie downstream beyond the region amplified by the Folmer primers.Typical DNA barcoding studies would inevitably conclude the presence of cryptic species in such cases.Rigorous Quality Control Against Numts Is Necessary in DNA Barcod-ing.According to the standard DNA barcoding protocol published by the Consortium for the Barcode of Life(CBOL, ),there appears to be a minimum amount of quality control involved in generating COI se-quences.By using simple protocols,it is easy to generate molecular species tags rapidly and inexpensively,which is one of the main goals of DNA barcoding initiative(22).However, molecular evolutionary processes such as the nuclear integra-tion of mtDNA present challenges to every step of this standard protocol,and without specific quality control mea-sures in place,the integrity of DNA barcode sequences is seriously compromised(3).Despite obvious shortcomings because of numts,the proponents of DNA barcoding have argued that‘‘numts have proven a minor limitation to using mitochondrial barcode in groups studied so far.’’Hebert et al.(16)also suggested that the taxonomic implication of numts is small.Our study clearly demonstrates that coamplification of numts with the mtDNA orthologs is not only a major limitation of DNA barcoding,but also has significant taxonomic impli-cations.Reported cases of numts are ever increasing and numts should not be treated as mere nuisance any more.In a showcase study of DNA barcoding,Hebert et al.(16)found that13individuals of skipper butterfly Astraptes fulgerator had heterozygous sequences and concluded that they were numts because the nonmitochondrial sequences of these13individ-uals(obtained by subtracting‘‘typical sequences’’from the ambiguous region)were highly similar among each other.The rationale behind this conclusion was that numts would be conserved among individuals because numts represent an ancient molecular event whereas the corresponding mtDNA sequences would be more variable.However,empirical studies of numts suggest that substitution occurs at different rates once mtDNA has been integrated into nucleus(23).Brower (24)argued against the claim by Hebert et al.(16)and suggested that the13haplotypes that shared heterozygosity were unlikely to be numts,but likely to be heteroplasmic mtDNA at best.We downloaded these questionable sequences from GenBank(AY666889,AY666943,AY666968,and AY667044)and looked for any sign of compositional bias and other indicators of numts.In comparison with other DNA barcodes Hebert et al.(16)used,these presumed numts had identical nucleotide composition and translated amino acid sequences as the orthologous mtDNA sequences and had no indels or in-frame stop codons.In other words,these presumed numt sequences were fully functional copies of mtDNA and are likely heteroplasmic mtDNA.With careful examination of the sequences in question,Hebert and colleagues could have easily avoided their incorrect inference.How to Control for Numts.Among the few barcoding studies that did attempt to control for numts(25),researchers extracted DNA from tissues known to be rich in mitochondria and amplified slightly longer fragments(750bp)based on the idea that numts are shorter than the barcode amplicon(26). However,muscle tissues still do contain nuclear DNA that can harbor numts and the size of numts can be highly variable and not necessarily smaller than the typical700-bp size of DNA barcodes(10).Several methods have been suggested as means to avoid numt coamplification,including RT-PCR,long PCR, and mtDNA enrichment(8).However,these methods are often tedious,time-consuming and expensive,and their effi-ciency is often not high enough to totally avoid numts(27). Recent studies have questioned the universal utility of the Folmer region in DNA barcoding(28),and especially when a large number of numts are suspected in this region,it would be worthwhile to analyze additional markers other than COI gene.We recommend a number of steps that researchersSong et al.PNAS͉September9,2008͉vol.105͉no.36͉13489E V O L U T I ON。

花青素的转运

ABCC1,an ATP Binding Cassette Protein from Grape Berry, Transports Anthocyanidin3-O-Glucosides W OARita Maria Francisco,a,b Ana Regalado,b,1Agnès Ageorges,c Bo J.Burla,a,2Barbara Bassin,a Cornelia Eisenach,a Olfa Zarrouk,b Sandrine Vialet,c Thérèse Marlin,c Maria Manuela Chaves,b Enrico Martinoia,a and Réka Nagy a,1,3,4a University of Zurich,Institute of Plant Biology,8008Zurich,Switzerlandb Instituto de Tecnologia Química e Biológica,2780-157Oeiras,Portugalc UnitéMixte de Recherche1083-SPO Sciences for Enology,Institut National de la Recherche Agronomique,Sci Oenol UMR1083, 34060Montpellier,FranceORCID ID:0000-0002-9663-5371(RN).Accumulation of anthocyanins in the exocarp of red grapevine(Vitis vinifera)cultivars is one of several events that characterize the onset of grape berry ripening(véraison).Despite our thorough understanding of anthocyanin biosynthesis and regulation,little is known about the molecular aspects of their transport.The participation of ATP binding cassette(ABC)proteins in vacuolar anthocyanin transport has long been a matter of debate.Here,we present biochemical evidence that an ABC protein,ABCC1, localizes to the tonoplast and is involved in the transport of glucosylated anthocyanidins.ABCC1is expressed in the exocarp throughout berry development and ripening,with a signiﬁcant increase at véraison(i.e.,the onset of ripening).Transport experiments using microsomes isolated from ABCC1-expressing yeast cells showed that ABCC1transports malvidin3-O-glucoside.The transport strictly depends on the presence of GSH,which is cotransported with the anthocyanins and is sensitive to inhibitors of ABC proteins.By exposing anthocyanin-producing grapevine root cultures to buthionine sulphoximine, which reduced GSH levels,a decrease in anthocyanin concentration is observed.In conclusion,we provide evidence that ABCC1 acts as an anthocyanin transporter that depends on GSH without the formation of an anthocyanin-GSH conjugate.INTRODUCTIONAnthocyanins represent the largest class ofﬂavonoids(Welch et al.,2008).They constitute one of the most important families of secondary metabolites and are widely distributed in plants. They have been shown to be synthesized as protective com-pounds in response to abiotic stresses,such as UV,cold,and drought,but also to attract pollinators.However,the importance of anthocyanins extends beyond the plant kingdom.During the last decades,anthocyanins were reported to be efﬁcient anti-oxidants and regulators of signaling pathways involved in many human diseases(Winkel-Shirley,2001;Grotewold,2006;Wallace, 2011).The health-promoting effects mostly depend on the con-sumption of high levels of dietary anthocyanins.Therefore, anthocyanin-rich extracts became sought after in the food industry, and numerous attempts have been made to improve the quantity of bioactive anthocyanins in dietary products.A study directly linking anthocyanin content to beneﬁcial effects on health was published recently,showing that feeding cancer-susceptible mice with anthocyanin-rich tomato(Solanum lycopersicum)ex-tended their life span(Butelli et al.,2008).Like many other secondary metabolites,anthocyanins are synthesized in the cytosol and accumulate in the vacuoles, mostly of the epidermal tissues of fruits,leaves,andﬂowers.It has been suggested that the structural enzymes involved in anthocy-anin biosynthesis form a complex via protein–protein interaction, which is anchored to the endoplasmic reticulum membrane (Saslowsky et al.,2005).To date,more than635anthocyanins have been identiﬁed.The aglycone forms(anthocyanidins) differ from each other by different hydroxylation and methyla-tion patterns.The derivatives of the most common anthocyanidins in nature(cyanidin,delphinidin,peonidin,petunidin,pelargonidin, and malvidin)represent over90%of the anthocyanins identiﬁed to date(Wallace,2011).Once the biosynthesis is completed, including the additional chemical modiﬁcations(glycosylation, methylation,and acylation;Winkel-Shirley,2008),anthocya-nins are transported into the vacuole.A report has shown that vacuolar accumulation ofﬂavonoids is a prerequisite for their biosynthesis and thus fulﬁls a regulatory function during syn-thesis(Marinova et al.,2007a).The mechanisms by which anthocyanins are transported into the vacuole have long been debated.To date,two major transport models have been pro-posed:membrane vesicle–and membrane transporter–mediated transport(Grotewold and Davies,2008;Zhao and Dixon,2010). Scientiﬁc evidence supports both models.The vesicle-mediated transport system is mostly based on microscopy observations (Irani and Grotewold,2005;Zhang et al.,2006;Gomez et al., 2011).Two distinct vesicular structures were described as having1These authors contributed equally to this work.2Current address:Division of Internal Medicine,University Hospital,Zurich,Switzerland.3Current address:Promega,Wallisellenstrasse55,8600Dubendorf,8006Zurich,Switzerland.4Address correspondence to reka.nagy@.The author responsible for distribution of materials integral to theﬁndingspresented in this article in accordance with the policy described in theInstructions for Authors()is:Réka Nagy(reka.nagy@).W Online version contains Web-only data.OA Open Access articles can be viewed online without a subscription./cgi/doi/10.1105/tpc.112.102152The Plant Cell,Vol.25:1840–1854,May2013,ã2013American Society of Plant Biologists.All rights reserved.a putative role in anthocyanin transport.Cytoplasmic anthocyanin bodies,called anthocyanoplasts,are vesicular structures sur-rounded by membranes,which gradually fuse together.Within vacuoles,anthocyanic vacuolar inclusions have been observed. These are dynamic,nonmembrane surrounded structures that, despite being associated with proteins and membranous sub-stances,have a role in storage rather than in transport of antho-cyanins.The anthocyanoplasts have been shown to colocalize with protein storage vacuoles and to transport anthocyanins in the trans-Golgi network(Zhao and Dixon,2010;Pourcel et al., 2010).It is likely that transporters might also be needed to load the vesicles with anthocyanins during membrane vesicle-mediated transport.Concerning the transporter-mediated model,biochemical, molecular,and genetic evidence support the involvement of both multidrug and toxic extrusion(MATE)and the ATP bind-ing cassette(ABC)proteins in the transport of anthocyanins (Goodman et al.,2004;Marinova et al.,2007b;Gomez et al.,2009; Zhao and Dixon,2009;Zhao et al.,2011).MATE transporters comprise a large family of transporters,widely distributed in all living organisms.Theﬁrst experimental evidence for the in-volvement of a MATE transporter inﬂavonoid transport came from a genetic screen in Arabidopsis thaliana aiming to identify the different steps ofﬂavonoid biosynthesis.Among other identiﬁed genes,Transparent testa12(TT12)was shown to en-code a MATE-type transporter involved in the biosynthesis of ﬂavonoids(Debeaujon et al.,2001).Further characterization demonstrated that the tonoplast-localized TT12was able to transport cyanidin3-O-glucoside(C3G)when heterologously expressed in yeast(Marinova et al.,2007b).When expressed in barrel medic(Medicago truncatula)hairy roots(HRs),Arabidopsis TT12as well as a MATE transporter of barrel medic,MATE1, transport the proanthocyanidin(PA)precursor epichatechin3-O-glucoside(Zhao and Dixon,2009).A second barrel medic MATE transporter,MATE2,was shown to have consistently different substrate speciﬁcity despite its high similarity to MATE1;it efﬁ-ciently catalyzes the vacuolar uptake of malonylated anthocyanins, while transporting glycosylated anthocyanidins at a lower rate (Zhao et al.,2011).More recently,two grapevine(Vitis vinifera) MATEs,AM1and AM3,have been described to speciﬁcally transport acylated anthocyanins,but not glucosylated ones, which are the predominant forms that accumulate in berries of most red grapevine cultivars(Gomez et al.,2009).Several lines of evidence also suggested the involvement of plant ABC transporters,in particular from the ABCC subfamily (formerly named multidrug resistance proteins[MRPs])in vacu-olarﬂavonoid sequestration(Klein et al.,2006).Complete ge-nome sequences of several organisms revealed that genes encoding ABCC-type proteins are abundant in plants.Among the ABC transporters,unequivocal vacuolar localization has only been shown for the ABCC-type proteins.However,functionally this subfamily of ABC transporters is only marginally charac-terized,and ABCC transporters might be responsible for many so far unknown functions.For a long time,ABCC-type proteins were considered as the classical GSH-S conjugate pumps (Ishikawa et al.,1997).However,in the last years it became evident that their function ranges from heavy metal sequestra-tion and detoxiﬁcation to chlorophyll catabolite and inositol hexa kis phosphate transport and ion channel regulation(reviewed in Kang et al.,2011).Furthermore,it was shown that rye(Secale cereale)ﬂavones that are conjugated to glucuronate are trans-ported into the vacuole by an ABC transporter(Klein et al.,1998). Later studies revealed that At-ABCC2is such a glucuronide conjugate transporter(Liu et al.,2001).Moreover,genetic studies in maize(Zea mays)indicated that maize anthocyanins are delivered to the vacuole by an ABCC-type transporter,MRP3 (Goodman et al.,2004);however,this study did not provide biochemical evidence that MRP3transports anthocyanins. Scientiﬁc evidence also designates glutathione S-transferases (GSTs)as crucial players in the vacuolar sequestration of an-thocyanins.The bronze coloration of the maize bronze-2(bz2) mutant was shown to be correlated with the cytosolic accu-mulation of oxidized and cross-linked anthocyanins.A rational basis for the bz2phenotype was obtained through the identiﬁ-cation of Bz2as a GST-encoding gene that might be capable of conjugating C3G with GSH(Marrs et al.,1995).Mutations in the GST-encoding genes in other plant species,such as petunia (Petunia hybrida;Alfenito et al.,1998)and Arabidopsis(Kitamura et al.,2004),also caused reduced anthocyanin accumulation and pigment mislocalization.More recently,Conn et al.(2008) showed that two grapevine GSTs are able to complement the maize bz2phenotype.The mechanisms underlying the in-volvement of GSTs in anthocyanin transport are still unknown. The initial explanation for their involvement in anthocyanin transport included that GSTs might conjugate anthocyanins with GSH,thus generating the molecules that will be recognized by tonoplast-localized transporters and are imported into the vacuole.However,there is no evidence for the presence of GSH-conjugated anthocyanins in planta(Mueller et al.,2000). Alternatively,it was suggested that GSTs might act as escort proteins/ligandins of anthocyanins that deliver these compounds to the transporter(Mueller et al.,2000).Using a grapevine HR system that produces anthocyanins but is silenced for a GST known to be required for efﬁcient anthocyanin accumulation, Gomez et al.(2011)observed that vesicles containing antho-cyanins were still visible,but anthocyanin accumulation in the large vacuole was impaired.This may be interpreted in two ways:Either vacuolar anthocyanin transport depends on a GST, or GST absence forces the accumulation of anthocyanins in vesicles due to an impaired fusion of anthocyanin-containing vesicles with the large,central vacuole.Recently,it was shown that TT19,an Arabidopsis GST,directly binds C3G without conjugating it with GSH,thus reinforcing the role of GSTs as carrier proteins for vacuolar anthocyanin sequestration(Sun et al.,2012).The recentﬁndings on the vacuolar sequestration of antho-cyanins suggest that chemical modiﬁcations of anthocyanins determine the preferential transport system.Due to the rele-vance of anthocyanins in grape ripening,and also because vacuolar sequestration of glucosylated anthocyanidins has only been partially characterized,we focused on elucidating whether and how ABCC-type transporters are involved in this process. Searching for Zm-MRP3homologs in the grapevine genome database,we identiﬁed ABCC1as having the highest protein sequence identity.Here,we report that the tonoplast-localized ABCC1transports anthocyanidin3-O-glucosides.The transport Transport of Glucosylated Anthocyanidins1841is preferential for malvidin3-O-glucoside(M3G),and it is strictly GSH dependent,which assigns an important role for GSH in the current model of anthocyanin transport into the vacuole. RESULTSPhylogenetic Analysis of Grapevine ABCC-Type Proteins Grapevine ABCC protein sequences from the available grape-vine protein databases were chosen as candidates to carry out phylogenetic studies.The study included all Arabidopsis ABCC proteins and MRP3,the maize ABCC described as being involved in the transport of anthocyanins into the vacuole(Goodman et al., 2004).The maximum likelihood phylogenetic analysis revealed that eight grapevine ABCC proteins group within the clade con-taining the maize MRP3(highlighted in dark gray,Figure1A;see Supplemental Data Sets1and2online).Among these,Vv-ABCC1and Vv-ABCC2cluster with At-ABCC10,and the other six form a distinct subclade(highlighted in light gray,Figure1A). While Vv-ABCC1and Vv-ABCC2are located on chromosome16, the other six proteins all cluster on chromosome2in a tandem arrangement(see Supplemental Table1online).Vv-ABCC1/ Vv-ABCC2share a higher amino acid sequence identity with Zm-MRP3than with any of the other six ABCCs(63%/59%), which suggests that these two proteins are candidates for anthocyanin transporters in grapevine.ABCC1Expression in Grape BerriesThe phylogenetic analysis of grapevine ABCCs revealed that eight ABCCs form a distinct clade with Zm-MRP3.In this clade, ABCC1and ABCC2share the highest protein sequence identity with Zm-MRP3(Figure1A).Given the presumed role of Zm-MRP3in anthocyanin transport(Goodman et al.2004),these two ABCCs were chosen to verify our assumption that grape-vine ABCC proteins are involved in anthocyanin transport. ABCC1encodes a protein of1480amino acids with a predicted molecular mass of;165kD and a calculated pI value of6.38 (Compute pI/MW tool at http://www.expasy.ch/tools/pi_tool. html).ABCC1exhibits the typical sequence characteristic of ABCC-type proteins,with a TMD0,two nucleotide binding do-mains each containing the Walker A,B,and ABC signature motifs, and two transmembrane domains(Figure1B).To determine when and in which berry tissues ABCC1is ex-pressed,quantitative RT-PCR analysis was conducted on RNA extracted from exocarp(skin)and mesocarp(pulp)at four dates, from day49to day97afterﬂowering(DAF).ABCC1was found to be expressed in the exocarp and mesocarp throughout berry development.However,while transcript levels decreased con-tinuously from day49to81in the mesocarp,a strong increase could be observed in the exocarp at68d,during the véraison(i.e., when sugars and anthocyanins started to accumulate)(Figure 2A).The expression of ABCC1was evaluated in several tissues and was expressed in all organs tested(Figure2B).Higher expression was observed in old leaves and berries(Figure2B). All attempts to amplify ABCC2cDNA from the exocarp were unsuccessful,suggesting that it might not be expressed in detectable amounts during the ripening stage.We were also interested in determining whether members of the subclade1 of the eight ABCCs within the Zm-MRP3–containing clade are expressed in berries.To this end,we conducted standard PCR analyses on exocarp tissues from different developmental stages. The analysis showed that some ABCC members are expressed in the exocarp during berry ripening(see Supplemental Figure1 online).ABCC1Localizes to the TonoplastTo determine the subcellular localization of ABCC1,full-length ABCC1cDNA was C-terminally fused to greenﬂuorescent protein (GFP)and expressed under the control of the cauliﬂower mosaic virus35S promoter.A g-TIP-RFP(for redﬂuorescent protein)was used as a control for tonoplast localization(Nelson et al.,2007). Agrobacterium tumefaciens carrying the P35S:VvABC1-GFP construct was inﬁltrated into tobacco(Nicotiana benthamiana) leaves(Figures3A to3H).Four days after inﬁltration,we ob-served the formation of small vacuoles(Figures3A and3E),with the GFP signal encircling these structures(Figures3B and3F). To conﬁrm that these small structures indeed corresponded to vacuoles,we performed coinﬁltration with the tonoplast marker g-TIP-RFP(Figure3G).Figure3H shows colocalization of both recombinant proteins around the edge of the small vacuoles. To further elucidate VvABCC1-GFP localization,protoplasts of transformed N.benthamiana leaves were subjected to gentle lysis(Figure3I).A bright-ﬁeld close-up of the released small vacuole in Figure3I is shown in Figure3J.VvABCC1-GFP and g-TIP-RFPﬂuorescence is observed around the released small vacuole(Figures3K and3L).Both signals colocalize(Figure 3M)at the tonoplast.Colocalization was further conﬁrmed by ﬂuorescence intensity over distance analysis(Figure3N)along the dotted line(Figure3M).Vacuolar fragmentation is a phenomenon described to occur in guard cells during different stages of stomata opening and closing(Gao et al.,2005),as a response to osmotic stress(Chang et al.,1996),or during senescence events(Kasaras et al.,2012).In this study,the small vacuoles may reﬂect a physiological adap-tation to any VvABCC1-GFP overexpression-induced changes in the cellular and/or vacuolar equilibrium.Therefore,we extended our VvABCC1localization analysis by performing transient expression in onion(Allium cepa)bulb epidermis cells by bio-listic particle delivery.Figure3O shows the brightﬁeld image of onion epidermis cell with the corresponding VvABCC1-GFP signal(Figure3P).Theﬂuorescence of the VvABCC1-GFP fu-sion protein showed a signal cytoplasmicallyﬁtting against, but not surrounding,the nucleus(arrows in Figures3P and3Q), which is indicative of tonoplast localization.Taken together, our results show that VvABCC1-GFP localizes to the vacuolar membrane in N.benthamiana plants,aﬁnding that is further supported by VvABCC1-GFP tonoplast localization in onion epidermis cells.ABCC1Mediates M3G TransportIt is known that ABCC transporters can transport a large number of chemically unrelated compounds(Kang et al.,2011).The observation that ABCC1is expressed in the exocarp of ripening1842The Plant Cellberries and its close relation to Zm-MRP3encouraged us to evaluate whether ABCC1is an anthocyanin transporter.For this purpose,we cloned the full-length cDNA encoding ABCC1(see Supplemental Table 1online)into the pNEV-Ura (Sauer and Stolz,1994)plasmid and expressed it in Saccharomyces cer-evisiae defective in the ABCC transporter Yeast Bile Transporter1(YBT1)(ybt1strain).Yeast cells transformed with the empty vector were used as control (pNEV).Total microsomal membrane vesi-cles were isolated from both yeast transformants.To assess the physiological intactness of the isolated vesicles,we decided to use the vesicles lumen acidi ﬁcation method.Acidi ﬁcation occurs only when vesicles are intact and is mainly the result of the activity of the plasma membrane and tonoplast H +-ATPases.Acidi ﬁcation leads to the accumulation of the weak base 9-amino-6-chloro-2-methoxyacridin (ACMA)and a concomitant quenching of ACMA ﬂuorescence (Casadio,1991).The addition of (NH 4)2SO 4restored the basal ﬂuorescence,indicating that the proton gradient formed across the membranes collapsed,further proof that the vesicles were intact.To elucidate whether and in what relative proportion vacuolar or plasma membrane vesicles were present in themixture,Figure 1.Phylogeny of Grapevine ABCC Proteins.(A)Maximum likelihood phylogeny of grapevine (Vv ),Arabidopsis (At )ABCC-type proteins,and Zm-MRP3/Zm-ABCC3.The clade comprising Vv-ABCC1,Vv-ABCC2,and Zm-MRP3is shaded in dark gray,and within this clade,the subclade comprising the six other Vv-ABCCs is shaded in light gray.Branch support values indicate nonparametric bootstrap values (in percentages of 1000replicates).The tree was estimated with PhyML 3.0from a trimmed protein sequence alignment.(B)Predicted ABC-type motives in Vv-ABCC1,At-ABCC10,and Zm-MRP3.The Walker A and B motifs as well the ABC signature are shown in bold letters.The three-character code is used to highlight conserved amino acids:*,full identity;:,one of the following strong groups is fully conserved (STA,NEQK,NHQK,NDEQ,QHRK,MILV,MILF,HY,and FYW);.,one of the following weaker groups is fully conserved (CSA,ATV,SAG,STNK,STPA,SGND,SNDEQK,NDEQHK,NEQHRK,FVLIM,and HFY).NBD,nucleotide binding domain.Bar =0.2amino acid substitutions per site.Transport of Glucosylated Anthocyanidins 1843and whether both were sealed,we performed the ACMA-quenching experiment in the presence of either vanadate (inhibitor of the plasma membrane H +-ATPases),ba ﬁlomycin A1(inhibitor of vacuolar H +-ATPases),or both.The results showed that both vanadate and ba ﬁlomycin reduced the ATP-dependent quench-ing to a similar extent,indicating that both plasma and vacuolar membrane vesicles were present and intact (see Supplemental Figure 2online).We conducted the initial transport studies with M3G,the major anthocyanin of grape berries (see Supplemental Table 2online).The transport experiments were performed at room temperature,with a standard substrate concentration of 0.5mM,using the rapid ﬁltration technique as reported by Tommasini et al.(1996).HPLC was used to quantify the anthocyanins taken up into the vesicles and to con ﬁrm that there is no alteration in the structure of the substrates during the transport experiments (Figure 4A).Signi ﬁcant M3G uptake into yeast microsomes could not be ob-served in the absence or presence of ATP.Knowing that ABCCs mainly transport organic anions and that for some animal ABC transporters GSH was shown to be cotransported with their sub-strates (König et al.,2003),we included the negatively charged GSH into our transport assays at a 5mM ﬁnal concentration.Under these conditions,M3G was taken up in a time-dependent manner by the yeast vesicles expressing ABCC1and uptake of M3G was ATP and GSH dependent (Figures 4B to 4D).To validate whether the transport was strictly ABC transporter driven,we included known ABC-type transporter inhibitors in the transport assays.In the presence of the sulphonylurea glibenclamide or probenecid,M3G transport was strongly reduced to 1and 4%,respectively,of the transport activity observed without inhibitors (Table 1).Vana-date completely abolished M3G transport,and the nonhydrolyzable ATP analog,adenyl-59-yl imidodiphosphate (AMPPNP),could not replace ATP,demonstrating that ATP hydrolysis is a prerequisite for the transport (Table 1).These results further con ﬁrm that ABCC1mediates M3G transport in an ABC-type fashion.M3G transport was not detectable when experiments were performed at 0°C.These observations are further proof that the transport data presented in Table 1are not due to nonspeci ﬁc permeability of yeast microsomes or to the binding of M3G to yeast vesicles.Substrate Speci ﬁcity of ABCC1To verify the substrate speci ﬁcity of ABCC1,additional transport experiments were performed with delphinidin-3-O -glucoside (D3G)and the aglycon delphinidin.D3G is one of the ﬁve major anthocyanins present in grape berries and is structurally the most distinct compared with M3G.Transport experiments were conducted as described for M3G.No uptake was observed when delphinidin was added as a substrate (Figure 5A).By contrast,ABCC1transported the 3-O -glucoside of delphinidin,although at a signi ﬁcantly lower rate compared with M3G (Figure 5B).As for M3G,D3G uptake was dependent on both ATP and GSH (Figure 5A).To investigate whether ABCC1could transport non-anthocyanin-type ﬂavonoids,we performed a set of trans-port experiments with the ﬂavan-3-ol epicatechin (Figure 6A).However,no transport was observed with epicatechin as sub-strate (Figure 6B).Uptake of Radiolabeled GSH in the Presence of M3G Transport of M3G and D3G was only observed in the presence of GSH (Figures 4and 5),but the mechanism behind the re-quirement for GSH was still unclear.Although it was suggested that GST would conjugate GSH with anthocyanins,the HPLC analyses following our transport assays did not reveal changes in the retention time of the substrates or additional peaks that could point to GSH-conjugated or modi ﬁed M3G or D3G (Fig-ures 4and 5).These results led us to hypothesize a similar mechanism as postulated in animals (i.e.,cotransport of a GSH substrate)(König et al.,2003).To test this hypothesis,co-transport experiments with M3G and radiolabeled GSH were performed.All transport experiments were performed in the presence of 5mM NH 4Cl to dissipate the proton gradient,thus inhibiting the endogenous yeast GSH transporters,which were described to depend on a proton gradient (Bourbouloux et al.,2000).No ATP-dependent GSH uptake into microsomes was detected when M3G was absent from the transport mixtures (Table 2).However,with 0.5mM M3G in the transport reaction,we observed a time and ATP-dependent uptake of GSH in Vv-ABCC1–carrying yeast microsomes.By contrast,microsomes isolated from yeast cells carrying the empty vector did not take up GSH (Table 2).Decrease in GSH Levels Alters the Anthocyanin Concentration in HRsTo further investigate and validate the mechanism indicating that GSH is required for anthocyanin transport,we used a transgenic anthocyanin-producing HR system (Cutanda-Perez et al.,2009).In this system,grapevine HRs transformed with the transcription factor Vv-MYBA1exhibit a constitutive synthesis and accumulation of anthocyanins.In addition,Vv-ABCC1isFigure 2.Quantitative Real-Time PCR Expression Pro ﬁle of ABCC1.(A)Expression pro ﬁle of ABCC1in grape berry tissues during fruit maturation.Transcript levels of ABCC1were normalized to the grapevine Actin gene.Results represent the mean 6SE of three replicates.Total anthocyanin content in grape berries during fruit maturation represented by the mean 6SE of six biological replicates is shown.(B)Transcript levels of ABCC1in various grapevine organs.Young leaves correspond to leaves with a fresh weight of 0.3g,and old leaves are fully expanded and had a mean fresh weight of 2.8g.Gene ex-pression was normalized to the grapevine EF1a reference gene.Results represent the mean 6SE of three replicates.1844The Plant Cellhighly expressed in transformed HRs from different grapevine cultivars compared with the HRs obtained with a wild-type Agrobacterium rhizogenes (Figure 7A).Transgenic MYBA1-HRs from Tempranillo cultivar grown on LGO medium (Torregrosa and Bouquet,1997)were transferred to fresh medium supplemented with different concentrations of buthionine sulfoximine (BSO),a compound that inhibits glutamyl-Cys synthetase,the enzyme responsible for the ﬁrst step in GSH synthesis.The growth rate of HRs was similar in control and BSO-containing media;however,2weeks after exposure to BSO,we observed a signi ﬁcantdecreaseFigure 3.ABCC1-GFP Localizes to the Tonoplast.(A)Bright-ﬁeld image of a tobacco epidermis cell transiently expressing ABCC1-GFP.Bar =20µm.(B)GFP signal encircling small vacuoles,which only occur in tobacco epidermis cells overexpressing VvABCC1-GFP.(C)Chlorophyll auto ﬂuorescence corresponding to the image in (E).(D)Bright-ﬁeld,chlorophyll,and GFP ﬂuorescence overlay image.(E)Bright-ﬁeld image of the VvABCC1-GFP overexpression-induced small vacuoles in a tobacco epidermis cell.Bar =10µm.(F)VvABCC1-GFP signal encircling small vacuoles.(G)Signal of the tonoplast marker g -TIP-RFP encircling small vacuoles.(H)Overlay image of VvABCC1-GFP and g -TIP-RFP ﬂuorescence showing colocalization of both dyes in small vacuole membranes.(I)Bright-ﬁeld image of a transiently transformed tobacco protoplast releasing a small vacuole (box).Bar =20µm.(J)Close-up bright-ﬁeld image of the small vacuole in (I).Bar =5µm.(K)VvABCC1-GFP signal at the vacuolar membrane.(L)Tonoplast marker g -TIP-RFP at the vacuolar membrane.(M)Overlay image of VvABCC1-GFP and g -TIP-RFP ﬂuorescence at the membrane encircling the released small vacuole.(N)Fluorescence intensity over distance plot of VvABCC1-GFP and g -TIP-RFP ﬂuorescence along the dotted line in M.(O)Bright-ﬁeld image of onion epidermis cells transiently expressing VvABCC1-GFP.Bar =50µm.(P)VvABCC1-GFP signal.(Q)Bright-ﬁeld and GFP ﬂuorescence overlay image showing a signal around the nucleus indicative of tonoplast localization (arrows in [P]and [Q]).Transport of Glucosylated Anthocyanidins 1845。

青春期男生长痘痘给建议英语作文

青春期男生长痘痘给建议英语作文Title: Tips for Teenage Boys Dealing with AcneIntroductionAcne is a common skin condition that many teenagers experience during their adolescent years. It can be frustrating and embarrassing, but there are ways to manage and treat acne. In this article, we will provide some tips for teenage boys who are dealing with acne.1. Follow a Good Skincare RoutineOne of the most important things you can do to prevent and treat acne is to establish a good skincare routine. This includes washing your face twice a day with a gentle cleanser, using a non-comedogenic moisturizer, and applying acne treatment products as needed. Avoid harsh scrubbing or washing your face too frequently, as this can irritate your skin and make acne worse.2. Avoid Touching Your FaceTouching your face can transfer bacteria and oils from your hands to your skin, which can lead to breakouts. It's important toavoid touching your face throughout the day, and to wash your hands regularly to keep them clean.3. Eat a Healthy DietEating a healthy diet can also help improve your skin health. Try to include plenty of fruits, vegetables, whole grains, and lean proteins in your diet, and limit your intake of sugary and processed foods. Drinking plenty of water can also help keep your skin hydrated and healthy.4. Get Regular ExerciseRegular exercise can help improve your overall health, including the health of your skin. Exercise can help reduce stress, which is a common trigger for acne breakouts. Just be sure to shower and change out of sweaty clothes as soon as possible after exercising to prevent clogged pores.5. Avoid Overwashing Your FaceWhile it's important to keep your face clean, overwashing can actually make acne worse. Washing your face more than twice a day or using harsh cleansers can strip your skin of its natural oils and lead to irritation. Stick to a simple skincare routine and be gentle with your skin.6. Talk to a DermatologistIf you're struggling to manage your acne on your own, it may be helpful to see a dermatologist. A dermatologist can recommend prescription-strength acne treatments, such as topical creams or oral medications, that can help clear up your skin. They can also provide guidance on how to properly care for your skin.ConclusionDealing with acne during your teenage years can be challenging, but with the right skincare routine and lifestyle habits, you can manage and treat your acne effectively. By following these tips, you can help improve the health of your skin and boost your confidence. Remember, acne is a common and treatable condition, so don't be afraid to seek help if you need it.。

“生命的奇迹：一片叶子的故事”

“生命的奇迹：一片叶子的故事”示例1:Title: The Miracle of Life: The Story of a LeafIntroduction:In the vast world of nature, every living organism has a unique story to tell. One such remarkable story is that of a simple leaf. This article explores the journey of a leaf, highlighting the miracles of life that unfold within its existence.英文回答 (English Answer):1. Birth:The story of a leaf begins with its birth. It all starts as a tiny bud on a branch, slowly unfurling its delicate green surface. This process is a miracle in itself - a new life emerging from the dormant winter. With each passing day, the leaf grows, absorbing sunlight and carbon dioxide, converting them into energy through the process of photosynthesis.中文回答 (Chinese Answer):1. 诞生:一片叶子的故事始于它的诞生。

它作为一个小小的蓓蕾出现在枝条上，慢慢展开它娇嫩的绿色表面。

这个过程本身就是一个奇迹——一个新的生命从沉睡的冬天中崭露头角。

洗澡翻译

洗澡翻译Taking a ShowerShowering is a daily routine for most people. It not only helps us maintain personal hygiene, but also provides a relaxing and refreshing experience after a long day. In this article, we will discuss the importance of showering and provide some tips for optimizing this activity.Showering not only cleans our bodies, but also has numerous health benefits. Firstly, it helps remove dirt, sweat, and bacteria from our skin, preventing infections and unpleasant odors. Showering also helps to unclog pores, preventing acne and other skin conditions. Furthermore, showering improves blood circulation, promoting healthier and glowing skin. It also helps to regulate body temperature and relieve muscular tension.To make the most out of your showers, here are some tips to keep in mind. Firstly, it is important to adjust the water temperature according to your preference. Avoid using excessively hot water, as it can strip away the natural oils from your skin, leading to dryness and irritation. On the other hand, extremely cold water may not effectively remove dirt and bacteria. Aim for lukewarm water to achieve a balance between cleanliness and skin health. Additionally, choose a suitable shower gel or soap that suits your skin type. If you have dry skin, opt for moisturizing products that can hydrate your skin while cleansing. If you have oily or acne-prone skin, look for products containing ingredients such as salicylic acid or tea tree oil to control oil production and preventbreakouts. Taking care of your skin during showering can lead to healthier and smoother skin.Furthermore, be mindful of the duration of your showers. A long, hot shower may seem enticing, but it can actually do more harm than good. Prolonged exposure to hot water can strip away the natural oils from your skin and leave it dry and irritated. Limit your showers to around 10-15 minutes to keep your skin hydrated and prevent excessive drying.Lastly, don't forget to moisturize your skin after showering. Applying a moisturizer immediately after showering can help lock in the moisture and keep your skin soft and supple. Look for moisturizers that are suitable for your skin type and apply them liberally all over your body.In conclusion, showering is not only a necessary part of maintaining personal hygiene, but also provides various health benefits. By following these tips, you can optimize your showering experience and keep your skin healthy and radiant. So, the next time you step into the shower, remember to take care of your skin and enjoy the refreshing and rejuvenating experience.。

关于去痘痘的作文英语

关于去痘痘的作文英语Title: Effective Ways to Get Rid of Acne。

Acne, a common skin condition affecting millions worldwide, can be both physically and emotionally distressing. However, with the right approach and proper care, it is possible to effectively manage and reduce acne breakouts. In this essay, we will explore some of the most effective methods to combat acne and achieve clearer, healthier skin.First and foremost, maintaining good hygiene is essential in preventing and treating acne. Washing the face twice daily with a gentle cleanser helps to remove excess oil, dirt, and impurities that can clog pores and lead to breakouts. It's important to use lukewarm water and avoid harsh scrubbing, as this can irritate the skin and exacerbate acne.In addition to regular cleansing, exfoliation isanother crucial step in acne care. Exfoliating the skin helps to remove dead skin cells and unclog pores, reducing the likelihood of acne flare-ups. However, it's important to choose a gentle exfoliant and avoid over-exfoliating, as this can strip the skin of its natural oils and cause further irritation.Furthermore, maintaining a healthy diet plays a significant role in managing acne. Consuming a balanceddiet rich in fruits, vegetables, whole grains, and lean proteins provides the body with essential nutrients that support skin health. Avoiding greasy, fried foods and excessive sugar can also help to reduce inflammation and prevent acne breakouts.In addition to diet, staying hydrated is crucial for maintaining clear, healthy skin. Drinking an adequate amount of water each day helps to flush out toxins from the body and keep the skin hydrated, reducing the risk of acne breakouts. Aim to drink at least eight glasses of water per day to keep your skin looking its best.Moreover, incorporating topical treatments into your skincare routine can help to target acne directly. Ingredients such as benzoyl peroxide, salicylic acid, and retinoids are commonly used to treat acne and can help to reduce inflammation, unclog pores, and promote cell turnover. It's important to start with a low concentration and gradually increase as tolerated to minimize irritation.In addition to topical treatments, prescription medications may be necessary for severe or persistent acne. Oral antibiotics, hormonal therapies, and isotretinoin are all options that your dermatologist may consider depending on the severity of your acne and your individual needs.It's essential to follow your dermatologist's recommendations closely and attend regular follow-up appointments to monitor your progress.Furthermore, managing stress levels is crucial in preventing acne breakouts. Stress can trigger hormonal changes in the body that can exacerbate acne, so finding healthy ways to cope with stress, such as exercise, meditation, or deep breathing exercises, can help to keepyour skin clear and healthy.In conclusion, while acne can be a challenging condition to manage, it is possible to achieve clearer, healthier skin with the right approach and proper care. By maintaining good hygiene, adopting a healthy diet, staying hydrated, using topical treatments, and managing stress levels, you can effectively reduce acne breakouts and improve the overall appearance of your skin. Remember to be patient and consistent with your skincare routine, anddon't hesitate to seek help from a dermatologist if needed. With dedication and perseverance, you can overcome acne and enjoy clear, radiant skin.。

如何保持毛孔通畅英文作文

如何保持毛孔通畅英文作文下载温馨提示:该文档是我店铺精心编制而成，希望大家下载以后，能够帮助大家解决实际的问题。

文档下载后可定制随意修改，请根据实际需要进行相应的调整和使用，谢谢!并且，本店铺为大家提供各种各样类型的实用资料，如教育随笔、日记赏析、句子摘抄、古诗大全、经典美文、话题作文、工作总结、词语解析、文案摘录、其他资料等等，如想了解不同资料格式和写法，敬请关注!Download tips: This document is carefully compiled by theeditor. I hope that after you download them,they can help yousolve practical problems. The document can be customized andmodified after downloading,please adjust and use it according toactual needs, thank you!In addition, our shop provides you with various types ofpractical materials,such as educational essays, diaryappreciation,sentence excerpts,ancient poems,classic articles,topic composition,work summary,word parsing,copyexcerpts,other materials and so on,want to know different data formats andwriting methods,please pay attention!Keeping pores clear is essential for maintaining healthy skin. Here are some tips to help you achieve this:1. Exfoliate regularly: Regular exfoliation helps remove dead skin cells and unclog pores. You can use a gentle scrub or a chemical exfoliant like salicylic acid. This will leave your skin feeling smooth and prevent the buildup of dirt and oil.2. Cleanse your face twice a day: Cleansing your face in the morning and evening is crucial for keeping your pores clear. Use a gentle cleanser that is suitable for your skin type. Avoid harsh soaps or cleansers that can strip your skin of its natural oils and cause irritation.3. Avoid heavy makeup: Wearing heavy makeup can clog your pores and lead to breakouts. Opt for lightweight, non-comedogenic products that won't block your pores. Make sure to remove your makeup thoroughly before going to bed toprevent pore congestion.4. Use a toner: Toning your skin after cleansing can help remove any remaining impurities and tighten your pores. Look for a toner that contains ingredients like witch hazel or tea tree oil, which have astringent properties.5. Moisturize daily: Moisturizing is essential for keeping your skin hydrated and preventing excess oil production. Choose a lightweight, oil-free moisturizer that won't clog your pores. This will help maintain the balanceof your skin and prevent pore congestion.6. Protect your skin from the sun: Sun damage can cause your pores to become enlarged and clogged. Always apply a broad-spectrum sunscreen with at least SPF 30 before going outside. Additionally, wearing a wide-brimmed hat and seeking shade can provide extra protection.7. Avoid touching your face: Touching your face with dirty hands can transfer bacteria and oil to your pores, leading to breakouts. Try to avoid touching your facethroughout the day and wash your hands regularly to keep them clean.8. Maintain a healthy diet: Eating a balanced diet that is rich in fruits, vegetables, and whole grains can help promote healthy skin. Avoiding greasy and processed foods can prevent excess oil production and keep your pores clear.9. Stay hydrated: Drinking enough water is essentialfor maintaining healthy skin. It helps flush out toxins and keeps your skin hydrated, preventing pore congestion. Aimto drink at least 8 glasses of water per day.10. Don't skip regular skincare routine: Consistency is key when it comes to maintaining clear pores. Stick to your skincare routine and be patient. It may take time to see results, but with regular care, you can achieve andmaintain clear pores.Remember, everyone's skin is unique, so it's importantto find a routine that works best for you. By following these tips and being consistent with your skincare routine,you can keep your pores clear and achieve healthy, glowing skin.。

硕士医学英语生殖系统

At the time of ovulation(排
tract(泌尿生殖道), the urethra as it passes through the and prostateuglarnod(l前o列g腺i)sts(泌尿科医师) are called on
These hormones are manufactured in interstitial
These hormones are manufactured in interstitial cells(睾丸间质细胞) located between the tubules. In both males and females, the gonads are stimulated by the hormones follicle stimulating hormone(促卵泡成熟激素) (FSH) and luteinizing hormone (LH)(促黄体生成激素), released from the anterior pituitary gland(垂体前叶) beneath the brain. Although these hormones are the same in both males and females, LH is called interstitial cell-stimulating hormone (ICSH) (促间质细胞激素)in males.
wherele layer of the uterine wall is the
epirdiedypmirtios ductive and urinary tracts share a common passage,
Although these hormones are the

Soot Formation in Combustion Processes (Review)

Combustion,Explosion,and Shock Waves,Vol.41,No.6,pp.727–744,2005Soot Formation in Combustion Processes(Review)UDC536.46 Z.A.Mansurov1Translated from Fizika Goreniya i Vzryva,Vol.41,No.6,pp.137–156,November–December,2005.Original article submitted April19,2005.A review is given of recent papers on the phenomenology,kinetics,and mechanismof soot formation in hydrocarbon combustion;the eﬀects of various factors on theformation of polycyclic aromatic hydrocarbons,fullerenes,and soot,low-temperaturesoot formation in coolﬂames,combustion in electricﬁeld,and the paramagnetism ofsoot particles from an ecological viewpoint are considered.Key words:soot formation,polycyclic aromatic hydrocarbons,fullerenes,combus-tion,nanocarbon tubes.INTRODUCTIONSoot formation is an important and constantly studied aspect of combustion,which has practical signif-icance in the production of technical carbon as an active ﬁller in rubber products and as a component of printing paints,as well as in energetic problems of hydrocarbon power plants and ecological issues[1–3].Investigation of soot formation and oxidation is of special importance for several reasons.On the one hand,soot is a commercial product,whose world pro-duction is estimated at107tons per year.Black soot (technical carbon)is used as aﬁller for elastomers(90% of technical carbon is utilized for this purpose,2/3of them in tyre production)and in copy machines and laser printers.On the other hand,soot is known to be one of the main environmental pollutants.For example, direct-injection diesel enginesﬁrst convert≈10–20%of the fuel to soot.To understand the complex soot forma-tion processes,one needs adequate models that should be tested by measuring particle concentration,parti-cle size distribution,volume fraction of soot,etc.[3]. It is important that the indicated parameters be mea-sured in situ by nondisturbing methods.Optical tech-niques,especially laser diagnostic methods are the most suitable for the indicated purposes.In addition to be-ing nondisturbing,they have high spatial and temporal resolutions,which is especially useful in studies of soot formation and oxidation in turbulentﬂames.1Al-Farabi Kazakh National State University,Almaty480078;anara@kazsu.kz.At present,a wealth of experimental material on soot formation has been accumulated and various phe-nomenological models have been proposed[1–11].How-ever,the soot formation mechanism is still not com-pletely understood.In even seemingly simple cases,for example,homogeneous hydrocarbon pyrolysis,there is no full understanding of the process because it involves many fast parallel reactions resulting in a new solid phase—soot particles(for example,the time of con-version of methane with a molecular weight of16amu to soot particles with a molecular weight of106amu is 10−4–10−2sec).Soot formation have been studied under various conditions:ﬂames(premixed and diﬀusion),high-temperature reactors,and shock tubes.1.PHENOMENOLOGYOF SOOT FORMATIONAccording to the laws of thermodynamics,in a pre-mixed fuel–air system at standardﬂame temperature, solid carbon should appear at a mixture ratio(ratio of carbon to oxygen atoms)C/O≈1[5],which is not always the case.Soot formation begins,except in deto-nations,at C/O ratios that are less than1and depend on the type of fuel.Some typical threshold parame-ters of soot formation(C/O)cr for almost adiabatically burningﬂames are presented in Table1[5,8].We note that in most of the Bunsen burnerﬂames,except in C2H2–air,soot is released at the cone tip.The diﬀerent values of(C/O)cr in burners of two types(Table1)are due to diﬀerences in temperature and in the process of formation of polycellularﬂame.0010-5082/05/4106-0727c 2005Springer Science+Business Media,Inc.727728MansurovTABLE 1Soot Formation Threshold (C/O)cr for PremixedFlames (p =1bar,t =293K)[5,8]Mixture(C/O)crBunsen Flat burner ﬂame C 2H 6–air 0.480.47C 3H 8–air 0.470.53∗C 3H 8–O 2—0.67∗C 2H 4–air 0.60.62C 2H 4–O 2—0.7C 4H 8–air 0.48—C 6H 6–air0.570.62∗Methylnaphthalene–air 0.42—C 2H 2–air—0.83Note.The values for multicellular ﬂames are marked by anasterisk.Fig.1.Soot threshold (C/O)cr versus ﬂame temper-ature for C 2H 4–air mixtures at various pressures and for C 2H 4–O 2,C 2H 2–air,and C 6H 6–air mixtures at p =1bar [12].The results presented in Table 1show that soot formation is controlled by the kinetics and occurs in ﬂames under oxidation conditions.In postﬂame gases with C/O ratios somewhat lower than the soot forma-tion threshold there are the same unburned hydrocar-bons present as in mixtures with ratios C /O >(C /O)cr .Therefore,the main processes of soot formation in the ﬂames occur in the main reaction zone,in which there is competition between the oxidation and formation of suﬃcient concentrations of higherhydrocarbons.Fig.2.Final volume fraction of soot versus excess of carbon for fuel–air ﬂames from ﬂat burners at normal pressure [12].The soot formation thresholds depend on temper-ature and pressure,as is illustrated in Fig.1[12].The curve shape in this ﬁgure,which is identical for the dif-ferent mixtures and pressure,shows that there is not one but two thresholds for the C/O ratio at two diﬀer-ent temperatures.With a pressure rise,the minimum of the (C/O)cr ratio is shifted toward the stoichiometric mixture (C /O =0.33for C 2H 4–air mixtures).In the lower-temperature region,the values of (C/O)cr increase as the temperature decreases.Below 1350–1400K,the burned gas contains a substance with a high molecular weight,but at such a low temperature,it cannot be transformed to soot for a time of contact t <0.1sec.For the indicated substance,the H/C ra-tio is approximately unity but its components are not identiﬁed.It should be noted that this low threshold temperature is similar to that observed on the center line of laminar diﬀusion ﬂames at the beginning of soot formation.In passing from the soot formation threshold in Fig.1,where the mass concentration of soot is about 10−9g/cm 3,toward high values of C/O,one observes a strong increase in the fraction of the ﬁnal volume of soot f v ∞with respect to the volume of the ﬂame gas.The curves in Fig.2qualitatively show how eﬀectively the excess of carbon over the corresponding soot formation thresholds is converted to soot for various fuels [12].Information on the conversion of burning hydrocar-bon fuels to polycyclic aromatic hydrocarbons (PAHs)and soot [11]is shown diagrammatically in Fig.3.PAHs are usually formed under conditions of rich mixtures and can have carcinogenic properties (as,for exam-Soot Formation in Combustion Processes(Review)729Fig.3.Schematic diagram of soot formation in homoge-neous systems or in premixedﬂames[11].ple,benzpyrene).It is generally agreed that PAHs are important precursors(nuclei)of soot particles with a molecular weight of500–2000amu.The further particle growth is due to the surface growth in reactions involv-ing many acetylene molecules and coagulation.Oxida-tion of soot particles occurs primarily in nonpremixed mixtures after the addition of oxygen-containing gases.2.SOOT FORMATIONIN DIESEL ENGINESSoot particles are formed in the region between the fuel-rich side of the reaction zone of the diﬀusion ﬂame and the fuel spray.The soot formation process can be regarded as a transition from the gas phase to a solid phase[5]with an extremely complex nature of conversion of the hydrocarbon fuel molecules containing a few carbon atoms to carbonaceous particles contain-ing a few millions of carbon atoms.Theﬁnest particles areﬁrst formed by coagulation of PAHs.After that,the soot concentration increases as a result of adsorption of PAHs and especially as a result of particle surface re-actions that increase this surface.This process involves hydrogen detachment and carbon attachment[13].The primary particles coagulate into large aggregates,which are not necessarily found in the diesel exhaust gases. Their detection strongly depends on the temperature and time of conversion of the particles in the oxidizing medium.Therefore,only a few thousandth of theini-Fig.4.Soot emission parameters for a four-cylinderdiesel engine of volume2.4liters[5].tially formed mass of soot is not completely oxidized and enter the diesel exhaust gases.The soot emission from diesel engines constitutes a considerable part of the atmospheric aerosol even under proper operating conditions and depends on the engine performance.In some regions of Germany,the soot con-tent in aerosols reaches20%.The soot emission from a four-cylinder diesel engine of volume2.4liters[5]is shown in Fig.4.It is evident that at2500rpm,the emission is relatively low but increases as the velocity becomes higher.Soot emission from diesel engines is inﬂuenced by the atomization and conﬁguration of the spray,the method of air supply,turbulence level,pressure,injec-tion time,and ignition delay[14].Studies of engine exhaust soot have shown that the particle size distribution depends weakly on the oper-ating conditions and type of engine.The mean parti-cle diameter is about200–300˚A.In soot samples from exhaust systems or those collected at low exhaust tem-peratures there are heavy hydrocarbons condensed into agglomerates and converted to tarry material;in this case,the H/C ratio increases to unity.The injection time,as mentioned above,is a very important factor.Early injection[15]allows one to in-ject more fuel,evaporate it,and mix it with air before ignition.High injection rates can also reduce soot for-mation.Increasing the air supply temperature reduces the ignition delay and,hence,promotes soot formation [14].As noted in[1],the real values of the C/O ratio should,whenever possible,be kept close to the critical values since the volume fraction and particle size of soot increase as the C/O ratio grows.730MansurovFig.5.Amount of soot(fraction of C atoms in sootparticles)versus pressure and temperature[17].3.PRESSURE EFFECTON SOOT FORMATIONThe pressure eﬀect on soot formation can be in-terpreted as the eﬀect of increased density of carbon atoms at elevated pressure.This assertion was veriﬁed in experiments on combustion of ethylene mixtures with oxygen and other oxidizers(p=1bar)[15].In these experiments,the reaction zone was spatially resolved. The density of carbon atoms in soot particles was three times higher than their density in the corresponding air ﬂames.The coagulation coeﬃcients in theseﬂames were constant.Observations of coagulation inﬂames with high densities of carbon atoms under normal pressure agree with theﬁndings of[8,15,16]obtained for C2H4–air ﬂames burning at high pressures,and support the as-sertion on the eﬀect of elevated density of carbon atoms.The volume fraction of soot increases with a rise in pressure and the C/O ratio,and the temperature depen-dence is described by a bell-shaped curve[17](Fig.5). The latter is due to two circumstances.Soot formation requires precursor radicals;therefore,this process does not go at low temperatures.In addition,soot precur-sors undergo pyrolysis and oxidation at elevated tem-peratures,so that the soot formation process is limited by the range of T=1000–2000K.Curves of carbon density versus C/O ratio for soot, PAHs,and C2H2at p=1,10,and70bar[18]are given in Fig.6.It is evident that pressure increases therateFig. 6.Carbon density versus the C/O ratio foracetylenes,PAHs,and soot at various pressures[18].of formation of PAHs and soot.The C/O ratio also inﬂuences the volume fraction of soot:an increase in the latter with increasing C/O ratio is also possible at high pressures.Above the particle formation threshold,the density of soot carbon increases.For large values of C/O,the increase in the carbon density slows down.The depen-denceρC(C/O)is stronger at p=10and70bar than at p=1bar.From Fig.6,it is evident that an increas-ing amount of carbon fuel is converted to soot as the pressure rises.In PAHs,the carbon density is two orders of mag-nitude lower than that in soot but the dependences of the carbon density of soot and PAHs on the C/O ra-tio at various pressure are very similar.From the data obtained,the following expression can be written:ρC(soot,PAH)≈[(C/O)−(C/O)cr]2.5–3at T>1700K and1 p 10bar.At pressures above10bar,most of the carbon is con-verted to CO and CO2.The most strikingﬁnding is the strong decrease in the C2H2concentration at p>10bar.At p=70bar, its concentration is below the detection limit.4.FULLERENES AND THEIRFORMATION DURING COMBUSTION As is known,in nature there are two allotropic forms of carbon—transparent solid diamond and lay-ered soft graphite.In1968,Sladkov with associated from the Institute of Petrochemical Processing of theSoot Formation in Combustion Processes(Review)731USSR Academy of Sciences synthesized a third al-lotropic form of carbon—carbine,a white substance with a density intermediate between the densities of di-amond and graphite.C60and C70fullerenes were identiﬁed in1985 [19]and obtained in macroscopic quantities in1990by graphite vaporization in an arc discharge.In1988and1991,C60and C70fullerene ions were extracted in signiﬁcant amounts fromﬂame and identiﬁed spec-troscopically[20,21].Howard[22]found considerable amounts of C60and C70in low-pressure premixed lami-nar benzene–oxygenﬂames.The greatest concentrationof C60+C70was20%of the resulting soot.The max-imum rate of formation of C60+C70was observed forp=69torr,C/O=0.989,and dilution with25%he-lium.In the fullerenes produced inﬂames,the C70/C60 ratio was in the range of0.26–8.8,whereas for graphitevaporization,C70/C60=0.02–0.18.Fullerenes are a class of exclusively carbon com-pounds with polyhedral closed-shell structure.Theywere identiﬁed as ionized particles in low-pressure fuel-richﬂat premixed acetylene and benzene–oxygenﬂames by molecular-beam sampling combined with mass-spectrometer analysis[20].Later,macroscopic quantities of various fullerenes,not only such as C60and C70[21,23]but also larger species up to C116,were extracted by solvents from the soot formed in low-pressure premixed laminar benzene ﬂames[24].The yield of C60and C70was20%of the weightof soot and0.5%of the weight of the starting fuel car-bon[25].The relationship between fullerenes and soot formation was theoretically established by Zhang et al.[26]after the discovery of fullerenes in experiments with laser vaporization of graphite[19].The reactions of fullerene and soot formation in an arc discharge have much in common with the reactions in rich fuelﬂames. Bulbous fullerene nanostructures were detected along with fullerenes in soot using high-resolution transmis-sion electron microscopy[27].Figure7gives concentration proﬁles of fullerenesand soot and the temperature proﬁle in a benzene–oxygen–argonﬂame(C/O=0.88)[23].It is evident that fullerenes are formed after the main stage of soot formation.As the C/O ratio increases from0.88to0.96, the fullerene concentration increases monotonically.Grieco et al.found[28]that in theﬂame reactionzone,the quantity of closed-shell structures in soot,as well as the fullerene concentration in the gas phase,in-creases with increasing residence time.At a distance of more than70mm above the burner,the fullerene concentration decreased,and at a distance between60 and120mm the concentration of closed-shellstructuresFig.7.Concentration proﬁles of fullerenes and sootand the temperature proﬁle in a benzene–oxygen–argonﬂame[23](H is the distance from the burner). decreased.The transition from amorphous to fullerene carbon can be explained by deposition gas-phase par-ticles,such as PAHs,on soot particles.The particle deposition is accompanied by internal redistribution of solid-state carbon and by reaction between gas-phase fullerenes and the growing soot particles,which is con-sistent with the consumption of fullerenes at a distance beyond70mm above the burner.Highly ordered nanos-tructures such as nanotubes and fullerene bulbs were detected in solid samples collected from the walls and top of the combustion chamber,indicating that they formed during the internal redistribution processes oc-curring in solid-state carbon for a time not exceeding 100msec.This conclusion is consistent with the for-mation of closed-shell species by intense thermal treat-ment of the initially disordered,insoluble part of the fullerene-containing soot generated in an electric arc discharge[29].In contrast to the proposed role of fullerene struc-tures as soot precursors[26],the results of[30]indicate parallel growth of gas-phase fullerene molecules along the vertical inﬂame.In addition,it is found[31]that the nucleation of soot particles is accompanied by de-position of fullerenes on them.Frenklach and Ebert[25]explain fullerene forma-tion by successive buildup of bent structures,i.e.,PAHs containingﬁve-and six-membered rings.They be-lieve that inﬂames bent PAHs should be less abundant732MansurovFig.8.Fullerene concentrations proﬁles on the axisof a premixed benzene–oxygen–argonﬂame[28].than planar PAHs,which is supported by measurements of concentrations of PAHs(including corannulene—a bent molecule)in fullerene-formingﬂames[32].Corannulene is a subsystem of almost all fullerenes and is therefore of special interest as a precursor of fullerenes.Corannulene occurs in detectable concen-trations in ethylene–airﬂames at atmospheric pressure, in which fullerenes have not been detected,but in large concentrations it was found in low-pressure fullerene-forming benzeneﬂames[33].The kinetic validity of the mechanism of fullerene formation by successive gas-phase reactions is shown by Baum et al.[34].Based on calculations for a plug-ﬂow reactor with experimental concentrations of various species as input data,the authors described the forma-tion of C60and C70fullerenes beginning withﬂuoran-thene and the subsequent growth by successive steps of hydrogen detachment and the addition of C2H2with corannulene as an intermediate compound.The formation of C60and C70fullerenes by de-tachment of hydrogen and the addition of C2H2was also tested by modeling a low-pressure fullerene-forming ﬂat premixed benzene-oxygen–argonﬂame at an equiv-alence ratio of2.4[34].The concentrations proﬁles of fullerenes[28]are characterized(Fig.8)by two local maxima,theﬁrst of which is smaller.Studying the evolution of the con-centrations of fullerenes,PAHs,and soot with increas-ing distance above the burner,Grieco et al.[28]draw the conclusion that theﬁrst maximum may be due to the reactive coagulation of PAHs,accompanied by in-tramolecular condensation,including dehydrogenation, rearrangement,and ringformation.Fig.9.Model for the formation ofﬁve-and six-membered rings through the linkage of two PAHs bythe zipper mechanism[37].Apparently,PAHs are mostly consumed in soot for-mation,and to a lesser extent in fullerene formation. Grieco et al.[28]concluded that the second concen-tration maximum,which is in the region of the most intense fullerene formation,cannot be attributed to re-actions involving PAHs alone,whose concentrations in this region are at or below the detection limit of ana-lytical equipment,but it can be explained by sequential attachment of acetylene to PAHs.The pathways of fullerene formation inﬂames have been extensively studied by Homann’s group using di-rect input of ionic and neutral species into a mass spectrometer[30,35,36].Experiments combining molecular-beam sampling with mass-spectral analysis were performed with low-pressure premixedﬂames of acetylene[37],benzene[38],butadiene and naphthalene [37]with oxygen.The important role of bimolecular reactions between two PAHs involving coordinated de-tachment of hydrogen(the so-called zipper mechanism) in the growth of fullerenes was explained[37].The re-action starts with a sandwich-type arrangement of two recondensed PAHs(Fig.9)and has a distinctive feature such as the formation of exactly12pentagons,which are larger or equal to coronene(C24H12),irrespective of the size of the PAHs.At the same time,by the zip-per mechanism,the pentagons should be brought to the thermodynamically most favorable positions,for exam-ple,by pyracylene rearrangement[39,40].Some ex-perimental evidence of the zipper mechanism has been obtained.Thus,hydrogenated species such as C60H x (1<x<6)[37]were predicted,and the shape and lo-cation of their concentration proﬁles agree with those of the precursors of the corresponding fullerenes.Further-more,a new class of carbon molecules—the so-called aromers,which are negatively charged ions produced by the reaction of two PAHs—was proposed.Aromers are represented as hydrogen-rich PAHs—species with a rather low level of structural packing,direct precursors of fullerenes[38].Soot Formation in Combustion Processes(Review)733The formation of fullerenes requires a number of single-step monomolecular reactions,such as cage clo-sure,C H bond rupture,and intramolecular rear-rangements,and,therefore,a high temperature of the process is preferred.In conclusion,a combined mecha-nism for fullerene formation can be presented.Taking into account the geometrical incompatibility of the ma-jority of aromers for the growth of fullerenes as theﬁnal products of the reaction between diﬀerent PAHs,it is assumed that the reactions with acetylene can lead to a further growth of the molecules up to the geometrical compatibility for required for cage closure.Finally,theﬂames suitable for the synthesis of macroscopic quantities of fullerenes[23,24]are char-acterized by higher values of C/O than theﬂames from which molecular-beam sampling can be performed [30,38].The processes of soot and fullerene formation along theﬂame axis at high C/O ratios,where substan-tial quantities of fullerenes are produced,are apparently similar,and,hence,the contribution of the condensed-phase formation pathways cannot be ruled out.5.FORMATION OF NANOCARBON TUBES AND NANOFIBERS IN FLAMESRecently,there have been a number of papers on the synthesis of carbon nanotubes usingﬂames as a heat source.The production of carbon nanomaterials(such as nanotubes and nanoﬁbers)inﬂames has a number of advantages over the production processes using elec-tricity.First,ﬂame as a heat source is far less expen-sive than electricity.Second,the synthesis inﬂames are more suitable for measurements than the methods based on electricity[41].Yuan et al.[42,43]found mul-tiwalled carbon nanotubes on a nickel–chromium wire and on a stainless steel mesh in diﬀusionﬂames.For C2H2diﬀusionﬂames,Vander Wal et al.[44] studied the relative contribution of the electronic struc-ture and chemical composition using Cu,Fe,and Ni,as catalysts and Al2O3,CaO,SiO2,and TiO2as supports. Those studies showed that the electronic interaction be-tween the metal catalyst and the support considerably inﬂuences the density,the homogeneity,and structure of the carbon nanotubes.In[45,46],a fuel-rich premixed ﬂame served as a heat source and a thin wire mesh of stainless steels(grade304)coated with cobalt was used as the catalyst.The postﬂame gas mixture was studied in equal proportions with various fuels to determine the optimal conditions for the growth of nanotubes in using hydrocarbons.The authors[45,46]came to the con-clusion the optimal molar fractions of CO and H2equal ≈0.105,which can be implemented using an ethylene–air mixture in a proportion of1.62.Fig.10.Radial temperature distribution showing three zones of formation of nanomaterials[51].Saveliev et al.reported[47]on the synthesis of carbon nanotubes in oxygen-rich opposed-ﬂow methane ﬂames.Straightened“bean-sprout-like”nanotube bun-dles were synthesized in a methane diﬀusionﬂame by Yuan et al.[48].In the experiments of Merchan-Merchan et al.[49],a thick layer of vertically aligned carbon nanotubes on the surface of a catalytic sam-ple was detected using an opposed-ﬂowﬂame with con-trolled electricﬁeld.The typical temperature for the synthesis of carbon nanotubes and nanoﬁbers in diﬀusionﬂames with cat-alytic methane is lower than the soot-formation temper-ature.Theﬁndings of[42,43]suggest that the forma-tion of carbon nanotubes and nanoﬁbers on a catalytic support occurs inside the soot-formation zone of a nor-mal diﬀusionﬂame.Unlike in diﬀusionﬂame,however, the formation of carbon nanotubes and nanoﬁbers on a catalytic support can occur outside the soot-formation zone,which is also located outside theﬂame front.Carbon atoms as a source of graphite layers,cat-alytic metals for the conversion of gas-phase carbon atoms to solid graphite layers,and heat sources for the activation of the catalytic metals are three impor-tant factors in the synthesis of carbon nanomaterials in ﬂames.Lee et al.[50]studied the synthesis of carbon nanotubes and nanoﬁbers on a metal support coated with Ni(NO3)2in an ethylene inverse diﬀusionﬂame as a heat source.Figure10shows the gas temperature distribution at theﬂame front[51],illustrating three zones of for-mation of nanomaterials using a support coated with Ni(NO3)2.The maximum gas temperature was1655K at3.5mm from theﬂame axis.In the temperature range of1400–900K,carbon nanotubes of diameter20–60mm734Mansurov Scheme No.1.formed.At T>1400K and T<900K,iron nanorods and carbon nanoﬁbers,respectively,were synthesized.6.FORMATION OF CARBON MACROPARTICLES DURINGPYROLYSIS OF HYDROCARBONSCarbon(soot)macroparticles are associated pri-marily with exhaust gas emissions from a powerful diesel engine or pyrolytic equipment.Diesel engine soot is an undesirable product that reduces the service life of engines,pollutes the environment,and is a hazard to man’s health since soot particles contain molecules of polyaromatic hydrocarbons.For example,for trucks of moderate power,the U.S.regulation limits the ex-haust of particles to0.05g/(kW·h)[52].Soot,however, is also produced commercially with strictly monitored operating conditions and is used in the production of trunks and gums.This kind of soot is called technical carbon.Studies of soot formation during pyrolysis of pure hydrocarbons and hydrocarbon mixtures under isother-mal conditions have shown that:1)sooty aerosols with high activation energies are formed[53–56];2)there are quantitative relations for the soot formation tendency of hydrocarbon[54–56];3)the inhibition of soot particle nucleation is observed during pyrolysis of hydrocarbon mixtures[53,56–58].The temperature and concentration of the hydro-carbon subjected to pyrolysis and the gas residence time in the pyrolytic system are the main factors determining the soot formation of carbon macroparticles.In the experiments of Shurupov[58],benzene, acetylene,carbon tetrachloride,and methane diluted with helium were pyrolized in a jet-ﬂow reactor under nearly isothermal conditions(at T=1373–1673K)with a gas residence time of up to125msec.The eﬀects of the temperature and initial concentration of the hy-drocarbons on the beginning of formation of disperse carbon and the number density of soot particles were studied.Empirical equations were obtained that de-scribe the induction period of soot formationτind for the isothermal pyrolysis of benzene,acetylene,carbon tetrachloride,and methane with variations in the con-centration of the substance subjected to pyrolysis and temperature:τind=(6.3±0.8)·10−12[C6H6]−0.8exp(26,700/T),τind=(3.2±0.5)·10−9[C2H2]−0.9exp(19,000/T),τind=(4.3±0.7)·10−9[CCl4]−0.9exp(18,700/T),τind=(9.0±1.5)·10−10[CH4]−0.9exp(21,600/T) (τind in seconds).The diﬀerences in the induction period of soot nu-cleation for diﬀerent hydrocarbons can be explained by the inhibition eﬀect of soot particle formation.The ex-periments showed that during pyrolysis of hydrocarbons diluted with helium,the concentration of soot particles is constant for contact times of10–125msec.7.SOOT FORMATION MECHANISMMany researchers regard polycyclic aromatic hy-drocarbons as precursors of soot particle nuclei.Thus, in an acetylene–oxygen diﬀusionﬂame,Homann and Wagner[8]found two types of polyaromatic molecules: 1)polycyclic aromatic compounds without sidechains:naphthalene,acenaphthalene,coronene, and phenanthrene;2)polycyclic aromatic compounds with side chains(amolecular weight of150–500).The concentration of polyaromatic compounds of the ﬁrst group remained almost unchanged in the soot-formation zone,whereas the concentration of PAHs of the second group increased sharply.In the zone of intense soot formation,one can dis-tinguish one more large group of molecules—poly-acetylenes,which are intermediate products from the formation of polyaromatic compounds.Homann and Wagner[59]proposed a model for the formation of poly-acetylenes by scheme No.1.。

怎么减少皮肤伤害英语作文

怎么减少皮肤伤害英语作文英文回答：In our relentless pursuit of flawless skin, we often overlook the relentless assault it endures daily. From environmental aggressors to our own habits, our skin faces a barrage of potential harm. Yet, with mindful actions, we can significantly reduce the damage and preserve its youthful glow.Sun exposure ranks high among the most detrimental factors for skin health. The ultraviolet (UV) rays emitted by the sun penetrate deep into the skin, damaging collagen and elastin fibers that provide elasticity and firmness. Over time, this damage manifests as wrinkles, fine lines, and age spots. To minimize sun damage, incorporate sunscreen into your daily routine, reapplying it liberally every two hours. Seek shade during peak sun hours and consider covering exposed skin with breathable fabrics or UV-protective clothing.Pollution plays a significant role in skin damage. Tiny particles suspended in the air can penetrate pores, clogging them and creating inflammation. These inflammatory reactions contribute to premature aging and the development of skin conditions such as eczema and psoriasis. To combat pollution's harmful effects, cleanse your skin thoroughly twice a day, using a gentle cleanser that won't strip your skin of its natural oils. Consider incorporating antioxidants into your skincare routine to neutralize the free radicals generated by pollution. Green tea extract, vitamin C, and ferulic acid are potent antioxidants that can help shield your skin from environmental damage.Our daily habits can inadvertently contribute to skin damage. Harsh soaps, excessive exfoliation, and improper shaving techniques can compromise the skin's protective barrier, making it more susceptible to external aggressors. Instead, opt for gentle, pH-balanced cleansers, exfoliate no more than twice a week with a mild scrub, and use a sharp razor with shaving cream to minimize irritation.Emotional stress can also take a toll on our skin. When we experience stress, our bodies release hormones like cortisol, which can trigger inflammation and breakouts. Chronic stress can lead to skin conditions like acne, eczema, and rosacea. To mitigate the impact of stress on your skin, engage in stress-reducing activities like yoga, meditation, or spending time in nature. Incorporating relaxation techniques into your routine can help regulate cortisol levels and promote skin health.By understanding the factors that contribute to skin damage and implementing mindful practices, we can effectively reduce the harm and maintain the vibrancy of our skin. Remember, your skin is a reflection of your overall health and well-being. Embrace these strategies as an investment in your skin's future, and you will reap the rewards of a radiant, youthful complexion.中文回答：在追求零瑕疵肌肤的过程中，我们常常忽视了皮肤每天都在承受的无情侵害。

松六齿小蠹成虫触角感器的扫描电镜观察

松六齿小蠹成虫触角感器的扫描电镜观察王全坡;张然然;陈辉【摘要】[目的]研究松六齿小蠹成虫触角感受器的形态、数量和分布特征,为明确松六齿小蠹触角感器的功能和寄主树木选择提供理论依据.[方法]运用扫描电子显微镜,对松六齿小蠹雌雄成虫触角外部形态与感器进行超微形态观察.[结果]松六齿小蠹雌、雄成虫的触角感器分为毛形感器(Ⅰ型和Ⅱ型)、锯齿形感器、刺形感器、芽形感器、锥形感器和槽纹感器等6类.其中毛形感器数量最多,约占总数的85％;锯齿形感器的分布最广,在触角各节均有分布.触角锤头部感器的种类和数量最多,6种感器均有分布,占全部感器数量的92.3％;柄节的感器数量最少,仅占3.4％.[结论]松六齿小蠹雌、雄成虫触角的形态及感器类型和分布无明显差异,其中毛形感器Ⅰ、槽纹感器为化学感器,具有感知嗅觉的作用;毛形感器Ⅱ、锯齿形感器为机械感器.【期刊名称】《西北农林科技大学学报（自然科学版）》【年(卷),期】2013(041)011【总页数】6页(P87-92)【关键词】松六齿小蠢;触角;感器;扫描电镜【作者】王全坡;张然然;陈辉【作者单位】西北农林科技大学林学院,陕西杨凌712100;西北农林科技大学林学院,陕西杨凌712100;西北农林科技大学林学院,陕西杨凌712100【正文语种】中文【中图分类】S763.38;Q964松六齿小蠹(Ips acuminatus Gyllenhal)隶属于鞘翅目(Coleoptera)小蠹科(Scolytidae)齿小蠹属(Ips)，是广泛分布于世界各地危害针叶树木的蛀干害虫[1]。

近年来，由于气候变化的影响，松六齿小蠹在阿尔卑斯山东西部的欧洲赤松林区大面积爆发，严重威胁着森林的健康[2-4]；其在我国主要危害华山松(Pinus armandi)、油松(Pinus tabulaeformis)、马尾松(Pinus massoniana)等松科植物[1]，也是秦岭巴山林区华山松小蠹虫的优势种群和华山松大小蠹的主要跟随种[5]。

祛痘护肤英语

祛痘护肤英语Acne Treatment and Skincare: Fabulous Tips for Clear and Healthy SkinIntroduction:Having clear and healthy skin is a goal for many individuals. Acne, a common skin condition, can be frustrating and affect one's self-confidence. In this article, we will explore effective strategies for treating and preventing acne, as well as tips for maintaining a healthy skincare routine.1. Understanding Acne:Acne is a skin condition characterized by the formation of pimples, blackheads, and whiteheads. It occurs when hair follicles become clogged with oil and dead skin cells. Understanding the causes and types of acne is crucial for its effective treatment.Types of Acne:- Comedonal Acne: Characterized by the presence of blackheads and whiteheads.- Inflammatory Acne: Results in inflamed lesions such as pustules, papules, or nodules.- Cystic Acne: The most severe form of acne, resulting in painful, deep cysts.2. Treating Acne:a) Cleansing and Exfoliating:Gentle cleansing twice a day is essential for removing excess oil, dirt, and bacteria from the skin. Avoid using harsh cleansers that can strip the skin of its natural oils. Exfoliating once or twice a week can help remove dead skin cells, preventing clogged pores.b) Topical Treatments:Topical treatments such as benzoyl peroxide or salicylic acid can be used to target specific acne lesions. These ingredients help reduce inflammation and unclog pores, promoting clearer skin. Apply these treatments as directed by a dermatologist.c) Oral Medications:For moderate to severe acne, oral medications such as antibiotics or isotretinoin may be prescribed. These medications work to reduce bacteria, inflammation, and oil production, resulting in improved skin condition.d) Professional Treatments:In severe cases, professional treatments like chemical peels, microdermabrasion, or laser therapy may be recommended by a dermatologist. These procedures can help improve acne scarring and promote overall skin health.3. Preventing Acne:a) Cleanse Properly:Avoid over-washing your face, as this can strip away essential oils and exacerbate acne. Use a gentle cleanser and lukewarm water to wash your face twice daily.b) Moisturize:Choose a non-comedogenic moisturizer to hydrate your skin without clogging the pores. Moisturizing is essential, even for oily skin types, as it helps maintain the skin's barrier function.c) Healthy Diet:A balanced diet rich in fruits, vegetables, whole grains, and lean proteins can contribute to healthy skin. Avoiding excessive consumption of sugary, greasy, and processed foods may help prevent breakouts.d) Stress Management:Stress can worsen acne flare-ups, so practicing stress-reducing techniques such as exercise, meditation, or deep breathing exercises can be beneficial for both physical and mental health.e) Hair and Makeup Products:Choose hair and makeup products labeled as "non-comedogenic" or "oil-free" to prevent clogged pores. Avoid touching your face excessively or resting your phone on your cheek, as these can transfer bacteria to the skin.4. Skincare Routine:a) Cleansing:Cleanse your face twice a day using a gentle cleanser, followed by patting dry with a clean towel.b) Toners and Serums:Apply a toner to balance the skin's pH levels and enhance absorption of subsequent products. Follow with a serum containing ingredients like vitamin C or niacinamide to address specific skin concerns.c) Moisturizing:Apply a moisturizer suitable for your skin type to keep the skin hydrated and supple. Don't forget to include sunscreen during the day to protect your skin from harmful UV rays.d) Weekly Treatments:Incorporate weekly treatments such as face masks or exfoliating treatments to promote skin renewal and radiance. Consult with a skincare professional to determine the most suitable products for your skin type.Conclusion:Achieving clear and healthy skin requires a combination of effective acne treatment, preventive measures, and a consistent skincare routine. By understanding the causes of acne and implementing the tips provided in this article, you can be on your way to achieving the radiant, blemish-free skin you desire. Remember to consult with a dermatologist for personalized advice and guidance on selecting the most appropriate acne treatment plan for your specific needs.。

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Formation and control of strip scale pores in hot rollingC.S.Li a,*,J.Z.Xu a ,X.M.He b ,X.H.Liu a ,G.D.Wang aaThe State Key Laboratory of Rolling and Automation,Northeastern University,Shenyang 110006,PR ChinabShanghai Baosteel Group Corporation,Shanghai 201900,PR ChinaReceived 21January 2000AbstractIn order to solve the problem of strip surface pores generated by the combination of the tertiary scale on strip surface with black oxide layers produced on the roll surface,the mechanism of strip scale pores formation was analyzed using several instruments such as the SEM,the AHM-2-HL optical microscope and the Oxford ISIS EDAX energy spectrometer.A method of control is advanced to reduce strip scale pores,which is effective and clearly bene®cial for enhancing the strip surface quality in practice.#2001Published by Elsevier Science B.V .Keywords:Formation;Control;Strip;Scale pores;Hot rolling1.IntroductionHot strip has been a subject to increasingly more stringent quality requirements during the past 30years.The require-ments relating to such parameters as thickness,width and pro®le tolerances are largely complied with today as a result of the use of perfectly operating equipment such as auto-matic gauge control (AGC),automatic width control (AWC),short stroke control (SSC)and continuously vari-able crown (CVC)equipment.An important quality feature of hot strip is the condition of its surface,which is subject to stringent requirements.Owing to roll wear and tertiary scale,the defect known as scale pores produced on the strip surface is a severe quality problem.During the hot rolling process,the strip surface will have a scale layer as it is rolled in each mill stand.The scale will be regenerated either after passing through the ®nishing descaler,or between each mill stand.Therefore,the strip surface condition in the roll bite will be dependent on the quantity and the characteristics of the scale formed before entering each mill stand.This scale is called tertiary scale because it is formed after the descaler but ahead of the ®nishing mill.Roll wear modi®es the original condition of the work rolls,changing their geometry and surface roughness.Thechanges in roll geometry (crown,diameter,shape)combined with the in¯uence of thermal expansion,affect the pro®le and ¯atness of the strip through the ®rst or the last stands of the mill.With regard to the effects on the surface condition of the rolls,two different conditions have to be taken into consideration.First,roll wear modi®es the product surface appearance.Second,when the wear is suf®ciently high,tertiary scale,developed after the descaler,may accumulate in the roll cavities and,after reaching certain limits,is removed from the roll and imprinted in the strip surface,generating the scale pores defect of hot strip rolling.Some references have discussed the mechanisms of roll wear [1±7]and of scale formation [8,9],but the mecha-nism of strip scale pores has not been clari®ed.This paper will further investigate the formation of strip scale pores.A method of control for strip scale pores will be advanced.2.Experimental material and methodsThe specimen material,taken from Shanghai Baosteel 2050mm hot rolling mill,in this paper is AP1057D5.The chemical composition of the material is shown in Table 1.Fig.1shows the pattern of the strip scale pores investigated by means of an AHM-2-HL optical microscope.It is clear that the scales pores are dispersed thin sandy scales,the color is black/brown,and the shape looks like pinholes.Fig.2shows an energy spectrum of the strip surface.The composition of the scale pores is determined to be Fe 2O 3byJournal of Materials Processing Technology 116(2001)201±204*Corresponding author.Fax: 86-24-23906472.E-mail address :lichangsheng@ (C.S.Li).0924-0136/01/$±see front matter #2001Published by Elsevier Science B.V .PII:S 0924-0136(01)01047-0means of a CAMBRIDGE S360X-ray Energy Spectrometer.In order to observe the pattern of the scales pores cross-section further,a CAMBRIDGE S360SEM was employed (see Fig.3).3.Results and discussions3.1.The formation mechanism of scale poresThe basic features of scale formation are as follows [4]:1.Ferrous elements under high temperature and oxidizing atmospheres generate scale.2.The scale layer consists of three elements,wustite (FeO),magnetite (Fe 3O 4),and hematite (Fe 2O 3),these being in different proportions at different temperatures.3.Each scale type has a different morphology,hardness and growth dynamics.The dynamics of scale layer formation is governed by the following three consecutive reactions:1.Transportation of oxidizing elements to the scale.2.Surface reaction.This reaction is of importance in both cases.The reaction rate is linear and dependent on the exchange potential between the atmosphere and the metal surface.3.Transportation of Fe 2 ions through the wustite,the speed for this step being parabolic.Table 2shows the density,peak stress,and the hardness value of the three kinds of oxide.The data shows that wustite is the softest scale,while hematite is signi®cantly harder than the work rolls (Vickers 410to 615).According to the theory of friction,the scale on the strip will slip between the roll surface and the strip surface in the deformation zone.There are four possible slippages:(i)between the scale and the roll surface,(ii)inside the scale,(iii)on the boundary between the strip matrix and the scale,and (iv)inside the strip.Actually,slippage will happen where there is minimum resistance.The position of the slippage surface depends on the composition of the scale and the matrix,the thickness of the scale,the rolling temperature,and the roll surface deterioration.The most likely position is the boundary between the strip matrix and the scale,as shown in Fig.4.Table 1Chemical composition of the strip investigated (wt.%)C Si Mn P S Other 0.1600.0770.7100.0240.0100.019Fig.1.The pattern of the scalepores.Fig.2.The scale pores spectrogram of the stripsurface.Fig.3.The pattern of the scale pores cross-section by SEM.Table 2Scale properties TypeDensity (g/cm 3)Peak stress (kg/mm 2)Vickers hardness FeO 5.670.04270±350Fe 3O 4 5.184420±500Fe 2O 35.2411000Fig.4.The pattern of the slippage between roll and strip.202 C.S.Li et al./Journal of Materials Processing Technology 116(2001)201±204In Fig.4,there are shown three areas,i.e.areas1,2,3. Area2is the key position where strip scale pores will be produced.In this area,the convex surface of the roll crushes into the strip surface.Because of the heavy cooling of the scale,the hardness of the scale becomes high in practice. When slippage between the roll and the strip occurs the scale will be deformed,and the strip matrix in contact will be furrowed.The more that the roll surface deteriorates,the more the effect.The scale will accumulate on the strip surface and is crushed into the strip matrix,resulting in the formation of scale pores on the strip surface.The formation of the scale pores is related to the falling of the roll surface oxide layers.Roll surface deterioration is due to three factors as follows:(i)contact fatigue arises betweenthe®nishing work rolls and the backup rolls,(ii)as a result of slippage,friction and wear occur between the work rolls and the strip,and(iii)because of cooling water and contact diathermancy,thermal fatigue occurs.If tertiary scale exists on the strip surface,and the roll surface is rough,thin scale will be crushed into the strip matrix.3.2.Control of strip scale poresThe present investigation is mainly focused on the mechanism of the formation of strip scale pores.How to prevent and how to control scale pores are important for practical production.The best method is to control the temperature of the roll and the temperature of the®nishing front stand strip by the following means:1.Keep the device of the descaler and the roll cooling water in good condition.Between F2and F3,cooling water is used to reduce the temperature of the strip.To lessen the amount of the tertiary scale,the atmosphere was isolated to the best of the available fixing abilities. The composition of the scale is different at different temperatures,and the hardness of the scale is different also.Fig.5shows the temperature from stand F1to stand F7measured in the Shanghai Baosteel2050mmhot rolling mill.The difference of the strip temperature between F3and F4is218C under F6to F7water cooling,and198C under F2to F3cooling water.Fig.6 shows that the temperature of the roll surface was reduced.It is clear that the oxide layers of the roll surface were decreased when the roll temperature was reduced.2.Great acceleration in power must be avoided.If the power acceleration is large,the strip temperature will be high and the roll surface will be rough,so that the strip matrix will be furrowed.3.High pressure water along production line was em-ployed to control tertiary scale.The temperature of RT4is controlled at970±10008C for some strip steel (e.g.,AR4158E5,AR3151E5,AR4247E5).4.Industrial applicationSome results were obtained by implementing modi®ca-tions,these being as follows:1.Increased rolling mill productivity.The downtime for roll changes was reduced from24times in1997to4 times in1999(Fig.7).Fig.5.The rolling temperature for differentstands.Fig.6.The work roll temperature for differentstands.Fig.7.Roll change delay times in different years(times).C.S.Li et al./Journal of Materials Processing Technology116(2001)201±2042032.The rejection of the product due to strip scale pores was reduced from 0.163%in 1997to 0.054%in 1999[1±8](Table 3).3.The rolling schedule length is now 75km without roll change,being increased from the rolling schedule length of 60km in 1997(Fig.8).5.Conclusions1.In hot strip rolling,when slippage between the roll and the strip occurs,the scale will be deformed,and the stripmatrix in contact will be furrowed.The more that the roll surface deteriorates,the greater is the furrowing effect.2.The scale accumulates on the strip surface,and is crushed into the strip matrix,thus scale pores are formed on the strip surface.The formation of the scale pores is related to the falling off of the roll surface oxide layers.3.The main methods to control scale pores are to keep the device of the descaler and the roll cooling water in good condition.The roll surface oxide layers and tertiary scale should be controlled well.References[1]C.E.Peterson,Iron Steel Eng.12(1956)98.[2]O.Kato,ISIJ Int.32(1992)1216.[3]S.-I.Araya,S.Uchida,S.Hamauzu,in:Proceedings of theInternational Conference on Steel Rolling,Chiba,Japan,1998,p.417.[4]J.C.Mascia,O.C.Marini,E.Ubici,Iron Steel Engr.6(1998)48.[5]Yoshikazu,ISIJ Int.32(1992)1194.[6]H.Noguchi,Trans.ISIJ 28(1998)479.[7]V .Lanteri,C.Thomas,J.Bocquet,in:Proceedings of the InternationalConference on Steel Rolling,Chiba,Japan,1998,p.423.[8]Y .Oike,ISIJ Int.32(1992)1211.[9]H.Chuanqing,Z.Kun,F.Mingqiang,Iron and Steel 32(1997)32(in Chinese).Table 3The rejections for scale poresYear (month)19971998199912345678Rejection (%)0.1630.1080.060.060.020.080.080.010.050.07Quantity (t)2522258936933229308Fig.8.Schedule length in different years (km).204C.S.Li et al./Journal of Materials Processing Technology 116(2001)201±204。