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Endotoxin removal from protein solutions

Journal of Biotechnology76(2000)97–119/locate/jbiotecReview articleEndotoxin removal from protein solutionsDagmar Petsch1,Friedrich Birger Anspach*Biochemical Engineering Di6ision,GBF-Gesellschaft fu¨r Biotechnologische Forschung mbH,Mascheroder Weg1,D-38124Braunschweig,GermanyReceived14April1999;received in revised form17August1999;accepted23August1999AbstractEndotoxins liberated by gram-negative bacteria are frequent contaminations of protein solutions derived from bioprocesses.Because of their high toxicity in vivo and in vitro,their removal is essential for a safe parenteral administration.A general method for the removal of endotoxins from protein solutions is not available.Methods used for decontamination of water,such as ultraﬁltration,have little effect on endotoxin levels in protein solutions. Various techniques described in the patent literature are not broadly applicable,as they are tailored to meet speciﬁc product requirements.Besides ion-exchangers and two-phase extraction,afﬁnity techniques are applied with varying success.Also,taylor-made endotoxin-selective adsorber matrices for the prevention of endotoxin contamination and endotoxin removal are discussed for this purpose.After giving an overview of the properties of endotoxins and the signiﬁcance of endotoxin contamination,this review intends to provide an overall picture of the various methods employed for their removal.Avenues are pointed out how to optimise a method with regard to the speciﬁc properties of endotoxins in aqueous solution.©2000Elsevier Science B.V.All rights reserved.Keywords:Down stream processing;Lipopolysaccharides;Endotoxin removal;Two-phase extraction;Afﬁnity adsorption;Method optimizationAbbre6iations:BFGF,basicﬁbroblast growth factor;BSA,bovine serum albumin;CF,clearance factor;CIP,cleaning-in-place; DAH,diaminohexane;DEAE,diethylaminoethane;DOC,deoxycholate;EDTA,ethylenediaminetetraacetic acid;EU,endotoxin unit;FT-IR,Fourier transform-infrared;GMP,good manufacturing practice;HEPES,4-(2-hydroxyethyl)-piperazin-1-ethanesul-fonic acid;HSA,human serum albumin;IgG,immunoglobulin G;IgM,immunoglobulin M;KDO,2-keto-3-deoxyoctonic acid; LAL,Limulus amoebocyte lysate;NMR,nuclear magnetic resonance;PEI,poly(ethyleneimine);PLH,poly-L-histidine;PLL, poly-L-lysine.*Corresponding author.Tel.:+49-531-6181743;fax:+49-531-6181175.E-mail address:anspach@gbf.de(F.B.Anspach)1Present address:MPB Cologne GmbH,Eupener Straße161,D-50933Ko¨ln,Germany.0168-1656/00/$-see front matter©2000Elsevier Science B.V.All rights reserved.PII:S0168-1656(99)00185-6D.Petsch,F.B.Anspach/Journal of Biotechnology76(2000)97–119 981.IntroductionIn the course of this century bacterial endotox-ins turned out to be one of the most interesting and exciting molecules found in nature.Their peculiar structure,their chemical and physical diversity and their broad spectrum of biological activities has resulted in worldwide research in thisﬁeld.While the knowledge about chemical composition and structure of endotoxins is well developed,many questions remain to be answered about the role of endotoxins in human health, especially its pathophysiology.Closely related to this area is a technically very important aspect, the problem of removing undesirable traces of endotoxin from aqueous solutions,especially from parenterals.Bacterial endotoxins show strong biological ef-fects at very low concentrations in human beings and many animals when entering the blood stream, e.g.during a bacterial infection or via intravenous application of a contaminated medicament.This requires removing even minute amounts of endotoxin from such preparations. The threshold level of endotoxin for intravenous applications is set to5endotoxin units(EU)per kg body weight and hour by all pharmacopoeias (European Pharmacopoeia,1997).The term EU describes the biological activity of an endotoxin. For example,100pg of the standard endotoxin EC-5,200pg of EC-2and120pg of endotoxin from Escherichia coli O111:B4have an activity of 1EU(Kru¨ger,1989).It is taken as a rule of thumb that1EU corresponds to100pg of endo-toxin.Meeting this threshold level has always been a challenge in biological research and phar-maceutical industry(Berthold and Walter,1994). Endotoxins are very stable molecules,their bio-logically active part surviving extremes of temper-ature and pH in comparison to proteins(Sharma, 1986).Routinely,temperatures of180–250°C and acids or alkalis of at least0.1M must be chosen to destroy endotoxins in laboratory equipment. Thus,it is a challenge to remove endotoxin con-taminations from sensitive substances,such as proteins.Although common puriﬁcation protocols may reduce the endotoxin content below the threshold level,an absolute guarantee cannot be given.It may happen that one charge of theﬁnal product is accidentally contaminated and fails the quality control.This product has to be discarded;also, reprocessing is not ruled out speciﬁcally but is a costly alternative.Subjecting the product to trace decontamination is not allowed owing to GMP regulations except this is explicitly part of the validated process.The question,how endotoxin removal can be carried out in an economical way has occupied many investigators and has been—although not published—the reason for process rearrange-ments in many cases.However,this item has not yet been solved satisfactorily.This article intends to discuss relevant aspects regarding endotoxin removal from protein solutions and critically re-view existing approaches.First,an introduction in the chemical and biological properties of endotox-ins will be given as those are strongly inﬂuencing all removal techniques.2.Properties of endotoxins2.1.Origin of endotoxinsSince more than100years it is known that gram-negative bacteria carry a heat-stable toxin called endotoxin.This terminus was chosen by Richard Pfeiffer(1858–1945),a pupil of Robert Koch,to distinguish endotoxins from bacterial exotoxins,such as botulinum or tetanus toxins. Endotoxins are an integral part of the outer cell membrane of gram-negative bacteria and are re-sponsible for the organisation and stability(Vaara and Nikaido,1984).Approximately three-quarters of the bacterial surface consist of these molecules. As dominant surface structures they also partici-pate in the interaction of the bacterial cell with its environment and possible hosts.Only a few gram-negative bacteria,namely some Sphingomonas species,are devoid of endotoxin but carry other amphiphilic molecules replacing it(Kawahara et al.,1991).Apart from these exceptions endotoxins can be regarded as a characteristic attribute of gram-negative bacteria.D.Petsch,F.B.Anspach/Journal of Biotechnology76(2000)97–11999Although endotoxins areﬁrmly anchored within the bacterial cell wall(Raetz,1990),they are continuously liberated into the surrounding medium.Endotoxin release clearly does not hap-pen only with cell death but also during growth and division.Since bacteria can grow in nutrient-poor media such as water,saline and buffers, endotoxins are found almost everywhere.High concentrations are found where bacteria accumu-late or are to be used for technical purposes,such as in bioprocessing.2.2.Chemical and supramolecular structure Chemically endotoxins are lipopolysaccharides2 that consist of three biologically,chemically,ge-netically and serologically different parts(Fig.1). These are a non-polar lipid component,called lipid A,the so-called core oligosaccharide and a heteropolysaccharide representing the surface antigen(O-antigen).Since the pioneering work of Westphal and Lu¨deritz in1954(Westphal and Lu¨deritz,1954), numerous studies have been published about the structure and composition of each of the three endotoxin regions;a comprehensive description is provided by Rietschel(1984),and a review by Raetz(1990).The O-antigen is built up of a chain of repeat-ing oligosaccharide units(of three to eight monosaccharides each),which are strain speciﬁc and determinative for the serological identity of the respective bacterium.The bacterial O-antigens are an impressive example of nature’s versatility along a given chemical make-up.Some deﬁcient strains lack the O-antigen,such as E.coli K-12. This genetic defect does neither impair the viabil-ity of the microorganism nor the biological po-tency of endotoxin.The core oligosaccharide has a conserved struc-ture with an inner KDO-heptose region and an outer hexose region.In E.coli speciesﬁve differ-ent core types are known,Salmonella species share only one core structure.The most conservative part of endotoxin is lipid A,which,apart from few exceptions(Mayer and Weckesser,1984;Vaara and Nurminen,1999), shows very narrow structural relationship in dif-ferent bacterial genera.It consists of a b-1,6 linked disaccharide of glucosamine,covalently linked to3-hydroxy-acyl substituents with12–16 carbon atoms via amid and ester bonds;these may be further esteriﬁed with saturated fatty acids.This hydrophobic part of endotoxin adopts an ordered hexagonal arrangement,resulting in a more rigid structure compared with the rest of the molecule.Lipid A-deﬁcient strains show increased permeability of the outer cell membrane forFig.1.Schematic view of the chemical structure of endotoxin from E.coli O111:B4according to Ohno and Morrison(1989). Hep,L-glycero-D-manno-heptose;Gal,galactose;Glc,glucose;KDO,2-keto-3-deoxyoctonic acid;NGa,N-acetyl-galac-tosamine;NGc,N-acetyl-glucosamine.2Frequent synonyms of endotoxin are lipopolysaccharide and pyrogen.D.Petsch,F.B.Anspach/Journal of Biotechnology76(2000)97–119100Fig.2.Structures of endotoxin aggregates in aqueous solutions of different composition.The left symbol represents the endotoxin monomer;(open square)and(grey square)are hydrophilic sites,(black square)is lipophilic,(black circle)represent charged functional groups,(open circle)bivalent cations,such as Ca2+and Mg2+.periplasmic proteins(Nurminen et al.,1997)and a decline in cell viability(Mohan et al.,1994). Strains lacking lipid A or endotoxin are not known.The core region close to lipid A and lipid A itself are partially phosphorylated(p K1=1.3, p K2=8.2of phosphate groups at lipid A,Hou and Zaniewski,1990a);thus endotoxin molecules exhibit a net negative charge in common protein solutions.A certain microheterogeneity results from non-stoichiometrical substitutions.For example,phos-phate groups at lipid A and the core region may be substituted with arabinose,ethanolamine and phosphate in varying amounts.Also single sac-charide units of the O-antigen may be acetylated, sialylated or glycosylated.Beyond this,O-anti-gens vary concerning the number of repeating units,causing a certain heterogeneity of the endo-toxin population of each bacterium(Novotny, 1984).Active alterations of the ratio of endotoxin variants on the outer cell membrane enable bacte-ria to adopt to changing environmental conditions (phase variation,see Van Putten,1993).This has the consequence that a single chemical structure of the endotoxin molecule is hardly to be found even at the same bacterium.The molar mass of an endotoxin monomer as shown in Fig.1is about10kDa,though also 15–20kDa is reported owing to the variability of the oligosaccharide chain.Endotoxin molecules form aggregates with high stability,therefore pro-ducing a bewildering polymorphism.In the early days of endotoxin research the description of the supramolecular structure was rather phenomeno-logical.Electron microscopy photographs showed endotoxins as‘snake-,donut-or rod-likeﬁlaments andﬂat sheets’(Shands et al.,1967;Hannecart-Pokorni et al.,1973).Modern analytical methods, such as X-ray diffraction,FT-IR spectroscopy, neutron scattering,NMR(reviewed by Seydel et al.,1993)and also molecular modelling(Kas-towsky et al.,1992),revealed a more detailed image of the three-dimensional organisation of endotoxins.Although these studies did not convey a uniform picture,it is evident that endotoxins aggregate in lamellar,cubic and hexagonal in-verted arrangements,such as micelles and vesicles, with diameters up to0.1m m(Fig.2).The lipid A portion was identiﬁed as the major morphological determinant.It is proposed that aggregation is governed by non-polar interactions between neighbouring alkyl chains as well as to bridges generated among phosphate groups by bivalent cations(De Pamphilis,1971;Wang and Hollingsworth,1996).Endotoxin micelles and vesicles are much more stable than those of simple detergents.Thus,vesi-cles are even found in ultrapure water.Monomers have to be explicitly created by using detergents (e.g.Triton X-114),bile acids(e.g.deoxycholic acid)and chelators(e.g.EDTA).However,also proteins may shift equilibria,releasing endotoxin monomers from aggregates(Li and Luo,1997). The terminal state of these processes cannot be predicted;it depends on the properties of proteinsD.Petsch,F.B.Anspach/Journal of Biotechnology76(2000)97–119101(net charge,hydrophobicity)and solutions(pH, ionic strength).In summary it can be said that despite a com-mon general assembly and charge there is consid-erable chemical and physical heterogeneity.This complicates the development of a generally appli-cable method for endotoxin removal from protein solutions.2.3.Clinical aspects of endotoxin intoxication Biomedical research revealed the toxically ac-tive part being lipid A.Endotoxins or lipid A do not act directly against cells or organs but through activation of the immune system,espe-cially the monocytes and macrophages.These cells release mediators,such as tumour necrosis factor,several interleukins,prostaglandins,colony stimulating factor,platelet activating factor and free radicals(Pabst and Johnston,1989;Rietschel et al.,1994),having potent biological activity and being responsible for the adverse effects seen upon endotoxin exposure.These include affecting struc-ture and function of organs and cells,changing metabolic functions,raising body temperature, triggering the coagulation cascade,modifying haemodynamics and causing shock(Martich et al.,1993).Many approaches have been made to prevent or treat the deleterious effects of endotoxins on immune cells.These include the use of anti-endo-toxin antibodies(Ziegler et al.,1991),endotoxin partial structures for blocking endotoxin receptors (Lynn and Golenbock,1992)and mediator recep-tor antagonists.However,the interaction of endo-toxins with immune cells is not only mediated by speciﬁc receptors.Cell priming may also occur by non-speciﬁc intercalation of endotoxin molecules into the membranes of the target cells(Morrison, 1985).Finally,it should not be concealed that endo-toxins may also have beneﬁcial effects.They have been used in artiﬁcial fever therapy,to destroy tumours and to improve non-speciﬁcally the im-mune defence.The uncertainty about its role for the human health was once described by Bennett (1963):‘‘Endotoxin can cause fever,but how manyhuman fevers are endotoxic?Endotoxin cancause shock,but how often is shock in manendotoxic?Endotoxin can modify resistance toinfections,but how often does endotoxin inﬂu-ence the susceptibility of man?’’Endotoxins’profound toxicity,however,for-bade a safe use in man so far.Any superﬂuousendotoxin exposure must be strictly avoided toprevent complications.This is especially true forintravenously applied medicaments.Endotoxintesting has therefore become an important part oftheir quality assessment.3.Signiﬁcance of endotoxin contamination Considering the data in Table1,human beingsare obviously anytime in contact with endotoxinsand apparently can handle this without problems.As long as endotoxins get into contact with theskin or digestion system,they are tolerated quitewell.Though,certain lung diseases are known tobe linked to endotoxin inhalation,for examplewith cigarette smoke or in crowded animal hous-ings(Dressel et al.,1991).In order to prevent theoutbreak of endotoxin-caused reactions,limitswereﬁxed for breath(20ng m−3$200EU m−3, Hasday et al.,1996)and intravenous applications(5EU kg−1body weight and hour;EuropeanPharmacopoeia,1997).In a production process,the source of endotoxin contamination must beconsidered—is it released from within the processor is it introduced by non-sterile process condi-tions?Furthermore,purpose and application oftheﬁnal product must be considered—for exam-ple,is the product to be used as diagnostic ortherapeutic and in which form,as ointment orparenteral?Of course,an exceptionally low endotoxin con-centration is not required in every case.Sincemany pharmaproteins are administered in a lowdose,modest endotoxin contents may complywith regulatory demands, e.g.according to theEuropean Pharmacopoeia(1997),for insulin10EU mg−1are allowed,for a-interferon even100EU mg−1.However,serum albumin or mono-clonal antibody preparations are administered inD.Petsch,F.B.Anspach/Journal of Biotechnology76(2000)97–119 102amounts of several hundred or thousand milli-grams per kg body weight;high endotoxin levels are out of question.With these proteins,methods are required to remove residual endotoxin traces that remain after using common puriﬁcation trains.3.1.Endotoxins in biotechnologyModern biotechnology offers a number of methods to produce proteins.Among these are microbial bioprocesses for the expression of hu-man proteins,such as growth hormones and inter-ferons,and culture techniques with mammalian cells,yeast and fungi for proteins exhibiting post-translational modiﬁcations,such as monoclonal antibodies.As today bioprocesses are employed to a large extent for the production of pharma-ceutical proteins,endotoxins became an impor-tant issue in manufacturing processes. Endotoxin concentrations involved in biotech-nological processes greatly depend upon the source of the product.They range from much less than100EU ml−1in cell culture supernatants toTable1Common endotoxin concentrations in everyday life and in protein solutions of different origina From Mu¨ller-Calgan(1989).b Measured in our laboratory.Fig.3.Average concentrations of proteins and endotoxins from different origin before and after puriﬁcation.D.Petsch,F.B.Anspach/Journal of Biotechnology76(2000)97–119103Table2Reduction of endotoxin concentration during puriﬁcation of bFGF,according to Rantze(1996)BFGFPuriﬁcation procedure Endotoxins(EU ml−1)(g l−1)0.4Filtrate of cell ho-\1500000 mogenateFractogel EMD-SO30.3\23000650(S)1.3Heparin-Sepharose14CL-6BPEI-Sepharose B0.02104vealed less than0.22EU ml−1while1.2×107EU ml−1were measured in the cell homogenate. Other examples are known with relatively high remaining endotoxin levels despite incorporation of afﬁnity steps.For example,Table2shows the progress of endotoxin removal during puriﬁcation of a recombinant basicﬁbroblast growth factor (bFGF)from a high cell density cultivation of E. coli.Although protein purity was greater than 99%after serial chromatography using a cation-exchange and a heparin afﬁnity sorbent,theﬁnal product exhibited an endotoxin content of14EU ml−1.In another example,a microbial contami-nation was accidentally introduced to a puriﬁed murine IgG1preparation(p I=5.5).After imme-diate sterileﬁltration,endotoxin and IgG1content were about100EU ml−1and3mg ml−1,respec-tively.Reprocessing of the IgG1solution using IgG1-selecive methods(anion-exchange,hydro-phobic interaction,and protein A afﬁnity chro-matography)was not successful.Also chromatography with endotoxin adsorbers based on polymyxin B or histidine were ineffective to reduce endotoxins to an acceptable limit when large sample volumes were applied onto columns. With small sample volumes a certain reduction of endotoxins was observed,however,at the expense of large product losses(results not published). For both proteins the endotoxin content was ﬁnally further reduced by speciﬁcally addressing the properties of the proteins and endotoxin molecules.With bFGF the endotoxin-selective sorbent based on poly(ethyleneimine),(PEI)-Sep-harose,was successful in the negative chromato-graphic mode,i.e.retention of bFGF was avoided while endotoxin was adsorbed(Table2).With IgG1only special endotoxin adsorbers were suc-cessful(see Table4).In the pharmaceutical industry several alterna-tive routes are known to come out with low-endo-toxin products.However,their diversity indicates the dilemma in endotoxin removal.The proce-dures listed in Table3were developed for pharmaproteins,taking advantage of characteris-tics of the production process,tailored to suit speciﬁc product requirements.Though,each pro-cedure addresses the problem in a completely different way;none of them turns out to bemore than1000000EU ml−1in supernatants of high cell density bacterial cultivations(Fig.3). Ideally,endotoxins should be absent when the product source is a cell culture supernatant and endotoxin-free media and buffers are used throughout.However,an endotoxin-free environ-ment can hardly be realised.Especially the water quality is an often discussed problem(Bommer and Ritz,1987).On the one hand the use of (expensive)endotoxin-free water(water for injec-tion)is uneconomical due to the large quantities needed.On the other hand,a signiﬁcant endo-toxin amount can be introduced into the produc-tion stream through accumulation.Also,if the product source is human blood,endotoxin con-tamination can be a serious problem owing to former or acute bacterial infections of blood donors.3.2.Endotoxin remo6al with product puriﬁcation Common puriﬁcation protocols that include several chromatographic steps,such as ion ex-change,hydrophobic interaction chromatography and gelﬁltration,may provide sufﬁcient endo-toxin clearance.Generally,the high endotoxin concentrations in the beginning(Fig.3)can be reduced to about100EU ml−1without special treatment.Even much lower remaining endotoxin contents may be realised, e.g.Bischoff et al. (1991),puriﬁed recombinant a1-antitrypsin in a three-step procedure,employing ultraﬁltration, anion-exchange and immobilised metal chelate afﬁnity chromatography.Theﬁnal product re-D.Petsch,F.B.Anspach/Journal of Biotechnology76(2000)97–119 104broadly applicable, e.g.anion-exchange chro-matography is potentially useful for the decon-tamination of basic proteins,such as urokinase(Green-Cross,1986)or bFGF.However,decon-tamination of acidic proteins would be accompa-nied by a substantial loss of the product due toadsorption(Hou and Zaniewski,1990b;Anspachand Hilbeck,1995).For small proteins,such asmyoglobin(M r:18000Da),ultraﬁltration can be useful to remove large endotoxin aggregates.With large proteins,such as immunoglobulins(M r:150000Da)ultraﬁltration would not be effective.Furthermore,ultraﬁltration will fail if interactions between endotoxins and proteins cause endotoxin monomers to permeate with proteins pick-a-pack through the membrane.In view of the large variety of products,expect-ing a generally applicable endotoxin clearancemethod is possibly utopian.On the other hand thediversity of applied techniques indicates that thespecial properties of endotoxin molecules are of-ten not considered in the development stage ofpuriﬁcation protocols.3.3.The problem of low remaining endotoxinconcentrationsAt theﬁnal stages in puriﬁcation trains thetarget protein is found at concentrations in the gl−1range while endotoxins are present in m g l−1amounts(Fig.3).The situation resembles the futile effort to search for a needle in a haystack.A further problem is the detection of these low concentrations.Routine analysis in amounts of picograms per millilitre is not possible by direct detection techniques.Today many cases are known—although not published—in which a pharmaceutically promising new substance was regarded pyrogenic while it turned out later that the origin of pyrogenicity was not the substance but endotoxins.These escaped insensitive detec-tion methods and thus their removal could not be properly addressed(Dinarello et al.,1984). Various bioassays are available to measure en-dotoxins,such as the rabbit test and the Limulus amoebocyte lysate(LAL)assay(Pearson,1985), the chicken embryo lethality assay(Galanos et al., 1971),and the galactosamine-primed mice lethal-ity test(Galanos and Freudenberg,1993).Among these,the LAL assay is most sensitive and most economical.The assay uses the blood coagulation system of the horseshoe crab Limulus polyphemus(or rela-tives,such as Tachypleus tridentatus,Tachypleus gigas or Tachypleus rotundicauda).In vitro,traces of endotoxin activate the isolated coagulation sys-tem either to initiate a gel formation(gel-clot variant)or to cleave a chromogen from a syn-thetic substrate to be read spectrophotometrically (chromogenic variant).A sensitivity of0.02EUTable3Examples of patented procedures for removal of endotoxins from protein solutionsProduct ReferenceProcedureKoyama et al.,1983Superoxid dismutase UltraﬁltrationMyoglobin Toyo-Soda,1989Cu-Zn-Superoxid dismutase Anion-exchange chromatography Green-Cross,1986Urokinase Nippon-Kayaku,1991aAdsorption on quaternised chitosanTNF,IL-1Dainippon-Pharmaceuticals,1987Nippon-Kayaku,1991bCu-Zn-Superoxid dismutase Adsorption on non-polar polymers,such as Amberlit XADCatalaseHydrophobic interaction chromatography Merck-USA,1987Hepatitis B surface antigenPertussis vaccine Sucrose gradient centrifugation Takeda Chemicals,1988Extraction with bile saltsImmunoglobulins Centocor,1988Lipocortine Extraction with detergents and HIC Behringwerke,1990Pepsin digestion removes endotoxin-binding Fc-fragmentImmunoglobulins Zimmermann,1982D.Petsch,F.B.Anspach/Journal of Biotechnology76(2000)97–119105ml−1can be accomplished with this assay(end-point method);the kinetic method is even more sensitive.Although the LAL assay has proved to be a sensitive and reliable method for endotoxin monitoring,many substances interfere with the coagulation cascade,causing false negative or pos-itive results.Such interferences are usually concen-tration dependent and may be avoided by dilution of the sample;however,this causes a loss of sensitivity.Also,it may fail occasionally,for exam-ple in complex biologicalﬂuids,such as blood, solutions of cationic proteins and with liposome-encapsulated endotoxin(Dijkstra et al.,1987; Petsch et al.,1998a;Pool et al.,1998).Owing to this,research carries on looking for alternative, sensitive bioassays.A whole blood stimulation assay is a recent development(Hartung and Wen-del,1996),which is currently in prevalidation stage at the Paul–Ehrlich Institute in Langen,Germany.3.4.Interactions of endotoxins and proteins Endotoxins show a remarkable capability to interact with other substances,including proteins. Molecular recognition can be assumed for interac-tions with anti-endotoxin antibodies and proteineous endotoxin receptors(e.g.CD14,CD16, CD18;Morrison et al.,1994).Many other interact-ing proteins,such as lysozyme(Ohno and Mor-rison,1989),lactoferrin(Elass-Rochard et al., 1995),and transferrin(Berger and Berger,1988), are basic proteins(p I\7).Electrostatic interac-tions can be assumed as the main driving force. However,other mechanisms must additionally exist as interactions with neutral(haemoglobin, Kaca et al.,1994)and even acidic proteins(p I B7) are known,taking place also at low ionic strength. Still it is controversially discussed how these inter-actions take place.With serum albumins fatty acid binding domains might be involved(David et al., 1995).Generally hydrophobic interactions with proteins are conceivable,however,strong evidence that these govern the interaction mechanism is missing.It is more probable that competition of protein-bound carboxylic groups and endotoxin-bound phosphoric acid groups about Ca2+may result in dynamically stable calcium bridges be-tween proteins and endotoxins.Regardless the mechanism that proves most signiﬁcant,these interactions result in a masking of endotoxin molecules and consequently to a partial escape from removal procedures.A typical example is described by Karplus et al.(1987). They failed to reach the endotoxin limit if decon-tamination of bovine catalase was done with the afﬁnity sorbent Polymyxin B–Sepharose using the standard protocol.In another study,Petsch et al. (1998a)found that the removal of small amounts of endotoxins can be more difﬁcult from basic proteins than from acidic proteins.Owing to protein–endotoxin interactions,en-dotoxin removal from protein solutions requires techniques that can generate strong interactions with endotoxins,such as afﬁnity chromatography. Alternatively,a speciﬁc dissociation of protein–endotoxin complexes may improve the availability of endotoxin molecules,e.g.Karplus et al.(1987) employed the surfactant octyl-b-glucopyrannoside to dissociate human IgG-endotoxin complexes and to support endotoxin adsorption to Polymyxin B–Sepharose.However,this practice implies the next problem,the removal of residual surfactant.4.Selective removal of endotoxinsThe most secure way to avoid any microbial contamination and with it the release of endotoxin is absolute sterility during the production and downstream processes.Yet,if a decontamination method is to be employed,it must ensure a high recovery of the target product.Data collected in our laboratory suggest that it must be strictly distinguished between the removal of endotoxin from protein-free and protein-containing solutions (Anspach and Hilbeck,1995;Petsch et al.,1998b). In a protein-free solution,methods can be em-ployed that take advantage of the different size of endotoxin and water as well as salt and other small molecules.4.1.UltraﬁltrationGelﬁltration chromatography reveals that more than80%of the endotoxin activity of a。

野生和人工种植罗布麻种子形态及解剖特征比较

第15期收稿日期:2012-08-27基金项目:吉林省科技发展计划重点项目(20110909)作者简介:陈凤清(1962-),女,吉林白城人,副教授,博士,主要从事生物化学和分子生物学的研究工作,(电话)0436-*******(电子信箱)congjianmin@;通讯作者,丛建民(1974-),男,副教授,博士,主要从事野生植物资源、表观遗传学的研究工作,(电话)0436-*******。

罗布麻(Apocynum venetum L.)为夹竹桃科罗布麻属植物,在我国主要分布在西北、东北及华东等地区[1]。

其茎皮纤维可作为纺织、造纸等工业的原材料;花芳香,是良好的蜜源植物[2-4]。

自古以来,罗布麻就被国人誉为“仙草”[1,5,6],其根、茎、叶入药具有治疗高血压[5,7]、心力衰竭等功效[5]。

罗布麻可以适应极端恶劣环境,生命力极强,有防风固沙、保持水土的作用。

20世纪50年代起,国内外学者对罗布麻资源、引种、人工种植等做过不少工作,但对罗布麻种子表面形态特征和种子内部结构的研究甚少。

现在对罗布麻的开发利用已产业化,但人为过度无序地开发造成罗布麻面积大幅减少,对生态环境造成严重影响。

吉林省西部地区盐碱、干旱、沙漠化严重,近年来野生罗布麻分布逐渐减少。

试验对吉林省西部地区野生和人工种植罗布麻种子表面结构特征和种子内部结构进行了比较研究,以期为罗布麻品种的鉴定、栽培驯化及药用提供理论依据。

1材料与方法1.1材料野生罗布麻种子于2011年10月采自吉林省白城镇赉巨宝山,人工种植罗布麻种子于2011年10月采自白城师范学院试验田。

1.2方法1.2.1罗布麻种子形态特性测定罗布麻种子长野生和人工种植罗布麻种子形态及解剖特征比较陈凤清,丛建民,林碧云,潘娜,王国强(白城师范学院,吉林白城137000)摘要:对比研究了吉林省白城地区野生罗布麻(Apocynum venetum L.)种子与人工栽培罗布麻种子的形态特征和解剖特征。

定向突变生物合成

定向突变生物合成英文回答：Directed mutagenesis is a technique used in molecular biology to introduce specific changes or mutations into a DNA sequence. This technique allows scientists to study the effects of these mutations on gene function and protein structure. There are several methods available for directed mutagenesis, including site-directed mutagenesis and random mutagenesis.Site-directed mutagenesis is a targeted approach where specific nucleotides in the DNA sequence are changed to create a desired mutation. This can be achieved using techniques such as PCR-based mutagenesis oroligonucleotide-directed mutagenesis. For example, if I want to introduce a point mutation in a gene to study its effect on protein function, I can design a pair of primers that contain the desired mutation and use them in a PCR reaction to amplify the gene with the mutation.Random mutagenesis, on the other hand, introduces random mutations throughout the DNA sequence. This can be done using techniques such as error-prone PCR or chemical mutagenesis. Random mutagenesis is useful when studying the effects of a large number of mutations or when trying to create a library of variants with different properties.Once the mutations are introduced, the mutated DNA sequences can be cloned into an expression vector and used to produce mutant proteins. The mutant proteins can then be purified and characterized to determine their functional and structural properties. For example, if I introduce a mutation in a protein involved in enzyme catalysis, I can compare the catalytic activity of the mutant protein to the wild-type protein to understand the role of the mutated residue in the reaction.Directed mutagenesis has a wide range of applicationsin biotechnology and biomedical research. It can be used to study the function of specific genes and proteins, identify key amino acid residues involved in protein-proteininteractions, and engineer proteins with improved properties. For example, directed mutagenesis has been used to create enzymes with enhanced catalytic activity, antibodies with improved binding affinity, and receptors with altered ligand specificity.中文回答：定向突变是分子生物学中一种用于在DNA序列中引入特定改变或突变的技术。

白来航鸡STING基因克隆、生物信息学及组织表达特性分析

中国畜牧兽医 2024,51(4):1372-1381C h i n aA n i m a lH u s b a n d r y &V e t e r i n a r y Me d i c i n e 白来航鸡S T I N G 基因克隆㊁生物信息学及组织表达特性分析王艳,张颖,赵雅妮,易林,史珍,周长明,周宛蓉,姜莉莉,樊兆斌(菏泽学院药学院,菏泽274015)摘要:ʌ目的ɔ克隆白来航鸡干扰素基因刺激因子基因(s t i m u l a t o ro f i n t e r f e r o n g e n e s ,S T I N G )C D S 区序列并进行生物信息学和组织表达分析,为阐明S T I N G 基因在抗病毒免疫应答中的作用奠定基础㊂ʌ方法ɔ采用P C R 扩增并克隆白来航鸡S T I N G 基因C D S 区,测序后对其编码氨基酸序列进行相似性比对及系统进化树构建,利用生物信息学预测S T I N G 蛋白的理化特性及结构功能,并利用实时荧光定量P C R 技术检测S T I N G 基因在鸡心脏㊁肝脏等14个组织中的表达情况㊂ʌ结果ɔ白来航鸡S T I N G 基因C D S 区序列全长1140b p ,编码379个氨基酸㊂相似性比对和系统进化树分析结果表明,白来航鸡S T I N G 基因与原鸡的相似性最高(99.7%),亲缘关系最近,与冠小嘴乌鸦亲缘关系最远㊂S T I N G 蛋白为酸性㊁亲水性蛋白,分子质量为42.625k u ,等电点(pI )为6.67,不稳定系数为69.26,脂肪系数为105.01㊂该蛋白大部分在线粒体和内质网上合成,含有跨膜结构,不含信号肽㊂S T I N G 蛋白二级结构包括α-螺旋(54.62%)㊁延伸链(10.29%)㊁β-转角(3.43%)及无规则卷曲(31.66%)㊂蛋白互作分析表明,白来航鸡S T I N G 蛋白与N F K B 1㊁D D X 41㊁c G A S ㊁T B K 1等蛋白存在相互作用㊂实时荧光定量P C R 结果显示,S T I N G 基因在白来航鸡组织中广泛表达,其中在肺脏中表达量最高,且显著高于其他组织(P <0.05);在胸肌中表达量最低㊂ʌ结论ɔ本研究成功克隆了白来航鸡S T I N G 基因,其C D S 区序列全长1140b p ,编码379个氨基酸㊂白来航鸡S T I N G 蛋白为酸性㊁亲水性蛋白,含有跨膜结构㊂S T I N G 基因在白来航鸡肺脏中表达量最高㊂研究结果为深入探究白来航鸡S T I N G 基因编码蛋白功能提供了材料㊂关键词:白来航鸡;S T I N G 基因;克隆;生物信息学;表达中图分类号:S 831;Q 78文献标识码:AD o i :10.16431/j.c n k i .1671-7236.2024.04.005 开放科学(资源服务)标识码(O S I D ):收稿日期:2023-10-20基金项目:山东省自然科学基金项目(Z R 2019M C 036㊁Z R 2023Q C 302);菏泽学院大学生创新训练项目;菏泽学院新兽药工程重点实验室联系方式:王艳,E -m a i l :1722804585@q q .c o m ;张颖,E -m a i l :3082291255@q q.c o m ㊂王艳和张颖对本文具有同等贡献,并列为第一作者㊂通信作者姜莉莉,E -m a i l :l y j l l f x y l 024@163.c o m ;樊兆斌,E -m a i l :438321212@q q.c o m C l o n i n g ,B i o i n f o r m a t i c s a n dT i s s u eE x pr e s s i o nC h a r a c t e r i s t i c s o f S T I N G G e n e i n W h i t eL e gh o r nC h i c k e n s WA N G Y a n ,Z H A N G Y i n g ,Z H A O Y a n i ,Y IL i n ,S H I Z h e n ,Z H O U C h a n g m i n g,Z H O U W a n r o n g,J I A N GL i l i ,F A NZ h a o b i n (C o l l e g e o f P h a r m a c y ,H e z eU n i v e r s i t y ,H e z e 274015,C h i n a )A b s t r a c t :ʌO b j e c t i v e ɔI nt h i ss t u d y ,t h eC D Sr e g i o ns e qu e n c eo f s t i m u l a t o ro f i n t e r f e r o n g e n e s (S T I N G )g e n e i n W h i t eL e g h o r nc h i c k e n sw a s c l o n e da n da n a l y z e db y bi o i n f o r m a t i c s a n d t i s s u e e x p r e s s i o n ,w h i c h l a i da f o u n d a t i o nf o re l u c i d a t i n g th er o l eo f S T I N G g e n e i na n t i v i r a l i m m u n e r e s p o n s e .ʌM e t h o d ɔT h eC D S r e g i o n o f S T I N G g e n e i n W h i t eL e g h o r n c h i c k e n sw a s a m p l i f i e db y P C Ra n d c l o n e d .A f t e r s e q u e n c i n g ,t h es i m i l a r i t y o f t h ee n c o d e da m i n oa c i ds e qu e n c eo f S T I N G g e n ew a sc o m p a r e da n dt h e p h y l o g e n e t i ct r e e w a sc o n s t r u c t e d .T h e p h y s i c o c h e m i c a l p r o pe r t i e s a n d s t r u c t u r a lf u n c t i o no f S T I N G p r o t e i nw e r e p r e d i c t e db y b i o i n f o r m a t i c s ,a n d t h e e x pr e s s i o no f4期王艳等:白来航鸡S T I N G基因克隆㊁生物信息学及组织表达特性分析S T I N G g e n e i n14t i s s u e ss u c ha sh e a r t a n d l i v e ro f W h i t eL e g h o r nc h i c k e n sw e r ed e t e c t e db y R e a l-t i m e q u a n t i t a t i v eP C R.ʌR e s u l tɔT h e s e q u e n c eo f t h eC D Sr e g i o no f S T I N G g e n e i n W h i t e L e g h o r nc h i c k e n sw a s1140b p i n t o t a l l e n g t h,e n c o d i n g379a m i n o a c i d s.S i m i l a r i t y a l i g n m e n t a n d p h y l o g e n e t i c t r e e a n a l y s i s s h o w e d t h a t S T I N G g e n e i n W h i t eL e g h o r nc h i c k e n sh a d t h eh i g h e s t s i m i l a r i t y w i t h G a l l u s g a l l u s a n d t h e c l o s e s t g e n e t i cr e l a t i o n s h i p,a n d t h ef a r t h e s t g e n e t i c r e l a t i o n s h i p w i t h C o r v u s c o r n i xc o r n i x.S T I N G p r o t e i n i n W h i t eL e g h o r n c h i c k e n sw a s a n a c i d i c,h y d r o p h i l i c p r o t e i n w i t ha m o l e c u l a rw e i g h to f42.625k u,a n i s o e l e c t r i c p o i n t(p I)o f6.67,a ni n s t a b i l i t y c o e f f i c i e n t o f69.26,a n da f a t c o e f f i c i e n to f105.01.S T I N G p r o t e i n w a ss y n t h e s i z e d m o s t l y o nm i t o c h o n d r i a a n de n d o p l a s m i c r e t i c u l u m,c o n t a i n e da t r a n s m e m b r a n e s t r u c t u r e,a n dn o s i g n a l p e p t i d e.T h es e c o n d a r y s t r u c t u r e o fS T I N G p r o t e i ni n c l u d e d a l p h a h e l i x(54.62%), e x t e n d e dc h a i n(10.29%),b e t at u r n(3.43%)a n dr a n d o m c o i l(31.66%).P r o t e i ni n t e r a c t i o n a n a l y s i s s h o w e d t h a t t h e r ew e r e i n t e r a c t i o n sb e t w e e nS T I N Ga n dN F K B1,D D X41,c G A S,T B K1 a n do t h e r p r o t e i n s.T h er e s u l t so fR e a l-t i m e q u a n t i t a t i v eP C R s h o w e dt h a t S T I N G g e n e w a s w i d e l y e x p r e s s e d i n t h e t i s s u e s o fW h i t eL e g h o r o n c h i c k e n s,w i t h t h e h i g h e s t e x p r e s s i o n i n l u n g, w h i c hw a s s i g n i f i c a n t l y h i g h e r t h a n t h a t i no t h e r t i s s u e s(P<0.05),a n d t h e l o w e s t e x p r e s s i o n i n p e c t o r a l i s m u s c l e.ʌC o n c l u s i o nɔI nt h i ss t u d y,S T I N G g e n ei n W h i t e L e g h o r nc h i c k e n s w a s s u c c e s s f u l l y c l o n e d,t h e t o t a l l e n g t ho f t h eC D Sr e g i o nw a s1140b p,e n c o d i n g379a m i n oa c i d s. S T I N G p r o t e i n i n W h i t e L e g h o r n c h i c k e n s w a s a n a c i d i c,h y d r o p h i l i c p r o t e i n w i t h a t r a n s m e m b r a n e s t r u c t u r e.T h e r e w a st h eh i g h e s te x p r e s s i o no f S T I N G g e n ei nl u n g o f W h i t e L e g h o r o n c h i c k e n s.T h er e s u l t s p r o v i d e d m a t e r i a l s f o r t h e i n-d e p t hs t u d y o f t h e f u n c t i o no f t h e p r o t e i ne n c o d e db y S T I N G g e n e i n W h i t eL e g h o r nc h i c k e n s.K e y w o r d s:W h i t eL e g h o r n c h i c k e n s;S T I N G g e n e;c l o n i n g;b i o i n f o r m a t i c s;e x p r e s s i o n先天性免疫是机体抵御病原微生物的第一道防线㊂宿主编码模式识别受体(P R R s)对来自病原体核酸的识别启动了复杂的信号转导途径,最终产生Ⅰ型干扰素(I F N-Ⅰ)和促炎细胞因子,并激活机体的天然免疫反应[1]㊂环鸟腺苷酸合成酶(c G A S)作为D N A识别受体,能识别并结合胞质双链D N A(d s D N A)[2]㊂当胞质中存在异常d s D N A时,c G A S能催化合成第二信使2 ,3 -c G AM P(c G AM P),结合并有效激活干扰素基因刺激因子基因(s t i m u l a t o r o f i n t e r f e r o n g e n e s,S T I N G),随后S T I N G招募T A N K结合激酶(T B K1)和I K B 激酶(I K K)使其磷酸化,激活导致产生I F N-Ⅰ和干扰素调节因子3(I R F3)㊁核因子κB(N F-κB)等细胞因子的表达[3]㊂S T I N G的功能主要体现在c G A S-S T I N G-I F N 信号通路中㊂c G A S-S T I N G通路除了构成有效的防御组织损伤和病原体入侵的监测系统外,还在自噬㊁翻译㊁代谢稳态㊁细胞凝聚㊁D N A损伤修复㊁衰老和细胞死亡中发挥作用[4]㊂然而c G A S-S T I N G通路的异常或过度激活可导致原发性发病机制和多种自身免疫性疾病[5]㊂家禽马立克病病毒(M D V)和鸡新城疫病毒(N D V)均靶向S T I N G接头蛋白,从而阻碍c G A S-S T I N G通路介导抗病毒天然免疫的应答[6-7]㊂禽痘病毒(F W P V)可以刺激鸡巨噬细胞中的c G A S-S T I N G通路,上调I F N相关因子表达,使宿主有效防御F W P V感染;禽白血病病毒(A L V)通过其编码的P15蛋白抑制激活c G A S-S T I N G通路,从而有助于病毒复制和持续感染[8]㊂S T I N G在细胞因子诱导㊁自噬诱导㊁代谢调节和内质网应激等方面发挥重要功能[9],且在健康和疾病中发挥着至关重要的作用,广泛参与各种细胞过程㊂近年来,靶向S T I N G的激动剂成为国内外抗肿瘤药物研发的新热点,S T I N G激动剂在乳腺癌㊁结肠癌㊁肝癌㊁黑色素瘤及淋巴瘤等肿瘤模型中都展现出了强有力的抗肿瘤活性[10]㊂目前国内外对鸡S T I N G在抗病毒免疫中的研究鲜有报道㊂本研究以白来航鸡为研究对象,克隆S T I N G基因C D S 区,运用在线软件对该基因编码蛋白进行生物信息学分析,并采用实时荧光定量P C R技术检测S T I N G基因在白来航鸡不同组织中的表达特征,以期为深入探究S T I N G在抗病毒免疫应答中的作用提供参考依据㊂3731中国畜牧兽医51卷1 材料与方法1.1 材料供试动物选取同一批次的3只3周龄健康的白来航鸡(菏泽市某养殖场)㊂颈部放血处理后,无菌采集每只白来航鸡的心脏㊁肝脏㊁脾脏㊁胰脏㊁肺脏㊁肾脏㊁脑㊁胆㊁腺胃㊁十二指肠㊁回肠㊁盲肠㊁直肠㊁胸肌共14个组织置于超低温备用㊂T r i z o l ㊁P r i m e S c r i p t T MR T R e a g e n t K i t w i t h gD N AE r a s e r ㊁D L 2000D N A M a r k e r ㊁限制性内切酶E c o R Ⅰ㊁H i n d Ⅲ均购自宝日医生物技术(北京)有限公司;S Y B R P r e m i x E x T a q T M (2ˑ)购自T a K a R a 公司;T 4D N A 连接酶㊁pE T -28a (+)载体㊁大肠杆菌D H 5α感受态细胞㊁T I A N p r e p Mi n i P l a s m i dK i t 均购自天根生化科技(北京)有限公司㊂1.2 方法1.2.1 引物设计及合成根据G e n B a n k 中原鸡S T I N G 基因序列(登录号:K P 893157.1),使用P r i m e rP r e m i e r 5.0软件设计普通P C R (下划线处为E c o R Ⅰ和H i n d Ⅲ酶切位点)和实时荧光定量P C R 引物,以G A P DH 为内参基因,引物信息见表1㊂引物均由生工生物工程(上海)股份有限公司合成㊂表1 引物序列信息T a b l e 1 P r i m e r s e q u e n c e i n f o r m a t i o n 基因G e n e s引物序列P r i m e r s e qu e n c e s (5'ң3')退火温度A n n e a l i n gt e m pe r a t u r e /ħ产物长度P r o d u c tl e n g t h /b p 用途A p p l i c a t i o n S T I N G F :C C G G A A T T C A T G C C C C A G G A C C C G T C A A C C 62.81140普通P C R R :C C C A A G C T T C T G G G C T C A G G G G C A G T C A C T S T I N G F :G C C C C A G G A C C C G T C A A C C A G 60.0114实时荧光定量P C R R :A G C A C C A C G A A G C A C A C A G C C AG A P DHF :G A C G T G C A G C A G G A A C A C T A 60.0122实时荧光定量P C RR :A T G G C C A C C A C T T G G A C T T T1.2.2 总R N A 提取及反转录利用T r i z o l 法提取各组织总R N A ,溶于D N a s e /R N a s e -f r e ed d H 2O ,利用紫外分光光度计及琼脂糖凝胶电泳检测R N A 浓度及纯度,将R N A 反转录为c D N A ,―20ħ保存备用㊂1.2.3 S T I N G 基因C D S 区克隆及测序以白来航鸡脾脏组织c D N A 为模板进行P C R 扩增㊂P C R 反应体系20μL :c D N A 模板0.5μL ,上㊁下游引物(10μm o l /L )各0.5μL ,P C R M a s t e r M i x10μL ,d d H 2O8.5μL ㊂P C R 反应程序:94ħ预变性5m i n ;94ħ变性30s ,62.8ħ退火30s ,72ħ延伸75s ,共35个循环;72ħ延伸7m i n ㊂P C R 产物用1.0%琼脂糖凝胶电泳检测,取鉴定正确的扩增产物回收纯化后连接p E T -28a (+)载体,转化大肠杆菌D H 5α感受态细胞,均匀涂布于含卡那霉素的L B 固体培养基上,过夜培养14h 后筛选出圆滑白色单菌落并进行菌液P C R 鉴定,将挑选得到的阳性克隆菌液送生工生物工程(上海)股份有限公司测序㊂1.2.4 生物信息学分析使用M e g A l i g n 软件将白来航鸡与G e n B a n k 数据中已公布的原鸡(G a l l u s ga l l u s ,登录号:N P _001292081.2)㊁盔珠鸡(N u m i d am e l e a g r i s ,登录号:X P _021265823.1)㊁火鸡(M e l e a g r i s g a l l o pa v o ,登录号:X P _010717095.1)㊁白尾雷鸟(L a g o pu s l e u c u r a ,登录号:X P _042749793.1)㊁日本鹌鹑(C o t u r n i x j a po n i c a ,登录号:X P _015731739.1)㊁凤头朱鹮(N i p po n i a n i p po n ,登录号:K F Q 92075.1)㊁冠小嘴乌鸦(C o r v u s c o r n i xc o r n i x ,登录号:X P _039415878.1)的S T I N G 基因氨基酸序列进行相似性比对,并利用M e ga 7.0软件构建系统进化树㊂利用P r o t P a r a m 在线软件(h t t ps :ʊw e b .e x p a s y .o r g /p r o t pa r a m /)分析S T I N G 蛋白理化性质;利用P r o t S c a l e 在线软件(h t t p s :ʊw eb .e x p a s y .o r g /pr o t s c a l e /)分析S T I N G 蛋白亲/疏水性;利用S i g n a l P4.1(h t t ps :ʊs e r v i c e s .h e a l t h t e c h .d t u .d k /s e r v i c e s /S i g n a l P -4.1/)㊁T MHMM (h t t ps :ʊs e r v i c e s .h e a l t h t e c h .d t u .d k /)在线软件分别预测S T I N G 蛋白信号肽与跨膜区;利用S M A R T (h t t p :ʊs m a r t .e m b l -h e i d e l b e r g .d e /s m a r t /b a t c h .p l )和P S P R TⅡ(h t t p :ʊp s o r t .h g c .j p/f o r m 2.h t m l )在线软件分别预测S T I N G 蛋白结构域和亚细胞定位;利用47314期王艳等:白来航鸡S T I N G基因克隆㊁生物信息学及组织表达特性分析N e t P h o s3.1在线软件(h t t p s:ʊs e r v i c e s.h e a l t h t e c h.d t u.d k/se r v i c e.p h p?N e t P h o s-3.1)预测S T I N G 蛋白磷酸位点;利用Y i n O Y a n g1.2(h t t p s:ʊs e r v i c e s.h e a l t h t e c h.d t u.d k/s e r v i c e s/Y i n O Y a n g-1.2/)和N e t N G l y c1.0(h t t p s:ʊs e r v i c e s.h e a l t h t e c h.d t u.d k/s e r v i c e s/N e t N G l y c-1.0/)分别预测S T I N G蛋O-糖基化和N-糖基化位点;通过P r a b i S O P MA(h t t p s:ʊn p s a.l y o n.i n s e r m.f r/c g i-b i n/s e c p r e d_s o p m a.p l)和S W I S S M O D E L(h t t p s:ʊs w i s s m o d e l.e x p a s y.o r g/i n t e r a c t i v e)在线软件分别预测S T I N G蛋白二级结构和三级结构;利用S T R I N G12.0在线软件(h t t p s:ʊv e r s i o n-12-0. s t r i n g-d b.o r g/)和C y t o s c a p e软件进行蛋白互作分析㊂1.2.5白来航鸡S T I N G基因组织表达特性检测以鸡14个组织c D N A为模板,利用实时荧光定量P C R检测S T I N G基因在白来航鸡各组织中的相对表达情况,以G A P DH作为内参基因㊂P C R 反应体系10μL:c D N A0.5μL,上㊁下游引物(10μm o l/L)各0.3μL,S Y B R P r e m i x E x T a q T M (2ˑ)5μL,d d H2O3.9μL㊂P C R反应程序:95ħ2m i n;95ħ15s,60ħ1m i n,共45个循环;熔解程序:95ħ15s,60ħ1m i n;95ħ15s㊂每个组织样品设3个重复,采用2―әәC t法计算相对表达量㊂1.3数据统计分析通过S P S S26.0软件中单因素方差分析对基因表达量进行显著性检测,采用L S D和邓肯式法进行多重比较,利用G r a p h P a dP r i s m8.0软件作图㊂结果用平均值ʃ标准差表示,P<0.05表示差异显著㊂2结果2.1白来航鸡S T I N G基因C D S区序列克隆1.0%琼脂糖凝胶电泳显示,在约1140b p附近出现清晰条带(图1),与预期条带大小符合㊂应用D N AMA N软件分析测序结果显示,本试验克隆的白来航鸡S T I N G基因C D S区片段长为1140b p,编码379个氨基酸,碱基组成分别为: A(17.54%)㊁G(30.35%)㊁T(18.16%)㊁C(33.95%), G C含量高于A T含量,说明白来航鸡S T I N G基因C D S区的D N A双链较稳定㊂M,D L2000D N A M a r k e r;1~3,S T I N G基因P C R扩增产物;4,阴性对照M,D L2000D N A M a r k e r;1-3,P C Ra m p l i f i c a t i o n p r o d u c t s o f S T I N G g e n e;4,N e g a t i v e c o n t r o l图1白来航鸡S T I N G基因P C R扩增结果电泳图F i g.1E l e c t r o p h o r e t i c m a p o fP C Ra m p l i f i c a t i o nr e s u l t so f S T I NG g e n e i n W h i t eL e i g h o r n c h i c k e n s2.2生物信息学分析2.2.1相似性比对及系统进化树构建相似性比对结果显示,白来航鸡S T I N G基因编码氨基酸序列与原鸡㊁灰胸竹鸡㊁环颈雉㊁盔珠鸡㊁火鸡㊁白尾雷鸟㊁日本鹌鹑㊁朱鹮和冠小嘴乌鸦的相似性分别为99.7%㊁93.7%㊁89.1%㊁87.3%㊁90.2%㊁87.3%㊁83.6%㊁63.9%和62.0%(图2)㊂采用M e g a7.0软件中M L法构建系统进化树,结果显示,白来航鸡与原鸡之间的亲缘关系最近,与灰胸竹鸡的亲缘关系次之,朱鹮和冠小嘴乌鸦形成另一个分支,与冠小嘴乌鸦亲缘关系最远(图3)㊂2.2.2理化特性分析白来航鸡S T I N G蛋白分子式为C1897H3032N530O541S22,分子质量为42.625k u,理论等电点(p I)为6.67,推测该蛋白为酸性蛋白㊂组成白来航鸡S T I N G蛋白的20种氨基酸中,L e u 所占比例最高(17.2%),而A s n和M e t含量最低(1.3%),其中带负电荷的氨基酸(A s p和G l u) 42个,带正电荷的氨基酸(A r g和L y s)40个㊂在哺乳动物网织红细胞内的半衰期为30h,脂肪系数为105.01㊂S T I N G蛋白的不稳定系数为69.26,高于阈值40,表明该蛋白不稳定㊂2.2.3亲/疏水性预测白来航鸡S T I N G蛋白在第34位氨基酸处分值最高(3.500),在第257位氨基酸处分值最低(―2.789)(图4),总平均亲水性G R A V Y为―0.041,即S T I N G为亲水性蛋白㊂5731中国畜牧兽医51卷图2 不同物种S T I N G 基因氨基酸序列相似性比对F i g .2 S i m i l a r i t y a l i g n m e n t o f a m i n o a c i d s e q u e n c e o f S T I NG g e n e a m o n g d i f f e r e n t s pe c i es 图3 基于S T I N G 基因氨基酸序列构建的系统进化树F i g .3 P h y l o g e n e t i c t r e e b a s e do na m i n o a c i d s e qu e n c e o f S T I N G g e ne 图4 白来航鸡S T I N G 蛋白亲/疏水性预测F i g .4 H y d r o p h i l i c i t y a n dh y d r o p h o b i c i t yp r e d i c t i o no f S T I NG p r o t e i n i n W h i t eL e gh o r n c h i c k e n s 67314期王艳等:白来航鸡S T I N G 基因克隆㊁生物信息学及组织表达特性分析2.2.4 信号肽及跨膜区预测白来航鸡S T I N G 蛋白不含信号肽(图5),推测该蛋白不是分泌型蛋白;该蛋白含4个跨膜区,分别位于第27―42㊁57―73㊁101―112及124―141位氨基酸处(图6),属于跨膜蛋白㊂图5 白来航鸡S T I N G 蛋白信号肽预测F i g .5 S i g n a l p e p t i d e p r e d i c t i o no fS T I NG p r o t e i ni n W h i t e L e gh o r n c h i c k e ns 图6 白来航鸡S T I N G 蛋白跨膜区预测F i g .6 T r a n s m e m b r a n e r e g i o n p r e d i c t i o no f S T I NG p r o t e i n i n W h i t eL e gh o r n c h i c k e n s 2.2.5 结构域预测及亚细胞定位预测结果显示,白来航鸡S T I N G 蛋白具有1个保守结构域T M E M 173,位于第50―342位氨基酸处㊂白来航鸡S T I N G 蛋白大部分在线粒体和内质网上合成,在细胞核中合成较多,在细胞质中合成最少㊂2.2.6 修饰位点预测白来航鸡S T I N G 蛋白丝氨酸㊁酪氨酸㊁苏氨酸磷酸化位点分别有25㊁3及5个(图7);存在43个O -糖基化磷酸位点(图8),不存在N -糖基化磷酸位点㊂图7 白来航鸡S T I N G 蛋白磷酸化位点预测F i g .7 P h o s p h o r y l a t i o n s i t e p r e d i c t i o no f S T I NG p r o t e i n i n W h i t eL e gh o r n c h i c k e n s 2.2.7 二级结构及三级结构预测白来航鸡S T I N G 蛋白二级结构包括α-螺旋(54.62%)㊁延伸链(10.29%)㊁β-转角(3.43%)㊁无规则卷曲(31.66%)(图9)㊂白来航鸡S T I N G 蛋白三级结构主要以α-螺旋为主(图10),与二级结构预测结果一致㊂2.2.8 蛋白互作分析将白来航鸡S T I N G 蛋白序列导入S T R I N G12.0在线数据库,结果显示,该蛋白与N F K B 1㊁D D X 41㊁c G A S ㊁T B K 1等蛋白存在互作(图11)㊂7731中国畜牧兽医51卷图8 白来航鸡S T I N G 蛋白O -糖基化位点预测F i g .8 O -g l y c o s y l a t i o n s i t e p r e d i c t i o no f S T I N G p r o t e i n i n W h i t eL e gh o r n c h i c k e ns h ,α-螺旋;e ,延伸链;t ,β-转角;c ,无规则卷曲h ,A l ph ah e l i x ;e ,E x t e n d e d c h a i n ;t ,B e t a t u r n ;c ,R a n d o mc o i l 图9 白来航鸡S T I N G 蛋白二级结构F i g .9 S e c o n d a r y s t r u c t u r e p r e d i c t i o no f S T I NG p r o t e i n i n W h i t eL e gh o r n c h i c k e ns 图10 白来航鸡S T I N G 蛋白三级结构F i g .10 T e r t i a r y s t r u c t u r e p r e d i c t i o no fS T I NG p r o t e i ni n W h i t eL e gh o r n c h i c k e ns 图11 白来航鸡S T I N G 蛋白互作分析F i g .11 P r o t e i ni n t e r a c t i o n a n a l y s i so fS T I NG p r o t e i ni n W h i t eL e gh o r n c h i c k e n s 87314期王艳等:白来航鸡S T I N G 基因克隆㊁生物信息学及组织表达特性分析2.3 白来航鸡S T I N G 基因组织表达分析通过实时荧光定量P C R 检测S T I N G 基因在白来航鸡14个组织中的表达情况,结果显示,S T I N G 基因在白来航鸡肺脏中的表达量最高,且显著高于其他组织(P <0.05);在胸肌中的表达量最低(图12)㊂肩标不同字母表示差异显著(P <0.05);肩标相同字母表示差异不显著(P >0.05)V a l u e sw i t hd i f f e r e n t l e t t e r s u p e r s c r i p t sm e a n s i g n i f i c a n t d i f f e r e n c e (P <0.05);W h i l ew i t h t h e s a m e l e t t e r s u p e r s c r i p t sm e a n n o s i gn i f i c a n t d i f f e r e n c e (P >0.05)图12 S T I N G 基因在白来航鸡不同组织中的相对表达量F i g .12 T h e r e l a t i v e e x p r e s s i o no f S T I NG g e n e i nd i f f e r e n t t i s s u e s o fW h i t eL e gh o r n c h i c k e n s 3 讨论S T I N G 是固有免疫系统I F N -Ⅰ信号通路中的一个重要接头蛋白,定位在内质网上,可激活T B K I ㊁I K K ㊁I R F 3及N F -κB 通路,强烈刺激I F N -Ⅰ和白介素等免疫炎症因子的产生[11]㊂I F N -β在S T I N G 通路中分泌量最高,它不仅能够消灭癌细胞,还能通过促使树突细胞成熟实现抗原递呈,从而将固有免疫应答与获得性免疫应答相联系[12]㊂S T I N G 基因缺失使小鼠胚胎成纤维细胞(S T I N G -/-M E F s)极易受到负链病毒感染,如水疱性口炎病毒(V S V )和1型单纯疱疹病毒(H S V -1)[13]㊂本研究成功克隆白来航鸡S T I N G 基因C D S 区序列,为后期研究禽类S T I N G 蛋白抗病毒作用机制提供基础数据㊂系统进化树显示,白来航鸡与原鸡亲缘关系最近,与冠小嘴乌鸦亲缘关系最远,表明该基因在不同物种间保守性具有一定差异㊂白来航鸡S T I N G 蛋白为酸性㊁亲水性蛋白㊂以上结果表明该基因在不同物种的发育进化过程中表现出了种内相似性和种间差异性㊂白来航鸡S T I N G 蛋白无信号肽,说明该蛋白不是分泌蛋白,结合蛋白的亚细胞定位推测其主要存在于细胞质或线粒体中㊂另外,白来航鸡S T I N G 蛋白具有4个跨膜结构域㊂在S T I N G 二聚体中,8个T M 螺旋被分成由T M 2和T M 4组成的中间层和由T M 1和T M 3组成的外围层,其中1个S T I N G 分子的T M 1和另1个分子的T M 2㊁T M 3和T M 4被堆积在一起,形成结构域包裹的结构,这种保守的结构特征对蛋白的稳定性和功能至关重要[14]㊂白来航鸡S T I N G 蛋白存在T M E M 173保守结构域,预测结果显示其可促进先天免疫信号传导,与刘健新等[15]对山羊S T I N G 蛋白结构域的预测结果一致㊂白来航鸡S T I N G 蛋白中含有43个O -糖基化磷酸位点,这些修饰位点可能影响S T I N G 蛋白的空间构象㊁合成㊁运输㊁定位等生物过程,不存在N -糖基化磷酸位点,从而对该蛋白的不稳定性产生影响㊂S T I N G 蛋白共有33个潜在磷酸化位点,该蛋白本身的磷酸化是下游信号转导所必需的,其中含有的2个保守的丝氨酸和苏氨酸位点是T B K 1的磷酸化靶点㊂另外,S T I N G 磷酸化有助于其从细胞质到高尔基体的适当易位,开启免疫反应,且还参与防止S T I N G 过度激活和维持体内平衡[16]㊂蛋白质棕榈酰化是一种功能强大且保守的翻译后修饰,脂肪酸链通过可逆的硫酯键与蛋白质的半胱氨酸残基连接[17]㊂在高尔基体中,S T I N G 的2个半胱氨酸残基(C ys 88和9731中国畜牧兽医51卷C y s91)被棕榈酰化,激活其下游信号通路,这是S T I N G激活所必需的翻译后修饰[18]㊂蛋白互作预测结果显示,S T I N G蛋白与N F K B1㊁D D X41㊁c G A S㊁T B K1等蛋白存在相互作用㊂S T I N G蛋白可与c G A S结合形成第二信使c G A M P,使得S T I N G招募并激活T B K1,进而诱导I F N-Ⅰ和其他调节因子[19]㊂研究表明,当树突状细胞在感染H S V-1或腺病毒后,D D X41与S T I N G直接相互作用介导T B K1㊁N F-κB和I R F3的激活[20]㊂推测S T I N G 通过与这些蛋白互作,从而参与免疫及抗病毒过程㊂通过对白来航鸡S T I N G基因的组织表达特性分析可知,S T I N G基因的分布具有组织特异性,其中在肺脏中表达量最高,而石树端等[21]研究发现鸭S T I N G基因在腺胃中表达水平最高,表明家禽中m R N A组织分布存在一定差异;其次是脾脏,提示S T I N G基因广泛分布于宿主的免疫器官;此外, S T I N G基因在回肠㊁十二指肠等肠道组织中也有表达,因为肠道中的微生物菌群是启动免疫的重要因子,肠道微生物与宿主的互作建立机体的适应性免疫系统,可有效维持免疫防御作用[22];在胸肌中表达量最低,与金洁[7]研究结果一致㊂研究表明, S T I N G基因表达量降低可以提示肝癌和胃癌患者的预后不良[23-24],而注射S T I N G激动剂可使人乳头瘤病毒相关肿瘤组织消退[25]㊂另外,通过抑制S T I N G通路可以阻断衰老细胞释放促炎信号,从而改善老年神经退行性疾病[26]㊂因此,抑制或激活S T I N G信号通路,可为疾病治疗提供新的途径和特异性治疗靶点[21]㊂本试验通过对白来航鸡S T I N G 基因编码蛋白进行生物信息学和组织表达特性分析,为揭示S T I N G基因在固有免疫系统中的作用提供数据,并为后期探究S T I N G编码蛋白的生物学功能提供切实的理论基础㊂4结论本研究成功克隆了白来航鸡S T I N G基因C D S 区序列,大小为1140b p,编码379个氨基酸㊂白来航鸡与原鸡的亲缘关系最近,与冠小嘴乌鸦亲缘关系最远㊂S T I N G蛋白属于酸性㊁亲水性蛋白,存在跨膜结构,无信号肽,主要在细胞质和线粒体中发挥作用㊂S T I N G基因在白来航鸡肺脏中表达量最高,在胸肌中表达最低㊂参考文献(R e f e r e n c e s):[1] G U O Y,J I A N GF,K O N G L,e t a l.O T U D5p r o m o t e si n n a t e a n t i v i r a l a n d a n t i t u m o r i m m u n i t y t h r o u g hd e u b i q u i t i n a t i n g a n ds t a b i l i z i n g S T I N G[J].C e l l u l a r&M o l e c u l a r I m m u n o l o g y,2021,18(8):1945-1955.[2]J I A N G M,C H E N P,W A N G L,e t a l.c G A S-S T I N G,a ni m p o r t a n t p a t h w a y i n c a n c e r i m 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[17] WA N G Y,L U H,F A N GC,e t a l.P a l m i t o y l a t i o na s as i g n a lf o r d e l i v e r y[J].A d v a n c e si n E x p e r i m e n t a lM e d i c i n e a n dB i o l o g y,2020,1248:399-424. [18]胡燕萍,许锦霞,徐阿慧,等.宿主c G A S-S T I N G信号通路调控机制的研究进展[J].中国预防兽医学报,2022,44(12):1344-1351.HU YP,X UJX,X U A H,e t a l.R e c e n t a d v a n c e s i nt h e r e g u l a t i o n m e c h a n i s m o f h o s t c G A S-S T I N Gs i g n a l i n gp a t h w a y[J].C h i n e s e J o u r n a l o f V e t e r i n a r yM e d i c i n e,2022,44(12):1344-1351.(i nC h i n e s e) [19] Z HA N GC,S HA N G G,G U IX,e t a l.S t r u c t u r a l b a s i so f S T I N G b i n d i n g w i t h a n d p h o s p h o r y l a t i o n b yT B K1[J].N a t u r e,2019,567(7748):394-398.[20] MA T ZK M,G U Z MA N R M,G O O D MA N A G.T h er o l eo fn u c l e i ca c i ds e n s i n g i nc o n t r o l l i n g m i c r o b i a la n da u t o i m m u n ed i s o r d e r s[J].I n t e r n a t i o n a lR e v i e wo f C e l l a n d M o l e c u l a rB i o l o g y,2019,345:35-136.[21]石树端,高全新,程玉强,等.S T I N G基因荧光定量检测方法的建立及其在鸭组织中的分布[J].畜牧与兽医,2017,49(7):69-73.S H I S D,G A O Q X,C H E N G Y Q,e t a l.E s t a b l i s h m e n t o f R e a l-t i m e P C R f o r d e t e c t i o n o fS T I N G g e n ea n di t st i s s u ed i s t r i b u t i o ni nd u c k[J].A n i m a lH u s b a n d r y&V e t e r i n a r y M e d i c i n e,2017,49(7):69-73.(i nC h i n e s e)[22]李淑珍,常文环,刘国华,等.肉鸡肠道微生物及其调控[J].动物营养学报,2020,32(7):2989-2996.L IS Z,C HA N G W H,L I U G H,e t a l.I n t e s t i n a lm i c r o b i o t ao fb r o i l e rc h i c k e n sa n di t sr e g u l a t i o n[J].C h i n e s e J o u r n a l o f A n i m a lN u t r i t i o n,2020,32(7):2989-2996.(i nC h i n e s e)[23] B U Y,L I UF,J I A Q A,e t a l.D e c r e a s e d e x p r e s s i o no fTM E M173p r e d i c t s p o o r p r o g n o s i si n p a t i e n t s w i t hh e p a t o c e l l u l a r c a r c i n o m a[J].P L o S O n e,2016,11(11):e0165681.[24] S O N G S,P E N G P,T A N G Z,e t a l.D e c r e a s e de x p r e s s i o n of S T I N G p r e d i c t s p o o r p r og n o s i s i np a t i e n t s w i t h g a s t r i cc a n c e r[J].S c i e n t i f i cR e p o r t s,2017,7:39858.[25]张展,刘朝晖.S T I N G信号通路在H P V相关恶性肿瘤中的作用[J].国际妇产科学杂志,2023,50(1):30-34.Z HA N G Z,L I U Z H.R o l e o f S T I N G s i g n a l i n gp a t h w a y i n H P V-r e l a t e d m a l i g n a n t t u m o r s[J].J o u r n a l o f I n t e r n a t i o n a lO b s t e t r i c s a n dG y n e c o l o g y,2023,50(1):30-34.(i nC h i n e s e)[26] G U L E N M F,S AM S O N N,K E L L E R A,e t a l.c G A S-S T I N G d r i v e s a g e i n g-r e l a t e d i n f l a m m a t i o n a n dn e u r o d e g e n e r a t i o n[J].N a t u r e,2023,620(7973):374-380.(责任编辑晋大鹏)1831。

体外哺乳动物细胞基因突变试验的英语

体外哺乳动物细胞基因突变试验的英语Gene mutation is a crucial process in the evolution of species, as it introduces genetic diversity and drives adaptation to changing environments. In the field of molecular biology, researchers often conduct gene mutation experiments on mammalian cells to study the effects of specific genetic changes on cellular function. One commonly used method for studying gene mutations in mammalian cells is the in vitro mammalian cell gene mutation assay.The in vitro mammalian cell gene mutation assay is a widely accepted and standardized test for assessing the mutagenic potential of chemicals and other substances. This assay is based on the principle that mutations in specific genes can lead to changesin cellular phenotype, such as altered growth characteristics or resistance to certain toxins. By exposing mammalian cells to a test substance and then monitoring for genetic changes, researchers can determine the mutagenic potential of the substance in question.To conduct an in vitro mammalian cell gene mutation assay, researchers typically start by selecting a suitable mammalian cell line for the experiment. Commonly used cell lines include Chinese hamster ovary (CHO) cells, L5178Y mouse lymphoma cells, and TK6 human lymphoblastoid cells. These cell lines are chosen for their sensitivity to genetic changes and their ability to accurately reflect the mutagenic potential of test substances.Once a cell line has been selected, researchers expose the cells to varying concentrations of the test substance and incubate them for a specified period of time. During this incubation period, the cells are allowed to replicate and divide, giving them the opportunity to accumulate genetic mutations. After the incubation period, researchers can assess the presence of gene mutations by performing molecular analyses, such as polymerase chain reaction (PCR) or DNA sequencing.The results of an in vitro mammalian cell gene mutation assay can provide valuable information about the potential mutagenic effects of a test substance. If the test substance induces a significant increase in the frequency of gene mutations in the treated cellscompared to untreated controls, it may be considered mutagenic. This information is important for assessing the safety of chemicals and other substances, as mutagenic compounds have the potential to cause genetic damage and increase the risk of cancer.In conclusion, the in vitro mammalian cell gene mutation assay is a powerful tool for studying the mutagenic potential of chemicals and other substances. By exposing mammalian cells to test substances and monitoring for genetic changes, researchers can gain valuable insights into the effects of specific genetic mutations on cellular function. This assay plays a crucial role in assessing the safety of chemicals and informing regulatory decisions to protect human health and the environment.。

糙皮侧耳

我们曾先后基的差异表达基因进行了研究，并构建了糙皮侧耳菌丝体和子实体原基基因差异表达谱。
本研究在筛选糙皮侧ptidase基因全长cDNA序列，并对其进行了定量分析。
Yike I ，2011年，Mycopathologia .概述了天冬氨酸蛋白酶在真菌病理生理过程中的作用。 Park SK等，2006年，Fungal Genetics and Biology.利用RFLP, Northern blot， QTL（数量性状遗传位点）图谱绘制等方法研究了天冬氨酸蛋白酶在糙皮侧耳蛋白质新陈代谢中的促进作用以及在子实体发育中的潜在作用。
糙皮侧耳天冬氨酸蛋白酶基因的克隆及定量分析
研究背景
糙皮侧耳（Pleurotus ostreatus）又称平菇，是世界上第三大人工栽培的食用菌。
糙皮侧耳由双核菌丝扭结成团发育为原基即子实体分化发育是其生活史中最重要的过程。
糙皮侧耳菌丝体和原基形态比较
迄今为止,国内外对糙皮侧耳及其它一些担子菌由双核菌丝扭结成团发育为原基的过程所需的营养及外界环境条件研究得较为深入与透彻。
Aspartic peptidase 5′端克隆电泳图
cDNA 5′端序列比对结果
Species Laccaria bicolor Coprinopsis cinerea Aspergillus clavatus Penicillium marneffei E value 2e-163 1e-159 2e-74 4e-65 Max ident 74% 75% 69% 68% Gene aspartic peptidase endopeptidase aspartic endopeptidase aspartic endopeptidase

丙肝病毒分子分型方法

HCV genotypes in Northern Italy: a survey of 1368 histologically proven chronic hepatitis C patients
( 4)限制性片段长度多态性分析 ( RFLP)法:
用能识别特定酶切位点的限制性内切酶将 PCR 扩增的片段切割成不同长度的片段
（9）Taqman探针实时PCR测试法
这一方法用到了Taqman探针(针对5’ UTR) 结合荧光实时PCR
M Lindh，Harmoun C．Genotyping of hepatitis C virus by Taqman real—time PCIK．Journal of Clinical Virology，2005；34：108．
（2）基因型特异性引物PCR法
Okamoto等首先采用分型的效果仍比较差需使用7对引物
Okamoto H et al. J Gen Virol. (1992) Typing hepatitis C virus by polymerase chain reaction with type-specific primers: application to clinical surveys and tracing infectious sources.
该方法常用的酶为HaeIII、RsaI 、Mva I 、 Hinf I、Scrf I
该方法检测基因型的精确度为97％不能区分所有的HCV基因亚型
也不能识别在泰国和越南发现的变异基因型，
Davidson F, Simmonds P, Ferguson JC, et al. Survey of major genotypes and subtypes of hepatitis C virus using RFLP of sequences amplified from the 5′non-coding region. J Gen Virol, 1995, 76:1197-1204.

Dynamic Transcriptome Landscape of Maize Embryo and

Dynamic Transcriptome Landscape of Maize Embryo and Endosperm Development1[W][OPEN]Jian Chen2,Biao Zeng2,Mei Zhang,Shaojun Xie,Gaokui Wang,Andrew Hauck,and Jinsheng Lai*State Key Laboratory of Agro-biotechnology and National Maize Improvement Center,Department of Plant Genetics and Breeding,China Agricultural University,Beijing100193,People’s Republic of ChinaMaize(Zea mays)is an excellent cereal model for research on seed development because of its relatively large size for both embryo and endosperm.Despite the importance of seed in agriculture,the genome-wide transcriptome pattern throughout seed development has not been well ing high-throughput RNA sequencing,we developed a spatio-temporal transcriptome atlas of B73maize seed development based on53samples from fertilization to maturity for embryo, endosperm,and whole seed tissues.A total of26,105genes were found to be involved in programming seed development,in-cluding1,614transcription factors.Global comparisons of gene expression highlighted the fundamental transcriptomic repro-gramming and the phases of development.Coexpression analysis provided further insight into the dynamic reprogramming of the transcriptome by revealing functional transitions during bined with the published nonseed high-throughput RNA sequencing data,we identiﬁed91transcription factors and1,167other seed-speciﬁc genes,which should help elucidate key mechanisms and regulatory networks that underlie seed development.In addition,correlation of gene expression with the pattern of DNA methylation revealed that hypomethylation of the gene body region should be an important factor for the expressional activation of seed-speciﬁc genes,especially for extremely highly expressed genes such as zeins.This study provides a valuable resource for understanding the genetic control of seed development of monocotyledon plants.Maize(Zea mays)is one of the most important crops and provides resources for food,feed,and biofuel (Godfray et al.,2010).It has also been used as a model system to study diverse biological phenomena,such as transposons,heterosis,imprinting,and genetic diversity (Bennetzen and Hake,2009).The seed is a key organ of maize that consists of the embryo,endosperm,and seed coat.Maize seed development initiates from a double fertilization event in which two pollen sperm fuse with the egg and central cells of the female gametophyte to produce the progenitors of the embryo and endosperm, respectively(Dumas and Mogensen,1993;Chaudhury et al.,2001).The mature embryo inherits the genetic information for the next plant generation(Scanlon and Takacs,2009),whereas the endosperm,which is storage tissue for the embryo,persists throughout seed devel-opment and functions as the site of starch and protein synthesis(Sabelli and Larkins,2009).Elucidation of the genetic regulatory mechanisms involved in maize seed development will facilitate the design of strategies to improve yield and quality,and provide insight that is applicable to other monocotyledon plants.A key means to explore the mechanisms of seed de-velopment is to identify gene activities and functions. Genetic studies have uncovered a number of genes that play major roles in governing embryogenesis and ac-cumulation of endosperm storage compounds,such as Viviparous1,KNOTTED1,Indeterminate gametophyte1, Shrunken1(Sh1),Opaque2(O2),and Defective kernel1 (Chourey and Nelson,1976;McCarty et al.,1991;Smith et al.,1995;Vicente-Carbajosa et al.,1997;Lid et al., 2002;Evans,2007).Furthermore,the activity of some genes has also been extensively studied.Typical exam-ples are zein genes that encode primary storage proteins in endosperm.Woo et al.(2001)examined zein gene expression and showed that they were the most highly expressed genes in endosperm based on EST data,where-as their dynamic expression patterns were revealed in a later study(Feng et al.,2009).Nevertheless,informa-tion on the global gene expression network throughout seed development is still very limited.The transcriptome is the overall set of transcripts, which varies based on cell or tissue type,develop-mental stage,and physiological condition.Analysis of transcriptome dynamics aids in implying the function of unannotated genes,identifying genes that act as critical network hubs,and interpreting the cellular pro-cesses associated with development.In Arabidopsis (Arabidopsis thaliana),the genes expressed in devel-oping seed and its subregions at several develop-ment stages have been analyzed with Affymetrix GeneChips(Le et al.,2010;Belmonte et al.,2013).In1This work was supported by the National High Technology Re-search and Development Program of China(863Project,grant no. 2012AA10A305to J.L.)and the National Natural Science Foundation of China(grant no.31225020to J.L.).2These authors contributed equally to the article.*Address correspondence to jlai@.The author responsible for distribution of materials integral to the ﬁndings presented in this article in accordance with the policy de-scribed in the Instructions for Authors()is: Jinsheng Lai(jlai@).[W]The online version of this article contains Web-only data.[OPEN]Articles can be viewed online without a subscription./cgi/doi/10.1104/pp.114.240689maize,microarray-based atlases of global transcription have provided insight into the programs controlling development of different organ systems (Sekhon et al.,2011).Compared with microarray,high-throughput RNA sequencing (RNA-seq)is a powerful tool to com-prehensively investigate the transcriptome at a much lower cost,but with higher sensitivity and accuracy (Wang et al.,2009b).Several studies have taken ad-vantage of the RNA-seq strategy to interpret the dy-namic reprogramming of the transcriptome during leaf,shoot apical meristem,and embryonic leaf develop-ment in maize (Li et al.,2010;Takacs et al.,2012;Liu et al.,2013).To date,only two studies have focused on identi-fying important regulators and processes required for embryo,endosperm,and/or whole seed development in maize based on a genome-wide transcriptional pro-ﬁle produced by RNA-seq (Liu et al.,2008;Teoh et al.,2013).However,these studies were limited by the low number of samples used and they did not provide an extensive,global view of transcriptome dynamics over the majority of seed development stages.Here,we pres-ent a comprehensive transcriptome study of maize em-bryo,endosperm,and whole seed tissue from fertilization to maturity using RNA-seq,which serves as a valu-able resource for analyzing gene function on a global scale and elucidating the developmental processes of maize seed.RESULTSGeneration and Analysis of the RNA-seq Data SetTo systematically investigate the dynamics of the maize seed transcriptome over development,we generated RNA-seq libraries of B73seed tissues from different developmental stages,including 15embryo,17endo-sperm,and 21whole seed samples (Fig.1).Utilizing paired-end Illumina sequencing technology,we gen-erated around 1.9billion high-quality reads,80.2%of which could be uniquely mapped to the B73referencegenome (Schnable et al.,2009;Supplemental Table S1).The genic distribution of reads was 66.6%exonic,25.6%splice junction,and 2.6%intronic,leaving about 5%from unannotated genomic regions,demonstrating that most of the detected genes have been annotated.Uniquely mapped reads were used to estimate nor-malized transcription level as reads per kilobase per million (RPKM).To reduce the in ﬂuence of transcrip-tion noise,genes from the B73ﬁltered gene set (FGS)were included for analysis only if their RPKM values were $1.Considering that our purpose was not to identify minor differential expression of genes between two time points of development,but to provide an atlas of gene expression pro ﬁle across tissues using time se-ries biological samples,we only randomly selected 12samples to have a biological replicate (Supplemental Fig.S1)to assess our data parisons of bio-logical replicates showed that their expression values were highly correlated (average R 2=0.96).For the samples with biological replicates,we took the average RPKM as the expression quantity.To further evaluate the quality of our expression data,we compared the transcript abundance patterns of a number of selected genes with previously measured expression pro ﬁles (Supplemental Fig.S2).For example,LEAFY COTYLE-DON1(LEC1),which functions in embryogenesis,was mainly expressed in the early stage of the embryo (Lotan et al.,1998).Globulin2(Glb2)had high expres-sion late in embryogenesis,in accordance with its func-tion as an important storage protein in the embryo (Kriz,1989).Similarly,O2,a transcription factor (TF)that regulates zein synthesis (Vicente-Carbajosa et al.,1997),and Fertilization independent endosperm1(Fie1),a repressor of endosperm development in the absence of fertilization (Danilevskaya et al.,2003),were almost ex-clusively expressed in the endosperm.Expression pat-terns of these selected genes were identi ﬁed exclusively in their known tissue of activity,indicating that the embryo and endosperm samples were processed well.In total,we detected 26,105genes expressed in at least 1of the 53samples (Supplemental Data Set S1).The distribution of these genes is revealed by aVennFigure 1.Overview of the time series maize seed samples used for RNA-seq analysis.The photographs show the changes in maize embryo,endosperm,and whole seed during development.The 53samples shown here were used to generate RNA-seq libraries.Bar =5mm.Transcriptome Dynamics of Maize Seeddiagram (Fig.2A),which shows that 20,360genes were common among all three tissue types.The number of genes detected in endosperm tissue during the devel-opmental stages was lower and much more variable compared with embryo or whole seed tissue,and a greater number of genes were expressed in the tissue during the early and late phases.Several thousand fewer expressed genes were detected 14d after polli-nation (DAP)in the endosperm compared with 6or 8DAP (Fig.2B;Supplemental Data Set S2).In addition,the median expression level in the embryo was roughly 2-fold greater than that of the endosperm from 10to 30DAP (Fig.2C).Of the 1,506genes unique to the whole seed samples,451were present in RNA-seq data of 14and/or 25DAP pericarp tissue (Morohashi et al.,2012).Considering that the maternal tissue is the vast majority of the content of the early seed (Márton et al.,2005;Pennington et al.,2008),we inferred that most of these genes might be expressed exclusively in maternal tissue such as the pericarp or nucellus.Moreover,1,062genes in the embryo and endosperm with low expression were not detected in whole seed samples (Fig.2C).Division of Development Phases by Global Gene Expression PatternsTo gain insight into the relationships among the dif-ferent transcriptomes,we performed principal compo-nent analysis (PCA)on the complete data set,which can graphically display the transcriptional signatures and developmental similarity.The ﬁrst component(40.7%variance explained)separated samples based on tissue identity and clearly distinguished embryo from endosperm samples,with whole seed samples located in between (Fig.3A).The second component (24.4%variance explained)discriminated early,mid-dle,and late stages of development for all three tissues (Fig.3A).The wider area occupied by endosperm sam-ples than embryo demonstrates stronger transcriptome reprogramming in developing endosperm,which is mainly attributable to drastic changes in the early and late stages.Moreover,whole seed samples of 0to 8DAP and 30to 38DAP clustered closely to the embryo,but 10to 28DAP samples were close to the endosperm.Cluster analysis of the time series data for the tissues grouped samples well along the axis of developmental time (Fig.3,B –D).Embryo samples from 10to 20DAP and 22to 38DAP were the primary clusters,which correspond to morphogenesis and maturation phases of development (Fig.3B).This is consistent with the embryo undergoing active DNA synthesis,cell division,and differentiation,and then switching to synthesis of storage reserve and desiccation (Vernoud et al.,2005).Expression differences in endosperm samples resulted in three primary clusters,which correspond to early,middle,and late phases of development (Fig.3C).The earliest time point (6and 8DAP)is an active period of cell division and cell elongation that terminates at about 20to 25DAP (Duvick,1961).The samples of 10to 24DAP formed one subgroup and 26to 34DAP formed another subgroup,suggesting that they mark the period forming the main cell types and maturation of endo-sperm,respectively (Fig.3C).The two subgroupsformFigure 2.Analysis of global gene expres-sion among different samples.A,Venn diagram of the 26,105genes detected among embryo,endosperm,and whole seed.B,Number of genes expressed in each of the samples.C,Comparison of expression levels of genes detected in embryo and endosperm tissues.Chen et al.a larger cluster for active accumulation of storage com-pounds during 10to 34DAP.The distinct cluster of 36and 38DAP is in accordance with the end of storage compound accumulation in the endosperm and the ac-tivation of biological processes involved in dormancy and dehydration.In whole seed tissue,a primary clus-ter was formed from the earliest time points with 0to 4DAP and 6to 8DAP samples as subgroups,separating the nucellus degradation as well as endosperm syncy-tial and cellularization phases from the rapid expansion of the endosperm and development of embryonic tissues (Fig.3D).After 10DAP,the embryo and endosperm dominate the formation of seed,as shown in Figure 1and morphological observation (Pennington et al.,2008).As effected by both embryo and endosperm,10to 28DAP whole seed samples clustered together and 30to 38DAP formed another group (Fig.3D).These results con ﬁrmthat the expression data successfully captured the char-acteristic seed development phases and should there-fore contain valuable insights about corresponding changes in the transcriptome.Integration of Gene Activity and Cellular Function across Development PhasesThe PCA and hierarchical clustering analysis graph-ically display the relationship among different sam-ples,but do not indicate the detailed cellular ing the k-means clustering algorithm,we classi ﬁed the detected genes into 16,14,and 10coexpression modules for embryo,endosperm,and whole seed,re-spectively,each of which contains genes that harbor similar expression patterns (Fig.4).We then used Map-Man annotation to assign genes to functionalcategoriesFigure 3.Global transcriptome relationships among different stages and tissues.A,PCA of the RNA-seq data for the 53seed samples shows ﬁve distinct groups:I for embryo (light red),II for endosperm (light blue),and III to V for early (III),middle (IV),and late (V)whole seed (light purple).B to D,Cluster dendrogram showing global transcriptome relationships among time series samples of embryo (B),endosperm (C),and whole seed (D).The y axis measures the degree of variance (see the “Materials and Methods”).The bottom row indicates the developmental phases according to the cluster dendrogram of the time series data.au,Approximately unbiased.Transcriptome Dynamics of Maize Seed(Supplemental Fig.S3).Thus,we can aggregate genes over continuous time points and obtain a view of func-tional transitions along seed development.According to the cluster analysis results,most mod-ules of the embryo can be divided into middle (10–20DAP)and late (22–38DAP)stages (Fig.4A).The middle stage,best represented by modules C1to C7,is typi ﬁed by the overrepresentation of glycolysis,tri-carboxylic acid cycle,mitochondrial electron transport,redox,RNA regulation,DNA and protein synthesis,cell organization,and division-related genes.This is consis-tent with the high requirement of energy during em-bryo formation.The late stage represented by C8to C12exhibited up-regulation of the cell wall,hormone me-tabolism (ethylene and jasmonate),stress,storage pro-teins,and transport-related genes,which coincides with the maturation of the embryo.The modules C13to C16included genes that were broadly expressed across the time points sampled and were related to hormone me-tabolism (brassinosteroid),cold stress,RNA process-ing and regulation,amino acid activation,and protein targeting.All of the 14coexpression modules of endosperm can be roughly divided into early (6–8DAP),middle (10–34DAP),and late (36–38DAP)stages (Fig.4B).The early stage (represented by modules C1to C4)isexempli ﬁed by high expression of hormone metabo-lism (gibberellin),cell wall,cell organization and cycle,amino acid metabolism,DNA,and protein synthesis –related genes,which is consistent with differentiation,mitosis,and endoreduplication.Genes in the tricar-boxylic acid cycle and mitochondrial electron transport are also overrepresented and related to energy de-mands at that time.The middle stage (best represented by C5to C8,in which different modules have distinct pro ﬁles)is the active storage accumulation phase and exhibits high expression of carbohydrate metabolism genes,as expected.Increased expression of protein degradation-related genes around 26to 34DAP in C7and C8coincides with the process of endosperm matu-ration.Genes involved in protein degradation,second-ary metabolism,oxidative pentose phosphate,receptor kinase signaling,and transport were up-regulated in the late stage in modules C9to C14during the con-cluding phase of endosperm maturation.Ten DAP and later time points of whole seed sam-ples re ﬂect the additive combination of embryo and endosperm expression.Genes that are active early in development (0–8DAP)in clusters C1to C4are ex-pected to be related to maternal tissue,which is the bulk of the seed at that time.A group of genes are highly expressed in C1at 0DAP,but theirexpressionFigure 4.Coexpression modules.A to C,Expression patterns of coexpression modules of embryo (A),endosperm (B),and whole seed (C),ordered according to the sample time points of their peak expression.For each gene,the RPKM value normalized by the maximum value of all RPKM values of the gene over all time points is shown.Chen et al.drops rapidly by2DAP,suggesting that they have functional roles that precede pollination.This group includes photosynthesis light reaction members and some TFs involved in RNA regulation.Genes related to cell wall and protein degradation,signaling,nucle-otide metabolism,DNA synthesis,cell organization, and mitochondrial electron transport are overrepre-sented in C2to C4,which has increased expression after pollination,in accordance with the degradation of nucellus tissue and development of embryonic tissues. The expression patterns and functional categories of the1,506genes detected only in whole seed samples are shown in Supplemental Figure S4.Because these genes tend to be expressed at high levels mainly before 8DAP,they are presumed to have functions in early seed development.Together,these data show that the transition of major biochemical processes along the developmental time axis of the seed is produced partly by highly coordi-nated transcript dynamics.TF Expression during Seed DevelopmentOf the2,297identiﬁed maize TFs(Zhang et al.,2011), 1,614(70%)are included in our analysis(Supplemental Data Set S3),which accounts for6.18%of the total number of genes detected in seed tissue.The num-ber of TFs detected in the different samples is shown in Supplemental Figure S5A.Their proportion to the total genes expressed in each tissue time point was always greater in embryo than endosperm samples (Supplemental Fig.S5B).Shannon entropy has been used to determine the speciﬁcity of gene expression, with lower values indicating a more time-speciﬁc pro-ﬁle(Makarevitch et al.,2013).The Shannon entropy of TFs was signiﬁcantly lower than all other genes in both embryo(P=2.3310210)and endosperm(P,1.53 1023),indicating that TFs tended to be expressed more time speciﬁcally than other genes(Supplemental Fig.S5, C and D).The number of TFs from each family used in the seed development program,along with the proportion of members present in the coexpression modules rel-ative to the total members of the family expressed in the tissue,is shown in Supplemental Figure S6. Enrichment of these TF families in the coexpression modules based on observed numbers was evaluated with Fisher’s exact test.Signiﬁcant TF family enrich-ment was identiﬁed for speciﬁc coexpression modules. For example,12auxin-response factor TFs(38.7%) were expressed in embryo module C2during mor-phogenesis and one-half of the detected members of the WRKY family were active in endosperm module C12late in endosperm development.The WRKY family has been reported to be mainly involved in the physiological programs of pathogen defense and se-nescence(Eulgem et al.,2000;Pandey and Somssich, 2009).Twenty-one MIKC family TFs(56.8%)were pres-ent in whole seed module C2,implying an important role in regulating genes involved in response to fertil-ization.The developmental speciﬁcity of the detected TFs makes them excellent candidates for reverse ge-netics approaches to investigate their role in grain production.Tissue-Speciﬁc Genes of SeedIdentiﬁcation of uncharacterized tissue-speciﬁc genes can help to explain their function and understand the underlying control of tissue or organ identity.To gen-erate a comprehensive catalog of seed-speciﬁc genes, results from this study were compared with25pub-lished nonseed RNA-seq data sets(Jia et al.,2009;Wang et al.,2009a;Li et al.,2010;Davidson et al.,2011;Bolduc et al.,2012),including root,shoot,shoot apical meristem, leaf,cob,tassel,and immature ear(Supplemental Table S2).In total,we identiﬁed1,258seed-speciﬁc genes,in-cluding91TFs from a variety of families(Supplemental Data Set S4).To gain further insight into the spatial expression trend in the developing seed,we divided these genes into four groups:embryo speciﬁc,endo-sperm speciﬁc,expressed in both embryo and endo-sperm,and other as only expressed in whole seed (Table I).The dynamic expression patterns of these genes reﬂect their roles in corresponding development stages(Supplemental Fig.S7).The largest numbers of seed-speciﬁc genes were observed in the endosperm, consistent with a study in maize using microarrays (Sekhon et al.,2011),perhaps reﬂecting the speciﬁc function of endosperm.We compared the distribution of tissue-speciﬁc genes and TFs in embryo and endosperm coexpression mod-ules to identify important phases in the underlying transcription network.Coexpression modules with an enrichment of tissue-speciﬁc genes or TFs may provide insight about uncharacterized genes and preparation for subsequent developmental processes.A feature of gene activity is shown in Figure5.Fisher’s exact test (P,0.05)was used to determine modules with sig-niﬁcant enrichment of tissue-speciﬁc genes and TFs in the embryo and endosperm.In the embryo,TFs and tissue-speciﬁc genes were signiﬁcantly enriched in the late phase,suggesting a speciﬁc process during matu-ration.In the endosperm,we observed that TFs and tissue-speciﬁc genes were overrepresented in the mid-dle phase,which conforms to the role of endosperm inTable I.Total number of detected seed-speciﬁc genes and TFsData are presented as n.Tissue Type Speciﬁc Genes Speciﬁc TFs Embryo24923Endosperm74259Embryo and endosperm2196Other a483Total1,25891a These genes only detected expression in whole seeds.Transcriptome Dynamics of Maize Seedstorage compound accumulation and to speci ﬁc pro-gress at this phase.To gain further insight into the functional signi ﬁ-cance of tissue-speci ﬁc genes,overrepresented gene ontology (GO)terms were examined using the WEGO online tool (Ye et al.,2006;Supplemental Fig.S8).All overrepresented GO terms were observed for themiddle embryo development phase,including the bio-synthetic process,cellular metabolic process,macro-molecule,and nitrogen compound metabolic process.Similarly,overrepresented GO terms for the endosperm were mostly observed in the early phase,including the macromolecule,nitrogen compound metabolic pro-cess,DNA binding,and transcription regulator.GenesFigure 5.Distribution and enrichment of genes,tissue-speciﬁc genes,TFs,and tissue-speciﬁc TFs in coexpression modules of embryo and endosperm.A and B,Bars indicate the percentage of all detected genes (green),tissue-speciﬁc genes (blue),TFs (red),and tissue-speciﬁc TFs (purple)observed in a coexpression module (C)or in the development phase (Total)relative to the total number of each group detected across samples for embryo (A)and endosperm (B).The number of genes represented by the percentage is shown on the right y axis.Enrichment for tissue-speciﬁc genes and TFs was evaluated with Fisher’s exact test based on the number of genes observed in each coexpression module,whereas enrichment for tissue-speciﬁc TFs was evaluated based on the number of TFs observed in each coexpression module.Asterisks represent signiﬁcant enrichment at a false dis-covery rate #0.05.Chen et al.involved in the nutrient reservoir class were enriched in the middle phase.The oxidoreductase class was overrepresented in late phase,and is known to be in-volved in maturation (Zhu and Scandalios,1994).The Expression of Zein Genes in EndospermZeins are the most important storage proteins in maize endosperm and are an important factor in seed quality.According to Xu and Messing (2008),there are 41a ,1b ,3g ,and 2d zein genes.In order to explore their expression pattern,we ﬁrst con ﬁrmed the gene models by mapping publicly available full-length com-plementary DNAs of zein subfamily genes to B73bac-terial arti ﬁcial chromosomes,and then mapped these back to the reference genome.Because some of these zein genes were not assembled in the current B73ref-erence genome or were only annotated in the working gene set,we con ﬁrmed a ﬁnal set of 35zein genes in the FGS of the B73annotation,including 30a ,1b ,3g ,and 1d zein genes (Supplemental Table S3).About three-quarters (26)of these were in the list of the 100most highly expressed genes in the endosperm,based on mean expression across all endosperm samples (Supplemental Table S4).The distribution of these most highly expressed genes clearly showed that the 26zeins and 4starch synthesis genes were actively expressed in the middle phase of endosperm devel-opment,characteristic of storage compound accumu-lation (Fig.6A).Previous research has shown that the zein genes con-stitute approximately 40%to 50%of the total tran-scripts in the endosperm (Marks et al.,1985;Woo et al.,2001),but these results are based on EST data from a single tissue or pooled tissues and only a few zein genes were assessed.Thus,we reevaluated the transcriptomic contribution of zein genes across endosperm develop-ment using our RNA-seq data,which is able to overcome the high structural similarity among them,especially in the a family (Xu and Messing,2008).Zein genes stably accounted for about 65%of transcripts from 10to 34DAP,with 19-kD a zeins (approximately 42%),22-kD a zeins (approximately 8%),and g zeins (approximately 10%)representing the most abundant transcripts (Fig.6B).The expression of different members within agivenFigure 6.Analysis of highly expressed genes in the endosperm.A,The distribution of the 100most highly expressed genes in the endosperm ordered by mean expression in different modules.B,The dynamic transcript levels of different zein gene family members in the endosperm as reﬂected by their percentage among all detected gene transcript levels.C,Heat map showing RPKM values of 35zein genes in the different development stages of the endosperm.+,Having intact coding regions;2,with premature_stop;N,no;Y ,yes.Transcriptome Dynamics of Maize Seed。

免疫实验中遇到的种属中英文对照(超级全)(最新整理)

毒蜥(毒蜥属)
Polyoma virus
多瘤病毒
Goose
鹅
Alligator
鳄鱼
Crocodile
鳄鱼
Non-Human Primate
非人灵长类
Xenopus/Amphibian
非洲爪蟾蜍/两栖动物
Xenopus laevis
非洲爪蟾蜍光滑的
Baboon
狒狒
Opossum (Didelphimorphia)
斑马鱼(Brachydanio rerio)
Zebrafish (Danio rerio)
斑马鱼(Danio rerio)
Zebrafishfinch (Taeniopygia guttata)
斑胸草雀(Taeniopygia guttata)
Prunus persica
Rat (Rattus)
大鼠(鼠属)
Atlantic Salmon (Salmo salar)
大西洋鲑鱼(鲑)
Mummichog
底鳉鱼
Griffonia simplicifolia (syn.Bandeiraea simplicifolia)
豆科灌木植物加纳谷物
Viper
毒蛇
Gilamonster (Heloderma suspectum)
球形节杆菌（A.球形节杆菌）
Arthrobacterium ureafaciens (A.ureafaciens)
节杆菌（a.ureafaciens）
Aspergillus
曲霉
Aspergillus niger
黑曲霉
Aspergillus ochraceus
赭曲霉
Aspergillus oryzae

无胸腺裸鼠详细资料大全

无胸腺裸鼠详细资料大全无胸腺裸鼠（Nude Mouse，简称裸鼠）目前已成为医学生物学研究领域中不可缺少的实验动物模型。

它是先天性胸腺缺陷的突变小鼠, 由于第VIII连锁群内裸 *** 点基因发生纯合而形成的突变小鼠。

特别是在肿瘤学、免疫学、药品与生物制品的安全性评价及有效药品的筛选等实验方面，它有着特殊的价值。

裸鼠在科学研究中所以能成为有巨大潜力的试验模型，是由于nu基因有独特的遗传特性。

经过世界各国实验动物遗传育种学家的努力，目前已将nu基因导入不同近交系动物，成为系列动物模型，仅小鼠模型一种，已建立了二十余种近交系裸鼠。

由于它们具有不同的遗传背景和nu基因的遗传特性，使医学生物学研究者获得一种极其宝贵的试验材料。

基本介绍•中文学名：无胸腺裸鼠•拉丁学名：Nude Mouse•别称：裸鼠•界：动物界•门：脊索动物门•纲：哺乳纲•亚纲：真兽亚纲•目：齧齿目•科：鼠科•族：族鼠•属：属鼠基本概念,发展情况,生理特征,医学研究,概况,组织移植,生存条件,繁殖,饲料条件,动物特性,研究过程,特点,套用,基本概念裸鼠因无胸腺，T—淋巴细胞缺陷，抵抗力低下，常因消耗性疾病而死亡。

裸鼠是随着近代医学发展，特别是肿瘤学和免疫学研究的需要，在近十几年中发展起来的动物模型。

它是先天性胸腺缺陷的突变小鼠，是由于第Ⅶ连锁群（Linkage group）内裸 *** 点的等位基因发生纯合而形成的突变小鼠新品种。

裸鼠（纯合子nu/nu突变鼠）主要表现为无毛（但可看到一种细毛组织学证明有被毛滤泡）以及缺乏正常胸腺。

杂合子小鼠（nn/+）各方面表现都正常。

小鼠中有若干突变基因，它可产生一种为无毛的表现型（phenotype），例如无胸腺裸鼠（Nude、）裸鼠（Naked）、无毛鼠（Hairless）、无鼻毛鼠（Rhinol），不要把这些突变鼠基因相互混淆。

裸鼠的唯一特性是胸腺缺陷表现型，因此，不能将“裸鼠”与“无毛鼠”两词交换使用。

海洋生物技术

⑷其他用途
海洋生物技术除在海洋动物、海藻和微生物方面取得成绩外，还有更为广阔的用途。例如，纯种保存技术，实现了能对部分海洋生物成功地进行冷冻保存。将海产显花植物大叶藻的种子进行冷冻，对鲑科鱼类、贝类、甲壳类的生殖细胞进行收集和保存。再如，成功地进行了生物反应器和传感器的研究。人们成功地制成了氨生物传感器，其基本工作原理是用双层醋酸纤维膜将硝化细菌固定化，以溶解氧探针作为换能器。制成的恒温水浴控感器，其工作原理是以磷酸缓冲液，控制溶液的pH值，用于废水或河水品质监测。
4.美国在1992财政年度用于海洋生物技术研究与开发的投入约4400万美元,民间估计投入 2500万美元，以后逐年增加政府的投入 5. 1996年,我国将海洋生物技术纳入国家高技术研究发展计划(863计划), ； 6.美国、日本、挪威、澳大利亚等发达国家先后制定了国家发展计划，把海洋生物技术研究确定为21世纪优先发展领域；

Ohira等(2003) 克隆了日本对虾的蜕皮抑制激素(MIH)基因，并研究了重组MIH的生物活性；
Adachi(2003)发现雄激素Ⅱ一睾酮在雌
鱼卵母细胞生长中起着调节作用；

Wong等(2003)研究了金鲷芳香化酶在性逆转中的分子调控机理；

Thompson等(2003)介绍了海洋脊索动物异体住囊虫细胞周期调节的模式构成；

二定义：
运用海洋生物学与工程学的原理和方法，利用海洋生物或生物代谢过程生产有用物质或定向改良海洋生物遗传特性所形成的高技术。
三海洋生物技术的研究：
<一>兴起和发展： 1.海洋生物技术起始于20世纪80年代，是传统海洋生物学发展的一门新兴研究领域 ; 2.日本1988年举办第一次国际海洋技术会议（IMBC）并定为海洋生物技术元年; 3.日本政府每年在海洋生物技术研究与开发上投入7000万到1亿美元,企业投资达2.8亿～ 4亿美元。

基因敲除的原理

《基因敲除的原理》基因敲除可以说是基因组学、细胞分离培养以及转基因技术的组合。

那么基因敲除的原理是什么呢？基因敲除的方法有哪些呢？在此，做个小结，以供大家学习。

一.概述：基因敲除是自80年代末以来发展起来的一种新型分子生物学技术，是通过一定的途径使机体特定的基因失活或缺失的技术。

通常意义上的基因敲除主要是应用DNA 同源重组原理，用设计的同源片段替代靶基因片段，从而达到基因敲除的目的。

随着基因敲除技术的发展，除了同源重组外，新的原理和技术也逐渐被应用，比较成功的有基因的插入突变和iRNA ，它们同样可以达到基因敲除的目的。

二.实现基因敲除的多种原理和方法：1.利用基因同源重组进行基因敲除基因敲除是80年代后半期应用DNA 同源重组原理发展起来的。

80年代初，胚胎干细胞(ES细胞)分离和体外培养的成功奠定了基因敲除的技术基础。

1985年，**证实的哺乳动物细胞中同源重组的存在奠定了基因敲除的理论基础。

到1987年，Thompsson**建立了完整的ES细胞基因敲除的小鼠模型[1]。

直到现在，运用基因同源重组进行基因敲除依然是构建基因敲除动物模型中*普遍的使用方法。

(1)利用同源重组构建基因敲除动物模型的基本步骤：①. 基因载体的构建：把目的基因和与细胞内靶基因特异片段同源的DNA 分子都重组到带有标记基因(如neo 基因，TK 基因等)的载体上，成为重组载体。

基因敲除是为了使某一基因失去其生理功能，所以一般设计为替换型载体。

②.ES 细胞的获得：现在基因敲除一般采用是胚胎干细胞，*常用的是鼠，而兔，猪，鸡等的胚胎干细胞也有使用。

常用的鼠的种系是129及其杂合体，因为这类小鼠具有自发突变形成畸胎瘤和畸胎肉瘤的倾向，是基因敲除的理想实验动物。

而其他遗传背景的胚胎干细胞系也逐渐被发展应用。

③.同源重组：将重组载体通过一定的方式(电穿孔法或显微注射)导入同源的胚胎干细胞(ES cell)中，使外源DNA 与胚胎干细胞基因组中相应部分发生同源重组，将重组载体中的DNA 序列整合到内源基因组中，从而得以表达。

病毒诱导的植物基因沉默详解

病毒诱导的植物基因沉默详解上个世纪20年代，科学家发现植物与病毒之间存在交叉保护现象：被病毒侵染后的植物可能产生对该病毒株系和相近株系的抗性。

但这种抗性也可能存在“恢复”的现象。

直到上世纪90年代，这些现象的产生机制才被逐渐阐释清楚：是由于病毒基因发生了转录后基因沉默而致使表达受到抑制的结果。

这种现象因此被称为“病毒诱导的基因沉默（virus-induced gene silencing, VIGS）”。

基于这种机制的启发，人们尝试将植物基因片段插入到病毒载体z 中，侵染植物达到实现基因表达的抑制。

经过多年的研究与发展，该技术已经逐渐成熟，并广泛用于植物基因功能研究和植物遗传改良应用。

图1.诱导的植物基因沉默实例。

VIGS的作用机制VIGS的作用机制与另一种常用的基因沉默技术——RNA干扰（RNAi）有很多相似之处。

相较于RNAi，基因沉默具有快速、高效、通量高等优点。

VIGS是利用携带目的基因的cDNA 片段的病毒载体侵染植物，病毒在植物体内的复制和转录能特异性诱导和插入片段序列同源的mRNA降解或者诱导其被甲基化等修饰，导致其不能正常翻译，从而引起植物表型或者指标发生变化。

具体地，病毒在植物体内的复制和表达过程中会形成双链RNA（double-strandedRNA, dsRNA）。

dsRNA首先被Dicer类似物（DCL，如DCL4）的RNase-III家族特异性核酸内切酶切割成小分子干扰RNA（small interfering RNA, siRNA）。

siRNAs进一步扩增，并以单链形式与Argonatute（AGO）RNA结合蛋白和RNase结合形成RNA诱导的沉默复合体（RNA-inducedsilencingplex，RISC）。

RISC能与同源RNA特异性互补结合，导致同源mRNA降解，发生转录后水平的基因沉默。

或者，RISC能与细胞核内的同源DNA相互作用导致其被甲基化修饰，发生转录水平的基因沉默。

野生蕉ω-3脂肪酸去饱和酶基因FAD7密码子偏性分析

野生蕉ω-3脂肪酸去饱和酶基因FAD7密码子偏性分析赖志宸;林玉玲【摘要】The codon bias of ω-3 fatty acid desaturase gene, FAD7, of Musa itinerans was analyzed using CodonW, SPSS19.0, MEGA5.0, and the EMBOSS online program.It was also compared with the FAD7s from different plants and genomes of model organisms.The results showed the codon bias of FAD7 in M.itinerans to be low and leaning toward the synonymous codons with G or C.And, it appeared that the codons CUC, CAG and AGG received a high usage frequency in the gene.In comparison with the FAD7s of different plants, the components of its monocotyledons were bias toward the use G or C, while dicotyledons the opposite.A cluster analysis on the FAD7s based on codon usage bias and CDS showed a same result.Hence, the dicotyledons and monocotyledons in the gene could be satisfactorily differentiated.Based on the frequency of codon usage, the prokaryotic expression system seemed to be suitable for heterologous expression of the FAD7 in M.itinerans.%为了解FAD7的密码子使用特性,试验采用CodonW、SPSS19.0、MEGA5.0等软件和EMBOSS在线程序对野生蕉FAD7密码子偏性进行分析,同时与其他单/双子叶植物FAD7和模式生物基因组进行比较.结果显示,野生蕉FAD7密码子偏性水平较弱,密码子组成和结尾偏好使用G 或C,而CUC、CAG和AGG等在该基因中具有较高的使用频率;物种间FAD7比较结果发现,单子叶植物FAD7密码子组成明显偏好G或C,而双子叶植物则完全相反;基于密码子使用频率FAD7聚类结果与CDS分类结果一致,均能将单/双子叶植物区分开来;此外,大肠杆菌原核表达受体系统可作为野生蕉FAD7基因异源表达理想试验体系.研究结果为野生蕉FAD7后续结构和功能研究提供了科学依据.【期刊名称】《福建农业学报》【年(卷),期】2017(032)005【总页数】5页(P503-507)【关键词】野生蕉;密码子偏性;ω-3脂肪酸去饱和酶;聚类分析【作者】赖志宸;林玉玲【作者单位】电子科技大学计算机科学与工程学院,四川成都 611731;福建农林大学园艺植物生物工程研究所,福建福州 350002;福建农林大学园艺植物生物工程研究所,福建福州 350002【正文语种】中文【中图分类】S668.1密码子是生物体中基因编码蛋白质的基本结构单元，自然界中大部分氨基酸均对应多个密码子，即为同义密码子(Synonymous codon)。

The Genetics of Caenorhabditis elegans

THE GENETICS OF CAENORΉABDITIS ELEGANSS. BRENNERMedical Research Council Laboratory of Molecular Biology,Hills Road, Cambridge, CB2 2QH, EnglandM a n u s cr i p t r ec ei v e d D ec em b er10,1973ABSTRACTMethods are describ ed for the isolation, complementation and mapping ofmutants of Caenorhabditis elegans,a small free-living nematode worm. About300 EMS-induced mutants affecting behavior and morphology have b een char-acteriz ed and about one hundred genes have b een defined. Mutations in 77 of thesealter the movem ent of the animal. Estimates of the induced mutation frequency ofboth the visible mutants and X chromosome lethals suggests that, just as inDrosophila, the genetic units in C. elegans a r e l a r g e.HOW genes might specify the complex structures found in higher organisms is a major unsolved problem in biology. Many of the molecular mechanisms involved in gene expression in prokaryotic microorganisms have already been found to exist in a relatively unmodified form in eukaryotic cells. The genetic code is universal and the mechanism of protein synthesis is much the same in both kinds of organisms. There are, by contrast, great differences in the organization of the genetic material. The chromosomes of higher organisms are complex structures that contain histones and other proteins in addition to DNA and the genetic units are much larger than their counterparts in simple prokaryotes (JUDD, SHED and KAUFMAN 1972). Although there are many theories suggesting how the extra DNA might be used for complex genetic regulation (BRITTEN and DAVIDSON 1969; GEORGIEV 1969; CRICK 1971), the problem is still opaque. We know very little about the molecular mechanisms used to switch genes on and off in eukaryotes. We know nothing about the logic with which sets of genes might be connected to control the development of the assemblages of different cells that we find in multicellular organisms.These questions arise in a particularly acute form in elaborate structures like nervous systems. In one sense, all neurons resemble each other; they must be excitable, able to transmit electrical signals and produce and respond to chemical transmitters. They must be equipped with very similar, possibly commonly specified biochemical machinery. Yet, in another sense, they are all very different; cells are located at specific places and are connected to each other in definite ways. How is this complexity represented in the genetic program? Is it the outcome of a global dynamical system with a very large number of interactions? Or are theredefined subprograms that different cells can get a hold of and execute for them-selves? What controls the temporal sequences that we see in development?Genetics 77: 71-94 May 1974BRENNER72 S.One experimental approach to these problems is to investigate the effects of mutations on nervous systems. In principle, it should be possible to dissect the genetic specification of a nervous system in much the same way as was done for biosynthetic pathways in bacteria or for bacteriophage assembly. However, one surmises that genetical analysis alone would have provided only a very general picture of the organization of those processes. Only when genetics was coupled with methods of analyzing other properties of the mutants, by assays of enzymes or in vitro assembly, did the full power of this approach develop. In the same way, the isolation and genetical characterization of mutants with behavioral alterations must be supported by analysis at a level intermediate between the gene and behavior. Behavior is the result of a complex and ill-understood set of computations performed by nervous systems and it seems essential to decompose the problem into two: one concerned with the question of the genetic specification of nervous systems and the other with the way nervous systems work to produce behavior. Both require that we must have some way of analyzing the structure of a nervous system.Much the same philosophy underlies the work initiated by BENZER on be-havioral mutants of Drosophila (for review, see BENZER 1971). There can be no doubt that Drosophila is a very good model for this work, particularly because of the great wealth of genetical information that already exists for this organism. There is also the elegant method of mosaic analysis which can be powerfully applied to find the anatomical sites of genetic abnormalities of the nervous system (HOTTA and BENZER 1972).Some eight years ago, when I embarked on this problem, I decided that what was needed was an experimental organism which was suitable for genetical study and in which one could determine the complete structure of the nervous system. Drosophila, with about 105 neurons, is much too large, and, looking for a simpler organism, my choice eventually settled on the small nematode, C a e n o r h a b d i t i s e l e g a n s.Extensive work on the nutrition and growth of this and related nematodes had been done by DOUGHERTY and his collaborators (see DOUGHERTY et al. 1959), and there was a classical study of its sexual cycle by NIGON (1949). C.elegans is a self-reproducing hermaphrodite, each animal producing both sperm and eggs. The adults are about 1 mm in length and the life cycle for worms grown on Escherichia coli is 3½ days at 20°. It has a small and possibly fixed number of cells (about 600, excluding the reproductive system) of which about one-half are neurons. Occasionally cultures are found with a few males. Such males may be maintained by mating them with the hermaphrodites. NIGON (1959) found that males contained one less chromosome than the hermaphrodites and that the chromosome constitution is 5AA + XX in the latter and 5ΑΑ + XO in males. Using two strains of C.elegans differing in reproductive capacity at 25°, FATT and DOUGHTERY (1963) were able to show mendelian segregation of a single locus controlling heat tolerance. More recently, DION and BRUN (1971) studied two spontaneous mutants in the Bergerac strain of C. elegans.Our work on this organism has been concentrated so far on two lines: the development of methods for determining the structure of the nervous system, whichGENETICS OF C. elegans 73will be described elsewhere, and establishing the basic genetic features of C. elegans, which is the subject of this and the accompanying paper (SULSTON and BRENNER 1974). This paper reports the characterization of large number of mutants, mostly affecting behavior. About one hundred genes have been mapped onto six linkage groups. The methods used are given in some detail, mainly because hermaphrodite genetics has special technical problems.MATERIALS AND METHODSMedia: 1. NG agar: 3 g NaCl, 2.5 Bactopeptone (Difco) and 17 g Bacto-agar (Difco) are dissolved in 975 ml distilled water. After autoclaving, 1 ml cholesterol in ethanol (5 mg/ml), 1 ml M CaCL2, 1 ml M MgSO4 and 25 ml M potassium phosphate buffer (pH 6.0} are added in order.2. M 9 buffer: 6 g Na2HP04, 3 g KH2PO4, 5 g NaCl and 0.25 g MgSO4•7H2O perlitre.3. S buffer: 0.1 M NaCl and 0.05 M potassium phosphate (pH 6.0).4. Standard bacteriological media are used for growth and maintenance of bacterial strains.Nematode strains: The nematode used in this work is the Bristol strain of Caenorhabditis elegans. It was originally sent by the late Professor E. C. DOUGHERTY as an axenic culture, but it was transferred to a strain of Escherichia coli B.After some passages on solid media, a culture was found which contained a large number of males. These males could be maintained by mating with hermaphrodites. From this stock, a hermaphrodite was isolated and its progeny used to establish two lines: one, a line of hermaphrodites propagating by self-fertilization; the other, a time with males. These are the founder stocks and carry the code name N2; all mutants have been isolated in these strains.Maintenance of stocks: Stocks are maintained on NAG plates seeded with OP50, a uracil-requiring mutant of E. coli, and incubated at 15°. 9 cm petri dishes are used and cultures require subculturing every 10 days or so. Male cultures are maintained by adding 6 or 7 males to a similar number of hermaphrodites on a seeded NG plate. Several of these stocks, staggered with respect to their subculturing, are held, so that active males are always available for crosses.A uracil-requiring strain of E. coli is used to prevent overgrowth of the bacterial lawn. The medium has limited uracil, and the bacteria cannot grow into a thick layer which obscures the worms.These plates are the working stocks for genetical and other experiments. The canonical stocks of the mutants are held frozen in liquid nitrogen. Many experiments on long-term maintenance were carried out without much success. DR. J. SULSTON discovered that the worms could be stored in liquid nitrogen, provided that glycerol was present and that the initial squeezing took place slowly. The standard method used is as follows: worms are washed off the surface of a petri dish culture using about 1.5 ml of S or M 9 buffer. To 1 ml of this suspension is added 1 ml of a 30% solution of glycerol in S buffer, and after mixing, four 0.5 ml aliquots are dispensed into small plastic tubes. These are placed in the holder provided with the Linde liquid nitrogen refrigerators at a level in the vapor phase giving a cooling rate of about 1°/min. After two hours or more, the tubes are mounted in canes and submerged in the liquid nitrogen. The next day, one of the four tubes is removed, thawed, and the contents poured on an NG plate. The plate is examined after a day to make sure that there are viable growing worms. The remaining three cultures are then stored: one in one refrigerator as a master stock, the other two in a different refrigerator as the canonical stocks. If, at any time, the last of these is used, it is immediately replaced so that the master stocks are only used in emergency. With the wild type and most mutants, it is mostly the early larval stages that survive freezing and thawing; eggs do not survive at all. This method has proved completely reliable.Plate stocks can become contaminated with bacteria and moulds. Cultures may be rendered monoxenic in the following way: A culture containing many eggs is suspended in 1.5 ml M 9 buffer. 1.5 ml of 4% glutaraldehyde in M 9 buffer is added and the suspension allowed to stand at 4° for 4 hours. A few drops of a culture of E. coli is spread over a half sector of a 9-cm NG74 S. BRENNERplate. The glutaraldehyde-treated suspension is briefly centrifuged, and the sediment taken up in 0.1 to 0.2 ml of M 9 buffer and applied to the edge of the uninoculated sector. If necessary, theplate is tilted so as to confine this to one side. The glutaraldehyde kills the worms and most contaminants but does not penetrate the eggs. After one day, these hatch and the larvae cross over to the bacterial lawn. The agar with the debris may then be removed.Induction of mutation with ethyl methanesulphonate (EMS): The animals are washed off the plate in M 9 buffer, and to 3 ml of the suspension is added 1 ml of freshly prepared 0.2 M ethyl methanesulphonate in M 9 buffer (final concentration 0.05 M). The standard treatment is for 4 hours at room temperature. The suspension is then taken up into a pipette and the worms allowed to concentrate by sedimentation. 0.2-0.5 ml is dripped onto the surface of an NG plate to absorb the excess fluid. The worms move out and can then be picked to initiate clones.Handling and observation of animals: Mass transfers of animals on plate cultures are carried out with paper strips. Single animals can be manipulated using a sharpened wooden stick or toothpick, sterilized by autoclaving. Observations of the plates are made using a dissecting microscope illuminated from below.EXPERIMENTS AND RESULTSHermaphrodite genetics: Self-fertilizing hermaphrodites have manyboratorythe animals are driven tox osome by the automatic segregation of heterozygous animals. Thefs used simply as a device to construct Isolation of mutants: Ethyl methanesulphonate (EMS) is a potent mutagen in C. elegans, penetrating the animals readily. We consider a young adult individual treated with this agent. Mature sperm have already been produced and stored, and the ovary is manufacturing eggs. Any mutation produced in such an animal will not appear in the homozygous form in the progeny because there are no cells at this stage that can give rise to both sperm and eggs. If the mutagen acted only on non-replicating gametes, such as sperm or oocytes, then the mutation in the F l progeny would be independent. On the other hand, if it reacted with oogonia then clones of F l animals would occur, but they would still be heterozygotes. Such clones have been detected but there are also many single events suggesting that both kinds of gametes are susceptible to the mutagen. It can also be shown that primordial germ cells are susceptible to mutagenic action. Newly hatched larvae treated with EMS produce large clones of mutants in their proheterozygous but homozygotes have bee foGENETICS OF C. elegans 75 In most of the experiments, mutants have come from the clones produced bymutagenized adults. Although the F1 progeny are heterozygous for induced mutations,a detectable fraction are abnormal in appearance or movement. Such variantshave been picked with the intention of isolating dominant or semidominant mutants,but in all cases these have produced wild-type progeny or segregated acoincidental recessive mutant with an unrelated phenotype. These animals arelikely to be mosaics in which the EMS-induced mutations become fixed afterfertilization and then only in cells that produce somatic structures. Semidominantmutants can, however, be isolated from the progeny of mutagenized young larvae; in thiscase the induced mutation becomes fixed during the development of the gonad in the treatedparent.Mutants with a dominant effect are rare in C. elegans and the vast majority ofthe mutants found are recessive. These emerge in the F2 progeny, from thesegregation of any F1 heterozygotes. By this stage, the plate, initiated by a singleparent, contains a very large number of individuals (up to 105), but not all ofthese have to be screened. One-quarter of the offspring of a heterozygous F l arehomozygous and if the number of F l animals is a few hundred then only about athousand of the F2 need to be examined before the plate is discarded as containing nomutants. In practice, mutants are so abundantly produced by EMS that this doesnot arise, and in fact, it is often advantageous to remove the parent before it haslaid all f its eggs to reduce the number of F1 animals to about 50. 30 to 40 platesare used at a time, and only one mutant is ultimately selected from each plate. 550mutants isolated in this way are dealt with in this paper; they comprise the M set.This mass isolation procedure is not altogether satisfactory. Since, at the time of picking,the plates contain large numbers of worms, mostly young, there is a bias against mutantsthat grow slowly or express their phenotypes fully only in the adult form. A morelaborious but more accurate procedure is to pick the mutants from the segregants of Flprogeny of mutagenized parents, putting single F1 animals on separate plates. Inpractice, only 5 such Fl clones are initiated from a given plate to ensure independentorigin; any repeats found (which has happened only once) are considered members of aclone and are discarded. On such plates, one-fourth of the progeny of any heterozygousF1 are homozygous mutants, and this widens the range of identifiable phenotypes. From318 such plates, 69 mutants were isolated, comprising the S set.Phenotypes of mutants: Most of the mutants have been selected by inspection.Although emphasis has been placed on mutants defective in movement to findgenes controlling the structure or function of the nervous system, we have alsocollected mutants with morphological abnormalities and with differences in sizeand shape. It is our general practice not to classify the mutants by differentnames corresponding to phenotype descriptions, but to put them into broad cate-gories and then characterize them genetically. A general description of thephenotypes now follows, and special comments on the individual mutants canbe found in Table 5.Uncoordinated mutants: The wild type (Figure 1a) displays a smooth sinuousmovement on the agar surface. It is important to realize that the motion of thebody is confined to the dorsoventral plane, and that, on plates, the animals are76S. ΒR ΕΝΝΕRFigure 1. --Photomicrographs of C. elegans and some of its mutants. a: wild type, b: dumpy (d γp-1), c: s m a l l (sma-2), d : long (lon-1) . The scale is 0.1 mm.lying on their sides. The head can move in all directions, but surface tension restrains it to the surface as well. The animals can reverse with the same wave-like motion. Reverse motion can be stimulated either by tapping the surface of the plate in front of the animal or by touching the tip of its head. Any mutant that shows any detectable defect in this normal pattern of behavior is called "unco-ordinated". As may be expected, this covers a very wide range of phenotypes from paralysis to quite small aberrations of movement. Although there are mutants with strikingly singular properties, most of the phenotypes are very difficult to describe. In general, the pulsating pharyngeal movements are not affected in the mutants, even in severely paralyzed animals. Paralysis of the pharynx would probably be lethal since the animals would be unable to feed. In some paralyzed and semiparalyzed mutants the vulva is also affected, and eggs are not laid. Progeny hatch inside the parent and ultimately devour it. Much of the animal's capacity for and control of motion is dispensable, because it does not require active males for propagation and because laboratory conditions can supply adequate sources of food.Roller mutants: The body of the animal rotates around its long axis as the animal moves. The effect is to force the animal to move in a circle, and such mutants are easily . recognized by the craters they inscribe in the bacterial lawn. When such animals reverse, the hand of rotation becomes opposite.Careful observation with a series of mutants shows that they all have the same hand of rotation of the body but that the way they move in circles on the plate differs,and this appears to be controlled by the movement of the head. In liquid media it can be shown that wave propagation in roller mutants is helical, rather than planar. In most of the mutants the phenotype is clearly expressed only in the adult form.GENETICS OF C. elegans 77Dumpy and small mutants: These animals are shorter than the wild type;dumpy mutants (Figure 1b) have the same diameter, but the small mutants (Figure 1c) arethinner and at least one seems to be an accurately scaled-down form of the wild type. Some of the dumpy mutants are also rollers. In many of the mutants the phenotype becomesexpressed only in the later stages of growth, whereas others have altered larvae as well.Long mutants: These mutants are longer and thinner than the wild type (Figure1d)Blistered mutants: In such mutants, fluid-filled blisters appear on thecuticle. Often the entire cuticle separates and the resulting single blister can squeeze the animal to death. This phenotype is expressed only in adults, the earlier larval stages appearing quite normal.Abnormal mutants: In these mutants there are clear-cut aberrations inthe morphology of the animal. They comprise a large and heterogeneous range of phenotypes on which not much work has, as yet, been done. Many of these abnormal mutants have variable phenotypes, and the mutations show low penetrance. One common variable abnormal phenotype is an animal with a notched head. All clones of such mutants contain animals that range from an apparently normal phenotype to very severe notching.The distribution of phenotypes in the M and S sets of mutants is shown inTable 1. Most of the mutants fall into the major phenotypic classes, except for six in the M set, labelled residual. These have special properties and will be described elsewhere.TABLE 1Phenotypes, linkage and summary of mapping for M and S mutantsAutosomal Sex-linked Unassigned Set PhenotypeLocate Not located Located Nat located Dominant Other Tota l M Uncoordinated 173 39 41 59 9 43364 D um p y a n d sm a l l 71 8 5 24 2* 0 110Long 5 0 4 1 0 0 10Roller 2 2 0 1 2 0 7 Blistered 8 0 0 0 1 0 9Abnormal 0 17 0 3 0 2444 Re sidua l .. .. .. .. .. .. 6259 66 50 88 14 67 550S Uncoordinated14 4 1 9 2 16 46 D um p y a n d sm a l l11 0 0 1 0 0 12 Long0 0 0 0 1 0 1 Roller1 1 0 1 0 0 3 Blistered0 0 0 0 0 0 0 A b n o r m a l0 1 0 1 0 5 726 6 1 12 3 21 69* Recessive lethals.78 S. BRENNERCharacterization of the mutants:Many of the mutants picked turn out to besterile and are discarded. Some do not breed true, producing mixed progeny. These are of two types: dominant mutants and mutants with variable penetrance. These canbe distinguished by picking 8 to 12 individuals onto separate plates and observingtheir progeny. Some dominant mutants persist as heterozygotes. They segregate⅓wild-type animals and are thus either lethal when homozygous or have a closely linked independent lethal mutation. If any mutant segregates a second,independent mutant, the double is picked and the two mutations are separated laterby recombination.Most mutants with variable phenotypes are not further analyzed. The re-mainder are then crossed with wild-type males. The presence of males in the progeny indicates that mating was successful. The mutants are classified accordingto the rules given in Table 2. The distinction between dominance and semi-dominance can be a matter of fine judgment, but there are definitely cases inwhich the heterozygote cannot be distinguished from the parental homozygous. For these, no assignment of linkage can be made and they are included in theunassigned class in Table 1. It is also at this stage in the analysis that mutantsare found with phenotypes that cannot be rapidly or reliably distinguished fromwild type on plates containing both. Such marginal mutants are withdrawn and these, together with the mutants with variable phenotypes, account for most ofthe mutants labelled as unassigned in Table 1.Very occasionally, the rules given in Table 2 break down. For example, among theresidual mutants there are two which were initially classified as autosomal recessives but which later turned out to be sex-linked with no expression in themale.Genetic complementation: Many of the mutants studied here have such severedefects in movement that males carrying such mutations in the homozygous formcannot mate efficiently with hermaphrodites. Autosomal recessives are, however, easily tested for complementation, by using heterozygous males. A mutant her-maphrodite (m1/m1, say) is mated with wild-type males. The heterozygous off-spring males (+/m1) are then crossed with tester hermaphrodites (m2/m2). In theresulting progeny only males are scored, since no distinction can be madebetween hermaphrodites produced by self-fertilization and those produced by fertilization with a male sperm carrying a non-complementary mutation. OfTABLE 2Classification of mutants: progeny of cross with wild-type malesPhenotype of progeny♀♂Typewild wild autosomal recessivewild mutant sex-linkedrecessive intermediate intermediate autosomalsemidominant intermediate mutant sex-linkedsemidominant mutant mutant dominantGENETICS OF C. elegans79these males, one-half must be wild type with genetic constitution +/m2 since the male contributes one wild-type allele. The other half are m1/m2males, and if these have the mutant phenotype then the two mutations do not complement each other. If all of the males are wild type then the two mutants complement.This method cannot be used for sex-linked mutants. For these, tester strains are constructed containing another mutant, suitably chosen to allow the progeny of any cross to be distinguished from those produced by selfing. For example, in the case of sex-linked uncoordinated mutants, doubles are constructed with an autosomal dumpy mutant. The sex-linked mutant to be tested is crossed with wild-type males and the resulting hemizygous males are then crossed with the double. Large numbers of such males must be used because they express the defect and mate very poorly. These crosses are then examined for non-dumpy hermaphrodites, which can only be produced by mating. If these are uncoordinated then the two mutations are non-complementing, whereas the presence of wild-type progeny signifies complementation. Complementation tests on sex-linked mutants are difficult and there are some mutants in which the movement defect is so severe as to prevent these strains from being used as donors. Only a limited number of the sex-linked mutants isolated have been studied.Location of mutants on linkage groups: As the number of complementation groups increased, allocation of new mutants involved more experiments, even if the testers were judiciously selected by phenotype. It became more effective first to locate the mutant on a linkage group and then to test it only with linked mutants. Sex-linked mutants are, of course, detected directly at the time of the initial backcross; special methods are required for the autosomal mutants. Consider the segregation of two recessive alleles a and b in the tr an s configuration. Table 3A shows the distribution of phenotypes in the progeny. If the mutants are unlinked, then the recombination frequency, p, is 0.5, and the phenotypes will show the classical 9:3:3:1 segregation ratios. Linked mutants can segregate by recombination, but the exact pattern will depend o n the recombination frequencies in each of the germ lines in the hermaphrodite. An initial experiment with two distant sex-linked markers yielded the doubly recessive homozygote (AB in Table 3A) in the progeny of the trans heterozygote). This proved that recombination occurs in both germ lines and additional experiments, reported below, show that the recombination frequencies are approximately the same in both.The trans configuration is used to identify the linkage group of a new mutant. Heterozygotes are constructed containing the mutant trans to tester mutants on the different linkage groups. The phenotype of the tester is chosen such that the double homozygote can be distinguished from both of the parents. Thus uncoordinated mutants are crossed with dumpy testers and morphological mutants with uncoordinated testers. The double heterozygotes are constructed as follows: The mutant (a/a) is crossed with wild-type males and the heterozygous male offspring (+/a) then mated with tester hermaphrodites (b/b). The wild-type hermaphrodites produced by this cross are of two types, ++/+b and a+/+b. Five of these are picked onto separate plates and allowed to produce progeny. Clones that segregate only one of the parental types are discarded. The remaining80 S. BRENNERT A B L E 3Patterns of segregation of phenotypes from doubly heterozygous hermaphroditesa andb are mutant recessive alleles and A and B their corresponding phenotypes. W is wild type phenotype.plates are then inspecte v d for the occurrence of the double homozygote. In practice th is scored as the ratio AB/A, which is p 2. This is a very sensitive test for linkage; the With these methods 285 autosomal recessive mutants have been allocated to complementation groups, 259 of the M set and 26 of the S set (Table 1). Most of the mutants that have not been allocated have phenotypes that are difficult to score in crosses with other markers either because of variable expression or be-cause the double cannot be readily distinguished from one of the parents. For example, some uncoordinated mutants have phenotypes which are obscured in a dumpy background so that the dumpy uncoordinated double is very similar to the dumpy tester. Table 1 also shows that relatively fewer of the sex-linked mutants have been allocated because of the difficulties of performing the crosses. The sample is biased toward mutants which can mate and, as will be pointed out later, many of the genes characterized by a single mutant rely for their distinction on mapping results rather than on exhaustive complementation tests.Table 4 shows the distribution of the M and S set mutants. The 259 M auto-somal mutants define a total of 77 genes, 56 unc, 14 sp γ and sma, 1 lon; 2 rol and。

大珠母贝组织蛋白酶B基因克隆及其表达分析

大珠母贝组织蛋白酶B基因克隆及其表达分析作者：徐扬王姿曼李俊辉梁飞龙邓岳文杨创业来源：《南方农业学报》2021年第05期摘要：【目的】分析组织蛋白酶B基因（CatB）在大珠母贝（Pinctada maxima）不同组织中的表达模式，明确CatB在大珠母贝中的功能作用，为培育生长快且抗逆性强的大珠母贝提供基础资料。

【方法】利用RACE克隆大珠母贝CatB基因，利用ExPASy ProtParam、ExPASy ProtScale、NPS@SOPMA、SWISS-MODEL及SignalP 4.1等在线软件进行生物信息学分析，并以实时荧光定量PCR检测CatB基因在大珠母贝外套膜（套膜区、边缘膜区和中央膜区）、肝胰腺、鳃、足和闭壳肌等组织中的表达情况。

【结果】大珠母贝CatB基因cDNA序列全长1365 bp，其开放阅读框（ORF）1026 bp，5'非编码区（5'-UTR）长度81 bp，3'非编码区（3'-UTR）长度258 bp，共编码341个氨基酸残基。

大珠母贝CatB蛋白分子量为37.73 kD，理論等电点（pI）为6.66，脂溶性系数为67.48，不稳定指数为31.18，亲水性平均系数（GRAVY）为-0.451，为稳定的亲水性蛋白;在第89～337位氨基酸存在一个Pept-C1结构域。

大珠母贝CatB蛋白二级结构以无规则卷曲为主，占51.03%，α-螺旋占26.98%，β-转角占9.09%，延伸链占12.90%;三级结构与马氏珠母贝（P. fucata martensii）CatB蛋白结构相似。

大珠母贝CatB氨基酸序列与马氏珠母贝CatB氨基酸序列（ADX32985.1）的相似性高达90.91%;与长牡蛎（Crassostrea gigas，XP_011428258.1）、海湾扇贝（Argopecten irradians，ANG56311.1）、褶纹冠蚌（Cristaria plicata，AEF32260.1）的CatB氨基酸序列相似性分别为79.47%、65.38%和62.18%。

零代码、无实验复现最新8+SCI，傻瓜式剩下高招！（附详细操作教程）

零代码、无实验复现最新8+SCI，傻瓜式剩下高招！（附详细操作教程）解螺旋公众号·陪伴你科研的第2590天无代码生信复现大家好，我是Jerry，今天我给大家分享一篇最新的单基因泛癌生信文章，该文章是发表于Frontiers in Immunology杂志上，最新影响因子为5.6分。

该篇文章的分析内容非常充实，值得大家学习和借鉴！！文章题目Systematic Pan-Cancer Analysis Identifies TREM2 as an Immunological and Prognostic Biomarker数据解读本篇文章属于单基因的泛癌分析，作者通过UCXC Xena网站下载TCGA-33种肿瘤的RNA-seq、体细胞突变和相关的临床数据。

同时，作者也从CCLE数据库中下载肿瘤细胞系的数据，并根据组织的来源计算出在21种组织内的表达水平。

此外，作者从正常组织的测序数据库（GTEx数据库）中下载31种组织的基因表达谱。

值得注意的是，作者选用癌组织和匹配的正常组织进行表达差异分析。

注：不同版本的TCGA数据，有可能获取的患者的样本例数不一致，因为TCGA数据库一直处于更新过程中。

所以看到生信文章中TCGA里同一个癌种的患者样本数不一致，大家也不要奇怪，有可能就是版本不同导致的。

大家做生信分析的时候，尽量选择TCGA数据库中的最新版本的数据进行下载。

复现工具◆仙桃学术工具（/products）◆ Human Protein Atlas database （/）Figure 1: TREM2的差异表达Figure 2: TREM2在正常组织和肿瘤组织内的基因表达对比Figure 3: TREM2的表达与总生存期之间的相关性；Figure 4: TREM2的表达与特定疾病生存期的相关性；Figure 5: TREM2的表达与无进展生存期的相关性；Figure 6: TREM2的表达与年龄的相关性；Figure 7: TREM2的表达与肿瘤分级的相关性；Figure 8: TREM2的表达与肿瘤内突变评分、微环境不稳定性（MSI）和错配修复的相关性；Figure 9: TREM2的表达与肿瘤微环境相关系数最高的5种肿瘤Table 1: TREM2的表达与不同肿瘤内免疫细胞浸润之间的相关性Figure 10: TREM2的表达与肿瘤内不同免疫细胞浸润的相关性；Figure 11: TREM2与免疫相关基因的共表达热图；文章复现Figure 1TREM2的差异表达复现步骤：仙桃学术生信工具网址：/products1. 进入主页，选择高级版，点击“立即使用”注：免费版和基础版都可以进行统计和可视化，由于高级版功能最全，这里选择高级版作为范例2. 选择分析工具，点击表达差异下属的非配对样本条目，根据本文作者的分析，选择囊括正常样本和肿瘤样本的泛癌数据集，即XENA-TCGA_GTEx数据集。

为什么缺氧时癌细胞还能生长

为什么缺氧时癌细胞还能生长大量研究已证实肿瘤缺氧，即肿瘤部分区域含有极低浓度的氧气，的确与更加侵袭性的肿瘤行为和更差的预后相关联。

这似乎表明肿瘤不会屈服于缺氧，相反肿瘤过量增加血液供应，因而经常会导致缺氧，从而给肿瘤发送生长和转移的信号以便寻找新的氧气源。

比如，缺氧性膀胱癌可能转移到肺部，而这经常是致命性的。

当缺乏氧气时，健康细胞将会减缓生长。

但令人惊奇的是，缺氧是恶性肿瘤的特征。

在最新版“自然通讯”杂志上发表的两篇文章显示，歌德大学和吉森大学的研究人员已经寻找出癌细胞是如何成功避免生长抑制的遗传程序的。

我们很早就知道，PHD蛋白质（脯氨酰羟化酶域蛋白）在缺氧调节中发挥着关键作用。

它可以控制低氧诱导转录因子（HIFs）的稳定性，而HIFs可以使细胞适应缺氧环境。

歌德大学的AmparoAcker-Palmer教授和吉森大学的TillAcker教授所领导的两个团队目前发现了一种特殊的PHD蛋白质，PHD3，它可以控制表皮生长因子受体（EGFR）。

在健康的细胞中，PHD3可以对外界压力产生反应，比如当氧气不足时，它可以刺激表皮生长因子受体摄入到细胞内部，从而使生长信号下调。

“我们发现，PHD3作为支架蛋白，可以结合到中央适配器蛋白如EPS15和Epsin1上，从而促进表皮生长因子受体摄入到细胞内。

”Acker-Palmer说。

而这一过程在肿瘤细胞中由于PHD3的损失而被打乱。

从而引起表皮生长因子受体的内化被抑制，造成EGFR信号的过度活化，进而使细胞生长不受控制。

该研究小组已经证明PHD3的损失是人类恶性脑肿瘤（胶质瘤）增长的关键因素。

肿瘤细胞因此可以在缺氧环境下抵制抑制生长的信号。

“这一发现在临床上具有重要意义，因为它展示了除了基因扩增外另一种抑制EGF受体过度活化的机制。

我们可以通过EGFR抑制剂来抑制其反应。

”吉森大学神经病理学家TillAcker解释说。

“我们的工作表明了PHD3在目前两个红火的研究领域上的一个意想不到的新功能：氧气测量和EGFR信号，”Acker-Palmer解释说。

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Curriculum VitaeMargaret C. Werner-WashburneAddress:Department of BiologyUniversity of New MexicoAlbuquerque, NM 87131(505)277-9338FAX (505)277-0304E-mail: maggieww@WEB: /biology/maggieww/Public_Html/Maggieww.html Educational History:B.A., 1971, Stanford University; Stanford, California, EnglishM.S., 1979, University of Hawaii; Honolulu, Hawaii, BotanyPh.D., 1984, University of Wisconsin-Madison; Madison, Wisconsin, Botany with minor in BiochemistryThesis title: Enterokinase Inhibitors in Plants. Sanford Siegel, advisor.Dissertation title: L-Aspartate transport into pea chloroplasts. Kenneth Keegstra, advisor Post doctoral fellow, 1984-1988, U Wisconsin-Madison. Advisor: Betty Craig Employment History – Academic positions:Regents’ Professor, 2009 – presentFull member, UNM Cancer Center, 1990- presentAdjunct full professor: Computer Sciences and Microbiology and Molecular Genetics Professor, 2002-presentVice Chair, Biology, 1997 -1998Associate Professor of Biology, 1994 - 2002Assistant Professor of Biology, 1988-1994NIH Post Doctoral Fellow, 1984-1988, University of Wisconsin, MadisonGraduate student/NIH Trainee, 1979-1984, University of Wisconsin, MadisonResearch and Teaching assistant, 1976-1979, University of Hawaii, Honolulu Employment History - Concurrent positions:Contractor: Sandia National Laboratories, Biosensors, 2009 –Consultant: in Genomics, including the Computer Sciences/Evolutionary Computing and Biosensors, Sandia National Laboratories, 1999-2004Interim director, Southwest Genomics and Biotechnology Alliance, 1999 – 2001Program director, Microbial Genetics, National Science Foundation, 1998-1999 Professional Recognition:Academic Awards and Honors:Harvard Foundation – 2011 Scientist of the Year AwardEE Just Medal; ASCB 2010Elected SACNAS Board of Directors, 2010Sarah Belle Brown Award for Public Service, UNM, 2010Academic Awards and Honors:Outstanding Faculty of Color Award from Graduate Students of Color, UNM, 201020 Women Who Make a Difference in NM – ABQ Journal, 2009Regents’ Professor, UNM, 2009Elected: AAAS Biological Sciences Steering committee – 2008-2012Andreoli-Woods Lecturer, Cal State LA, 2007Mujeres Valerosas award, Hispanic Women’s Council, 2006AAAS Fellow, 2006Special presidential service award - SACNAS, 2006SACNAS Distinguished Scientist Award, 2005EE Just Lecturer, American Soc for Cell Biology, 2005NIGMS MORE (Minority Opportunities in Research) Division Working Group, 2005 -2006 Presidential Award for Excellence in Science, Math, and Engineering Mentoring, 2004 Proclamation for service to NM from NM House of Representatives, March 2003NHGRI Genome Research Review Panel, NHGRI, 2001- 2005AAAS Latina Scientist 2003Special service award, National Science Foundation, 1999Outstanding program officer, BIO/National Science Foundation, 1999UNM Regents’ Lecturer, 1997-2000UNM Alumni Faculty Award for outstanding teaching and service to students, 1995 Presidential Young Investigator Award, NSF, 1990-1996Sigma Xi Honor Society, 1984Fellowships:Postdoctoral Research Fellow, 1984-1987, National Institute of HealthPostdoctoral Award, 1984, National Science Foundation, (not activated)Predoctoral Trainee, 1981-1984, National Institute of HealthAdvanced Opportunity Fellow, 1979-1981, University of WisconsinResearch Grant, 1978, Pacific Tropical Botanical Gardens, HawaiiRegents Scholar, 1974-1975, University of MinnesotaElizabeth Crosset Scholar, 1967-1971, Stanford UniversityResearch Funding:VectorBase: NIAID Resource CenterP.I. Dr. W. Gelbart and EBI, Imperial College, London(MWW, co-PI) 9/1/09 – 8/31/14, NAIAD contract(Approximately $150K per year – for UNM Annotation Center)Flybase: A Drosophila Genomic and Genetic Database NHGRIP.I. Dr. W. Gelbart, Harvard (MWW co-PI)1/1/10 -12/31/13 (approx $300,000 per year) ($2.0 M total) (for UNM AnnotationCenter)Flybase: A Drosophila Genomic and Genetic Database NHGRIP.I. Dr. W. Gelbart, Harvard (MWW co-PI)3/1/09 -12/31/09 (approx $250,000)Environnmental sensing using genome-scale proteomics.Contract with Sandia National Labs4/1/09-9/31/11 ($25,000 per year)Chemical Screen of TOR pathway GFP-fusion proteins in S. cerevisiaeP.I. M. Werner-Washburne, NIH R03 MH086450-014/1/09 – 3/31/11 ($25,000)UNM-IMSD 5R25-GM060201P.I. M. Werner-Washburne, NIH-MORE3/1/09-2/28/13 (approx $700 K per year direct) ($2.4 M total, directs)The biogenesis and survival of vegetative, quiescent yeast cellsP.I. M. Werner-Washburne, NSF MCB 06458543/1/07 – 2/28/12 ($660K)Genomic analyses of quiescent and non-quiesent cells in yeast stationary-phase cultures P.I. M. Werner-Washburne, NSF MCB 04456316/1/05 – 5/31/08 ($130,000)UNM-IMSD 5R25GM060201P.I. M. Werner-Washburne, NIH-MORE1/1/05-12/31/08 (approx $550K per year direct) (total $3M)Presidential Award for Excellence in Science, Math, and Engineering Mentoring P.I. M. Werner-Washburne, NSF5/1/04 – 4/30/06 ($10K) NSF – EHR 0342901SACNAS Genomics Training GrantCo-PI M. Werner-Washburne (P.I. Luis Haro)T32- 9/1/04 – 8/31/08 ($250,000 direct) ($1.5M total)Compendium of Gene Expression in Stationary-Phase YeastP.I. M. Werner-Washburne, NIH- NIGMS RO1 GM67593RO1 – 7/1/02-6/30/07 ($1.2M direct over four years) ($1.8 total)DNA printing- Sandia National Laboratories, PI Susan Brozik10/1/01 – 9/30/02 ($100K)Supplement to National Institutes of Health- National Human Genome Research Institute grant ($30,000) plus supplement to Initiative for Minority Student Development grant ($30,000) for a microarray scanner. 9/1/01Microarray analysis (adaptation of visualization software for analysis of complex, genome-scale datasets) and cell-based biosensor (using genomic approaches), Sandia National LaboratoriesCo-PI, M. Werner-Washburne11/30/00-9/30/01(approx. $90K)Exit from stationary phase: gene expression and its consequencesP.I. M. Werner-Washburne, National Science Foundation3/1/01-1/31/03 ($460K total for three years)A two-hybrid system for use in non-dividing yeast cellsP.I. M. Werner-Washburne; National Institutes of Health – National Human Genome Research Institute R21- HG022621/1/01 – 12/31/02 ($200K – direct costs)Microarray analysis of expression during exit from stationary phase in yeast.P.I. M. Werner-Washburne; Sandia National Laboratories1999; ($25,000)Study of gene expression in non-dividing cells.P.I. M. Werner-Washburne; Sandia National LaboratoriesJune 1 – August 31, 2000 ($13,000)The role of Snz and Sno proteins in Saccharomyces cerevisiae.P.I. M. Werner- Washburne; National Science FoundationSeptember 1, 1998 – August 31, 2001 ($110K/yr) ($450K total)Understanding the silence: cells in stasis (production of a video for PBS broadcast).P.I. M. Werner- Washburne; National Science Foundation -Special supplement for integrating research and education;Sept, 1996 - August, 1998; Award: $50,000 plus contract with Los AlamosNational Laboratories for animation: $34KDevelopmental regulation of signal transduction: Bcy1p in stationary-phase yeast.P.I. M. Werner- Washburne; National Science Foundation;Sept, 1996 - August, 2000 Award: $240K plus supplementsCharacterization of a novel, stationary-phase gene in the yeast Saccharomyces cerevisiae.P.I. M. Werner- Washburne; National Science Foundation;May, 1995 - April, 1998 Award: $380KThe Neurospora Genome Sequencing Project.co-P.I. M. Werner-Washburne. National Science Foundation, HRD (for studenttraining) August, 1995 - July, 1998 Award: $372K plus $300K matching fundsThe role of gene regulation in starvation-induced arrest in the yeast Saccharomyces cerevisiae.P.I. M. Werner-Washburne. Presidential Young Investigator Award, NSF.July, 1990 - June, 1996. Award: $450KEstablishment of a molecular biology facility.P.I. M. Werner-Washburne. National Science Foundation, NSF-HRD.Sept, 1992- Sept, 1996. Award: $328K plus $300K in matching funds.The role of gene regulation in starvation-induced arrest in the yeast Saccharomyces cerevisiae.P.I. M. Werner- Washburne; National Science Foundation, Microbial GeneticsNov, 1989 - Apr, 1995. Award: $600K, including supplementsScholarly achievements:Articles in refereed journals (cited > 4500 times in scientific literature):A Multiple network learning approach to capture system-wide condition-specific responsesS Roy, T Lane, and M Werner-WashburneBioinformatics (in press) (2011)The proteomics of quiescent and non-quiescent cell differentiation in yeast stationary- phase culturesG.S. Davidson,R.M. Joe,S. Roy, O. Meirelles, C.P. Allen, M.R. Wilson, P.H. Tapia,E.E. Manzanilla, A.E. Dodson,S. Chakraborty, M.Carter,S.Young, B. Edwards, L. Sklar,M. Werner-WashburneMolecular Biology of the Cell 22: 988-998 (2011)Encapsulation of S. cerevisiae in Poly(glycerol) Silicate Derived Matrices: Effect of Matrix Additives and Cell Metabolic Phase on Long-Term Viability and Rate of GeneExpressionJC Harper, DM Lopez, EC Larkin, ME Economides, SK McIntyre, TM Alam, MS Tartis, M Werner-Washburne, CJ Brinker, SM Brozik, DR WheelerChemistry of Materials (in press) (2011)Exploiting Amino Acid Composition for Predicting Protein-protein Interactions S. Roy, A.D. Martinez, H. Platero, T. Lane, and M. Werner-WashburnePLoSONE doi:10.1371/journal.pone.0007813 (2009).SSD1-V regulates transcript levels of many longevity genes in budding yeast and extendschronological life span in purified quiescent cellsLihong L., Y. Lu, Li-X.Qin, Z.Bar-Joseph, M.Werner-Washburne, and L.L. BreedenMolecular Biology of the Cell 20: 3851-3864 (2009)Scalable learning of large networksSushmita Roy, Sergey Plis, Margaret Werner-Washburne, and Terran LaneIET Systems Biology 404-U157 (2009)Fungal genome sequencing and bioenergy.S.E. Baker, J. Thykaer, W.S. Adney, T.S. Brettin, F.J. Brockman, P. D’Haeseleer, A.D.Martinez, R.M. Miller, D.S. Rokhsar, C.W. Schadt, T. Torok, G. Tuskan, J. Bennett, R.M.Berka, S.P. Briggs, J. Heitman, J.Taylor, B.G. Turgeon, M. Werner-Washburne, M.E. Himmel. Fungal Biology Reviews 22:1-5 (2008)A system for generating transcription regulatory networks with combinatorial control of transcription. S. Roy, M. Werner-Washburne, and T. Lane. Bioinformatics 24: 1318-1320; doi:10.1093/bioinformatics/btn126 (2008)Characterization of differentiated quiescent and non-quiescent cells in yeast stationary-phase cultures. A.D. Aragon, A.L. Rodriguez, O. Meirelles, S. Roy, G.S. Davidson, C.Allen, R. Joe, P. Tapia, D. Benn, and M. Werner-Washburne. Mol Biol Cell 19:1271-1280 (2008)A Hidden-state Markov Model for Cell Population DeconvolutionS. Roy, T. Lane, C. Allen, A.D. Aragon, M. Werner-Washburne. Journal ofComputational Biology 13:174901774 (2006)Multivariate curve resolution of time course microarray dataPeter D Wentzell, Tobias K Karakach, Sushmita Roy, M Juanita Martinez, Christopher P Allen, and Margaret Werner-Washburne. BMC Computational Biology 7:343 (2006)Isolation of quiescent and non-quiescent cells from Saccharomyces cerevisiae stationaryphase culturesChris Allen, Sabrina Büttner, Anthony D. Aragon, Jason A. Thomas, Osorio Meirelles,Jason E. Jaetao, Don Benn, Stephanie W. Ruby, Marten Veenhuis, Frank Madeo, and Margaret Werner-WashburneJ Cell Biology 174:89-100 (2006)Cell-Directed Assembly of the Bio-Nano InterfaceHK Baca, C Ashley, E Carnes, D Lopez, J Flemming, D Dunphy, S Singh, Z Chen, NLiu, H Fan, GP López, SM Brozik, M Werner-Washburne, CJ BrinkerScience 313: 337-341 (2006)Release of sequestered mRNA is responsible for a massive, non-transcriptional increase in mRNA after oxidative stress in S. cerevisiae stationary-phase culturesAnthony D. Aragon, Gabriel A. Quiñones, Edward V. Thomas, Sushmita Roy, andMargaret Werner-WashburneGenome Biology 2006 65/2: 357-360 (2006)An Automated, Pressure-Driven Sampling Device for Harvesting from Liquid Cultures for Genomic and Biochemical AnalysesAnthony D. Aragon, Gabriel A. Quiñones, Chris Allen, Jason Thomas, Sushmita Roy,George S. Davidson, Peter D. Wentzell, Brian Millier, Jason E. Jaetao, Angelina L. Rodriguez, and Margaret Werner-WashburneJournal of Microbiological Methods doi:10.1016/j.mimet.2005.08.015 (2005)Hyperspectral Microarray Scanning: Impact on the Accuracy and Reliability of GeneExpression DataJerilyn A. Timlin, David M. Haaland, Michael B. Sinclair, Anthony D. Aragon, M. Juanita Martinez, Margaret Werner-WashburneBMC Genomics 6:72 (2005)A Packed Micro Column Approach to a Cell-Based BiosensorJeb H. Flemming, Helen K. Baca, Margaret Werner-Washburne,Susan M. Brozik, Gabriel P. López Sensors & Actuators 113: 376-381 (2005)Inferring genetic networks from microarray dataS. Martin, G. Davidson, E. May, J-L Faulon, and M. Werner-Washburne3rd International IEEE (CSB), pg. 566-569 (2004)Sleeping Beauty: Quiescence in Saccharomyces cerevisiaeJoseph V. Gray, Gregory A. Petsko, Gerald C. Johnston, Dagmar Ringe, Richard Singer, and Margaret Werner-Washburne Microbiol. Mol. Biol. Rev. 68:187-206 (2004)Genomic analysis of stationary phase and exit in Saccharomyces cerevisiae: transcriptional profiling and analysis of essential genes. M. J. Martinez, AB Archuletta, AL Rodriquez, P Wentzell, AD Aragon, CP Allen, S Roy, and M. Werner-WashburneMolec Biol of the Cell 15: 5295-5305 (2004)Design, Construction, Characterization, and Application of a Hyperspectral Microarray Scanner. Michael B. Sinclair, Jerilyn A. Timlin, David M. Haaland, and Margaret Werner-WashburneApplied Optics 43:2079-2088 (2004)Hyperspectral Imaging of Biological Targets: The Difference A High Resolution Spectral Dimension And Multivariate Analysis Can Make. Jerilyn Timlin, David Haaland, Michael Sinclair, Monica Manginell, Susan Brozik, M. Juanita Martinez, Margaret Werner-Washburne, John Guzowski:ISBI 2004: 1529-1532 (2004)Genomics in Neurospora crassa: From One-Gene-One-Enzyme to 10,000 GenesEdward L. Braun, Donald O. Natvig, Margaret Werner-Washburne and Mary Anne Nelson. Elsevier Applied Mycology & Biotechnology series, Fungal Genomics 4:295-313 (2004)The Genome Sequence of the Filamentous Fungus Neurospora crassaJames E. Galagan1, Sarah E. Calvo1, Katherine A. Borkovich2, Eric U. Selker3, Nick D.Read4, William FitzHugh5, Li-Jun Ma1, Serge Smirnov1, Seth Purcell1, Bushra Rehman1, Timothy Elkins1, Reinhard Engels1, Shunguang Wang1, Cydney B. Nielsen1, Jonathan Butler1, David Jaffe1, Matthew Endrizzi1, Dayong Qui1, Peter Ianakiev1, Deborah Bell-Pedersen6, Mary Anne Nelson7, Margaret Werner-Washburne7, Claude P.Selitrennikoff8, John A. Kinsey10, Edward L. Braun11, Alex Zelter4,26, Ulrich Schulte12,Gregory O. Kothe3, Gregory Jedd13, Werner Mewes9,17, Chuck Staben14, Ed Marcotte15, David Greenberg16, Alice Roy1, Karen Foley1, Jerome Naylor1, Nicole Stange-Thomann1, Robert Barrett1, Sante Gnerre1, Michael Kamal1, Manolis Kamvysselis1, Cord Bielke9, Stephen Rudd17, Dmitrij Frishman17, Svetlana Krystofova2, Carolyn Rasmussen18,Robert L. Metzenberg19, David D. Perkins19, Scott Kroken20, David Catcheside21, Weixi Li 14, Robert J. Pratt6, Stephen A. Osmani23, Colin P.C. DeSouza24, Louise Glass18, Marc J. Orbach25, J. Andrew Berglund3, Rodger Voelker3, Oded Yarden26, Mike Plamann27, Stephan Seiler27, Jay Dunlap22, Alan Radford28, Rodolfo Aramayo6, Donald O. Natvig7, Lisa A. Alex29, Gertrud Mannhaupt9, Daniel J. Ebbole30, Michael Freitag3, Ian Paulsen16, Matthew S. Sachs31, Eric S. Lander1,32, Chad Nusbaum1 & Bruce Birren1Nature 422: 859 - 868(2003)Identification and removal of contaminating fluorescence from commercial and in-houseprinted DNA microarrays.M. Juanita Martinez, Anthony D. Aragon, Angelina Rodriguez, Jose Weber, David Haaland, Jerilyn Timlin, Michael Sinclair, and Margaret Werner-Washburne NAR 31:e18 (2003)Visual comparison of multiple genome-scale datasets.Werner-Washburne, M, B. Wylie, E. Fuge, J. Galbraith, J. Weber, and G.S. Davidson.Genome Research 12: 1564-1573 (2002)The genomics of stress responses.A. Gasch and M. Werner-Washburne,Functional and Integrative Genomics 2:181-192 (2002)Analysis of the pdx-1 (snz-1/sno-1) region of the Neurospora crassa genome: correlation of pyridoxine-requiring phenotypes with mutations in two structural genes.Bean, L.E., W.H. Dvorachek, Jr., E.L. Braun, A. Errett, G.S. Saenz, M.D. Giles, M.Werner-Washburne, M.A. Nelson, and D.O. Natvig.Genetics 157: 1067-1075 (2001)Characterization of the SNO and SNZ gene families in Saccharomyces cerevisiae: two highly conserved, co-regulated gene families involved in a response to nutrientlimitation.P.A. Padilla, E.K.Fuge, M.E. Crawford, A. Errett, M. Werner-WashburneJ. Bact. 180: 5718-5726 (1998)Yeast bcy1 mutants with stationary phase-specific defects.V. M. Peck, E.K. Fuge, P. A. Padilla, M.A. Gomez, and M. Werner-WashburneCurrent Genetics 32: 83-92 (1997)Expressed sequences from the conidial, mycelial and sexual stages of Neurospora crassa.M. A. Nelson, S. Kang, E. L. Braun, M. E. Crawford, P. L. Dolan, P. M. Leonard,J Mitchell, A. M. Armijo, L. Bean, E. Blueyes, T. Cushing, A. Errett, M. Fleharty,M Gorman, K. Judson, R. Miller, J. Ortega, I. Pavlova, J. Perea, S. Todisco, R. Trujillo,J. Valentine, A. Wells, M. Werner-Washburne, S Yazzie, and DO Natvig.Fungal Genetics and Biology 21: 348-363 (1997)A stationary-phase gene in Saccharomyces cerevisiae is a member of a novel, highly conserved genefamily.Edward L. Braun, Edwina K. Fuge, Pamela A. Padilla, and Margaret Werner-WashburneJournal of Bacteriology 178: 6865-6872 (1996)Stationary Phase in Saccharomyces cerevisiaeMargaret Werner-Washburne, E. L. Braun, M.E. Crawford, and V.M.PeckMolecular Microbiology 19:1159-1166 (1996)Protein synthesis in long-term stationary phase cultures of Saccharomyces cerevisiae.Edwina K. Fuge, Edward L. Braun, and Margaret Werner-WashburneJournal of Bacteriology 176:5802-5813. (1994)Stationary phase in the yeast Saccharomyces cerevisiae.M. Werner-Washburne, E. Braun, G.C. Johnston, and R. A. Singer.Microbiological Reviews 57:383-401. (1993)Isolation and characterization of AAP1; a gene encoding an alanine/arginineaminopeptidase in yeast.Dan Caprioglio, Christopher Padilla, and Margaret Werner-Washburne.Journal of Biological Chemistry 268:14310-14315. (1993)Heat-shock mediated cell-cycle blockage and G1 cyclin expression in the yeast Saccharomyces cerevisiae.A.Rowley,B. Butler, M. Werner-Washburne, G.C. Johnston, and R.A. Singer.Molecular and Cellular Biology 13:1034-1041. (1992)The translation machinery and 70 kilodalton heat shock protein cooperate in protein synthesis.R. J. Nelson, T. Ziegelhoffer, C. Nicolet, M. Werner-Washburne, and E. A. Craig.Cell 71: 97-105.(1992)Bcy1, the regulatory protein of cAMP- dependent protein kinase, is differentially modified in response to the physiological status of the cell.M. Werner-Washburne, David Brown, and Edward Braun.Journal of Biological Chemistry 266:19704-19709. (1991)Yeast Hsp70 RNA levels vary in response to the physiological status of the cell.M. Werner-Washburne, J. Becker, J. Kosic-Smithers, and E. A. Craig.Journal of Bacteriology 171: 2680-2688. ( 1989)Expression of members of the S.cerevisiae HSP70 multigene family.M. Werner-Washburne and E.A.Craig.Genome 31 :684-689. (1989)SSC1, an essential member of the yeast HSP70 multigene family, encodes a mitochondrial proteinE.A. Craig, J. Kramer, J. Shilling, M. Werner-Washburne, S. Holmes, J. Kosic-Smithers,and C.M. Nicolet.Molec. Cell Biol .9:3000-3008. (1989)A subfamily of stress proteins facilitates translocation of secretory and mitochondrial precursorpolypeptides.R. Deshaies, B.D. Koch, M. Werner-Washburne, E. Craig, and R. Schekman.Nature 332:800-805. (1988)Complex interactions among members of an essential subfamily of Hsp70 genes in yeast.M. Werner-Washburne, D.Stone, and E.A. Craig.Mol. Cell Biol. 7: 2568-2577.(1987)L-Aspartate transport into pea chloroplasts. Kinetic and inhibitor evidence for multiple transport systems.M. Werner-Washburne and K. KeegstraPlant Physiol. 78: 221-227. (1985)Precursors to two chloroplast proteins bind to the outer chloroplast membrane prior to import.K. Cline, M. Werner-Washburne, T.H. Lubben, and K. Keesgtra.J.Biol. Chem. 260: 3691-3696. (1985)Adenine nucleotide translocase dependent anion transport in pea chloroplasts.G.Woldegiorgis, S.Voss, E.Shrago, M. Werner-Washburne, and Kenneth Keegstra.Biochem. Biophys. Acta 810: 340-345.(1985)Thermolysin is a suitable protease for probing the surface of intact pea chloroplasts.K.Cline, M. Werner-Washburne, J. Andrews, and K. Keegstra.Plant Physiol. 75: 675-678. (1984)The chloroplast envelope: Is it homologous with the double membranes of mitochondria and gram-negative bacteria?K. Keegstra, M. Werner-Washburne, K. Cline, and J. Andrews.J. of Cellular Biochem. 24: 55-68. (1983)Analysis of pea chloroplast inner and outer envelope membrane proteins by two-dimensional gel electrophoresis and their comparison with stromal proteins.M. Werner-Washburne, K. Cline, and K. Keegstra.Plant Physiol. 73: 569-575. (1983)An adenine nucleotide-phosphoenolpyruvate counter-transport system in C3and C4 plant chloroplasts.G. Woldegiorgis, S. Voss, E. Shrago, M. Werner-Washburne, and K. Keegstra.Biochem. Biophys. Res. Comm. 116:945-951. (1983)Survey of plants for enterokinase inhibitors.M. Werner-Washburne, u, and H. Ako.Biochem. Biophys. Res. Com. 92: 1243-1249. (1980)Articles appearing as chapters in edited volumes:Still waters run deep: Aging and the survival of quiescent and non-quiescent cells instationary-phase culturesM. Werner-Washburne, S. Roy, and G.S. DavidsonAging in Yeast, 2011 (in press)Robust methods for microarray analysisGeorge S. Davidson, Shawn Martin, Kevin W. Boyack, Brian N. Wylie, Juanita Martinez, Anthony Aragon, Margaret Werner-Washburne, Mónica Mosquera-Caro, Cheryl Willman Genomics and Proteomics Engineering in Medicine and Biology, IEEE ed. Metin Akay; pp.99-130 (2007)Stationary phase in the yeast Saccharomyces cerevisiae.E. K. Fuge and M. Werner-Washburne. 1997In "Yeast Stress Responses", Hohmann S and Mager WH, eds., Molecular Biology Intelligence Unit, R.G. Landes Company, Austin, TX (1997), pp. 53-74Molecular requirements for protein translocation into the endoplasmic reticulum.R.Deshaies, J.Rothblatt, S.Sanders, M. Werner-Washburne, E.Craig, and R.Schekman.p. 187-198. In Bissell, Deho, Sironi, Torriani (ed.), Gene Expression and Regulation: The Legacy of Luigi Gorini. Elsevier, N.Y. (1988)Metabolite transport across the chloroplast envelope.M. Werner-Washburne and K. Keegstra.Oxford Surveys of Plant Mol. and Cell Biol. 2:123-145. (1985)Dicarboxylate transport into isolated chloroplasts. The evidence for multiple carrier systems. M. Werner-Washburne and K. Keegstra.In Proc. 14th Steenbock Symposium "Nitrogen Fixation and CO2 Metabolism." P.W.Ludden and J.E. Burris, eds. Elsevier Publ., N.Y. pp. 389-397.(1985)Other writings (video/patents/government reports/articles):FLOseq – a novel approach for proteomic analysisInvention Disclosure 1/5/2010A Perfect MixFindings Magazine (NIGMS- NIH)Fall, 2003/biology/maggieww/Public_Html/PerfectMix.pdfInteragency Report on the Federal Investment in Microbial GenomicsCoordinated and partially written by M. Werner-WashburneBiotech. Research Working Group, subcommittee of the NSTC Biotechnology Committee Office of Science and Technology Policy, 2000/html/microbial/2000microbial/nsf00203_1.htmFollowing the Coil of LifeAlbuquerque Tribune, January 1, 2000/biology/maggieww/Public_HTML/followingthecoil.htmSouthwest Genomics and Biotechnology Alliance White Paper(/swgaba), July 1999The mystery of an ancient geneNSF/PBS/LANL supported video (28 minutes)Produced by Denise Wallen and Larry Walsh.December, 1997. Shown nationally, April 1998.Invited or refereed talks/presentations to professional meetings and seminar assemblies: (past 5 years)The genomics of yeast stationary phase: birth, death, and wild flings in a flaskMIT, BostonApril, 2011Can You Imagine?Albert Einstein Science Conference – Harvard FoundationApril, 2011Quiescence in YeastSACNASAnnaheim, CA, Oct 2010From Post Doc to ProfessorSACNASAnnaheim, CA, 2010Musings on…mentor ingUWA Genome Sciences Departmental RetreatSeattle, Sept 2010The proteomics of quiescent and nonquiescent cell differentiation in S. cerevisiae stationary-phase culturesYeast Genetics and Molecular Biology MeetingVancouver, Aug 2010Complexity in yeast stationary-phase cultures and the implications for studies of aging University of Arizona (postponed due to other emergency)January 2010Aging and Stationary Phase in YeastBuck Institute on AgingMarch, 2010Holistic Mentoring: What I Learned From My GrandmotherUNM Mentoring InstituteNov 18, 2009From Nano to Terra: the biological revolution rumbles in yeast, a well-behaved organism INCBN IGERT, UNMNov 2, 2009What’s in a genome? What’s in MY genome?Osher Life Long Learning InstituteUNM, Continuing EducationSept 28, 2009Functional genomics: Insights into the complexity of yeast stationary phaseDuke UniversityApril 1, 2009Inference of Functional Networks of Condition-Specific Response: A Case Study of Quiescence in YeastS. Roy, T. Lane, D. Martinez, M. Werner-Washburne (delivered by S. Roy)Pacific Symposium on BiocomputingKona, Hawaii, January 2009Mentoring for students and facultyFor Peer Mentoring, the Mentoring Institute3 presentations at UNM in 2008Computer Sciences and the Quiescent State in YeastUNM CS DepartmentJanuary, 2008Quiescence: How yeast cells plan to live forever!New Mexico TechSocorro, NM, Nov 2007Changing your road to success to a path with heartCalStateLAAndreoli-Woods LectureLos Angeles, CA, May 2007Genomics of the quiescent state in yeastU Washington Genome CenterSeattle, Washington, March 2007MentoringU Washington Department of BiologySeattle, Washington, March 2007Genomics of the quiescent state in yeastU MontanaMissoula, MT, April, 2007The “Omics” Revolution in the Biological Sciences – The Necessity for the Interdisciplinary ApproachC. Allen, A.D. Aragon, S. Roy, O. Meirelles, and M. Werner-Washburne IPSI 2006, Lake Bled, SloveniaGenomic approaches to study the quiescent stateNational Association of Biology TeachersAlbuquerque, October 2006Phylogenomics presentationLANL- UNM Bioenergy Center GroupSeptember, 2006Isolation of quiescent cellsUNM PREP programSeptember, 2006Characterization of quiescent and non-quiescent cells from yeast SP culture Yeast Genetics and Molecular Biology MeetingPrinceton, NJ, July 2006MentoringNSF-QEM WorkshopAlbuquerque, March 2006Microarray Analysis for Biological Processes in YeastNew Mexico Bioinformatics SymposiumSanta Fe, March 2006Quiescence and genomicsSouthwest Junior Science and Humanities SymposiumAlbuquerque, March 2006Quiescence in yeast: When you come to a fork in the road, take it。