29842.核盤菌病毒ssdrv的539;末端克隆及侵染性全長cdna克隆的構(gòu)建

1、華中農(nóng)業(yè)大學(xué)碩士學(xué)位論文核盤菌病毒SsDRV的5末端克隆及侵染性全長cDNA克隆的構(gòu)建姓名：羊國根申請學(xué)位級別：碩士專業(yè)：植物病理學(xué)指導(dǎo)教師：姜道宏20090601核盤菌病毒SsDRV的5味端克隆及侵染性傘長eDNA克降的構(gòu)建AbstractInthisstudythefull—lengthgenomicsequenceofSclerotiniasclerotiorumdebilitationassociatedRNAvirus(SsD

2、RV)modifiedAeDNAinfectiousvectorofSsDRVwasconstructed，andtheinfectiouseDNAvectorwastransformedintoSclerotiniasclerotiorumstrainEp一1PNA367(virusfree)Previouslyaputativefull—lengtheDNAofSsDRVwasclonedontopCAMBIAl301vectorn

3、amedasp1301SsDRV(Xieetal，2006)Inthisstudyp1301SsDRVwasusedtotransformConiothyriumminitansamycoparasiteof&sclerotiorumwithAgrobacteriumtumefaciensmediatedtransformation105transformantswereobtained；someofthemwerecomfirmedP

4、CRamplificationThecolonymorphologyconidialproductionandparasiticalabilityofsometransformantswerestudied，andtheresultssuggestedthatthephenotypesoftransformantswerenotsignificantlyaffectedTheviraleDNAcouldbedetectedfromtes

5、tedtransformants，butdsRNAcouldnotbeextractedfromthemwithcelluloseCF11Thus，thefull—lengtheDNAcloneofSsDRVWasindoubtThefulllengtheDNAofSsDRVWasreanalyzed，andtheresultssuggestedthattheformersplicedeDNAWasincomplete，andthese

6、quenceof5’endtheremightbemissedAnadditional82ntlengthfragmentatthe5’terminusWasclonedfromtotalRNAofstrainEp1PNwithSMART(SwitchingMechanismAt5’endoftheRNATranscript，SMART)techniqueThus，thegenomeofSsDRVwasrespliced，andthef

7、ull—lengthsequenceis5500nucleotide(excludingpolyA)withonlyoneopenreadingframe(174nt一5274nt)，thereisacapstructureat5’endFurtheranalysisshowedthatthesequenceisvarableatthe5’untranslationregion，andhasapolyAstructureat3’endT

8、hesecondarystructuresof5UTRand3UTRwerepredicted，andthreehairpinstructureswerededucedat5’UTRandatRNAknotstructureat3UTRBasedonthemodifiedgenomicsequenceofSsDRVanewfulllengthinfectiouseDNAvectornamedpTSVH，Wasconstructedbyi