煙草黃酮醇合成酶基因的分離及其序列分析.pdf

1、華中科技大學碩士學位論文煙草黃酮醇合成酶基因的分離及其序列分析姓名：黃偉偉申請學位級別：碩士專業(yè)：生物化學與分子生物學指導教師：何光源20070208華中科技大學碩士學位論文AbstractFlavonoidscomprisethemostcommongroupofpolyphenolicplantsecondarymetabolites.Therearediversegroupofplantscontainingflavoneisof

2、lavoneflavonolchalconeetc.Theyplaykeyrolesinbiologicalactivities.Flavonolsareanimptantpartofflavonoidsproducedbyflavonolsynthase.AccdingtotheconservedcDNAsequenceofflavonolsynthase(FLS)genesapairofprimerswasdesigned，flav

3、onolsynthasegenewasclonedfromtobacco(NicotianatabacunL.)leavesusingRTPCRRACEmethods.ThefulllengthcDNAsequencewas1186bpincludedawholeF，whichencoded333residues.TheaminoacididentityamongFLSsfromdifferentspecieswashighrangin

4、gfrom47%to87%.Theenzymebelongstotheclass2oxoglutaratedependentdioxygenases.SomesimilardomainstoIsopenicillinIIsynthase2OGFe(II)oxygenasesuperfamilywerefoundwhensearchingtheproteinconserveddomainsinGenBank.Polypeptidealig

5、nmentsofFLSswith2oxoglutaratedependentdioxygenasesrelated2oxoglutarateindependentenzymesrevealedeightstrictlyconservedaminoacidsinthreeregions.Homologymodelingwasusedtopredictthetertiarystructure.Theresultsofhomologymode

6、lingindicatedthatthemainchainofFLScontained11βstrsofwhichfmedajellyrolldoublestredβhelixtopology.Thejellyrollfmedahyhobiccavityoneendofwhichfmedtheactivesite.Fourstrictlyconservedaminoacids(His219His275Asp221Arg285)werei

7、nthehyhobiccavityothers(Gly65His72Pro205Gly259)inturn.LiketheotherdioxygenasesofthisclassflavonolsynthasecDNAencodedeightstrictlyconservedaminoacidresidueswhichincludedtwohistidines(His219His275)oneacidicaminoacid(Asp221

8、)residuefFe(II)codinationanarginine(Arg285)proposedtobind2oxoglutaratetherestfour(Gly65His72Pro205Gly259)requiredfproperfoldingoftheflavonolsynthasepolypeptide.Keywds:TobaccoFlavonolsynthaseRACEPolypeptidealignmentsHomol