亞麻木質(zhì)素合成關鍵酶CAD基因的克隆與分析.pdf

1、中國農(nóng)業(yè)科學院碩士學位論文亞麻木質(zhì)素合成關鍵酶CAD基因的克隆與分析姓名：黃海燕申請學位級別：碩士專業(yè)：作物遺傳育種指導教師：王玉富20080501AbstractFlaxisaveryimportantsourceofnaturalfibresinthetextileindustryplaysallimportantroleinChineseeconomyandpeople’SlifeLigninisdifficulttoberemo

2、vedduringrettingprocessLigninhasagreaterimpactonthefiberqualityandperformanceofthetextilesImprovingattributeofflaxfibreviageneengineering，carrymgoutlowlignmbreeding，decreasingthecontentofligninandalteringitscomponentssee

3、mtobegoodwaystosolvertheproblemsInthispapertheflaxCADgeneWasclonedbyRTPCRusingapairofprimersweredesignedfromthesequencesofexistingCADgenesAntisenseCADgeneexpressionvectorofflaxhasbeenconstructed。Theresultsareasfollows：(1

4、)HighqualityflaxRNAwasextractedbyPlantRNAIsolationRegentandreversetranscribedintoeDNAusingSuperscriptRTKitThisisallefficientmethodforcloningtargetgenesfromplantwhichcontainsrichmultifarioussecondarymetabolitesuchaspectin

5、andpolysaccharide(2)AflaxeDNAfragmentWasamplifiedbyReverseTranscriptionPolymersChainReaction(RTPCR)usingapairofprimersweredesignedfromthesequencesofexistingCADgenesSequencinganalysisshowedthatthePCRproductswere477bpwith9

6、5％identitytotheCADgeneregisteredintheGenBankTheresultprovedthattheclonedgenefragmentshouldbeCADgenefragmentoftheflax(3)ThefulllengthsequenceoffalxCADgenecDNAwasclonedbyRACESequencinganalysisshowedthatthePCRproductswere89

7、6bp嘶t1192％identitytotheCADgenesequenceregisteredintheGenBank(4)TointroduceproperrestrictionsitesforsubsequentcloningintotheplantexpressionvectorboththepGEM一7Zf(一)vectorandthepGEMTCADwerecutbyEcoRItoreleasetheCADgenefragm

8、entandthepGEM一7Zf()fragment，ThetwofragmentwerejoinedtogethertoproduceanewplasmidpGEM一7Zf(一)一CAD(5)ThepGEM一7Zf()一CADplasmidandthebinaryvectorpBI121werecutbyXbaIandBamHIteleasetheCADgenefragmentandthelargefragmemsofpBll21A