Transcriptome analysis.docx

Tmnscrip1.onMu11i1.y×h光拄花生EM中索的代妣径的输艮盘分析目的，I找到与，索合成相关的不因2attHua分析皿«合成相网3明PKS基因在其中的作用InstructionWW第段：C6句)花生疮痂出凡客花生抡诟病(即加*“川MMWm("cBitaiKniir1.c1.Jcnkin)111940Bi1.iiucour1.泞次在阳关的巴西发现之后,日本根州宽山等I处9和阿根廷(GionhAH,1985)等国家也相维报道,并在各花生产区流行成灾近年来花?fc疮卷病在中国广东.广西、福建、山东和江字等主R花生产区相继发生他书.造成血枳OM产.危害产W.礁：段：花生疮痂桁曲同类桁吼式演相关研究)花生疮琬病第是引欣桐再发生的物麻粉,盯性阶段为麴)堂腔牌,痂粪腔留本的生理特性Materia1.%a><!nv(h<dsStniinxjndcu1.tureconditions健株择木来源,培养方法,并H经过叁定确定其种虢MeasuremenUoftoxincontentsandsp(ru1.ath>n测量海表令最及f1.ifft的方法RNAPrCPn11ionftRNA的提双以及CDNA文库构建ToiaJRNAwasextractedfmfreshsc1.cttia1.ponderg喀TR1.zo11RC昨cn(试剂公司)accordingtoxnanutc1.urvr*sns1.rudx>ns.AMif1.ktcn1.atx>un1.ofIu1.a1.1.<Nofm>!k¼IhanIOggwithOD2W,OD2SOva1.uesofI.Hto2.2Wused.Thewimp1.ewaxPuriRcdandImaIcdwithF>Naseinordertorr11>vcPrO(CinandDNAContaminunts.Samp1.efort11incrip<oncana1.ysiswaspreparedasperI1.1.umiiumanufacturer'sinstructionsWhCrCmag½ticbeadswitho1.igo<dT)wereusedtoi%o1.a(eJxi1.yadeny1.a1.ednRNA<pc1.y(AHRNA)fromUieIuU1.RNA.Hugnien1.atiDnbufferCimxistingnfdiva1.entca1.in115was;nkkxit<xintcr1.inj:ERNAtosh<v1.fragmentsof20()to700nuc1.eotidesin1.ength.TheseSbOnfragmentswereusedastemp1.atescosynthesizehcfirM-stra11dCDNAusingrandombcxamcr-primcr.TheSCCOndrtnmdCDNAW不synthesizedusingk11cr.dNTPs.RNacH.andDMApo1.ymerase1.ThePnMIUegwerePUrinedandreso1.vedwithQIAquickPCRPurificationKit(试剂盒公诃>andEBbut1.erforendrePantfiOnandUi1.infA.respective1.y.Puri1.1.cdCDMAfragmentswereconnectedwithWJUCrKinWadaptersa11<1.j»dCkxtmPh(IrCKdto%e1.evtsuiUb!etrag11>eHforK'Ranp1.ifica1.n.AgI1.em2100BxQ1.UdyZeraidApp1.iedBk>yaenH1.SICPomrPiU"RcaI-IutKPCRSy%½11WefVUMfduqun1.iIicahmiMdqua1.1.ka1.!ofthesamp1.eIibran.'forqua1.itycontro1.PairedYndCDNA1.ib11r>,w5constmctcdandMx1.I1.cIHrCdusing防序公司)Transvriptunwaa1.yxi转录fn测序以*39为材料，以谓控方素产生的主要环境闪索光照作为处理手区，分别以累暗处理和光照处理的碗姝作为林聚班的试发殂和对照机,DH1.aprocesi!数1«处理方法ToavoidnegativeimpactonWbM:qucn1.bioinfma1.>csana1.ysis,11wreadswerefi1.teredbytheremova1.ofraidswithaduptcrs.unknownnuc1.eo1.idcs1.argerthan10%.and1.owqua1.ityreadswheremorethanha1.fofthebases'qua1.itiesa1.essIhanfive(Q<5).Basecompositionandqua1.itydis1.ributi<mWenrkIerniincdasqua1.itycn(1.I1.ieub1.awdc1.eantvadsvre1.1.enapfd1.IhegenometiiIgc11c<sequence%nfSaauTMo2KinWShortOtideArw1.y*Package2(S0AP2)(241whereUPtofivebasesmismatcheswerea1.1.owedinthea1.ignment.Thea1.ignmentdataWagusedcocuku1.a(cdMribtionofreadsonreferencegenesandmapping11uio为了避免对防后的生物信息学分析产'1.T、和影响,通过用珏配券去除读数.未知核甘酸大了10%,以及低质量读数过池取始读数(rawre汕),其中一半以上的做旗步质小于5(QW5),戡砒组成和侦M分布核确定为质Itt控制，然后将茨得的c1.earread映射到使用短彩核甘酸分析包2(S0AP2)的Swm的基因蛆和庙闪序列,北中在对准中允许多达5个WU蛀H配.对数楙M了计算参考韭因读数的分布和映射比例.Bioinfurma1.ioh>t¼1.1.)cn<>%<>ax%cmb!y<1.unnu1.a1.i<m功算生物侑息学分析Fora1.ternativesp1.icingana1.ysis,junctionsitesweredetectedbyTopHutwithdefau1.tp<aramctc11i.JuiKiionsiteswhich<tvidcinfrnaiiononIXHHk1.afieSandcmbiutonofdHcrencexonsinaIraiiMrnp1.Dr1.be¼unegeneWereutcdIUdistinguishtheIypeofaJIerm1.nv、p1.inge¼entinc1.udingexondipping,intr<>11reniM>n,a1.tcma1.ivc5'sp1.icesite,andIkCgtiVC3'sp1.icesite.Fornove1.transcriptsprediction,genemode1.sfoundinirue里CniCregions200basePaini(bp)uwayfromstreamordouiMreangenes)wc<vthoughtcobepotentia1.CaMdaIc.Amember“theSOAP(栩耳？O'的分析包工SOApsnPwasuedtodetect疝嗒MnUCk。IiIfePOiynwrPhiun(SN冏).SNPvWCrCidentifiedonthecnmcnsiwsequenceIhmugh(beccwnjxriM>nwiththereferenceRCnnmCof1.rhin<xxrtisTM02Mrain.Genemode1.swerea1.ignedtoSwiwProt.TrtMB1.andNCBInr(B1.ASTPcut-offc-vaJ>ck5)andtheirfunctiona1.c1.assificationwasnnnoutcdbyGeneOnto1.ogy(GO).CI1.ftICrofOftho1.ogousGfOUPS<COG>.andKyotoEncyc1.opediaofGCnCandGCnOme9(KEGG1.PathWny".Conf1.rma1.kmfuni>cncexpressionusisrcH-ti11H*(unnthatiMrreversetranscriptionPCR使用实时荧光定址IjCR砒定韭因表达AIU1.1.yUofgcne%invo1.vedinmt，MKinrym<*UM)4i5m对PKSAUMyHj分析光感应基因分析Kewit('hHictcrhtic<sc,riptknoftheproductionOCtoxinnn<U)OrV光调控卜炳烧的点发的产生及产胞的特性描述RNAMH1.UenCin*aHM*Mt11wntRNA测序评估：测序的数1«分析.RNAsamp1.ecIrac1.odfrnm1.rix0,<Mi%ckm1.iumwaxXiibjcc1.cdtohh-thr(Hghpu1.IHuminiisequencinginordertogainanoverviewofthefingusIranMJripcome.OVCr5GbofruwreadswereobtainedfromeachCDNA1.ibrary.TheSCqUenCi1.喀assessmentrevea1.edagoodsequencingqua1.ityadahgn11e1.tvsu1.1.¼.Inkta1.52.933.332vadsWi1.h90bpinIeiig1.iiwereobtainedafterfi1.(enngIhe11wrcak.TheCICanreadweremappedtothegenomeAndgcno*c<enccxof1.rtiinoccnxi$(Tab1.e1).Themappingresu1.tsprovidennovera1.1.asscssmcnof(besequencing.Ac<mq1of65.4HIX1.46.1%c1.eanreadswtcab1.etomaptothe1.rhirxxxrot½gc>nandgenerespective1.yandabout6X.0and74.0%ofIberetptx1.ivemappedreudsIbepeftx1.mu1.chIyiUXXJ1.IiiiMiia1.ch).UIan0.5%IIK1.pPedivadsaremu1.hp1.iDna1.chwhi1.emajorityauuena1.ehwherthereadsWtnIrmappedtounique(on1.yone)positionsofthegenomeorgene.TherundomncssofmRNAfragmentationduringexperimenta1.settingwaseva1.uatedWi1.dtherandomdisibutinofreadsinreferencecncs.Apoorfadomncssoffragm