Modular organization of genes required for complex polyketide biosynthesis

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以大肠杆菌为异源表达复杂的聚酮类化合物生物合成抗菌肽yersiniabactin

A PPLIED AND E NVIRONMENTAL M ICROBIOLOGY,Nov.2003,p.6698–6702Vol.69,No.11 0099-2240/03/$08.00ϩ0DOI:10.1128/AEM.69.11.6698–6702.2003Copyright©2003,American Society for Microbiology.All Rights Reserved.Biosynthesis of Yersiniabactin,a Complex Polyketide-Nonribosomal Peptide,Using Escherichia coli as a Heterologous HostBlaine A.Pfeifer,1Clay C.C.Wang,1Christopher T.Walsh,2and Chaitan Khosla3,4* Chemical Engineering,1Chemistry,3and Biochemistry,4Stanford University,Stanford,California94305,and Department of Biological Chemistry and Molecular Pharmacology,Harvard Medical School,Boston,Massachusetts021152Received20June2003/Accepted18August2003The medicinal value associated with complex polyketide and nonribosomal peptide natural products hasprompted biosynthetic schemes dependent upon heterologous microbial hosts.Here we report the successfulbiosynthesis of yersiniabactin(Ybt),a model polyketide-nonribosomal peptide hybrid natural product,usingEscherichia coli as a heterologous host.After introducing the biochemical pathway for Ybt into E.coli,biosynthesis was initially monitored qualitatively by mass spectrometry.Next,production of Ybt was quantiﬁedin a high-cell-density fermentation environment with titers reaching67؎21(mean؎standard deviation)mg/liter and a volumetric productivity of1.1؎0.3mg/liter-h.This success has implications for basic andapplied studies on Ybt biosynthesis and also,more generally,for future production of polyketide,nonribosomalpeptide,and mixed polyketide-nonribosomal peptide natural products using E.coli.Polyketides and nonribosomal peptides represent two natu-ral product classes with tremendous therapeutic value(1). Erythromycin(a polyketide antibiotic),vancomycin(a nonri-bosomal peptide antibiotic),and epothilone(a mixed poly-ketide-nonribosomal peptide antitumor agent)represent just a few of these important natural compounds.Harnessing this medicinal relevance,however,is often limited by the rudimen-tary knowledge or poor growth and culturability proﬁles asso-ciated with the original microbial hosts.In addition,the com-plex structure of these compounds limits efﬁcient synthetic production and leaves microbial fermentation as the only economical route to these plex nonribosomal peptides and polyketides derive from complex or modular en-zymatic systems that show the potential for controlled manip-ulation,given the appropriate cellular host(7).To this end,an initiative to transfer the genetic and biochemical pathways responsible for these natural products from their original mi-crobial hosts to heterologous(and often more amenable)hosts has been implemented(12).This transfer would then offer the best of both worlds:an ideal(or signiﬁcantly better)and po-tentially universal heterologous host for the controlled produc-tion of therapeutic natural products.However,this simple con-cept faces signiﬁcant challenges posed by the nature of the complex natural products being produced.To generalize,the heterologous host must generate active enzymatic complexes (either a polyketide synthase[PKS]or nonribosomal peptide synthetase[NRPS])and the substrates that these complexes use for polyketide or nonribosomal peptide biosynthesis.The individual gene size and overall gene number for most modular PKS or NRPS systems pose problems for successful and coor-dinated gene expression(a problem compounded by the need for posttranslational modiﬁcation of PKS and NRPS via phos-phopantetheinylation).In addition,the individual PKS or NRPS will need a pool of intracellular substrates to biosynthe-size the respective natural product;PKS systems require small acyl coenzyme A substrates,while NRPS complexes use both proteinogenic and nonproteinogenic amino acids.These sub-strates may or may not be native to the heterologous host. Recently,the use of Escherichia coli for the heterologous pro-duction of complex polyketide natural products has been de-scribed(11).E.coli offers many advantages as a heterologous host,and the successful production of a complex polyketide opens future biosynthetic potential for other polyketide prod-ucts with this“user-friendly”organism.There also exists the potential to use this heterologous host to generate other com-plex natural products such as nonribosomal peptides or mixed polyketide-nonribosomal peptides.A candidate system to test the heterologous potential of E. coli further is that for yersiniabactin(Ybt),a siderophore nat-urally produced by Yersinia pestis(the causative agent for bu-bonic plague)during times of iron starvation(9).Ybt is bio-synthesized intracellularly by the modular Ybt synthetase composed of four primary enzymes that were recently used to reconstitute Ybt production in vitro(8)(Fig.1).Initially,YbtE adenylates a salicylate unit for addition to the second biosyn-thesis enzyme,HMWP2.HMWP2consists of two multidomain NRPS modules;as theﬁrst module accepts the activated sa-licylate unit through an aryl carrier protein,an adenylation domain attaches two cysteine units through thioester bonds to theﬁrst and second peptidyl carrier proteins.HMWP2then catalyzes the successive addition and cyclization of each cys-teine unit as the growing Ybt chain is then passed to HMWP1. HMWP1contains both a polyketide and nonribosomal peptide module.The ketosynthase domain initially accepts the partial Ybt chain from HMWP2.The PKS portion of HMWP1then catalyzes the addition of a malonate unit(preloaded onto the acyl carrier protein through the acyltransferase domain),and the resultant enzyme-tethered product is reduced(through an NADPH-dependent ketoreductase domain)and methylated (through theﬁrst methyltransferase domain using S-adenosyl-methionine as a substrate).The HMWP1NRPS module then catalyzes the addition,cyclization,and methylation of aﬁnal*Corresponding author.Mailing address:Department of Chemical Engineering,Stanford University,Stanford,CA94305-5025.Phone and fax:(650)723-6538.E-mail:ck@.6698cysteine unit (presumably loaded through the A domain of HMWP2)before the ﬁnal thioesterase domain catalyzes the release of Ybt from HMWP1.YbtU acts as a reductase,re-ducing the second thiazoline ring of the growing enzyme-teth-ered product during biosynthesis.YbtT (not depicted in Fig.1)represents an auxiliary enzyme thought responsible for in vivo biosynthetic editing (3).HMWP1,as indicated above,has both an NRPS function (through the addition of a cysteine unit)and a PKS activity (through the incorporation of a malonyl coen-zyme A unit).Hence,Ybt is a mixed polyketide-nonribosomal peptide natural product.In this work,we report the heterologous production of Ybt using E.coli and the implications for this success in both basic and applied studies.Successful production of Ybt further ex-pands the potential to use E.coli as a route to therapeutic polyketide,nonribosomal peptide,and mixed polyketide-non-ribosomal peptide products.For Ybt production,the high cell density titers achieved compare favorably to the industrial pro-duction of other heterologously produced complex natural products.A robust route to Ybt opens the possibility of using this high-af ﬁnity molecular ligand to speci ﬁcally target or in-hibit pathogenic microbes dependent upon Ybt for survival.MATERIALS AND METHODSStrains and plasmids.E.coli strain K207-3[BL21(DE3)⌬prpRBCD ::T7pro-moter-sfp -T7promoter-prpE panD ::panD25A ygfG ::T7promoter-accA1-T7pro-moter-pccB -T7terminator]was used for all experiments (K207-3is a derivative of BAP1[11]).Plasmids containing the genes for Ybt biosynthesis as well as ybtT were provided on individual expression plasmids as described previously (8).Standard molecular biology protocols were then used to couple these individual genes on multicystronic expression plasmids.pBP198(carbenicillin resistant and derived from pET21c [Novagen,Milwaukee,Wis.])contained (in order)HMWP2and ybtU ,with each gene under the control of its own T7promoter.Likewise,pBP205(kanamycin resistant and derived from pET28a)contained ybtE followed by HMWP1,with each gene under individual T7promoters.An additional plasmid,pBP200,contained ybtT under a T7promoter on a chloram-phenicol-resistant plasmid derived from pGZ119EH (5,10).In vivo gene expression and biosynthesis.Strains K207-3/pBP198/pBP205and K207-3/pBP198/pBP205/pBP200were used for Ybt biosynthesis with all plasmids or plasmid combinations introduced to K207-3via electroporation.All cultures used Luria-Bertani (LB)broth media and contained 100␮g of carbenicillin/ml,50␮g of kanamycin/ml,and 34␮g of chloramphenicol/ml where needed.Cul-tures (typically 5to 10ml)were inoculated 3%(vol/vol)with a previous starter culture,and growth was carried out at 37°C on a rotary shaker (250rpm)to an optical density at 600nm (OD 600)between 0.6and 0.8.Cultures were then cooled at 20°C for 10min.At this point,75␮M isopropyl-␤-D -thiogalactopyranoside (IPTG)was added together with 1mM salicylate.The culture was then incubated between 12and 30h at either 13or 22°C on a rotary shaker (200rpm).For analysis,samples were centrifuged and 5mM FeCl 3was then added to the resultant supernatant.The supernatant was then either extracted with ethyl acetate or submitted to the Stanford mass spectrometry facility for liquid chro-matography-mass spectrometry (LC-MS)analysis directly.Samples extracted were done so twice with an equal volume of ethyl acetate each time.These samples were dried and also submitted for LC-MS analysis.The gradient used for the LC-MS was a linear method from 2to 98%acetonitrile (balance water)with Ybt-Fe 3ϩobserved by MS at ϳ60%acetonitrile.The fragmentation patternforFIG.1.The Ybt synthetase and its substrates needed to reconstitute Ybt production in E.coli .ArCP,aryl carrier protein;A,adenylation;PCP,peptidyl carrier proteins;Cy,cyclization;KS,ketosynthase;ACP,acyl carrier protein;AT,acyltransferase;KR,NADPH-dependent ketoreductase;MT,methyltransferase;SAM,S -adenosylmethionine;TE,thioesterase.See the text for details on biosynthesis.V OL .69,2003YERSINIABACTIN PRODUCTION IN E.COLI 6699Ybt-Fe3ϩwas also determined and compared to previous reports(2,4).Finally,samples taken experimentally were compared by retention times and fragmen-tation patterns to an authentic sample of Ybt-Fe3ϩ.Negative controls includedK207-3/pBP198,K207-3/pBP205,and K207-3/pBP198/pBP205without added sa-licylate.Cell pellets were sonicated,clariﬁed,and analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis to examine the extent of gene expres-sion for both positive and negative controls.High cell density fed-batch fermentation for yersiniabactin puriﬁcation and quantiﬁcation.The fermentation procedure derives from a similar effort used for complex polyketide production(10).Brieﬂy,the fermentation medium(termedF1medium)contained KH2PO4at1.5g/liter,K2HPO4at4.34g/liter,(NH4)2SO4at0.4g/liter,MgSO4at150.5mg/liter,glucose at5g/liter,trace metal solution at1.25ml/liter,and vitamin solution at1.25ml/liter.The feed medium contained(NH4)2SO4at110g/liter,MgSO4at3.9g/liter,glucose at430g/liter;trace metalsolution at10ml/liter;vitamin solution at10ml/liter.The trace metals solutionconsisted of FeCl3⅐6H2O at27g/liter,ZnCl2⅐4H2O at2g/liter,CaCl2⅐6H2O at 2g/liter,Na2MoO4⅐2H2O at2g/liter,CuSO4⅐5H2O at1.9g/liter,H3BO3at0.5 g/liter,and concentrated HCl at100ml/liter.The vitamin solution consisted ofriboﬂavin at0.42g/liter,pantothenic acid at5.4g/liter,niacin at6g/liter,pyri-doxine at1.4g/liter,biotin at0.06g/liter,and folic acid at0.04g/liter.Fed-batch aerated fermentations were conducted by use of an Applikon3LBiobundle system(Applikon Inc.,Foster City,Calif.).A starter culture of K207-3/pBP198/pBP205was grown in1.5ml of LB medium(with100mg of carben-icillin/liter and50mg of kanamycin/liter).After reaching late exponential phaseat37°C and250rpm,the culture was centrifuged and resuspended in50ml of LB(at100-mg of carbenicillin/liter and50mg of kanamycin/liter).The culture grewovernight at30°C and200rpm to stationary phase,was centrifuged,and wasresuspended in20ml of phosphate-buffered saline for inoculation into the3-litervessel containing2liters of F1medium.Growth was conducted at37°C with pHmaintained throughout the experiment at7.1with1M H2SO4and concentratedNH4OH.Aeration was maintained at2.8liters/min with agitation controlled at600to900rpm to maintain dissolved oxygen over50%of air saturation.Thefermentation apparatus including a salt solution[KH2PO4,K2HPO4,and(NH4)2SO4]was autoclaved,whereas the additional components(MgSO4,glucose,tracemetals,and vitamins)wereﬁlter sterilized and added aseptically prior to inocu-lation along with carbenicillin at150mg/liter and kanamycin at75mg/liter.Thefeed medium was alsoﬁlter sterilized.Once the glucose was exhausted from thestarting medium(as indicated by a sudden decrease in the oxygen requirementof the culture),the temperature was reduced to22°C,and IPTG(75␮M)andsalicylate(0.160g/liter)were added.At that point a peristaltic pump started todeliver0.1ml of the feed medium/min,and samples were typically taken twicedaily thereafter.Initially,aﬁnal fermentation broth was extracted with ethyl acetate(2liters,twice).The extract was dried,resuspended in water and10%acetonitrile,andloaded onto a preparatory high-performance liquid chromatography(HPLC)instrument.A10to100%acetonitrile(balance water)gradient at an8-ml/minﬂow rate was used to obtain pure Ybt(isolated as an Fe3ϩchelate).Due to Ybtacid sensitivity,no triﬂuoroacetic acid was used in the HPLC puriﬁcation pro-cess.The Ybt fractions eluted at75%acetonitrile,and these fractions were pooled,dried,and conﬁrmed to contain Ybt-Fe3ϩby MS.The remaining puriﬁed prod-uct was resuspended in water and quantiﬁed at385nm by using the knownextinction coefﬁcient(εϭ2884)for Ybt-Fe3ϩ(2).This initial puriﬁed batch ofYbt-Fe3ϩwas then used to generate a calibration curve to quantify productionfrom subsequent fermentation time point samples that wereﬁrst clariﬁed beforedirectly loading the fermentation broth onto a preparatory HPLC instrument foranalysis.RESULTSGene expression and biosynthesis.Based upon earlier polyketide biosynthetic schemes,the potential to produce Ybt in E.coli was evaluated.The Ybt synthetase genes were intro-duced into E.coli strain K207-3by using the multicystronic plasmids previously developed(11).Initially,gene expression was analyzed at13or22°C based on previous work using similar postinduction temperatures(8,11).Qualitatively,the large gene products were more abundant at22°C(data not shown),and this postinduction temperature was subsequently used.After successful gene expression was conﬁrmed,in vivo ex-periments commenced for Ybt biosynthesis.The main differ-ence between these and previous experiments was the addition of salicylate upon induction with IPTG.The rest of the Ybt synthetase substrates were presumed to be native to E.coli. Isolation efforts indicated the production of Ybt found to be chelated to Fe3ϩ.This compound was analyzed by LC-MS (Fig.2)with LC retention times nearly identical to those indi-cated in previous reports(2,4)and an authentic Ybt-Fe3ϩstandard.Furthermore,MS and MS fragmentation patterns for the proposed Ybt-Fe3ϩcompound also matched those of previous reports and the authentic standard.Relative compar-isons showed increased Ybt production at22°C compared to that observed at13°C.Production could be eliminated by ex-cluding either of the two expression plasmids needed to recon-stitute the Ybt pathway or by omitting the addition of salicylate to cultures that had been induced for gene expression. Finally,the addition of YbtT,an enzyme thought to edit the biosynthetic production of yersiniabactin,resulted in increased levels of Ybt.Again,based on relative comparisons,the inclu-sion of YbtT increased yersiniabactin productionϳ2.5times over that observed for strains without this enzyme.High-cell-density yersiniabactin production and quantiﬁca-tion.A high-cell-density fermentation was performed to gen-erate Ybt in sufﬁcient quantities needed to readily quantify cellular productivity and overall titers.The fed-batch fermen-tation was initially used as a way to obtain pure Ybt.Upon completion of the fermentation run,Ybt was puriﬁed from the fermentation broth by using a combination of organic phase extraction and preparatory HPLC.The puriﬁed product was then quantiﬁed at385nm by using the known extinction coef-ﬁcient(2).This puriﬁed Ybt then served as a standard to quantify theﬁnal titers for subsequent fermentations.Figure3 shows the results for a typical fermentation run.Final titers of 67Ϯ21mg/liter were reached;the maximum speciﬁc produc-tivity of the recombinant E.coli strain was1.2Ϯ0.3mg/liter-h. Both parameters compare well to the biosynthesis of6-deoxy-erythronolide B in the same host(ﬁnal6-deoxyerythronolide B titer,95.2Ϯ7.7mg/liter;maximum6-deoxyerythronolide B speciﬁc productivity,1.1Ϯ0.007mg/liter-h)(10).DISCUSSIONThe development of E.coli-derived Ybt marks an important step forward for heterologously produced complex nonriboso-mal peptide products.Although E.coli has the ability to gen-erate simple nonribosomal peptide products(for example,the native E.coli siderophore enterobactin),Ybt biosynthesis rep-resents an advance due to the complex,modular nature of the Ybt biosynthetic pathway.This biochemical complexity is a hallmark for many important polyketide and nonribosomal peptides(including those examples cited at the beginning of this report),and the present example of heterologous produc-tion through E.coli helps support the notion that this host has the capacity to support production of both natural product classes.In addition,hybrid polyketide-nonribosomal peptide natural products like Ybt should also then be viable options for heterologous production using E.coli.Complex natural product heterologous biosynthesis requires both an active PKS or NRPS and intracellular substrates re-6700PFEIFER ET AL.A PPL.E NVIRON.M ICROBIOL.quired by these enzymes to catalyze their respective products.For Ybt,the yersiniabactin synthetase enzyme complex (con-taining two large,modular proteins in HMWP1and HMWP2)was introduced with multicistronic expression plasmids that facilitated the coordinate expression of all the needed biosyn-thetic genes upon induction with IPTG.Induction by IPTG also called for the expression of a chromosomally located sfp gene needed to activate the biosynthetic enzymesthroughFIG.2.LC-MS analysis of Ybt from K207-3/pBP198/pBP205.The top LC trace shows the Ybt-Fe 3ϩcompound eluting at 16.35min as analyzed by UV at A 210.The bottom trace presents the elution pro ﬁle for compounds within the mass range of 535to 536(molecular weight of Ybt-Fe 3ϩ,535).Finally,the MS pattern for the Ybt-Fe 3ϩpeak is presented.FIG.3.Representative fed-batch fermentation for Ybt production.The time courses of OD 600and Ybt titers for strain K207-3/pBP198/pBP205are shown.pBP198(carbenicillin resistant)contained HMWP2and ybtU ,whereas pBP205(kanamycin resistant)held ybtE and HMWP1.Together,pBP198and pBP205expressed the needed genes for complete Ybt reconstitution within the cellular environment of K207-3,a strain genetically engineered to support biosynthesis.V OL .69,2003YERSINIABACTIN PRODUCTION IN E.COLI 6701posttranslational modiﬁcation.(The sfp gene encodes an Sfp phosphopantetheinyltransferase that was transferred to K207-3from Bacillus subtilus to facilitate complex natural product biosynthesis[11].)Unlike most complex polyketide systems,many of the substrates needed for Ybt biosynthesis are native to E.coli.The exception was a salicylate unit used to initiate biosynthesis.To overcome this,salicylate was simply fed to the media after gene induction.Thus,for biosynthetic substrates that are not native to E.coli or that cannot be metabolically engineered for intracellular production,exoge-nous substrate feeding is yet another option.This study also indicated the enhanced effect that YbtT has on in vivo Ybt production.YbtT is thought to act as an auxil-iary thioesterase enzyme that edits the biosynthetic process, perhaps by removing“misprimed”units that have been incor-rectly attached to the Ybt synthetase complex.In these exper-iments,YbtT increased Ybt productionϳ2.5times.This result supports previous observations that YbtT increases Ybt pro-duction in vivo(3).Interestingly,analogous polyketide systems employing a YbtT analog also show an approximately twofold increase in in vivo polyketide production(10).Finally,the biosynthetic capabilities of this Ybt system were expanded by using the high-cell-density capabilities of E.coli in the context of a fed-batch ing this route pro-duced Ybt at levels comparable to those of heterologous host systems that produce the macrolide6-deoxyerythronolide B(6, 10)and highlights the potential for future heterologous(and therapeutic)polyketide,nonribosomal peptide,or mixed polyketide-nonribosomal peptide product biosynthesis in E. coli.Moreover,given the role of Ybt in establishing virulence for pathogenic microbes,its heterologous production(from a nonpathogenic organism)provides a source of this compound for inhibitor design or targeted drug delivery applications.For example,Ybt analogs,generated through manipulation of the modular Ybt synthetase,or Ybt-drug conjugates might present unique and high-afﬁnity ways to inhibit and target pathogens (such as Yersinia species or other pathogens[13])that have a distinct dependence on Ybt for survival.ACKNOWLEDGMENTSWe thank Sumati Murli at Kosan Biosciences for providing E.coli K207-3.Research in the authors’laboratories was supported by grants from the NIH(GM067937to C.K.and AI42708to C.T.W.).B.A.P.and C.C.C.W.were recipients of an ARCS predoctoral fellowship and an NIH postdoctoral fellowship,respectively.REFERENCES1.Cane,D.E.,and C.T.Walsh.1999.The parallel and convergent universe ofpolyketide synthases and nonribosomal peptide synthetases.Chem.Biol.6:R319–R325.2.Drechsel,H.,H.Stephan,R.Lotz,H.Haag,H.Zahner,K.Hantke,and G.Jung.1995.Structural elucidation of yersiniabactin,a siderophore from highly virulent Yersinia strains.Liebigs Ann.10:1727–1733.3.Geoffroy,V.A.,J.D.Fetherston,and R.D.Perry.2000.Yersinia pestis YbtUand YbtT are involved in synthesis of the siderophore yersiniabactin but have different effects on regulation.Infect.Immun.68:4452–4461.4.Haag,H.,K.Hantke,H.Drechsel,I.Stojiljkovic,G.Jung,and H.Zahner.1993.Puriﬁcation of yersiniabactin:a siderophore and possible virulence factor of Yersinia enterocolitica.J.Gen.Microbiol.139:2159–2165.5.Lessl,M.,D.Balzer,R.Lurz,V.L.Waters,D.G.Guiney,and nka.1992.Dissection of IncP conjugative plasmid transfer:deﬁnition of the trans-fer region Tra2by mobilization of the Tra1region in trans.J.Bacteriol.174:2493–2500.6.Lombo,F.,B.Pfeifer,T.Leaf,S.Ou,Y.S.Kim,D.E.Cane,P.Licari,andC.Khosla.2001.Enhancing the atom economy of polyketide biosyntheticprocesses through metabolic engineering.Biotechnol.Prog.17:612–617. 7.McDaniel,R.,A.Thamchaipenet,C.Gustafsson,H.Fu,M.Betlach,and G.Ashley.1999.Multiple genetic modiﬁcations of the erythromycin polyketide synthase to produce a library of novel“unnatural”natural products.Proc.A96:1846–1851.ler,D.A.,L.Luo,N.Hillson,T.A.Keating,and C.T.Walsh.2002.Yersiniabactin synthetase:a four-protein assembly line producing the non-ribosomal peptide/polyketide hybrid siderophore of Yersinia pestis.Chem.Biol.9:333–344.9.Perry,R.D.,P.B.Balbo,H.A.Jones,J.D.Fetherston,and E.DeMoll.1999.Yersiniabactin from Yersinia pestis:biochemical characterization of the siderophore and its role in iron transport and regulation.Microbiology 145:1181–1190.10.Pfeifer,B.,Z.Hu,P.Licari,and C.Khosla.2002.Process and metabolicstrategies for improved production of Escherichia coli-derived6-deoxyeryth-ronolide B.Appl.Environ.Microbiol.68:3287–3292.11.Pfeifer,B.A.,S.J.Admiraal,H.Gramajo,D.E.Cane,and C.Khosla.2001.Biosynthesis of complex polyketides in a metabolically engineered strain ofE.coli.Science291:1790–1792.12.Pfeifer,B.A.,and C.Khosla.2001.Biosynthesis of polyketides in heterolo-gous hosts.Microbiol.Mol.Biol.Rev.65:106–118.13.Welch,R.A.,V.Burland,G.Plunkett III,P.Redford,P.Roesch,D.Rasko,E.L.Buckles,S.R.Liou,A.Boutin,J.Hackett,D.Stroud,G.F.Mayhew,D.J.Rose,S.Zhou,D.C.Schwartz,N.T.Perna,H.L.Mobley,M.S.Donnenberg,and F.R.Blattner.2002.Extensive mosaic structure revealed by the complete genome sequence of uropathogenic Escherichia coli.Proc.A99:17020–17024.6702PFEIFER ET AL.A PPL.E NVIRON.M ICROBIOL.。

生态学专业术语英汉对照2

Ecology 生态学individuals 个体population 种群communities 群落ecosystems 生态系统behavioral ecology 行为生态学physiological ecology 生理生态学evolutionary ecology 进化生态学molecular ecology 分子生态学fitness 适合度natural selection 自然选择adaptation 适应genotype 基因型phenotype 表型phenotypic plasticity 表型可塑性offspring 后代genes 基因nongenetic factors 非遗传因素not inherited 不遗传conditions 条件resources 资源environmental variation 环境变异internal regulation 内调节homeostasis 稳态negative feedback 负反馈tolerance 耐受性temperature 温度not depletable 不能耗掉solar radiation 太阳辐射decouple 退耦niche 生态位habitat 栖息地multidimensional niche space多维生态位空间Fundamental niche基础生态位Realized niche 实际生态位Prey 猎物Foraging 觅食Dimension 轴或维Global wind pattern 地球的风型The circulation of oceans 洋流Rain 降雨Havoc['hævək]灾害Hurricane 飓风Latitude 纬度Irradiance[i'reidiəns,-si]辐射度Summer solstice 夏至Winter solstice 冬至Adiabatic cooling 绝热冷却Scale 尺度Coriolis effect 科里奥利效应Intertropical convergence zone热带辐合带Jet streams 急流Albedo 反照率Gulf stream 墨西哥湾流Lee of a continent 背风面Upwelling 上涌流Adiabatic lapse rate 绝热温度递减率Inversion 逆温Heat of condensation 凝结热Heat 热Temperature profiles温度剖面Relative humidity 相对湿度Saturated water 饱和水water vapor 水蒸汽microclimate 微气候thermal['θə:məl]conductivity 热传导chemical properties of water 水的化学特性penetration of light through water光线穿透水Energy transfer and water phases能量转化和水相Deplete 耗竭Ions 离子Electropositive 正电性的Electronegative 负电性的Beer’s law 比尔定律Heat capacity 热容量Maximum density 最大密度Latent heat of vaporization增发潜热Heat of fusion 溶解热Sublimation 升华Soil water 土壤水Field capacity 田间持水量The uptake of water by roots根对水的吸收Aquatic plants 水生植物Water availability 水的可利用性Plant productivity 植物生产力Permanent wilting point 永久萎焉点Potential evapotranspiration rate潜在蒸发蒸腾速率Capillary pores 毛细管孔隙Resource depletion zone 资源枯竭区Halophytes 盐生植物Water balance in fish 鱼类的水平衡Amphibians 两栖类Water conservation by terrestrial animals 陆生动物的水保持Mammalian 哺乳动物Kidneys 肾脏Bladder 膀胱Beavers 河狸Osmoregulation 渗透调节Countercurrent exchange 逆流调节Hypertonic 高渗的Homeotherms 恒温动物Poikilotherms 变温动物Ectotherms 外温动物Endotherms 内温动物Temperature thresholds 温度阀Mechanisms 机理Enzyme 酶The thermoneutral zone 热中性区Dehydration 脱水Rates of development and growth发育和生长速度Acclimation and acclimatization 驯化和气候驯化Developmental threshold Temperature 发育温度阀Physiological time 生理时间Vernalization 春化Species distribution 物种分布Evolved response 进化反应Mean temperature 平均温度Isotherm 等温线Radiant energy 辐射能Photosynthesis 光合作用Efficiency of radiant energy conversion 辐射能的转换效率Changes in the intensity of radiation 辐射强度的变化Strategic and tactical response of plants to radiation 植物对辐射的战略和战术响应Compensation point 补偿点Photosynthetically active radiation （PAR）光和活性辐射Efficiency of Photosynthesis 光合作用效率Photosynthetic capacity 光合能力Diurnal and annual rhythms of solar radiation 太阳辐射日节律和年节律Resource depletion zone 资源耗竭带Strategic difference 战略差异Tactical response 战术响应Transpiration 蒸腾Net assimilation 净同化量Nutrient sources 营养物资源Nutrient budgets 营养预算Terrestrial communities 陆地群落Aquatic communities 水生群落Geochemistry 地球化学Global biogeochemical cycles 全球生物地球循环Mechanical weathering 机械风化Chemical weathering 化学腐蚀Wetfall 湿降落Dryfall 干降落Rainout component 雨水冲失成分Washout component 水冲失成分Streamflow 溪流Denitrification 脱氮Endorheic内陆湖泊Biogeochemistry 生物地球化学Hydrosphere carbon 水圈的碳Weathering 风化作用Nitrogen cycle 氮循环Phosphorus 氮Sediment 沉积型Lithospheric 岩石圈Sulfur 硫The fate of matter in the community群落中物质的命运Producers 生产者Consumers 消费者Decomposers 分解者Autotrophs 自养生物Grazing mammals 草食哺乳动物Phytoplankton 浮游植物Zooplankton 浮游动物Bacteria 细菌Fungi 真菌Nonliving 无生命Food chains 食物链Primary and secondary production 初级和次级生产力Net Primary production 净初级生产力Aphotic zone 无光区Photic zone 透光区Primary consumers 初级消费者Secondary consumer 次级消费者Soil formation 土壤形成The soil profile 土壤剖面Primary classification：the great soil groups 主要分类：大土壤群Higher vegetation 高等植物Dynamic mixture 动态混合物Organic matter 有机质Cells 细胞Pedology 土壤学Subsoil 亚土壤Mineral soil 矿物质土壤Parent material 母质Soil series 土系Soil surveyor 土壤勘测员Succession 演替Ecosystem patterns 生态系统格局Soil horizons 土层Humic acids 腐植酸Great soil groups 土壤群Population size 种群大小Age and stage structure 年龄和时期结构Zygote 受精卵Unitary organism 单体生物Modular organism 构件生物Ramets 无性系分株Clone 无性系Genet 基株Evolutionary individuals 进化个体Immediate ecological impact 直接生态作用Stable age distribution 稳定年龄分布Age pyramid 年龄金字塔Stationary age distribution 固定的年龄分布Stage structure 时期结构Sizes classes 个体大小群Natality 出生率Mortality 死亡率Survivorship 存活率Life tables 生命表K-factor analysis k-因子分析The fecundity schedule 生殖力表Population growth 种群增长Density-independent Population growth 非密度制约性种群增长Density-dependent growth-the logistic equation 密度制约性种群增长：逻辑斯缔方程Life expectancy 生命期望Survivorship curve 存活曲线Cohort 同生群Age-specific survival rate 特定年龄存活率Key factors 关键因子Killing factor 致死因子Basic reproduction rate 基础繁殖率Carrying capacity 环境容纳量Estimating density 估计密度Mark release recapture 标记重捕法H3Density dependence密度制约Equilibrium population density 平衡种群密度Relative density相对密度Allee effect阿利效应Exactly compensating准确补偿Undercompensating补偿不足Overcompensating过度补偿H4Population fluctuations 种群波动Chaos 混沌Expanding and contracting populations 增长种群和收缩种群Stable limit cycle 稳定极限环I1Competition 竞争Predation 捕食Parasitism 寄生Mutualism互利共生Intraspecific competition种内竞争Interspecific competition种间竞争Exploitation competition利用性竞争Interference competition干扰性竞争Cannibalism 自相残杀Altruism 利他主义Commensualism 偏利共生Amensualism偏害共生I2Dispersal扩散Territoriality领域性Niche shift生态位转移Allelopathy异株克生Competive asymmetry 竞争不对称Scramble competition争夺竞争Contest competition格斗竞争Zero net growth isocline零增长等斜线Self-thinning自疏Inbreeding近亲繁殖Reproductive value繁殖价值Leks 求偶场I3Competitive exclusion 竞争排斥Limiting similarity 极限相似性Competitive release 极限释放Character displacement 性状替换Apparent competition 表观竞争Enemy-free space 无敌空间Highly heterogeneous 高度异质性Gaps 断层Probability refuge 隐蔽机率J1Herbivores 食草动物Carnivores 食肉动物Omnivores 杂食动物Chemical defences 化学防御Behavioral strategies 行为对策Specialists 特化种Generalist 泛化种Monophagous单食者Oligophagous寡食者Polyphagous 多食者Parasites 寄生者J2Predator switching 捕食者转换Profitability of prey 猎物收益率Plant defence 植物防御The ideal free distribution 理想自由分布Functional response 功能反应Superpredation 超捕食K1Parasites 寄生物Modes of transmission 传播方式Social parasites 社会性寄生物Helminth worms 寄生蠕虫Insects 昆虫Necrotrophs 食尸动物Parasitoids 拟寄生物The cellular immune response 细胞免疫反应Vectors 媒介Optimal habitat use 最佳生境利用Brood parasitism 窝寄生Evolutionary constraint 进化约束K2Immunity 免疫Cevolution协同进化Gene for gene 基因对基因Mimics 模仿Herd immunity 群体免疫Antigenic stability 抗原稳定L1Pollination 传粉Symbiotic 共生性Obligate 专性Lichens 地衣Outcrossing 异型杂交Mitochondria 线粒体Chloroplasts 叶绿体M1Reproductive values 生殖价Hypothetical organism 假定生物Migration 迁移Senescence衰老Diapause 滞育Dormancy 休眠Longevity 寿命Enormous variation 巨大变异Energy allocation 能量分配Semelparity 单次生殖Iteroparity 多次生殖Carrying capacity 容纳量Current/future reproduction当前/未来繁殖Habitat disturbance 环境干扰The current/future reproductive output 当前/未来繁殖输出A high/low cost of reproduction 高/低繁殖付出Seed bank 种子库Torper蛰伏Hibernation 冬眠Cryptobiosis 隐生现象Aestivation 夏眠Migration 迁徙Morphological forms 形态学性状Generations世代Mechanistic level 机制水平N1Cooperation 合作Grouping-benefits 集群-好处Altruism 利他行为Group defens e 群防御Inclusive fitness 广义适合度Eusociality 真社会性Hymenoptera 膜翅目Haplodiploid 单倍二倍体Venomous sting毒刺N2Sex 性The costs of inbreeding 近交的代价Self-fertilization 自体受精Sexual versus asexual reproduction 有性和无性生殖Sex ratio 交配体制Monogyny 单配制Polygyny 一雄多雌制Polyandry 一雌多雄制Inbreeding depression 近交衰退Hermaphrodite 雌雄同体Recombine 重组Rare type advantage 稀少型有利Equal investment 相等投入Local mate competition局域交配竞争Epigamic 诱惑性Intrasexual selection 性内选择Intersexual selection 性间选择O1Alleles 等位基因Polymorphism 多型Genetic drift 遗传漂变Genetic bottleneck 遗传瓶颈Rare species 稀有物种Extinction 灭绝Chromosome染色体Genotype 遗传型Phenotype 表现型Gene pool 基因库Gel electrophoresis 凝胶电泳O2Gene flow 基因流Differentiation 分化Sibling species 姊妹种Genetic revolution 遗传演变Peripheral isolates 边缘隔离PTransfer efficiencies 转换效率（net）primary productivity (净)初级生产力Respiratory heat 呼吸热Grazer system 牧食者系统Food chains 食物链Pathways of nutrient flow营养物流Food webs 食物网QCommunity structure 群落结构Community boundaries 群落边界Guilds同资源种团Community organization 群落组织Species diversity 物种多样性Energy flow 能量流Superorganism 超有机体Species-poor/rich 物种贫乏/丰富Biomass stability 生物量稳定性Tundra 冻原Island biogeography 岛屿生物地理学Turnover rate 周转率Source of colonists 移植者源Relaxation松弛Edgespecies 边缘物种Interior species 内部物种Corridor 走廊Greenways 绿色通道Community assembly群落集合Grazers 食草动物Carnivores 食肉动物Keystone species 关键物种Dominance control 优势控制Habitat affinity生境亲和力Prey switching 猎物转换RSuccession 演替Climax Community 顶级群落Pioneer species 先锋物种Primary succession 原生演替Alluvial deposit 冲积层Secondary succession 次生演替Acidifying effect 酸化作用Opportunistic机会主义Cellulose 植物纤维素Lignin 木质素Resource ratio hypothesis 资源比假说Fluctuations 波动Cyclic succession 循环演替Disturbance 干扰Patch dynamics板块动态Mini-succession 微型演替Cambium 形成层Neotropical forest 新热带雨林Priority effect 优先效应SVegetation 植被Ecotones 群落交错区Climate map 气候图Biomes 生物群系Heat budget 热量预算Zonation 分带Grassland 草地Primary regions 基本区域Desertification 荒漠化Arctic tundra 北极冻原Alpine tundra 高山冻原Permafrost 永冻层Coniferous boreal forest北方针叶林Temperature forest 温带森林Tropical forest 热带森林Salinization 盐渍化Primary saltwater regions 基本盐水区域Opens oceans 开阔海洋Continental shelves 大陆架The intertidal zone 潮间带Salt marsh 盐沼Mudflats淤泥滩Mangroves 红树林Pelagic 浮游生物Photic zone 有光带Phyto plankton 浮游植物Nekton 自泳动物Benthic 底栖Rocky shore 岩岸Zonation 分带Streams 溪流Ponds 池塘Environmental concerns 环境关系Catchment area 集水区Temperature inversion 温度逆转Biomanipulation 生物处理TThe goals of harvesting 收获目标Quota limitation 配额限制Environmental fluctuation环境波动Maximum possible yiel最大可能产量Net recruitment 净补充量Surplus yield 过剩产量Age structure 年龄结构Population data 种群数据Stable equilibrium 稳定平衡Harvesting effort 收获努力Gun licences 猎枪执照Rod licences钓鱼许可证Upwelling of cold water冷水上升流Fisheries 渔业Ocean productivity 大洋生产力The tragedy of the common公共灾难Overexploitation 过捕Pollution 污染Global decline 全球性下降By-catch 附带收获Community perturbations 群落扰动Oil spills 原油泄漏Eutrophication 富营养化Algal blooms 水华Red tides 赤潮Biomagnification 生物放大作用UPest 有害生物Natural enemies 天敌Ruderal 杂草型Economic/aesthetic injury level 经济/美学损害水平Cultural 栽培Biological control 生物防治‘Silent spring’寂静的春天Chemical toxicity 化学毒性Evolution of resistance抗性进化Microbial insecticide微生物杀虫剂Inoculation接种Augmentation扩大Inundation 爆发VRare species 稀有种Genetic diversity 遗传多样性Extinction 灭绝Endemic species 特有种Habitat fragmentation 生境片段化Insularization 岛屿化Biodiversity 生物多样性Strategies for conservation保育对策Antarctic treaty 南极协议Ecotourism生态旅游WAir pollution空气污染Acid rain 酸雨Water pollutants 水体污染物Soil pollution 土壤污染Acid deposition 酸降Pathogens病源体Chemical oxygen demand 化学需氧量Anaerobes 厌氧菌The greenhouse effect 温室效应Carbon dioxide 二氧化碳Ozone 臭氧Photochemical smog 光化学烟雾XOverview 概述Soil erosion 土壤侵蚀Soil compaction 土壤硬结Contour ploughing等高耕作Cover crops 覆盖作物No-till farming 免耕农业。

monocistronic and polycistronic名词解释

monocistronic and polycistronic名词解释Monocistronic and Polycistronic: An ExplanationMonocistronic and polycistronic are two terms used to describe the structure and organization of genetic material in living organisms, particularly in relation to messenger RNA (mRNA) molecules.Monocistronic refers to a genetic arrangement where an mRNA molecule carries the genetic code for a single protein. In simple terms, it means that one mRNA molecule encodes only one protein. This organization is commonly found in eukaryotes, such as animals and plants. Monocistronic mRNA is transcribed from a single gene and typically contains a single open reading frame (ORF), which is the sequence that is translated into a protein.On the other hand, polycistronic refers to a genetic arrangement where a single mRNA molecule carries the genetic code for multiple proteins. In this case, the mRNA contains multiple ORFs, each encoding a distinct protein. Polycistronic organization is commonly found in prokaryotes, such as bacteria. The polycistronic mRNA is transcribed from a gene cluster that contains several genes coding for related or functionally associated proteins.The key difference between monocistronic and polycistronic structures lies in the number of proteins encoded by a single mRNA molecule. Monocistronic mRNA produces one protein, while polycistronic mRNA produces multiple proteins. This variation in genetic organization has significant implications for gene regulation, expression, and overall cellular functionality.Monocistronic and polycistronic structures are differentially regulated in cells. Monocistronic mRNA expression is often tightly controlled, allowing for precise and specific regulation of protein production. In contrast, polycistronic mRNA expression canbe regulated in a coordinated manner, whereby all the genes within the cluster are transcribed and translated together. This coordination allows for the production of multiple proteins required for a particular biological process.Understanding the distinctions between monocistronic and polycistronic structures is essential in the fields of genetics and molecular biology. It aids in comprehending the complexity and diversity of gene regulation mechanisms across different organisms. The structural organization of genetic material plays a fundamental role in determining the functionality and diversity of living systems, and studying these concepts helps shed light on various biological processes.。

Fine mapping of a major quantitative trait loci, qSSP7 , controlling the number of spikelets per

Theor Appl GenetDOI 10.1007/s00122-008-0711-9ORIGINAL PAPERFine mapping of a major quantitative trait loci, qSSP7, controlling the number of spikelets per panicle as a single Mendelian factor in riceY. Z. Xing · W. J. Tang · W. Y. Xue · C. G. Xu ·Qifa ZhangReceived: 29 June 2007 / Accepted: 8 January 2008© Springer-Verlag 2008Abstract In our previous studies, one putative QTL a V ecting number of spikelets per panicle (SPP) was identi-W ed in the pericentromeric region of rice chromosome 7 using a recombinant inbred population. In order to de W ne the QTL (qSPP7), RI50, a recombinant inbred line with 70% of genetic background same as the female parent of Zhenshan 97, was selected to produce near-isogenic lines for the target region in the present study. In a BC2F2 popu-lation consisting of 190 plants, the frequency distribution of SPP was shown to be discontinuous and followed the expected Mendelian ratios (1:2:1 by progeny test) for single locus segregation. qSPP7 was mapped to a 0.4cM region between SSR marker RM3859 and RFLP marker C39 based on tests of the BC2F2 population and its progeny. Its additive and dominant e V ects on SPP were 51.1 and 24.9 spikelets, respectively. Of great interest, the QTL region also had e V ects on grain yield per plant (YD), 1,000 grain weight (GW), tillers per plant (TPP) and seed setting ratio (SR). Signi W cant correlations were observed between SPP and YD (r=0.66) and between SPP and SR (r=¡0.29) in the progeny test. 1082 extremely small panicle plants of a BC3F2 population containing 8,400 individuals were further used to W ne map the QTL. It turns out that qSPP7 co-segre-gated with two markers, RM5436 and RM5499 spanning a physical distance of 912.4kb. Overall results suggested that recombination suppression occurred in the region and posi-tional cloning strategy is infeasible for qSPP7 isolation. The higher grain yield of Minghui 63 homozygote as com-pared to the heterozygote suggested that Minghui 63 homo-zygote at qSPP7 in hybrid rice could further improve its yield.IntroductionWith the advent of DNA molecular markers, QTL mapping has become a routine strategy for the discovery of genes involved in complex quantitative traits. Thousands of QTL have been mapped for important agronomical traits in rice (/db/cmap/map_set_info?species_ acc=rice&map_type_acc=qtl). Although primary mapping populations including F2, recombinant inbred lines (RILs) and doubled haploid lines (DHs) have been widely used for QTL mapping in rice (Li et al. 1995; Yu et al. 1997; Xu et al 2004; Fan et al. 2005; Marri et al. 2005), QTL can only be localized to a genomic region (con W dential region) rather than a locus in those populations. Following the pri-mary mapping, advanced populations such as near isogenic lines (NIL) and chromosome segment substitution lines (CSSL) can be used to map QTL to a locus as a Mendalian factor by blocking the genetic background noise (Lin et al. 2002; Zhang et al. 2006; Wang et al. 2006). Based on this strategy, several QTLs have been isolated in tomato (Frary et al. 2000) and rice (Yano et al. 2000; Takahashi et al. 2001; Li et al. 2003) in recent years. The common aspect in all the QTL cloning work is to exploit high quality NILs as advanced mapping populations.Food shortage is one of the most serious global prob-lems. The United Nations Food and Agricultural Organiza-tion (FAO) estimates that 852 million people worldwideCommunicated by A. Paterson.Y. Z. Xing and W. J. Tang contributed equally to this work. Y. Z. Xing (&) · W. J. Tang · W. Y. Xue · C. G. Xu · Q. Zhang National Key Laboratory of Crop Genetic Improvementand National Center of Plant Gene Research,Huazhong Agricultural University, Wuhan 430070, Chinae-mail: yzhxing@; yzxing@Theor Appl Genetwere undernourished between 2000 and 2002 (http://www. /documents/show_cdr.asp?url_file0/docrep/007/y56 50e/y5650e00.htm). Rice is a staple food for many coun-tries. As the world population increases, rice production has to be raised by at least 70% over the next three decades to meet growing demands. In the long run, development of high yield varieties is one of the most important goals in rice cultivation. Unfortunately, yield improvement e Y ciency is deemed to be very low due to its complex property a V ected by number of spikelets per panicle (SPP), 1,000-grain weight (GW) and tillers per plant (TPP). Of these factors, SPP was shown to be highly correlated with yield and acts as a crucial component in determining rice yield (Hua et al. 2002). Therefore, dissection of its genetic basis would be of great value in breeding high yield variety. So far, QTLs for SPP have been mapped in lots of popula-tions, which were derived from inter-subspeci W c and intra-speci W c crosses (Li et al. 1997; Yu et al. 1997; Xing et al. 2002). Although many QTL controlling SPP were mapped in rice, only Gn1a, has been cloned. Gn1a, controlling grain productivity in rice, was elucidated to be a gene encoding cytokinin oxidase/dehydrogenase (OsCKX2), an enzyme that degrades the phytohormone cytokinin. Reduced expression of OsCKX2 causes cytokinin accumu-lation in in X orescence meristems and increases the number of reproductive organs, resulting in enhanced grain yield (Ashikari et al. 2005).In our previous studies, a QTL a V ecting the number of spikelets per panicle was repeatedly mapped to one pericen-tromeric region X anked by RFLP markers C1023 and R1440 on rice chromosome 7 in several primary mapping popula-tions (Yu et al. 1997, 2002; Xing et al. 2001, 2002). In the present study, we aimed to precisely estimate its genetic e V ect under near isogenic background and W ne map the QTL as a single Mendelian factor based on a large number of NILs. Materials and methodsDevelopment of NILIn our previous study, one QTL, qSPP7, was mapped to the interval between R1440 and C1023 on chromosome 7 using a recombinant inbred population derived from the cross between Zhenshan 97 and Minghui 63 (Xing et al. 2002). The recombinant inbred line 50 (RI50), was selected to backcross with the female parent Zhenshan 97 and a BC1F3 population was obtained with two sel W ngs. RI50 was homo-zygous for Minghui 63 alleles in the targeted QTL region on chromosome 7. Moreover, approximately 70 percent of RI50 genetic background was the same as that of Zhenshan 97 based on 221 polymorphic genome-wide markers (Xing et al. 2001). One line of BC1F3 population (BC1F3-120)with homozygous Minghui 63 alleles, showing uniform number of SPP, was selected to continue backcrossing with Zhenshan 97 to produce BC2F2 and BC2F3 populations. According to the genotypes of the X anking markers, one BC2F2 plant (BC2F2-SPP7) with homozygous Minghui 63 regions surrounding qSPP7 was chosen to backcross with Zhenshan 97 to produce the BC3F1. In order to screen the genetic background of BC2F1, 150 SSR markers evenly dis-tributed on 12 chromosomes were selected from all poly-morphic markers between Zhenshan 97 and Minghui 63 (McCouch et al. 2002). Eight chromosome fragments cov-ering about 150cM on chromosomes 1, 3, 5, 6, 7, 9 and 12 (two segments), were heterozygote in BC2F1. The markers situated in these 8 fragments were further used to screen several BC3F1 plants. Finally, the one with the least back-ground markers, that is, only three small regions on chro-mosomes 5, 9 and 12 were heterozygote besides about 40cM region containing the targeted QTL on chromosome 7 (Fig.1), was selected for BC3F2 production. The popula-tions of 190 BC2F2 and BC3F2 individuals were used to evaluate the gene e V ects, respectively. The BC3F1 plant was divided into several clones at seedling stage in order to harvest amounts of selfed seeds. 8,400 BC3F2 plants were used for W ne mapping the genes.Field trial and trait measurementThe 190 BC2F2 plants with RI50, Zhenshan 97 and Minghui 63 were sown on 17 May 2002 and transplanted to the W eld Fig.1The graphic genotype of the BC3F1 plant. The black and white chromosome segments were heterozygote and Zhenshan 97 homozy-gote, respectively. Only four chromosomes contained segments of donor parent, Minghui 63. The other eight chromosomes were W xed with Zhenshan 97Theor Appl Genetwith distance of 16.5cm between plants within a row, and of 25cm between rows on 11 June 2002 in Wuhan (East longitude 114°, North latitude 30°), where the average day length during the period between June and August is 13.5h. BC3F1 ratoons from Hainan were divided into seven individuals and transplanted in the W eld on 1 May 2003. Zhenshan 97, Minghui63, BC2F3-SPP7, the 190 BC2F3 families and the large BC3F2 population with 8,400 plants were sown on 19 May 2003. Plants were then transplanted to the W eld on 10 June 2003 in Wuhan. From each BC2F3 family, 16 healthy plants were selected for the progeny test. Field management essentially followed the normal agricul-tural practices, with fertilizer applied (per hectare) as fol-lows: 48.75kg N, 58.5kg P and 93.75kg K as the basal fertilizer; 86.25kg N at the tilling stage and 27.6kg N at the booting stage. Each plant was harvested individually at maturity.Yield per plant (YD) was scored as the total weight (g) of the grains from the entire plant. The number of tillers per plant (TPP) scored as the number of reproductive tillers for each plant. The number of spikelets per panicle (SPP) was measured as the number of spikelets divided by TPP. Seed setting rate (SR) was scored as the number of grains per panicle divided by SPP, 1000-grain weight (GW) as YD divided by the number of grains multiplied by 1,000. Five plants of Zhenshan 97, Minghui 63, BC2F3-SPP7 and BC3F1 were used to score the phenotype. The arithmetic mean of each homozygous BC2F3 family in the targeted region was regarded as the trait value of the genotype. For the heterozygote family (whose segregation ratio within each family––frequently distorted away from 1:2:1), the weighted mean of the 16 plants was regarded as the trait values of corresponding heterozygous BC2F2 plants. The weighted mean of trait value (y) was calculated by the for-mula: y=a/4+b/4+c/2, in which, a, b and c represent the trait mean value of Zhenshan 97 homozygote, Minghui 63 homozygote and heterozygote genotypes within a family, respectively.Genotyping of populationsGenomic DNA was extracted from 190 BC2F2 plants, 190 BC3F2 plants and 1,082 plants with small panicle (<96 spik-elets) out of 8,400 BC3F2 plants by using simple fast CTAB method (Murray and Thompson 1980). Co-dominant SSR markers located in the region were used to screen the poly-morphism between parents. The SSR genotyping was exe-cuted as described by Wu and Tanksley (1993).Linkage analysis and QTL analysisGenetic linkage map was constructed by using Mapmaker/ EXP 3.0 (Lincoln et al. 1992). The genetic distance was determined by Kosambi function. Due to very few regions segregated in the populations of the 190 BC2F2 plants and 190 BC3F2 plants, simple interval mapping were employed for QTL analyses using Mapmaker/QTL (Lander and Bot-stein 1989). The LOD thresholds ranged from 2.3 to 2.8 for the 5 traits, determined by 1,000 random permutations at a false positive rate of 0.05 for each trait. Performance of the self-pollinated progeny (BC2F3) was used to determine the individual BC2F2 genotypes at the QTL. Then he QTL was treated as a marker and were directly located into the link-age group by using Mapmaker/EXP 3.0.Fine mapping the gene using the extremely small panicle plantsBased upon the assumption that all 1082 small panicle (<96 spikelets) plants were homozygous for the recessive allele (Zhenshan 97) at the targeted locus, the recombination fre-quency between a marker and the gene locus was calculated by using of a maximum likelihood estimator: c=(N1+N2/ 2)/N (Allard 1956; Zhang et al. 1994), in which N is the total number of the extremely small panicle plants investi-gated, N1 is the number of plants homozygous for Minghui 63, and N2 is the number of plants heterozygous for the par-ents. 20 progeny plants of each recombinant were grown in the next normal rice growing season to re-evaluate its phe-notype.ResultsThe trait performance of Zhenshan 97, Minghui 63,BC2F3-SPP7, BC3F1Minghui 63 showed signi W cant higher trait values than Zhenshan 97 in SPP, GW, SR and YD; however, Zhenshan 97 had more TPP (Table1). Except SPP, no signi W cant di V erence was identi W ed in the other four traits between BC3F1 (ratoon) and BC2F3-SPP7 with homozygous Ming-hui 63 alleles in the targeted region. BC2F3-SPP7 and BC3F1 had much higher values in SPP and YD as compared to Minghui 63, the original parent of the population with higher trait values.Phenotypic variations in BC2F2 and BC2F3The frequency distribution of SPP showed discontinuous variation with a clear gap between 110 and 120 spikelets based on the progeny test in the BC2F2 population (Fig.2). According to the progeny test, the BC2F2 individuals could be classi W ed into three subgroups for SPP expressing uni-form large panicle, uniform small panicle and varied SPP among 16 plants within each family. The three subgroupsTheor Appl Genetcorresponded to the three genotypes, respectively: the homozygotes of Minghui 63 (MM) and Zhenshan 97 (ZZ),and heterozygote (MZ) at the targeted QTL. On average,the number of spikelets per panicle of genotype MM was 102 more than that of ZZ. Genotype ZM was intermediate to those of the homozygotes. As expected, the frequency distribution of SPP in the BC 2F 2 population followed theMendelian ratios (1:2:1 by progeny test) for single locus segregation ( 2=1.07, P =0.586). SPP displayed bimodal distribution in the 190 BC 3F 2 (Fig.2). The 190 plants were separated into two distinct groups: plants with small panicle (50 plants) which could be homozygous for Zhenshan 97alleles at the targeted gene locus and plants with large pani-cle (140 plants) which could be the mixture of homozyg-otes for Minghui 63 alleles and heterozygotes. Also,frequency distribution was also in agreement with the expected Mendelian ratio (1:3) for single locus segregation ( 2=0.11, P =0.740). In contrast, the other 4 traits showed continuous but not normal distribution (Fig.2). Therefore,it was impractical to clearly classify them into subgroups according to trait performance by the progeny test.The BC 2F 2 population was classi W ed into three sub-groups of ZZ, MM and MZ according to SPP performance in the progeny test, the average values of the W ve traits were calculated for the three subgroups. Paired t test was used to compare trait value di V erences between subgroups. The averaged values of GW and SR among three subgroupsTable 1Trait performance of Minghui 63, Zhenshan 97, BC 2F 3-SPP7and BC 3F 1 grown in the natural W eld in 2003Values within columns followed by the same letter are not statistically signi W cant (P =0.01) according to Duncan’s test*The trait value of BC 3F 1 was gotten from BC 3F 1 ratoon, which was divided into seven individuals and transplanted in the W eldPlant SPP GW (g)SR (%)TPP YD (g)Zhenshan 97109.2a 23.5a 72.7a 10.5b 19.6a Minghui 63148.6b 28.0b 77.1b 9.4a 29.9c BC 2F 3-SPP7185.4d 22.9a 74.2a 8.8a 27.7b BC 3F 1*165.2c23.8a75.3a9.1a26.9bFig.2Frequency distributions of the traits in BC 2F 2 population and BC 3F 2 population. Black, white and gray bars indicated the genotypes of heterozygous, homozygous for Minghui 63 alleles and homozygousfor Zhenshan 97 alleles in BC 2F 2 population at qSPP7, respectively.The three genotypes at qSPP7 were inferred by BC 2F 3 progeny testTheor Appl Genetindicated no signi W cant di V erence. On the contrary, signi W -cant di V erence existed between ZZ and the other two sub-groups (P =0.001) with regard to YD and TPP. MM genotype had the YD of 26.5g signi W cant higher than that of MZ, 24.2g (P =0.05).The correlation coe Y cients between yield and its four components were presented in Table 2. The correlation coe Y cient between SPP and YD was the highest (r =0.66)among all the investigated pair-tests, signi W cant negative correlations were observed between SPP and SR (r =¡0.29), between SPP and TPP (r =¡0.54). No signi W -cant correlation was detected between SPP and GW (r =0.03).Validation of QTL for SPPIn our previous studies, one putative QTL a V ecting SPP and YD was identi W ed in the pericentromeric region of rice chromosome 7, between RFLP markers C1023 and R1440(Fig.3a). In this study, QTL analysis was further performed in the same target region using BC 2F 2 and BC 3F 2 popula-tions.The genotypes of the corresponding BC 2F 2 plants at qSPP7 locus were easily determined on the performance of their progenies. Thus, qSPP7 treated as a marker waslocated in the locus between SSR marker RM3859 and RFLP marker C39, 0.2cM away from each marker (Fig.3b). qSPP7 showed partial dominance for SPP.In the BC 3F 2 population, one QTL with a LOD value of 56.1 for SPP was located in the interval between RM3859and C39 on chromosome 7. For simplicity, and hereafter,the QTL was referred as qSPP7, which was 0.2cM away from RM3859 and C39, and co-segregated with RM5436.Its additive and dominant e V ects were 59.8 and 28.3 spik-elets per panicle with a contribution of 74.2% to the pheno-typic variance.Accordingly, based on the BC 2F 2 population and its progenies, two major QTLs controlling YD and TPP, were respectively mapped to the same interval where qSPP7 was located (Table 3). Also, a minor QTL for SR was also detected in the same interval. Minghui 63 allele increased SPP and YD, but decreased TPP and SR. No QTL for GW was mapped. The QTL for SR expressed over-dominance,but the others showed partial dominance.High-resolution mapping of qSPP7The 190 BC 3F 2 plants could be divided into two subgroups with 112 spikelets per panicle as the boundary (Fig.2).Zhenshan 97 alleles at qSPP7 exhibited small SPP in con-trast to Minghui 63 alleles and heterozygous alleles. In order to avoid using heterozygous plants, 1,082 plants with extremely small panicle (<96 spiketes), which accounted for about 1/8 of 8,400 BC 3F 2 plants, were selected for map based gene cloning. Two SSR markers of RM5451 and RM445 X anking the QTL (Fig.3) were used to detect the recombinant events between markers and the targeted gene.Analysis of RM5451 identi W ed 66 recombination events between the marker and qSPP7 on one side, and analysis of RM445 detected 146 recombination events between theTable 2Correlation coe Y cient for yield traits calculated with BC 2F 3population *,**Signi W cant at the level of =0.05, 0.01 respectivelyTrait SPP SRYDGWSR ¡0.29**YD 0.66**0.22**GW 0.03¡0.050.20*TPP¡0.54**0.35**¡0.12¡0.03Fig.3Linkage maps showing the location of qSPP7 based on three populations. a Linkage map of chromosome 7 showing the QTL region based on the recombinant inbred lines from the cross between Zhenshan 97 and Minghui 63, the black bar indicated 1-LOD con W dence interval (Xing et al. 2001). b The QTL (qSPP7) location was mapped based on the 190 BC 2F 3 progeny test. The black dot indi-cates the centromere position. c The QTL (qSPP7) location was determined based on 1082 extreme small panicle BC 3F 2 plants. The black dot indicates the centromere positionTheor Appl Genetmarker and qSPP7 on the other side. Assay with four addi-tional markers of RM3859, RM1135, RM7110 and C39,which are more internal to qSPP7, identi W ed 4 recombina-tion events on the RM5451 side and 94, 88 and 4 recombi-nation events on the RM445 side, respectively (Fig.4).Using the formula given by Zhang et al. (1994), the recom-bination frequencies between RM3859 and qSPP7, and between C39 and qSPP7 were both calculated to be 0.18%,which equaled to 0.2cM from qSPP7 to both RM3859 and C39 (Fig.3c). Consequently, two SSR markers RM5436and RM5499 were chosen to screen the eight recombinants from each sides of the gene. However, no recombination event was left for the two markers. RM5436 and RM5499were located in the BAC clones of AP003756 and AP003836, respectively. The physical distance between the two markers is of 912.4kb (/Oryza_sativa_japonica/mapview?chr=7). Thus, the qSPP7was de W ned by RM3859 and C39, and co-segregated with a large region.DiscussionIn this study, a major QTL for SPP was W ne mapped to a 0.4cM region, which had e V ects on TPP, YD and SR as well. This mapping result bene W ted mainly from the use of near isogenic lines and should be of great value for transfer-ring the favorable alleles to rice varieties.The target region exhibited similar e V ects on SPP in both the F 2 population (Yu et al. 1997; 2002) and the RIL popu-lation (Xing et al. 2001; 2002) derived from the same cross between Zhenshan 97 and Minghui 63. The QTL-NIL pop-ulations in the present study revealed even richer informa-tion on this QTL. Besides the same direction of the genetic e V ect with Minghui 63 alleles increasing trait values across di V erent generations, the NIL population provided more accurate estimation of genetic e V ects on SPP due to the minimization of its genetic background noise. Obviously,these genetic e V ects in F 2 and RILs were underestimated.As expected, SPP frequency showed normal distribution in F 2 and RIL populations. In contrast, it expressed a bimodal or discontinuous SPP frequency distribution in the NILs,which could be used to determine the genotype of individ-ual plants at qSPP7 according to their progeny test. In fact,several examples of successful QTL cloning were bene W ted greatly from development of high quality NILs (Yamamoto et al. 2000; Lin et al. 2003) in the last decade. There were few heterozygous plants falling into the category of small number of SPP (Fig.2) in this study could cause false recombinants in the W ne mapping process. This problem was solved in two ways: W rst, only 1/8 of the plants with the smallest number of SPP were chosen from the 8,400 plants to screen the recombinants, which can greatly minimize the risk of false recombinants; second, the reliability of all screened recombinants were further con W rmed by progeny test, which showed uniform small number of SPP among 20 progeny plants. Hence, it is highly con W dent that all recombinants are authentic in this study.Although centromeres are often associated with a depression of meiotic recombination adjacent to theTable 3Genetic e V ects of the QTL region between RM3859 and C39on the four traits by Mapmaker/QTL A additive e V ect on the Minghui 63 allele, D dominant e V ect on the Minghui 63 allele, R 2 percentage of total phenotypic variance explained by the QTL*Genotypes of the corresponding BC 2F 2 plants at the gene locus were identi W ed by BC 2F 3 progeny test, and then the QTL treated as a single Mendelian factor was mapped to the linkage group. Its genetic e V ect estimated on the progeny data. Additive e V ects equal to the half of the trait value di V erence between two homozygotes, dominance e V ects equal to the trait value di V erence between heterozygote and the middle value of two homozygotesTraits Population LODA D D /A R 2 (%)SPP BC 2F 3*51.124.70.48BC 3F 256.159.828.30.4774.2YD (g)BC 2F 322.3 4.4 2.70.6143.2TPP BC 2F 332.4¡1.6¡1.00.6361.9SR (%)BC 2F 33.0¡1.6¡2.11.317.4Fig.4Genetic and physical map in the local region of qSPP7.Numbers of recombinants detected in 190 BC 2F 2 plants and 1082BC 3F 2 plants between the markers and qSPP7 were indicated in the parentheses in the top and bottom of markers, respectively. SSR mark-er physical position was given at two forms of physical distance andthe accession number of BAC clone contained the SSR marker (/Oryza_sativa_japonica/). The genetic positions of SSR markers were mined from the website http://rgp.dna.affrc.go.jp/cgi-bin/statusdb/stattable.pl?chr=7&lab=RGPTheor Appl Genetpericentromeric regions (Haupt et al. 2001), no recombina-tion suppression was reported in the pericentromeric region on rice chromosome 3 (Fan et al. 2006). Thus, it is possible to narrow down the QTL located in the pericentromeric regions to a candidate gene by map-based cloning in this study. Unfortunately, severe recombination suppression was observed in the targeted interval within pericentro-meric region (Fig.4). The recombinants between markers RM5451, RM445 and RM7110, and qSPP7 in the 1082 BC3F2 plants were about W ve-fold as those in the 190 BC2F2 plants, respectively. While, the recombinants in the 1082 BC3F2 plants decreased to four-fold as those in the 190 BC2F2 plants for markers RM3859 and C39, indicating the presence of light recombination suppression at the two marker loci. There was no recombinant in both two popula-tions for markers RM5436 and RM5499 covered a distance of more than 900kb. This indicated the presence of severe recombination suppression in the region between RM5436 and RM5499. No clear recombination suppression was observed in the regions between RM5451 and RM3859, RM1135 and RM7110. But clear recombination suppres-sion was occurred in the region between RM7110 and C39. Therefore, recombination suppression was occurred at least in the region between RM3859 and C39 although the right boarder of recombination suppression region could not be precisely de W ned due to lack of polymorphic markers in the large region between RM7110 and C39. The genetic dis-tance between RM3859 and C39 was 0.4cM based on mapping result in either 190 BC3F2 plants or 1082 extreme BC3F2 plants. RM3859 and C39 are located in the BAC clones AP004176 and AP004346, respectively. The physi-cal distance between these two clones was estimated to be as large as 2,400kb or so (/Oryza_ sativa_japonica/mapview?chr=7). The ratio of physical-to-genetic distance in this targeted region was about 6,000kb/ cM, which is 20 times higher than the estimated average ratio (250–300kb/cM) (Arumuganathan and Earle 1991). This is consistent with the ratio of 2740kb/cM, or 10 times higher than that of the rest of the genome reported by Wu et al. (2002). In tomato, it can be as high as 10- to 40-fold (Tanksley et al. 1992). In the present study, no recombi-nant was found between qSPP7 and markers in the large region of 912.4kb. Therefore, we failed to further narrow down the gene using a large population consisting of 1082 plants after qSPP7 was mapped to a 0.4-cM region using the small BC2F2 population consisted of 190 individuals. Taken together, there was no resolution di V erence between the large and small populations for QTL W ne mapping. That is to say, resolution of gene mapping could not be improved for a region with clear recombination suppression by enlarging a population even though there are lots of polymorphic markers in the region. This comes up with the conclusion that positional cloning strategy would not be feasible for isolation of the targeted gene, qSPP7.Besides GW, one QTL for YD and TPP and SR was simultaneously detected in the qSPP7 surrounding region. The QTL for SR expressed over-dominance, the QTL for other traits acted in partially dominant (Table3). These indicated that the homozygote with Minghui 63 alleles is promising to produce higher yield than does the heterozy-gote. Given the traits of yield components are highly corre-lated, ridge regression analysis was used for the phenotype data set collected from BC2F3 to build a regression equation of the form: YD=¡70.454+0.16£SPP+2.08£TPP+ 0.92£GW+39.23£SR. From the equation, the theoreti-cal YD can be easily predicted. For example, the additive and dominance e V ects of qSPP7 were estimated in BC2F3, the theoretical YD di V erence between genotypes MM and MZ at qSPP7 is 3.2g. This is well illustrated by the fact that the Minghui 63 homozygotes at qSSP7 showed larger average values in the traits than the heterozygotes in the BC2F3 progenies. Moreover, the averaged grain yield per plant of Minghui 63 homozygotes was 26.5g, which was signi W cantly higher (P=0.05) than heterozygotes of 24.2g in the BC2F3 progenies. This result was also consistent with the conclusion that heterozygotes were not always advanta-geous for F1 performance on the basis of an “immortalized F2” population produced from the same cross between Zhenshan 97 and Minghui 63 (Hua et al. 2002). Thus, the ideal genotype in the qSPP7 surrounding region in the hybrid should be Minghui 63 homozygotes for hybrid pro-duction. Markers X anking the QTL could be directly used for improvement of parent Zhenshan 97. Theoretically, the new hybrid Shanyou 63 with homozygous Minghui 63 alle-les at qSPP7 should exhibit higher YD than does its origi-nal hybrid Shanyou 63, an elite rice hybrid between Zhenshan 97 and Minghui 63 in last two decades in China. The new version Shanyou 63 is in the process of develop-ment.In summary, the QTL of qSPP7 will play a crucial role in developing high yielding rice varieties. E V orts in its iso-lation are worth being made in combination with bioinfor-matic strategy.Acknowledgments The authors thank Dr. Cheng JH for helpful lan-guage improvement. This work was supported by a grant from the Na-tional Natural Science Foundation of China (30470987), and a grant from the National Key Program on Basic Research and Development of China.ReferencesAllard RW (1956) Formulas and tables to facilitate the calculation of recombination values in heredity. Hilgardia 24:235–278 Arumuganathan K, Earle ED (1991) Nuclear DNA content of some important plant species. Plant Mol Biol Rep 9:211–215。

链格孢属真菌（Alternaria）属有丝分裂孢子真菌类群丝孢纲丝b...b-YNSTC

链格孢属真菌(Alternaria)属有丝分裂孢子真菌类群丝孢纲丝孢目，是半知菌暗色丝孢菌中一类非常常见的真菌。

链格孢属真菌是引起植物病害的重要真菌类群之一。

大部分链格孢属真菌寄生于不同种植物，特别是农作物，常引起包括玉米、小麦、马铃薯、苹果和番茄等几十种农作物的病害，而给农业造成重大经济损失。

有些链格孢属真菌是人和动物的条件致病菌，常引起多种疾病。

链格孢属真菌产生许多有毒代谢产物，包括腾毒素(tentoxin) ，链格孢毒素(altertoxins)，细交链孢菌酮酸(tenuazonic acid)，交链孢霉烯(altenuene)和交链孢酚(alternariol)等，其中有些毒素是其作用于植物的主要致病因子，通过毒素的作用不仅使植物产生症状，造成危害，而且人和动物摄入被污染的食品或饲料后可导致慢性或急性中毒。

有些毒素可用作除草剂和杀菌剂等，是有应用潜力的生物资源(Barkai-Golan, 2008)。

腾毒素是由一些链格孢属真菌产生的一个天然环四肽。

这个属的链格孢(A. alternata)，柑桔链格孢(A. citri) ，长柄链格孢(A. longipes) ，苹果链格孢(A. mali) ，葱链格孢(A. porri)和细链格孢(A. tenuis)的一些菌株已被报道产生腾毒素(Meyer, 1971; Liebermann, 1983; Kono, 1986; Suemitsu, 1990)。

从腐烂的西红柿和浓缩苹果汁中都检测到被链格孢属真菌污染而产生的腾毒素(Andersen, 2004; 何强，2010)。

腾毒素的化学结构是[cyclo-(L-MeAla-L-Leu-MePhe*(Z)∆+-Gly)] (Meyer, 1971)。

腾毒素的主要生物活性是选择性抑制一些高等植物叶绿体F1-ATP酶活性而导致植物萎黄病(Steele, 1976)。

其抑制机理是低浓度下抑制叶绿体F1-ATP酶中ATP水解和合成(Steele, 1976)，因此常用于研究F1-ATP酶的反应机制(Pavlova, 2004; Meiss, 2008)。

肾透明细胞癌舒尼替尼耐药的分子标志物筛选及相关生物学功能分析

第50卷第2期第142页2021年4月华中科技大学学报(医学版)A c t aM e dU n i vS c iT e c h n o lH u a z h o n gV o l .50 N o .2 P .142A pr . 2021*国家自然科学基金资助项目(N o .81927807)#共同第一作者刘郴郴,男,1995年生,医学硕士,E -m a i l :l i u c c 1325@126.c o m宋正帅,男,1988年生,医学博士,E -m a i l :867703966@q q.c o m ә通讯作者,C o r r e s p o n d i n g a u t h o r ,E -m a i l :x z h a n g@h u s t .e d u .c n 肾透明细胞癌舒尼替尼耐药的分子标志物筛选及相关生物学功能分析*刘郴郴1,2#, 宋正帅3#, 章小平1,2ә华中科技大学同济医学院附属协和医院1泌尿外科2泌尿外科研究所,武汉 4300223华中科技大学同济医学院附属武汉中心医院泌尿外科,武汉 430014摘要:目的通过公共数据库,利用生物信息学方法分析对舒尼替尼敏感或耐药的肾透明细胞癌(c c R C C )样本间的基因表达差异,筛选出舒尼替尼耐药相关分子并探讨介导获得性耐药的潜在信号通路和分子机制㊂方法从G E O 数据库中下载对舒尼替尼敏感或耐药的c c R C C 基因表达谱,利用G E O 2R 在线工具对不同表达水平基因进行筛选得到差异表达基因,构建差异基因的蛋白-蛋白互作网络,通过蛋白互作网络中的高连通性确定枢纽基因㊂对表达水平上调基因㊁下调基因以及枢纽基因进行分子功能㊁信号通路以及基因集富集分析(G S E A ),发掘耐药的潜在机制㊂基于T C G A 数据库,分析枢纽基因表达水平和c c R C C 患者总生存期及无病生存期之间的关系,并利用C o x 回归模型探讨靶基因和肾癌患者各种临床病理参数的相关性㊂结果筛选得到的舒尼替尼耐药样本差异表达基因中有上调基因38个,下调基因57个及10位枢纽基因,G O /K E G G 分析提示上调基因及前10位枢纽基因的生物过程富集在Ⅰ型干扰素信号通路㊁对病毒的防御反应;分子功能涉及2'-5'-o l i g o a d e n y l a t e 合成酶活性㊁双链R N A 结合㊁转移酶活性;信号通路富集在甲型流感㊁麻疹㊁单纯疱疹病毒感染和肠道免疫网络中产生I g A ;G S E A 结果显示舒尼替尼耐药组富集在复制应激㊁D N A 感应㊁干扰素激活等通路㊂生存分析表明I L 6㊁I S G 15㊁O A S L 高表达预示更差的生存期;I F I T 3㊁I S G 15㊁O A S L 的R O C 曲线下面积为0.7362~0.8914,且在肾癌组织中的表达明显上调,提示其具有潜在诊断价值,其中I S G 15的表达与c c R C C 的各种临床病理参数相关,可能促进肾癌的进展和转移㊂结论 I S G 15㊁I F I T 3㊁O A S L 是预测c c R C C 舒尼替尼耐药的潜在分子标志物,其中I S G 15的表达水平与c c R C C 的临床病理特征相关,是c c R C C 的独立预后因素,有望成为治疗c c R C C 的重要靶点㊂关键词:肾透明细胞癌; 舒尼替尼耐药; 分子标志物; 生物信息学中图分类号:R 737.11 D O I :10.3870/j.i s s n .1672-0741.2021.02.002S c r e e n i n g ofN e wB i o m a r k e r s f o r S u n i t i n i bR e s i s t a n c e i nC l e a rC e l lR e n a l C e l l C a r c i n o m a a n dA n a l y s i s o fR e l a t e dB i o l o gi c a l F u n c t i o n s L i uC h e n c h e n 1,2#,S o n g Z h e n g s h u a i 3#,Z h a n g X i a o p i n g1,2ә1D e p a r t m e n t o f U r o l o g y ,2I n s t i t u t e o f U r o l o g y ,U n i o n H o s p i t a l ,T o n g j iM e d i c a lC o l l e ge ,H u a z h o n g U n i v e r s i t y of S c i e n c e a n dT e c h n o l og y ,W u h a n430022,C h i n a 3D e p a r t m e n t o f U r o l o g y ,T h e C e n t r a lH o s p i t a l o f W u h a n ,T o n g j iM e d i c a lC o l l e ge ,H u a z h o n g U n i v e r s i t y of S c i e n c e o f T e c h n o l og y ,W u h a n430014,C h i n a A b s t r a c t O b je c t i v e T oa n a l y z et h e g e n e so fd if f e r e n t i a le x p r e s s i o nb e t w e e ns u n i t i n i b -s e n s i t i v ea n ds u n i t i n i b -r e s i s t a n t c c R C Cs a m p l e s b y b i o i n f o r m a t i c s a n a l y s i s b a s e d o n p u b l i c d a t a b a s e s a n d s c r e e n o u t r e l a t e dm o l e c u l e s t o e x p l o r e p o t e n t i a l s ig n a -l i n gp a th w a y s a n d m o l e c u l a rm e c h a ni s m s t h a tm e d i a t ea c q u i r e dd r u g r e s i s t a n c e .M e t h o d s T h e g e n ee x pr e s s i o n p r o f i l e sw e r e d o w n l o a d e d f r o mt h eG E Od a t a b a s e ,a n d t h e d i f f e r e n t i a l l y e x p r e s s e d g e n e sw e r e s c r e e n e du s i n g th eG E O 2R .T h e n t h e p r o t e i n -p r o t e i n i n t e r a c t i o nn e t w o r k so f t h ed i f f e r e n t i a l l y e x p r e s s e d g e n e sw e r ec o n s t r u c t e d ,a n dt h eh u b g e n e sw e r e i d e n t i f i e db y th e h i g hc o n n e c t i v i t y i n t h e p r o t e i n i n t e r a c t i o nn e t w o r k s .T h em o l e c u l a r f u n c t i o n ,s i g n a l i n gp a t h w a y a n d g e n e s e t e n r i c h m e n t a n a l y-s i sw e r e c a r r i e do u t f o r u p -r e g u l a t e d g e n e s ,d o w n -r e g u l a t e d g e n e s a n dh u b g e n e s t o e x p l o r e t h e p o t e n t i a lm e c h a n i s mo f d r u g re -s i s t a n c e .T h e r e l a t i o n s h i p b e t w e e nh u b g e n e e x p r e s s i o n l e v e l s a n d l if e t i m e o f c c R C C p a t i e n t sw a s a n a l yz e db a s e do n t h eT C G A d a t a b a s e .F i n a l l y ,t h e c o r r e l a t i o nb e t w e e n t h e s c r e e n e d g e n e s a n dv a r i o u s c l i n i c o p a t h o l o gi c a l f a c t o r so f c c R C C p a t i e n t sw a se x -p l o r e db y C o x r e g r e s s i o nm o d e l .R e s u l t s T h e r ew e r e 38u p -r e g u l a t e d g e n e s ,57d o w n -r e gu l a t e d g e n e s a n d10h u b g e n e s i n t h e d r u g -r e s i s t a n t s a m p l e s ,a n dG O /K E G Ga n a l y s i s i n d i c a t e d t h a t t h eu p -r e gu l a t e d g e n e s a n d t h e f i r s t 10h u b g e n e sw e r e e n r i c h e di n t h e t y p e I i n t e r f e r o n s i g n a l i n g p a t h w a y a n d t h e d e f e n s e r e s p o n s e t o t h e v i r u s.T h em o l e c u l a r f u n c t i o n s i n v o l v e d2'-5'-o l i g o a d e-n y l a t e s y n t h e t a s ea c t i v i t y,d o u b l e-s t r a n d e d R N A b i n d i n g a n dt r a n s f e r a s ea c t i v i t y.A n dt h es i g n a l i n gp a t h w a y so f t h o s e g e n e s w e r e e n r i c h e d i n i n f l u e n z aA,m e a s l e s,h e r p e s s i m p l e x i n f e c t i o n s,a n d i n t e s t i n a l i m m u n e n e t w o r k s t o p r o d u c e I g A.G S E Ar e s u l t s s h o w e d t h a tt h es u n i t i n i b-r e s i s t a n t g r o u p w a se n r i c h e di nr e p l i c a t i o ns t r e s s,D N A s e n s i n g,a n di n t e r f e r o na c t i v a t i o n p a t h-w a y s.S u r v i v a l a n a l y s i s s h o w e d t h a t h i g he x p r e s s i o n l e v e l so f I L6,I S G15a n dO A S L p r e d i c t e d p o o r e r s u r v i v a l.T h e a r e au n d e r t h eR O Cc u r v e s o f I F I T3,I S G15a n dO A S Lw e r e0.7362-0.8914,a n d t h e e x p r e s s i o n o f t h o s e g e n e sw a s s i g n i f i c a n t l y u p-r e g u-l a t e d i n c c R C Ct i s s u e s,s u g g e s t i n g t h e i r p o t e n t i a l d i a g n o s t i c v a l u e.T h e e x p r e s s i o n l e v e l o f I S G15c o r r e l a t e dw i t h v a r i o u s c l i n i c o-p a t h o l o g i c a l p a r a m e t e r s o f c c R C Ca n dm i g h t p r o m o t e c c R C C p r o g r e s s i o na n dm e t a s t a s i s.C o n c l u s i o n I S G15,I F I T3,a n dO A S L a r e e f f e c t i v e b i o m a r k e r s f o r p r e d i c t i n g s u n i t i n i b r e s i s t a n c e,i nw h i c h t h e e x p r e s s i o n l e v e l o f I S G15c o r r e l a t e sw i t h t h e c l i n i c a l a n d p a t h o l o g i c a l f e a t u r e s o f c c R C C,w h i c h i s e x p e c t e d t ob e a n i m p o r t a n t t a r g e t f o r t h e t r e a t m e n t o f c c R C C.K e y w o r d s c c R C C;s u n i t i n i b r e s i s t a n c e;b i o m a r k e r;b i o i n f o r m a t i c s肾癌是泌尿系统最常见的恶性肿瘤之一,其中又以肾透明细胞癌(c l e a r c e l l r e n a l c e l l c a r c i n o m a, c c R C C)占大多数(75%)[1-2]㊂约30%的肾癌患者在初诊时发现转移病灶以及30%的患者在行根治性肾切除术后发现复发是造成患者不良预后的主要原因[3-4]㊂目前针对进展期肾癌,随机临床试验证实靶向血管内皮生长因子(V E G F)等通路的抗血管生成药物,包括舒尼替尼㊁帕唑帕尼等,能够有效缓解疾病进展,并已得到广泛应用[5]㊂然而,目前仍缺乏能有效预测c c R C C靶向药物耐药性的分子标记物,因此,寻找和确认相应的分子标记物以预测疗效,探索耐药机制,是精准治疗的关键[1]㊂近年来,随着高通量芯片和生物信息学技术的发展,我们可以获取和分析肿瘤㊁肿瘤毗邻组织和正常组织在多种基因表达水平上的差异,从而发现肿瘤发展㊁侵袭㊁转移㊁复发以及靶向药物耐药等表型中的潜在分子机制㊂本研究基于T C G A㊁G E O等公共数据库,利用在线生物信息学工具和软件对数据进行筛选㊁分析㊁整理,得到c c R C C对舒尼替尼获得性耐药的分子标志物,为患者预后判断及治疗选择提供依据,并对其中潜在的信号通路及分子机制进行了探讨,为后续通过实验及临床数据进行进一步验证奠定了基础㊂1材料和方法1.1微阵列数据从G E O数据库中下载基因表达谱G S E76068㊂根据安捷伦G P L10558平台(I l l u m i n aH u m a n h t-12 v4.0表达芯片)提供的信息,G S E76068中共有24个样本,包括8个未处理的c c R C C样本,8个舒尼替尼敏感样本及8个舒尼替尼耐药样本㊂G S E76068中包含这24个样本的全基因组m R N A表达数据㊂1.2筛选差异表达基因(D E G s)利用基于R语言的G E O数据便捷在线分析工具G E O2R(h t t p s://w w w.n c b i.n l m.n i h.g o v/g e o/ G E O2r/),通过将 a d j.P<0.05 和 |l o g F C|>1 作为分界标准,得到舒尼替尼敏感样本和舒尼替尼耐药样本之间的D E G s㊂1.3蛋白-蛋白互作网络(P P I)及模块分析基于相应D E G s蛋白之间物理和功能联系,利用相互作用基因检索工具(S T R I N G,h t t p s:// s t r i n g-d b.o r g/c g i/n e t w o r k.p l)构建了P P I㊂同时根据最大交互数量ɤ5个,置信度ȡ0.4分的标准,用S T R I N G绘制并筛选出了10个枢纽基因㊂此外,利用分子复合体检测(M C O D E)插件,按照节点得分值=0.2,K值ȡ2,最大深度=100,在C y t o-s c a p e软件中构建了各模块的P P I网络㊂1.4D E G s和枢纽基因的G O和K E G G通路分析利用D A V I D(h t t p s://d a v i d.n c i f c r f.g o v/s u m-m a r y.j s p)数据库对D E G s和枢纽基因的基因本体(G O)和京都基因与基因组百科全书(K E G G)信号途径进行分析㊂D A V I D是一个免费开放的在线工具,为大规模的基因或蛋白质列表提供了系统全面的生物注释信息[6]㊂统计偏差的截止标准为P<0.05㊂1.5基因集富集分析(G S E A)为寻找c c R C C舒尼替尼耐药的潜在生物学功能和途径,进行了G S E A分析㊂16个c c R C C样本基因表达信息从G S E76068下载后,按舒尼替尼敏感和舒尼替尼耐药分为2组㊂选择A n n o t a t e d g e n e s e t s c2.c p.v6.2.s y m b o l s.g m t作为参考基因集,I l-l u m i n aH u m a n H T-12V4.0表达芯片作为芯片平台,并以P<0.05㊁F D R<0.25㊁基因大小ȡ100为分界标准㊂1.6枢纽基因的诊断㊁预后和临床病理价值分析从T C G A数据库中获取534例c c R C C患者总生存期(O S)和无病生存期(D F S)信息,以及患者年龄㊁性别㊁肿瘤T NM分期和G分级等临床信息(无特殊筛选标准),通过G r a p h P a dP r i s m6.0软件获得K a p l a n-M e i e r曲线,评估枢纽基因的预后价值㊂并通过受试者工作特征(R O C)曲线分析,确定这些枢纽基因对c c R C C的诊断价值㊂综合上述结果,重点评估靶基因I S G15的临床病理价值㊂㊃341㊃刘郴郴等.肾透明细胞癌舒尼替尼耐药的分子标志物筛选及相关生物学功能分析1.7统计学方法本研究采用G r a p h P a d P r i s m6.0(G r a p h P a d S o f t w a r e,I n c,U S A)和S P S S22.0(I B M S P S S,C h i-c a g o,I L)为统计分析软件,各组数据以平均值ʃ标准差表示,以P<0.05为差异具有统计学意义㊂采用非配对t检验分析c c R C C与正常肾组织之间枢纽基因表达的差异;通过M a n n-W h i t n e y检验统计目标基因和肾癌患者临床病理参数之间的联系;通过K a p l a n-M e i e r曲线和对数秩检验计算基因表达水平与患者O S㊁D F S的相关性,再结合O S和患者性别㊁年龄㊁T NM分期等变量做单因素C o x分析,并将筛选到的显著相关变量进行多因素C o x比例风险回归分析㊂2结果2.1筛选D E G s在G S E76068中,共有8个舒尼替尼敏感和8个舒尼替尼耐药的c c R C C样本㊂以P<0.05㊁|l o g F C|ȡ1为阈值,通过G E O2R分析出D E G s,其中包括表达上调基因38个,表达下调基因57个㊂2.2枢纽基因筛选与P P I网络构建根据连接度由高到低确定了10个枢纽基因,包括I L6㊁M X1㊁I S G15㊁I F I T1㊁O A S1㊁O A S2㊁I F I T3㊁I F I27㊁R S A D2㊁O A S L,其连接度依次为22㊁17㊁16㊁15㊁15㊁15㊁15㊁15㊁14㊁14㊂有趣的是,我们发现一半的枢纽基因属于干扰素相关的D N A损伤抗性标志基因(I R D S)[7]㊂根据S T R I N G查询的蛋白信息,我们分别构建了所有D E G s和前10位枢纽基因的P P I网络(图1A㊁1B);此外,C y t o s c a p e中的M C O D E插件识别出P P I网络中的前3个重要模块(图1C~1E)㊂2.3功能富集分析本研究通过D A V I D在线平台完成了G O功能和K E G G通路富集的分析㊂表1㊁2显示了D E G s的前5位基因本体类别㊂在生物过程(B P)方面,上调的D E G s 主要富集在Ⅰ型干扰素信号通路㊁对病毒的防御反应;在分子功能(M F)方面,上调的D E G s主要涉及2'-5'-o l i g o a d e n y l a t e合成酶活性㊁双链R N A结合㊁转移酶活性㊂此外,细胞成分(C C)分析显示,它们与胞质㊁线粒体有关㊂而K E G G途径主要富集在甲型流感㊁麻疹㊁单纯疱疹病毒感染和肠道免疫网络中产生I g A(表3)㊂其中图2A㊁2B给出了这些D E G s的G O和K E G G通路富集图㊂为了更全面地了解这些D E G s,我们对前10位枢纽基因进行了G O功能和K E G G通路富集㊂有趣的是如表4㊁5㊁6所示,这些枢纽基因的B P和K E G G通路富集在Ⅰ型干扰素信号通路㊁对病毒的防御反应㊁对病毒的反应㊁甲型流感㊁麻疹㊁单纯疱疹病毒感染等方面,与上调的D E G s和第1位模块基因的结果基本一致㊂A:D E G s互作网络;B:枢纽基因互作网络;C:模块1;D:模块2;E:模块3图1差异表达基因㊁枢纽基因和3个模块的蛋白-蛋白相互作用网络分析F i g.1P r o t e i n-p r o t e i n i n t e r a c t i o nn e t w o r ko f t h eD EG s,t o p10h u b g e n e s a n d3m o d u l a r a n a l y s e s㊃441㊃华中科技大学学报(医学版)2021年4月第50卷第2期表1 与c c R C C 舒尼替尼耐药相关上调表达基因G O 分析T a b l e 1 G e n e o n t o l o g y a n a l y s i s o f u p r e g u l a t e dd i f f e r e n t i a l l y e x pr e s s e d g e n e s a s s o c i a t e dw i t h s u n i t i n i b r e s i s t a n c e 类别项目数量%P 值生物过程(B P )G O :0060337~t y p e Ⅰi n t e r f e r o n s i g n a l i n gp a t h w a y 1338.23<0.01G O :0051607~d e f e n s e r e s p o n s e t ov i r u s 1441.17<0.01G O :0009615~r e s po n s e t ov i r u s 1135.29<0.01G O :0045071~n e g a t i v e r e g u l a t i o no f v i r a l g e n o m e r e pl i c a t i o n 823.52<0.01G O :0035457~c e l l u l a r r e s p o n s e t o i n t e r f e r o n -a l p h a 38.82<0.01细胞成分(C C )G O :0005829~c y t o s o l 1338.230.003G O :0005737~c y t o pl a s m 1647.050.007G O :0005739~m i t o c h o n d r i o n720.580.016G O :0005741~m i t o c h o n d r i a l o u t e rm e m b r a n e 38.820.023分子功能(M F )G O :0001730~2'-5'-o l i g o a d e n y l a t e s y n t h e t a s e a c t i v i t y38.82<0.01G O :0003725~d o u b l e -s t r a n d e dR N Ab i n d i n g 38.820.005G O :0016740~t r a n s f e r a s e a c t i v i t y 38.820.012G O :0005515~p r o t e i nb i n d i n g 2367.640.014G O :0005525~G T Pb i n d i n g411.740.030表2 与c c R C C 舒尼替尼耐药相关下调表达基因G O 分析T a b l e 2 G e n e o n t o l o g y a n a l y s i s o f d o w n r e g u l a t e dd i f f e r e n t i a l l y e x pr e s s e d g e n e s a s s o c i a t e dw i t h s u n i t i n i b r e s i s t a n c e 类别项目数量%P 值生物过程(B P )G O :0072593~r e a c t i v e o x y g e n s p e c i e sm e t a b o l i c p r o c e s s 40.06<0.01G O :0007155~c e l l a d h e s i o n 80.12<0.01G O :0006885~r e gu l a t i o no f p H 30.05<0.01G O :0033574~r e s po n s e t o t e s t o s t e r o n e 30.050.002G O :0071356~c e l l u l a r r e s p o n s e t o t u m o r n e c r o s i s f a c t o r 40.060.002细胞成分(C C )G O :0005615~e x t r a c e l l u l a r s pa c e 130.19<0.01G O :0005576~e x t r a c e l l u l a r r e g i o n 110.160.002G O :0005578~p r o t e i n a c e o u s e x t r a c e l l u l a rm a t r i x 50.070.003G O :0045121~m e m b r a n e r a f t40.060.011G O :0005577~f i b r i n o g e n c o m p l e x 20.030.020分子功能(M F )G O :0005178~i n t e g r i nb i n d i n g 60.09<0.01G O :0050840~e x t r a c e l l u l a rm a t r i xb i n d i n g 40.06<0.01G O :0008201~h e p a r i nb i n d i n g60.09<0.01G O :0001968~f i b r o n e c t i nb i n d i n g 30.040.001G O :0070051~f i b r i n o g e nb i n d i n g20.030.007表3 与c c R C C 舒尼替尼耐药相关差异表达基因K E G G 分析T a b l e 3 K E G G p a t h w a y a n a l y s i s o f d i f f e r e n t i a l l y e x pr e s s e d g e n e s a s s o c i a t e dw i t h s u n i t i n i b r e s i s t a n c e 类别项目数量%P 值表达上调基因h s a 05164:I n f l u e n z aA 514.70<0.01h s a 05162:M e a s l e s411.760.001h s a 04672:I n t e s t i n a l i m m u n e n e t w o r k f o r I g A p r o d u c t i o n 38.820.003h s a 05168:H e r p e s s i m pl e x i n f e c t i o n 411.760.004h s a 04060:C y t o k i n e -c y t o k i n e r e c e p t o r i n t e r a c t i o n 411.760.009表达下调基因h s a 05144:M a l a r i a40.06<0.01h s a 04360:A x o n g u i d a n c e 40.060.001h s a 04510:F o c a l a d h e s i o n40.060.026h s a 04668:T N Fs i g n a l i n gp a t h w a y30.040.045h s a 04380:O s t e o c l a s t d i f f e r e n t i a t i o n30.040.064㊃541㊃刘郴郴等.肾透明细胞癌舒尼替尼耐药的分子标志物筛选及相关生物学功能分析A :上调D E G s 的G O 分析;B :下调D E G s 的G O 分析;C :DE G s 的K E G G 通路分析图2 D E G s 的G O 和K E G G 通路富集分析F i g.2G Oa n dK E G G p a t h w a y s e n r i c h m e n t a n a l y s i s o fD E G s 表4 与c c R C C 舒尼替尼耐药相关枢纽基因G O 分析T a b l e 4 G e n e o n t o l o g y a n a l ys i s o f h u b g e n e s a s s o c i a t e dw i t h c c R C Cs u n i t i n i b r e s i s t a n c e 类别项目数量%P 值生物过程(B P )G O :0060337~t y p eⅠi n t e r f e r o n s i g n a l i n gp a t h w a y1317.564.78E -17G O :0051607~d e f e n s e r e s p o n s e t ov i r u s 1621.623.46E -16G O :0009615~r e s p o n s e t ov i r u s 1216.211.45E -12G O :0045071~n e g a t i v e r e g u l a t i o no f v i r a l g e n o m e r e pl i c a t i o n 912.165.91E -12G O :0006955~i m m u n e r e s p o n s e 1013.518.44E -05细胞成分(C C )G O :0005615~e x t r a c e l l u l a r s p a c e 1621.053.33E -04G O :0005576~e x t r a c e l l u l a r r e g i o n 1722.367.24E -04G O :0005829~c y t o s o l 2431.576.01E -03G O :0005739~m i t o c h o n d r i o n 1317.107.85E -03G O :0005737~c y t o p l a s m 3242.101.24E -02分子功能(M F )G O :0008201~h e p a r i nb i n d i n g 79.098.35E -05G O :0005178~i n t e g r i nb i n d i n g 67.791.10E -04G O :0001730~2'-5'-o l i g o a d e n y l a t e s y n t h e t a s e a c t i v i t y33.891.19E -04G O :0050840~e x t r a c e l l u l a rm a t r i xb i n d i n g45.192.12E -04G O :0005515~p r o t e i nb i n d i n g5267.535.49E -03表5 与c c R C C 舒尼替尼耐药相关的枢纽基因K E G G 分析T a b l e 5 K E G G p a t h w a y a n a l ys i s o f h u b g e n e s a s s o c i a t e dw i t h c c R C Cs u n i t i n i b r e s i s t a n c e 项目数量%P 值h s a 05144:M a l a r i a512.821.38E -04h s a 05164:I n f l u e n z aA 615.382.45E -03h s a 04360:A x o n g u i d a n c e 512.824.95E -03h s a 05162:M e a s l e s512.825.83E -03h s a 04060:C y t o k i n e -c y t o k i n e r e c e p t o r i n t e r a c t i o n 615.388.01E -03h s a 05323:R h e u m a t o i d a r t h r i t i s 410.251.22E -02h s a 05168:H e r p e s s i m pl e x i n f e c t i o n 512.821.74E -02h s a 04668:T N Fs i g n a l i n gp a t h w a y410.252.01E -02h s a 04672:I n t e s t i n a l i m m u n en e t w o r k f o r I gA p r o d u c t i o n 37.702.73E -02h s a 04145:P h a go s o m e 410.255.11E -02㊃641㊃华中科技大学学报(医学版) 2021年4月第50卷第2期表6 模块1K E G G 通路分析T a b l e 6 K E G G p a t h w a y a n a l y s i s o f t o p1m o d u l e 项目P 值h s a 05164:I n f l u e n z aA <0.01h s a 05160:H e p a t i t i sC 0.003h s a 05162:M e a s l e s0.003h s a 05168:H e r p e s s i m pl e x i n f e c t i o n 0.0062.4 基因集富集分析为深入了解G S E 76068样本中导致舒尼替尼耐药的潜在生物学途径和机制,我们进行了G S E A 分析㊂根据截止标准F D R<0.25,P <0.05,显示了12个功能基因组(图3)㊂该结果表明,舒尼替尼耐药组富集在胞质D N A 感知通路㊁T o l l 样受体信号通路㊁R I G -I 受体信号通路㊁干扰素信号通路㊁激活A T R 应对复制应激㊁G 2/M 检查点等㊂P a r e n t a l :舒尼替尼敏感组;S u n i t i n i b -R :舒尼替尼耐药组;以P <0.05且F D R<0.25为显著富集图3 G S E 76068样本中舒尼替尼敏感组和舒尼替尼耐药组之间的基因组富集分析F i g.3G e n e s e t e n r i c h m e n t a n a l y s i s b e t w e e ns u n i t i n i b r e s p o n s e a n d s u n i t i n i b r e s i s t a n c e g r o u p o fG S E 76068s a m p l e s 2.5 K a pl a n -M e i e r 生存分析在K a pl a n -M e i e r 分析的基础上,分析了枢纽基因的表达与c c R C C 患者O S ㊁D F S 之间的关系㊂结果显示,I L 6(H R =2.360,l o g-r a n k P <0.01)㊁I S G 15(H R =1.902,l o g -r a n k P <0.01)㊁O A S L (H R =1.842,l o g-r a n k P <0.01)的高表达预示c c R C C 患者更差的O S ;I L 6(H R =1.387,l o g-r a n k P =0.0463)㊁I S G 15(H R =1.74,l o g -r a n k P =0.0052)㊁O A S L (H R =2.195,l o g -r a n k P =0.0001)的高表达预示c c R C C 患者更差的D F S (图4㊁图5)㊂而I F I T 1的表达和更好的O S (H R =0.5598,l o g-r a n k P =0.0001)和D F S (H R =0.6368,l o g-r a n k P =0.0112)相关㊂患者总生存期信息来自T C G A 数据库;A :I F I T 1;B :I L 6;C :I S G 15;D :O A S L图4 枢纽基因对肾癌患者总生存(O S)的预后价值评估F i g.4P r o g n o s i s a s s e s s m e n t o f o v e r a l l s u r v i v a l b y H u b g e n e s ㊃741㊃刘郴郴等.肾透明细胞癌舒尼替尼耐药的分子标志物筛选及相关生物学功能分析患者无病生存期信息来自T C G A 数据库;A :I F I T 1;B :I L 6;C :I S G 15;D :O A S L图5 枢纽基因对肾癌患者无病生存期(D F S)的预后价值评估F i g.5P r o g n o s i s a s s e s s m e n t o f d i s e a s e f r e e s u r v i v a l b y h u b g e n e 2.6 枢纽基因的受试者工作特征(R O C )曲线分析我们通过R O C 曲线来评估枢纽基因是否具有潜在诊断价值㊂如图6,所示基因可以完全区分c c R C C 和配对的正常组织,曲线下面积(A U C )为0.7362~0.8914㊂此结果表明,这些枢纽基因可能是c c R C C 患者的有效诊断生物标志物㊂2.7 枢纽基因在c c R C C 组织和正常肾组织中的表达利用T C G A 数据库对c c R C C 和正常组织中枢纽基因的表达水平进行评估㊂如图6,与正常组织相比,其中部分基因如I S G 15㊁I F I T 3㊁O A S L 在c c R C C 组织中的表达明显上调㊂基于I S G 15较好的诊断和预后价值,我们进一步研究了它和c c R C C 患者临床病理参数之间的相关性㊂2.8 I S G 15的表达与c c R C C 的各种临床病理参数相关对T C G A 数据库中534例包含较完整信息的c c R C C 样本数据(其中2例缺少T ㊁N 分期,4例缺少M 分期,10例缺少G 分级信息)分析显示,I S G 15m R N A 表达上调与较高的病理分期分级㊁远处转移有显著的相关性(图7),I S G 15的表达水平随着肿瘤病理分期分级升高有增高的趋势㊂患者临床病理资料见表7,依据I S G 15表达水平降序排列,取中位均分为I S G 15高表达和低表达两组㊂I S G 15的高表达与这些临床病理参数之间有显著的相关性,与上述结果一致㊂为了进一步探讨I S G 15的预后价值,我们评估了其表达水平和患者各种临床病理因素与O S 相关性㊂单因素和多因素C o x 比例风险回归分析表明,I S G 15高表达是c c R C C 患者O S 的独立风险因素,可作为独立预后指标(表8)㊂A~C :基因表达水平分析;D ~F :基因表达R O C 曲线分析;患者基因表达水平信息均来自T C G A 数据库;**P <0.01图6 枢纽基因在c c R C C 患者与正常人肾组织表达水平对比及R O C 曲线分析F i g.6E x p r e s s i o n l e v e l a n dR O Cc u r v e a n a l y s i s o f h u b g e n e s i n c c R C C v s .n o r m a l r e n a l t i s s u e s ㊃841㊃华中科技大学学报(医学版) 2021年4月第50卷第2期A :T 分期;B :淋巴结转移;C :远处转移;D :G 分级;**P <0.01图7 I S G 15m R N A 水平与c c R C C 患者多种临床病理参数相关F i g.7T h eh i g hm R N Ae x p r e s s i o no f I S G 15w a s c o r r e l a t e dw i t hv a r i o u s c l i n i c o p a t h o l o g i c a l p a r a m e t e r s 表7 c c R C C 患者I S G 15m R N A 表达水平与临床病理参数的关系T a b l e 7 A s s o c i a t i o nb e t w e e n I S G 15m R N Ae x p r e s s i o na n d c l i n i c o p a t h o l o g i c a l pa r a m e t e r s o f p a t i e n t sw i t h c c R C C 参数例数I S G 15低表达I S G 15高表达P 值A g e (y e a r s )0.293 <60245129116 ȡ60287137150G e n d e r0.523 F e m a l e 1889098M a l e 344176168Ts t a ge <0.01 T 1o rT 2341193148 T 3o rT 419173118Ns t a ge 0.623 N 0o rN X 516259257 N 11679Ms t a g e 0.003 M 0o rM X 451239212 M 1792752G g r a d e0.019G 1o rG 2242134108 G 3o rG 4282127155表8 I S G 15m R N A 表达水平与患者总生存期的单因素和多因素回归分析T a b l e 8 U n i v a r i a t e a n dm u l t i v a r i a t e a n a l y s e s o f I S G 15m R N Ae x pr e s s i o na n d p a t i e n t o v e r a l l s u r v i v a l 变量单因素分析H R95%C I P 值多因素分析H R95%C I P 值A g e (y e a r s ) <60(n =243)1.7861.304~2.445<0.011.51041.098~2.0860.011ȡ60(n =281)G e n d e rF e m a l e (n =183)0.9420.692~1.2820.703 M a l e (n =341)Ts t a ge T 1o rT 2(n =335)3.1032.293~4.201<0.011.5731.097~2.2550.014 T 3o rT 4(n =189)Ns t a ge N 0o rN X (n =508)3.8462.082~7.104<0.012.8321.269~4.4720.007 N 1(n =16)Ms t a g e M 0o rM X (n =446)4.2923.147~5.853<0.012.5141.759~3.593<0.01 M 1(n =78)G g r a d e G 1o rG 2(n =245)2.6161.867~3.666<0.011.6941.178~2.4360.004 G 3o rG 4(n =279)I S G 15L o w (n =262)1.9541.431~2.667<0.011.3961.011~1.9290.043H i gh (n =262)㊃941㊃刘郴郴等.肾透明细胞癌舒尼替尼耐药的分子标志物筛选及相关生物学功能分析3讨论c c R C C是最常见的肾癌亚型,酪氨酸激酶抑制剂(t y r o s i n ek i n a s e i n h i b i t o r,T K I)类靶向药物,如舒尼替尼仍是进展期c c R C C治疗的基石[2,8]㊂事实证明,由于个体差异及肿瘤异质性等因素,不管放化疗㊁靶向治疗还是免疫治疗,总存在着一部分患者先天不敏感或对持续治疗耐受㊂所以,研究c c R C C舒尼替尼耐药的分子标志物和功能途径对c c R C C患者的治疗方式选择及生存预后评估具有重要意义㊂本研究利用多种生物信息学工具发现了一组具有代表意义的分子标志物,有望成为诊断甚至治疗靶点应用于临床,此外我们还分析了其中枢纽基因的诊断和预后价值,并探讨了导致c c R C C舒尼替尼耐药的潜在信号通路㊂3.1D N A感知器-Ⅰ型干扰素通路-I R D S轴可能导致c c R C C获得性耐药到目前为止,已有大量文献报道肾癌舒尼替尼耐药相关机制,包括T K I s被溶酶体螯合㊁缺氧诱导因子(H I F)等关键基因的突变和修饰㊁血管生成替代途径(A K T/m T O R)的激活㊁肿瘤异质性和微环境等,但仍缺乏共识[9]㊂在本研究中,G S E A分析表明D N A损伤反应相关基因集(激活A T R应对复制应激和G2/M检查点)得到显著富集㊂同时,根据G O/K E G G分析,我们发现生物过程(Ⅰ型干扰素信号通路㊁对病毒的防御反应)㊁信号通路(甲型流感㊁麻疹和单纯疱疹病毒感染)和基因集(干扰素信号传导)均得到富集(图2A㊁2B㊁3D),最重要的是,G S E A结果表明胞质D N A感知通路㊁T o l l样受体信号通路㊁R I G-I受体信号通路等经典D N A感应通路均被显著富集㊂据此,我们认为肿瘤细胞通过靶向药物治疗,在复制应激等因素下导致D N A损伤,D N A损伤产物被D N A感应蛋白感知并激活了干扰素信号通路(如c G A S-S T I N G信号通路)㊂在过去的几十年中,研究发现Ⅰ型干扰素(I F N-Ⅰ)来源于各种免疫细胞或肿瘤细胞,作为调节数百种下游细胞因子转录的强大免疫调节因子,参与肿瘤免疫㊁感染调控和组织损伤等过程㊂一般来说,I F N-Ⅰ和Ⅰ型干扰素途径对抑制感染和杀伤肿瘤细胞尤为重要,因此在20多年前,大剂量干扰素被用于治疗转移性肾癌,但疗效不甚理想,而且由于它的副作用使其并没有被广泛应用㊂目前,一些临床前试验已经证明了I F N-Ⅰ定向治疗肿瘤的可取性,但更多的是需要与其他疗法,如靶向治疗㊁放化疗和新型免疫疗法联合使用,才能取得更好的效果㊂然而,近期研究结果表明,Ⅰ型干扰素通路在肿瘤进展和获得性耐药中起着至关重要的作用,尤其是在长期和持续暴露于I F N-Ⅰ的情况下,干扰素相关D N A损伤抗性标志基因(I R D S)明显上调并发挥促癌促耐药作用[10-14]㊂例如J A K抑制剂R u x-o l i t i n i b可以克服非小细胞肺癌对顺铂的耐药性[15]㊂此外,c G A S/R I G-I/T L R4等D N A感应蛋白作为明星分子,在免疫调节途径中对癌症的进展和治疗具有决定性的影响,但与Ⅰ型干扰素信号通路一样, D N A感应蛋白也具有两面性㊂有研究表明,D N A 感应蛋白D D X41是肿瘤启动因子同时也是放疗的不良预后因子[16];另一项研究调查了在基因毒药物作用下,T L R4通路的激活和下游产物的上调保护了残余肿瘤细胞,并促进了肿瘤的转移[17]㊂有趣的是本研究中我们筛选的大部分枢纽基因如I S G15㊁O A S L㊁I F I T3在舒尼替尼耐药样本中上调,均属于I R D S基因,且这个独特的基因子集正是D N A感应和Ⅰ型干扰素信号通路的下游基因㊂综上所述,我们认为在舒尼替尼的作用下,c c R C C中细胞核酸的积累被D N A感应蛋白所感知,并引发了Ⅰ型干扰素通路的激活,继而导致I R D S的上调,促进了肿瘤获得性耐药㊂3.2I R D S是c c R C C获得性耐药的潜在分子标志Ⅰ型干扰素启动了数百个干扰素下游基因的表达,涉及抗病毒㊁抗肿瘤和抗炎功能[18]㊂越来越多的研究表明,在抗病毒反应的第二阶段,在持续低剂量I F Nβ刺激下,未磷酸化的转录因子I S G F3(U-I S G F3)高表达,该转录因子进一步介导一个特殊的干扰素刺激基因子集(I S G s)上调,发挥抵抗D N A 损伤和延长抗病毒效果的作用[7,12,19-22]㊂耐人寻味的是,正是该基因子集中的一部分驱动了放化疗的获得性耐受,并被统称为干扰素相关D N A损伤耐药基因(I R D S),且已经作为预测乳腺癌㊁头颈部肿瘤等恶性肿瘤的放化疗预后因子应用于临床[7,10,12,23]㊂到目前为止,已经发现了49个以上的基因属于I R D S基因㊂从我们富集的生物信息学结果来看, I R D S基因在舒尼替尼耐药组中显著上调㊂如前所述,越来越多的证据表明,细胞D N A损伤产物被c G A S/R I G-I/T L R4等感应蛋白感知,然后激活干扰素通路,促进I R D S的表达[14,24-26]㊂随后,该轴的激活所引发的I R D S上调通过D N A损伤修复和其他分子机制促进了肿瘤的生存[13,27]㊂因此我们有理由相信该过程同时促进了c c R C C的获得性耐药,㊃051㊃华中科技大学学报(医学版)2021年4月第50卷第2期但还需要更多的实验证据㊂我们的研究为I R D S基因I S G15㊁O A S L㊁I F I T3可作为肾癌获得性耐药的有效分子标志物及预后指标提供了线索㊂3.3I S G15是c c R C C潜在的诊断和预后标志物I S G15,即干扰素刺激基因15,1987年作为泛素样蛋白(U b l s)被发现,在各种细胞活动如稳定蛋白㊁调节细胞周期㊁应激反应和信号转导中发挥了重要作用,类似于泛素化,其与蛋白结合的方式被称为I S G y l a t i o n,是未被充分开发的翻译后蛋白修饰途径(P T M s)之一㊂几十年来,对I S G15的研究一直集中在抗病毒复制方面㊂直到最近,研究人员发现I S G15在乳腺癌㊁前列腺癌和胰腺癌等肿瘤发病㊁进展和耐药中发挥了重要作用[7,13,28-30]㊂本研究首次探讨了I S G15在c c R C C中的表达和临床病理参数之间的联系㊂我们发现,与正常肾组织相比,I S G15在c c R C C组织中表达上调,并与c c R C C患者的不良预后相关;多变量回归分析表明,I S G15表达水平是c c R C C的独立预后因素㊂基于此,我们认为I S G15能作为一个潜在的分子标志物用于c c R C C 患者的诊断和预后判断㊂参考文献[1] D iM a r t i n oS,D eL u c aG,G r a s s iL,e ta l.R e n a l c a n c e r:n e wm o d e l s a n d a p p r o a c h f o r p e r s o n a l i z i n g t h e r a p y[J].JE x p C l i nC a n c e rR e s,2018,37(1):217.[2]S i e g e lR L,M i l l e rK D,J e m a lA.C a n c e r s t a t i s t i c s,2019[J].C AC a n c e r JC l i n,2019,69(1):7-34.[3]M o t z e rRJ,B u k o w s k iR M,F i g l i nR A,e t a l.P r o g n o s t i cn o-m o g r a mf o rs u n i t i n i bi n p a t i e n t s w i t h m e t a s t a t i cr e n a lc e l lc a r c i n o m a[J].C a n c e r,2008,113(7):1552-1558.[4]N e r i c hV,H u g u e sM,P a i l l a rMJ,e t a l.C l i n i c a l i m p a c t o f t a r-g e t e d t h e r a p i e s i n p a t i e n t sw i t hm e t a s t a t i c c l e a r-c e l l r e n a l c e l lc a r c i n o m a[J].O n c oT a r g e t sT h e r,2014,7:365-374.[5]M o t z e rRJ,M c c a n nL,D e e nK.P a z o p a n i bv e r s u s s u n i t i n i b i nr e n a l c a n c e r[J].N e wE n g l JM e d,2013,369(20):1968-1970.[6]K a n e h i s a M,G o t oS,H a t t o r iM,e t a l.F r o m g e n o m i c s t oc h-e m i c a l g e n o m i c s:n e w d e v e l o p m e n t si n K E G G[J].N u c l e i cA c i d sR e s,2006,34(D a t a b a s e i s s u e):D354-D357.[7]W e i c h s e l b a u mRR,I s h w a r a nH,Y o o nT,e t a l.A n i n t e r f e r o n-r e l a t e d g e n e s i g n a t u r e f o rD N Ad a m a g e r e s i s t a n c e i s a p r e d i c-t i v em a r k e r f o r c h e m o t h e r a p y a n dr a d i a t i o n f o rb r e a s t c a n c e r[J].P N A S,2008,105(47):18490-18495.[8]A t k i n sM B,T a n n i rN M.C u r r e n t a n d e m e r g i n g t h e r a p i e s f o rf i r s t-l i n e t r e a t m e n t o fm e t a s t a t i c c l e a r c e l l r e n a l c e l l c a r c i n o m a[J].C a n c e rT r e a tR e v,2018,70:127-137.[9]M a k h o vP,J o s h i S,G h a t a l i aP,e ta l.R e s i s t a n c et os y s t e m i ct h e r a p i e s i nc l e a rc e l lr e n a lc e l lc a r c i n o m a:m e c h a n i s m sa n dm a n a g e m e n t s t r a t e g i e s[J].M o lC a n c e rT h e r,2018,17(7):1355-1364.[10] K h o d a r e vN N,B e c k e t tM,L a b a y E,e t a l.S T A T1i so v e r e x-p r e s s e d i n t u m o r s s e l e c t e d f o r r a d i o r e s i s t a n c e a n d c o n f e r s p r o-t e c t i o n f r o mr a d i a t i o n i n t r a n s d u c e d s e n s i t i v e c e l l s[J].P N A S,2004,101(6):1714-1719.[11] K h o d a r v eNN,M i n nAJ,E f i m o v aEV,e t a l.S i g n a l t r a n s d u c-e r a n d a c t i v a t o r of t r a n s c r i p t i o n1r eg u l a t e s b o th c y t o t o xi c a n dp r o s u r v i v a l f u n c t i o n s i n t u m o r c e l l s[J].C a n c e rR e s,2007,67(19):9214-9220.[12] D u a r t eC W,W i l l e y CD,Z h i D,e t a l.E x p r e s s i o ns i g n a t u r eo fI F N/S T A T1s i g n a l i n gg e n e s p r e d i c t s p o o rs u r v i v a lo u t c o m ei n g l i o b l a s t o m am u l t i f o r m e i nas u b t y p e-s p e c i f i cm a n n e r[J].P L o SO n e,2012,7(1):e29653.[13] B o e l e n s M C,W u TJ,N a b e tB Y,e ta l.E x o s o m et r a n s f e rf r o ms t r o m a l t ob r e a s t c a n c e rc e l l sr eg u l a t e s th e r a p y r e si s t-a n c e p a t h w a y s[J].C e l l,2014,159(3):499-513.[14] B u d h w a n iM,M a z z i e r i R,D o l c e t t i R.P l a s t i c i t y o f t y p e I i n t e r-f e r o n-m e d i a t e dr e s p o n s e s i nc a n c e rt h e r a p y:f r o m a n t i-t u m o ri m m u n i t y t o r e s i s t a n c e[J].F r o n tO n c o l,2018,8:322.[15]H u Y,H o n g Y,X u Y,e ta l.I n h i b i t i o no f t h eJ A K/S T A Tp a t h w a y w i t h r u x o l i t i n i b o v e r c o m e sc i s p l a t i n r e s i s t a n c ei nn o n-s m a l l-c e l l l u n g c a n c e r N S C L C[J].A p o p t o s i s,2014,19(11):1627-1636.[16] L a e n g l e J,S t i f t J,B i l e c zA,e t a l.D N Ad a m a g e p r e d i c t s p r o g-n o s i s a n d t r e a t m e n t r e s p o n s e i n c o l o r e c t a l l i v e rm e t a s t a s e s s u-p e r i o r t o i m m u n o g e n i cc e l ld e a t ha n d Tc e l l s[J].T h e r a n o s-t i c s,2018,8(12):3198-3213.[17] R a nS.T h e r o l eo fT L R4i nc h e m o t h e r a p y-d r i v e n m e t a s t a s i s[J].C a n c e rR e s,2015,75(12):2405-2410.[18] C h e o n H,H o l v e y-b a t e sE G,S c h o g g i n sJ W,e ta l.I F Nβ-d e-p e n d e n t i n c r e a s e s i nS T A T1,S T A T2,a n dI R F9m e d i a t er e-s i s t a n c e t ov i r u s e sa n d D N A d a m a g e[J].E M B OJ,2013,32(20):2751-2763.[19] W a n g W,Y i nY,X uL,e t a l.U n p h o s p h o r y l a t e d I S G F3d r i v e sc o n s t i t u t i v e e x p r e s s i o no f i n t e r f e r o n-s t i m u l a t ed ge n e s t o p r o-t e c t a g a i n s tv i r a l i n f e c t i o n s[J].S c iS i g n a l,2017,10(476):e a a h4248.[20] C h e o nH,H o l v e y-b a t e sEG,S t a r kGR.A b s t r a c t485:C h r o n-i c e x p o s u r et oal o w d o s eo f i n t e r f e r o n-βi n c r e a s e s l e v e l so fD N Ad a m a g e r e s i s t a n c e s i g n a t u r e g e n e s t h r o u g hu n p h o s p h o-r y l a t e d I S G F3[J].C a n c e rR e s,2014,74(19S u p p l):485.[21] C h e o nH,S t a r kG R.U n p h o s p h o r y l a t e dS T A T1p r o l o n g s t h ee x p r e s s i o n o fi n t e rf e r o n-i n d u c e di m m u n e r eg u l a t o r y g e n e s[J].P N A S,2009,106(23):9373-9378.[22] K h o d a r e vN N,R o i z m a nB,W e i c h s e l b a u m RR.M o l e c u l a r p a-t h w a y s:i n t e r f e r o n/s t a t1p a t h w a y:r o l e i n t h e t u m o r r e s i s t a n c e t o g e n o t o x i cs t r e s sa n da g g r e s s i v e g r o w t h[J].C l i n C a n c e rR e s,2012,18(11):3015-3021.[23] C h e o n H,Y a n g J,S t a r kG R.T h e f u n c t i o n so f s i g n a l t r a n s-d u ce r s a n d a c t i v a t o r s of t r a n s c r i p t i o n s1a n d3a s c y t o k i n e-i n-d u c i b le p r o t e i n s[J].J I n t e r fC y t o kR e s,2011,31(1):33-40.[24] L i u H,G o l j i J,B r o d e u rL K,e ta l.T u m o r-d e r i v e dI F Nt r i g-g e r sc h r o n i c p a t h w a y a g o n i s ma n ds e n s i t i v i t y t oA D A Rl o s s[J].N a tM e d,2018,25(1):95-102.[25] B h a t N,F i t z g e r a l d K A.R e c o g n i t i o no fc y t o s o l i c D N A b yc G A Sa n do t h e r S T I N G-de p e n d e n t s e n s o r s[J].E u r J I m m u-n o l,2014,44(3):634-640.[26] B r o n t eV.T u m o r sS T I N Ga d a p t i v ea n t i t u m o r i m m u n i t y[J].I m m u n i t y,2014,41(5):679-681.[27]S u nN K,H u a n g SL,C h a n g TC,e t a l.T L R4a n dN F k a p p a Bs i g n a l i n g i sc r i t i c a l f o rt a x o l r e s i s t a n c e i no v a r i a nc a r c i n o m ac e l l s[J].JC e l l P h y s i o l,2018,233(3):2489-2501.[28] D e s a i SD,R e e dRE,B u r k s J,e t a l.I S G15d i s r u p t s c y t o s k e l e-t a l a r c h i t e c t u r e a n d p r o m o t e sm o t i l i t y i nh u m a nb r e a s t c a n c e rc e l l s[J].E x p B i o lM e d(M a y w o o d),2012,237(1):38-49.[29] K i e s s l i n g A,H o g r e f eC,E r bS,e ta l.E x p r e s s i o n,r e g u l a t i o na n d f u n c t i o no f t h e I S G y l a t i o ns y s t e mi n p r o s t a t e c a n c e r[J].O n c o g e n e,2009,28(28):2606-2620.[30]A g a r w a lS,M c g o w e n K,J a n s s o n K,e ta l.A b s t r a c t2896:S T A T1d e p e n d e n t i n t e r f e r o n-r e l a t e dD N Ad a m a g e r e s i s t a n c es i g n a t u r e(I R D S)a s a s u r v i v a lm e c h a n i s mi nc a s t r a t e r e s i s t a n tp r o s t a t e c a n c e r(C R P C)[J].C a n c e rR e s,2017,77(13S u p p l): 2896.(2020-10-27收稿)㊃151㊃刘郴郴等.肾透明细胞癌舒尼替尼耐药的分子标志物筛选及相关生物学功能分析。

合成生物学相关文献(免费共享)

合成生物学相关文献（免费共享）摘要：通过将组成生物系统的各类单元模块化、标准化，合成生物学希望达成一种新的生物技术发展模式：即从主要开发里欧那个天然生物系统既有功能，变为用人工设计合成的生物系统来完成天然系统不能完成或者完成效率低的功能。

合成生物学通过开展生物元件或者器件、生物途径等多个层次的工程化研究来实现上述目标。

◆综述:1.Boyle PM,Silver PA.2009. Harnessing nature’s toolbox: regulatory elements forsynthetic biology. J R Soc Interface, doi；10.1098.rsif.f8.0521.focus2.McArthur IV GH，Fong SS.2010. Toward engineering synthetic microbialmetabolism. J Biomed Biotechnol,doi:10.1155/2010/459760。

综述了元器件工程（components engineering）、和途径工程（pathway engineering）的进展。

3.Andrianantoandro E,Basu S,Karig D,et al.2006.Synthetic biology:new engineeringrules for an emerging discipline. Mol Syst Biol,2:14-27。

◆合成生物学元器件工程：利用不同调控机制的人工调控器件:4.Boyle PM,Silver PA.2009. Harnessing nature’s toolbox: regulatory elements forsynthetic biology. J R Soc Interface, doi；10.1098.rsif.f8.0521.focus。

生物学基本名词详细解释(中英文对照)

生物学基本名词详细解释（中英文对照）/english/speciality/5091.html组织相容性Histocompatibility字面上讲是指不同组织共存的能力；严格地讲是指所有移植蛋白的一致性，这是阻止移植和器官排斥的需要。

组织相容性的分子基础是修饰几乎所有人类细胞表面的一套移植蛋白。

这些蛋白是由位于6号染色体上的一段称为主要组织相溶性复合体基因，MHC编码的。

这些蛋白高度多态。

例如，它们在不同的人中显示差异。

尽管很多人会有一些相同的MHC分子，极少数人有完全相同的MHC分子。

微小的差别导致这些蛋白质被移植受体的免疫系统识别为外来的而进行破坏。

对成功的移植来说这些蛋白质应该在供体和受体之间相匹配。

双胞胎相配的几率最高，接下来是兄弟姐妹。

在一般人群中只有10万分之一的比例是MHC匹配的，可以允许移植。

Literally, the ability of different tissues to “get along”; strictly, identity in all of the transplantation proteins, which is a requirement for the prevention of graft or organ rejection. The molecular basis of histocompatibility is a set of transplantation proteins that decorate the surface of nearly all human cells. These proteins are encoded by genes that are grouped on a part of chromosome 6 called the major histocompatibility complex, or MHC. These proteins are highly “polymorphic” i.e., they show variation in different individuals. Although many in dividuals may share some identical MHC molecules, a very low number share all the MHC molecules. The consequence of these minor differences is that these proteins are recognized by the transplant recipient’s immune system as being foreign, and so are targe ted for destruction (since the immune system’s job is to eradicate any foreign proteins or cells that invade the body). For successful transplantation these proteins ideally should be matched between donor and recipient. Twins have the highest rate of matc h, followed by siblings. In the general population only 1 in 100,000 individuals is sufficiently “MHC matched” to another person to allow transplantation.X射线结晶学X-ray Crystallography阐述蛋白质、DNA或其它生物分子的原子水平的三维结构的技术。

2022年考研考博-考博英语-南京师范大学考试全真模拟易错、难点剖析AB卷(带答案)试题号：91

2022年考研考博-考博英语-南京师范大学考试全真模拟易错、难点剖析AB卷（带答案）一.综合题(共15题)1.单选题The scientific and medical communities are very excited about the chances genetic research provides for getting rid of disease and prolonging human life. But those communities and policy makers also are careful about the scientific door they are opening as the project uncovers the mysteries of life.For the last few years, the genetic advances in the developing field of biotechnology have provides material for all kinds of work, but the developments of modern science in unlocking the secrets of the human genetic code have opened a world of possibilities for human health, as well as for the popular imagination.While European and Japanese researchers are making rapid progress in decoding human DNA, the leading organization for genetic research is in the United States, which began in 1990, is “unlocking the code”of the human body to learn how to defeat fatal diseases. Already, the Human Genome Project has become widely known and praised for finding the genes connected with terrible diseases as yet and making progress toward separating the genes that show a sign of breast cancer or AIDS.Once these genes are found and studied, researchers can develop new ways to attack infections, and genetic diseases. Medical companies are very interested in mapping the human genome, as they expect to develop a lot of new drugs for these illnesses.1. Why did the scientists work hard at mapping the human genome?2. Which country studied the genes most rapidly in the world?3. Which of the following is NOT true?4. The author suggests that the Human Genome Project can cause（）.5. The main idea of this article is about（）.问题1选项A.Because the human genome can destroy many illnesses.B.Because the human genome’s completion can help them get rid of many diseases.C.Because they wanted to be better known than others.D.Because the human genome can provide a lot of chances of work. 问题2选项A.Japan.B.Germany.C.The United States.D.China.问题3选项A.If the genes can be found, scientists can study many new ways to cure illnesses.B.The scientists have made great progress in connecting the genes with the cancers.C.Many medical companies show great interest in drawing the human genome map.D.The United States began the Genes Study early in the 19th century.问题4选项A.the policy makers to feel very worried and carefulB.the scientists to work harderC.many people to find work easilyD.a lot of companies to produce many new drugs问题5选项A.unlocking genetic codeB.the genes’ discoveryC.the great human genomeD.the genes and the scientists【答案】第1题:B第2题:C第3题:D第4题:B第5题:A【解析】1.推理判断题。

微生物次级代谢产物合成基因簇预测分析

18
Analysis tools of antiSMASH
NCBI BLAST+ HMMer 3, Muscle 3 Glimmer 3 FastTree
TreeGraph 2 Indigo-depict PySVG JQuery SVG
19
Pipeline for genomic analysis of secondary metabolites
PKS PKS PKS (neg.)
PKS PKS PKS
PKS_KS PKS_AT fabH
ene_KS mod_KS t2clf
SMART SMART This study
Yadav et al. (2009) Yadav et al. (2009) This study
Terpene Terpene ……….
gene prediction by Glimmer3 (prokaryotic data) or GlimmerHMM
(eukaryotic data) Transform the predicted results to EMBL format
21
Detection of gene clusters
nonribosomal peptides 非核糖体肽(NRP) bacteriocins 细菌素 aminocoumarins 基香豆素
butyrolactones 丁内酯
terpenes 萜烯 beta-lactams β-内酰胺 siderophores 铁载体
indoles 吲哚类
lantibiotics 羊毛硫抗生素
22
NRPS/PKS domain architecture analysis

医学遗传学名词解释

医学遗传学名词解释1.遗传印记（genetic imprinting）：来自父母双方的同源染色体或等位基因存在着功能上的差异；同一染色体或基因由不同亲体传给后代时可引起不同的表型，这种遗传现象叫做基因组印记。

2.罗伯逊易位（Robersonian translocation）：近端着丝粒染色体在着丝粒处发生断裂，在着丝粒处重接，这种易位方式成为罗伯逊易位，也称着丝粒融合3.遗传平衡定律（Hardy-Weinberg equilibrium law）：在一定条件下，（1）在一个很大的群体；（2）随机婚配而非选择性婚配；（3）没有自然选择；（4）没有突变发生；（5）没有大规模迁移。

群体的基因频率和基因型频率在一代一代繁殖传代中保持不变4.单亲二体（uniparental disomy）：单亲二体指来自父母一方的染色体片段被另一方的同源部分取代，或一个个体的两条同源染色体都来自同一亲体，前者成为节段性单亲二体。

5.微缺失综合征（microdeletion syndrome）：染色体缺失几个基因，该片段不能用传统细胞遗传学方法检测到，故称微缺失，表型取决于多个基因的剂量效应而不是一个基因的点突变，也称为连续基因缺失综合征。

6. 动态突变（dynamic mutation）：三核苷酸重复拷贝数在正常情况下有一定的变异范围，而扩展时就表现为疾病，这种突变不稳定，其拷贝数与病情正相关。

例如;亨廷顿舞蹈症 HD为（CAG）n 扩增；7.遗传咨询（genetic counseling）：由咨询医师及咨询者即患者及其家属就某种遗传病在一个家庭中发生复发风险和防治上所面临的问题进行讨论，是患者及其家属了解病情且在医师帮助下采取相应措施。

8.修饰基因（modifier gene）：某些基因对某种遗传性状并无直接影响，但可以加强或减弱与该遗传性状有关的主要基因的作用。

具有此种作用的基因即为修饰基因。

9.交叉遗传（cross inheritance）：在X连锁遗传中，男性的致病基因来至于母亲，将来只传给女儿，不存在从男性到男性的传递。

生态学专业术语英汉对照2

Ecology 生态学individuals 个体population 种群communities 群落ecosystems 生态系统behavioral ecology 行为生态学physiological ecology 生理生态学evolutionary ecology 进化生态学molecular ecology 分子生态学fitness 适合度natural selection 自然选择adaptation 适应genotype 基因型phenotype 表型phenotypic plasticity 表型可塑性offspring 后代genes 基因nongenetic factors 非遗传因素not inherited 不遗传conditions 条件resources 资源environmental variation 环境变异internal regulation 内调节homeostasis 稳态negative feedback 负反馈tolerance 耐受性temperature 温度not depletable 不能耗掉solar radiation 太阳辐射decouple 退耦niche 生态位habitat 栖息地multidimensional niche space 多维生态位空间Fundamental niche基础生态位Realized niche 实际生态位Prey 猎物Foraging 觅食Dimension 轴或维Global wind pattern 地球的风型The circulation of oceans 洋流Rain 降雨Havoc['hævək] 灾害Hurricane 飓风Latitude 纬度Irradiance [i'reidiəns,-si]辐射度Summer solstice 夏至Winter solstice 冬至Adiabatic cooling 绝热冷却Scale 尺度Coriolis effect 科里奥利效应Intertropical convergence zone热带辐合带Jet streams 急流Albedo 反照率Gulf stream 墨西哥湾流Lee of a continent 背风面Upwelling 上涌流Adiabatic lapse rate 绝热温度递减率Inversion 逆温Heat of condensation 凝结热Heat 热Temperature profiles温度剖面Relative humidity 相对湿度Saturated water 饱和水water vapor 水蒸汽microclimate 微气候thermal['θə:məl]conductivity 热传导chemical properties of water 水的化学特性penetration of light through water光线穿透水Energy transfer and water phases能量转化和水相Deplete 耗竭Ions 离子Electropositive 正电性的Electronegative 负电性的Beer’s law 比尔定律Heat capacity 热容量Maximum density 最大密度Latent heat of vaporization增发潜热Heat of fusion 溶解热Sublimation 升华Soil water 土壤水Field capacity 田间持水量The uptake of water by roots根对水的吸收Aquatic plants 水生植物Water availability 水的可利用性Plant productivity 植物生产力Permanent wilting point 永久萎焉点Potential evapotranspiration rate 潜在蒸发蒸腾速率Capillary pores 毛细管孔隙Resource depletion zone 资源枯竭区Halophytes 盐生植物Water balance in fish 鱼类的水平衡Amphibians 两栖类Water conservation by terrestrial animals 陆生动物的水保持Mammalian 哺乳动物Kidneys 肾脏Bladder 膀胱Beavers 河狸Osmoregulation 渗透调节Countercurrent exchange 逆流调节Hypertonic 高渗的Homeotherms 恒温动物Poikilotherms 变温动物Ectotherms 外温动物Endotherms 内温动物Temperature thresholds 温度阀Mechanisms 机理Enzyme 酶The thermoneutral zone 热中性区Dehydration 脱水Rates of development and growth发育和生长速度Acclimation and acclimatization 驯化和气候驯化Developmental threshold Temperature 发育温度阀Physiological time 生理时间Vernalization 春化Species distribution 物种分布Evolved response 进化反应Mean temperature 平均温度Isotherm 等温线Radiant energy 辐射能Photosynthesis 光合作用Efficiency of radiant energy conversion 辐射能的转换效率Changes in the intensity of radiation 辐射强度的变化Strategic and tactical response of plants to radiation 植物对辐射的战略和战术响应Compensation point 补偿点Photosynthetically active radiation （PAR）光和活性辐射Efficiency of Photosynthesis 光合作用效率Photosynthetic capacity 光合能力Diurnal and annual rhythms of solar radiation 太阳辐射日节律和年节律Resource depletion zone 资源耗竭带Strategic difference 战略差异Tactical response 战术响应Transpiration 蒸腾Net assimilation 净同化量Nutrient sources 营养物资源Nutrient budgets 营养预算Terrestrial communities 陆地群落Aquatic communities 水生群落Geochemistry 地球化学Global biogeochemical cycles 全球生物地球循环Mechanical weathering 机械风化Chemical weathering 化学腐蚀Wetfall 湿降落Dryfall 干降落Rainout component 雨水冲失成分Washout component 水冲失成分Streamflow 溪流Denitrification 脱氮Endorheic内陆湖泊Biogeochemistry 生物地球化学Hydrosphere carbon 水圈的碳Weathering 风化作用Nitrogen cycle 氮循环Phosphorus 氮Sediment 沉积型Lithospheric 岩石圈Sulfur 硫The fate of matter in the community 群落中物质的命运Producers 生产者Consumers 消费者Decomposers 分解者Autotrophs 自养生物Grazing mammals 草食哺乳动物Phytoplankton 浮游植物Zooplankton 浮游动物Bacteria 细菌Fungi 真菌Nonliving 无生命Food chains 食物链Primary and secondary production 初级和次级生产力Net Primary production 净初级生产力Aphotic zone 无光区Photic zone 透光区Primary consumers 初级消费者Secondary consumer 次级消费者Soil formation 土壤形成The soil profile 土壤剖面Primary classification：the great soil groups 主要分类：大土壤群Higher vegetation 高等植物Dynamic mixture 动态混合物Organic matter 有机质Cells 细胞Pedology 土壤学Subsoil 亚土壤Mineral soil 矿物质土壤Parent material 母质Soil series 土系Soil surveyor 土壤勘测员Succession 演替Ecosystem patterns 生态系统格局Soil horizons 土层Humic acids 腐植酸Great soil groups 土壤群Population size 种群大小Age and stage structure 年龄和时期结构Zygote 受精卵Unitary organism 单体生物Modular organism 构件生物Ramets 无性系分株Clone 无性系Genet 基株Evolutionary individuals 进化个体Immediate ecological impact 直接生态作用Stable age distribution 稳定年龄分布Age pyramid 年龄金字塔Stationary age distribution 固定的年龄分布Stage structure 时期结构Sizes classes 个体大小群Natality 出生率Mortality 死亡率Survivorship 存活率Life tables 生命表K-factor analysis k-因子分析The fecundity schedule 生殖力表Population growth 种群增长Density-independent Population growth 非密度制约性种群增长Density-dependent growth-the logistic equation 密度制约性种群增长：逻辑斯缔方程Life expectancy 生命期望Survivorship curve 存活曲线Cohort 同生群Age-specific survival rate 特定年龄存活率Key factors 关键因子Killing factor 致死因子Basic reproduction rate 基础繁殖率Carrying capacity 环境容纳量Estimating density 估计密度Mark release recapture 标记重捕法Density dependence密度制约Equilibrium population density平衡种群密度Relative density相对密度Allee effect阿利效应Exactly compensating准确补偿Undercompensating补偿不足Overcompensating过度补偿H4Population fluctuations 种群波动Chaos 混沌Expanding and contracting populations 增长种群和收缩种群Stable limit cycle 稳定极限环I1Competition 竞争Predation 捕食Parasitism 寄生Mutualism互利共生Intraspecific competition种内竞争Interspecific competition种间竞争Exploitation competition利用性竞争Interference competition干扰性竞争Cannibalism 自相残杀Altruism 利他主义Commensualism 偏利共生Amensualism偏害共生I2Dispersal扩散Territoriality领域性Niche shift生态位转移Allelopathy异株克生Competive asymmetry 竞争不对称Scramble competition争夺竞争Contest competition格斗竞争Zero net growth isocline零增长等斜线Self-thinning自疏Inbreeding近亲繁殖Reproductive value繁殖价值Leks 求偶场Competitive exclusion 竞争排斥Limiting similarity 极限相似性Competitive release 极限释放Character displacement 性状替换Apparent competition 表观竞争Enemy-free space 无敌空间Highly heterogeneous 高度异质性Gaps 断层Probability refuge 隐蔽机率J1Herbivores 食草动物Carnivores 食肉动物Omnivores 杂食动物Chemical defences 化学防御Behavioral strategies 行为对策Specialists 特化种Generalist 泛化种Monophagous单食者Oligophagous寡食者Polyphagous 多食者Parasites 寄生者J2Predator switching 捕食者转换Profitability of prey 猎物收益率Plant defence 植物防御The ideal free distribution 理想自由分布Functional response 功能反应Superpredation 超捕食K1Parasites 寄生物Modes of transmission 传播方式Social parasites 社会性寄生物Helminth worms 寄生蠕虫Insects 昆虫Necrotrophs 食尸动物Parasitoids 拟寄生物The cellular immune response 细胞免疫反应Vectors 媒介Optimal habitat use 最佳生境利用Brood parasitism 窝寄生Evolutionary constraint 进化约束Immunity 免疫Cevolution协同进化Gene for gene 基因对基因Mimics 模仿Herd immunity 群体免疫Antigenic stability 抗原稳定L1Pollination 传粉Symbiotic 共生性Obligate 专性Lichens 地衣Outcrossing 异型杂交Mitochondria 线粒体Chloroplasts 叶绿体M1Reproductive values 生殖价Hypothetical organism 假定生物Migration 迁移Senescence衰老Diapause 滞育Dormancy 休眠Longevity 寿命Enormous variation 巨大变异Energy allocation 能量分配Semelparity 单次生殖Iteroparity 多次生殖Carrying capacity 容纳量Current/future reproduction当前/未来繁殖Habitat disturbance 环境干扰The current/future reproductive output 当前/未来繁殖输出A high/low cost of reproduction 高/低繁殖付出Seed bank 种子库Torper蛰伏Hibernation 冬眠Cryptobiosis 隐生现象Aestivation 夏眠Migration 迁徙Morphological forms 形态学性状Generations世代Mechanistic level 机制水平Cooperation 合作Grouping-benefits 集群-好处Altruism 利他行为Group defens e 群防御Inclusive fitness 广义适合度Eusociality 真社会性Hymenoptera 膜翅目Haplodiploid 单倍二倍体Venomous sting毒刺N2Sex 性The costs of inbreeding 近交的代价Self-fertilization 自体受精Sexual versus asexual reproduction 有性和无性生殖Sex ratio 交配体制Monogyny 单配制Polygyny 一雄多雌制Polyandry 一雌多雄制Inbreeding depression 近交衰退Hermaphrodite 雌雄同体Recombine 重组Rare type advantage 稀少型有利Equal investment 相等投入Local mate competition局域交配竞争Epigamic 诱惑性Intrasexual selection 性内选择Intersexual selection 性间选择O1Alleles 等位基因Polymorphism 多型Genetic drift 遗传漂变Genetic bottleneck 遗传瓶颈Rare species 稀有物种Extinction 灭绝Chromosome染色体Genotype 遗传型Phenotype 表现型Gene pool 基因库Gel electrophoresis 凝胶电泳O2Gene flow 基因流Differentiation 分化Sibling species 姊妹种Genetic revolution 遗传演变Peripheral isolates 边缘隔离PTransfer efficiencies 转换效率（net）primary productivity (净)初级生产力Respiratory heat 呼吸热Grazer system 牧食者系统Food chains 食物链Pathways of nutrient flow营养物流Food webs 食物网QCommunity structure 群落结构Community boundaries 群落边界Guilds同资源种团Community organization 群落组织Species diversity 物种多样性Energy flow 能量流Superorganism 超有机体Species-poor/rich 物种贫乏/丰富Biomass stability 生物量稳定性Tundra 冻原Island biogeography 岛屿生物地理学Turnover rate 周转率Source of colonists 移植者源Relaxation松弛Edgespecies 边缘物种Interior species 内部物种Corridor 走廊Greenways 绿色通道Community assembly群落集合Grazers 食草动物Carnivores 食肉动物Keystone species 关键物种Dominance control 优势控制Habitat affinity生境亲和力Prey switching 猎物转换RSuccession 演替Climax Community 顶级群落Pioneer species 先锋物种Primary succession 原生演替Alluvial deposit 冲积层Secondary succession 次生演替Acidifying effect 酸化作用Opportunistic机会主义Cellulose 植物纤维素Lignin 木质素Resource ratio hypothesis 资源比假说Fluctuations 波动Cyclic succession 循环演替Disturbance 干扰Patch dynamics板块动态Mini-succession 微型演替Cambium 形成层Neotropical forest 新热带雨林Priority effect 优先效应SVegetation 植被Ecotones 群落交错区Climate map 气候图Biomes 生物群系Heat budget 热量预算Zonation 分带Grassland 草地Primary regions 基本区域Desertification 荒漠化Arctic tundra 北极冻原Alpine tundra 高山冻原Permafrost 永冻层Coniferous boreal forest北方针叶林Temperature forest 温带森林Tropical forest 热带森林Salinization 盐渍化Primary saltwater regions 基本盐水区域Opens oceans 开阔海洋Continental shelves 大陆架The intertidal zone 潮间带Salt marsh 盐沼Mudflats淤泥滩Mangroves 红树林Pelagic 浮游生物Photic zone 有光带Phyto plankton 浮游植物Nekton 自泳动物Benthic 底栖Rocky shore 岩岸Zonation 分带Streams 溪流Ponds 池塘Environmental concerns 环境关系Catchment area 集水区Temperature inversion 温度逆转Biomanipulation 生物处理TThe goals of harvesting 收获目标Quota limitation 配额限制Environmental fluctuation环境波动Maximum possible yiel最大可能产量Net recruitment 净补充量Surplus yield 过剩产量Age structure 年龄结构Population data 种群数据Stable equilibrium 稳定平衡Harvesting effort 收获努力Gun licences 猎枪执照Rod licences钓鱼许可证Upwelling of cold water冷水上升流Fisheries 渔业Ocean productivity 大洋生产力The tragedy of the common公共灾难Overexploitation 过捕Pollution 污染Global decline 全球性下降By-catch 附带收获Community perturbations 群落扰动Oil spills 原油泄漏Eutrophication 富营养化Algal blooms 水华Red tides 赤潮Biomagnification 生物放大作用UPest 有害生物Natural enemies 天敌Ruderal 杂草型Economic/aesthetic injury level 经济/美学损害水平Cultural 栽培Biological control 生物防治‘Silent spring’寂静的春天Chemical toxicity 化学毒性Evolution of resistance抗性进化Microbial insecticide微生物杀虫剂Inoculation接种Augmentation扩大Inundation 爆发VRare species 稀有种Genetic diversity 遗传多样性Extinction 灭绝Endemic species 特有种Habitat fragmentation 生境片段化Insularization 岛屿化Biodiversity 生物多样性Strategies for conservation保育对策Antarctic treaty 南极协议Ecotourism生态旅游WAir pollution空气污染Acid rain 酸雨Water pollutants 水体污染物Soil pollution 土壤污染Acid deposition 酸降Pathogens病源体Chemical oxygen demand 化学需氧量Anaerobes 厌氧菌The greenhouse effect 温室效应Carbon dioxide 二氧化碳Ozone 臭氧Photochemical smog 光化学烟雾XOverview 概述Soil erosion 土壤侵蚀Soil compaction 土壤硬结Contour ploughing等高耕作Cover crops 覆盖作物No-till farming 免耕农业。

多烯大环内酯类抗生素的研究

1.早期的研究通过基因缺失的方法探索各个基因在该类抗生素合成中的作用，以及确定新产物抗真菌活性，水溶性，毒副效应的影响。
缺失AmphL产21缺失AmphDIII产22。说明环外羧基的生成要早于C8羟基的合成以及大环糖基化反应 Byrne, B.; Carmody, M.; Gibson, E.; Rawlings, B.; Caffrey, P. 2003. Biosynthesis of deoxyamphotericins and deoxyamphoteronolides by engineered strains of Streptomyces nodosus. Chem. Biol. 2003, 10, 1215 – 1224.
5.水平基因转移也是聚酮类抗生素PKS进化的主要方式。
Jenke-Kodama H, Sandmann A, Mü ller R, Dittmann E (2005) Evolutionary implications of bacterial polyketide synthases. Mol Biol Evol 22:2027-2039.
对AmphNM以及AmphDII的缺失产25.海藻糖氨变为鼠李糖这表明糖基转移酶同时也具有对糖的灵活性。对AmphC中两个模块的缺失产生了26.说明对PKS进行改造已改变大环C原子数可以成功。 Carmody, M.; Byrne, B.; Murphy, B.; Breen, C.; Lynch, S.; Flood, E.; Finnan, S.; Caffrey, P. Analysis and manipulation of amphotericin biosynthetic genes by means of modified phage KC515 transduction techniques. Gene 2004, 343, 107 -115.

分子生物学第二章-染色体,染色质和核小体-chromosomes, chromatin, and the nucleosome

• 许多复杂性相近的生物体其基因组大小却显著不同：小麦基因组是水稻基因组的40 倍
• 基因的数目而不是基因组的大小与生物体的复杂性更密切相关
– Gene number（基因数目）: the number of genes included in a genome
Gene density（基因密度）: the average number of genes per Mb of genomic DNA
1.4 大肠杆菌的基因组几乎全部由基因组成
• 大肠杆菌单一染色体上绝大多数DNA都编码蛋白质或RNA，而大部分非编码序列都参与调控基因的转录
• 大肠杆菌复制起始区：指导整个复制机制的组装，非常小，仅占4.6Mb基因组中的几百个碱基对
1.5 较复杂的生物体基因密度降低
• Gene density（基因密度）: the average number of genes per Mb of genomic DNA
• 每个细胞中可以有多个完整拷贝的质粒存在
• 大多数真核生物细胞是二倍体（diploid), 一个染色体有两个拷贝
• 多倍体真核细胞（polyploid): 细胞中每条染色体都超过两个拷贝
• 巨核细胞：特化的多倍体细胞，每条染色体有128个拷贝，通过多倍体形式维持高水平新陈代谢，以生产大量的血小板
• 两种因素会导致真核生物基因密度的降低：
– 基因长度的增加
– 基因间隔区序列（ intergenic sequences）的增加
Increases in gene size（基因长度的增加的两个原因）:
(1) increase in the sequence of regulatory sequence（当生物体复杂性增加时，基因的调控序列的长度显著增长）

Gene Modification

Gene modification is a product of the high technology, people always argue that how the high technology impact on human beings, and the usual conclusion is it turns out to be a double-edged sword. GM is not an exception as well.As a product of human wisdom, on the one hand, GM help to increase the quantity of the agriculture industry. On the other hand, GM can improve the quality of the plants, like make the soybean plant strong enough to protect itself from the strong wind, or generate some noxious liquid to kill the pest. Furthermore, GM offers a way to make the agricultural products normative. All above aspects can benefit both the consumers and the farmers.However, GM is such a new technology to us, people can not utilize it as well as other mature ones. Lots of pain has been induced during the application of GM. Some farmers report pigs and cows became sterile from GM corn, some other researches found out that there was a gene from a Brazil nut which carried allergies into soybeans. What is more, there is a concern that the pregnant mothers eating GM foods may endanger offspring. Many people resist the GM food because of the potential risk mentioned above.All in all, GM still needs a long time to develop, and we expect that GM can do us more benefits to make our life more comfortable.。

赤霉素合成途径

赤霉素（Erythromycin）是一种广谱抗生素，常用于治疗细菌感染。

它是一种大环内酯类抗生素，具有良好的抗菌活性和耐受性，能够有效地抑制细菌的生长和繁殖。

赤霉素的合成途径涉及多个步骤和关键酶催化反应，下面将详细介绍赤霉素的合成途径。

1.赤霉素合成途径概述赤霉素的合成途径主要包括以下几个步骤：链延伸、环化、羟基化、脱水和脱氢等。

这些步骤通过一系列酶催化反应完成，最终得到赤霉素分子。

2.链延伸赤霉素的合成始于一个二十碳原子的多肽链。

首先，由核糖体在mRNA模板上进行蛋白质合成，形成多肽链。

该多肽链由氨基酸组成，其中包括丙氨酸、丝氨酸和谷氨酰胺等。

3.环化在链延伸后，需要对多肽链进行环化。

这一步骤由赤霉素合成酶（Erythromycin synthase）催化完成。

赤霉素合成酶能够将多肽链的两端连接起来，形成一个大环结构。

这个大环结构是赤霉素分子的骨架。

4.羟基化在环化后，需要对赤霉素分子进行羟基化修饰。

羟基化可以增加赤霉素分子的活性和稳定性。

羟基化是由多个酶催化的反应完成的，其中包括CYP450酶家族和脱氢酶等。

5.脱水脱水是赤霉素合成途径中的一个关键步骤。

在这一步骤中，通过脱水作用将赤霉素分子中的一部分水分子去除，使其变为更加稳定的形式。

脱水通常由特定的脱水酶催化。

6.脱氢最后一个步骤是脱氢反应，在这一步骤中，通过去除赤霉素分子中的氢原子，使其形成双键结构。

脱氢反应通常由NAD+或者NADP+等辅助因子参与。

7.合成途径调控赤霉素合成途径的调控是一个复杂而精细的过程。

在细菌中，赤霉素合成途径受到多个因素的调控，包括底物浓度、酶活性和基因表达等。

这些调控机制能够确保赤霉素的合成在合适的时机和条件下进行。

综上所述，赤霉素的合成途径包括链延伸、环化、羟基化、脱水和脱氢等多个步骤。

这些步骤通过一系列酶催化反应完成，最终得到赤霉素分子。

赤霉素合成途径的调控是一个复杂而精细的过程，能够确保赤霉素在适当的时机和条件下进行合成。