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《蓖麻磷脂酶c基因的克隆及其rnai表達(dá)載體的構(gòu)建》由會(huì)員上傳分享，免費(fèi)在線閱讀，更多相關(guān)內(nèi)容在學(xué)術(shù)論文-天天文庫。
1、摘要蓖麻是內(nèi)蒙地區(qū)非常具有區(qū)域特色的世界十大油料作物之一。蓖麻油由脂肪酸組成，主要成分是蓖麻油酸。磷脂酶C在控制蓖麻油酸生物合成過程中起關(guān)鍵性的作用，它催化2一油酰基卵磷脂形成卜?；?一油?；视?，使非羥基脂肪酸進(jìn)入三酰基甘油。通過基因工程手段設(shè)計(jì)并構(gòu)建含有磷脂酶C基因的RNAi表達(dá)載體，以期在植物體內(nèi)轉(zhuǎn)錄出含有部分磷脂酶C基因發(fā)夾結(jié)構(gòu)的dsRNA，發(fā)揮干擾磷脂酶C基因表達(dá)的作用，從而提高總蓖麻油含量。本試驗(yàn)以通蓖5號蓖麻為研究對象，提取其發(fā)育階段的種子總RNA，逆轉(zhuǎn)錄成cDNA，通過降落PCR得到長度分別為803bp和45
2、8bp的磷脂酶C基因片段C1和C2，采用Sanger雙脫氧鏈終止法對Cl和C2進(jìn)行測序，測序結(jié)果與Genbank中蓖麻磷脂酶C基因的mRNA進(jìn)行同源性分析，同源性分別達(dá)到99．88％和99．13％，成功克隆了蓖麻磷脂酶C基因。并采用一步法成功構(gòu)建了蓖麻磷脂酶C基因的RNAi表達(dá)載體，為通過RNAi干擾技術(shù)獲得總蓖麻油含量高的蓖麻新材料奠定了基礎(chǔ)。關(guān)鍵詞：蓖麻；磷脂酶C；RNAi；表達(dá)載體CloningofAPhospholipaseCandConstructionoftheRNAiExpresionVectorinCastor
3、AbstractCastorisoneofthetenleadingoilcropsintheworldandhasalsoregionalcharacteristicinInnerMongolia．Castoroiliscomposedoffattyacidanditsmainingredientisricinoleicacid．PhospholiDaseCplaysacriticalroleinricinoleicacidbiosynthesis，anditcatalyzes2一oleoyllecithintoform1-
4、acyl一2一oleoyl—sn—glycerol，whichistheprocessofnon—hydroxyfattyacidintotriacylglycer01．ThisresearchaimsatdesigningandbuildingRNAiexpressionvectorofphosphoiipaseCbygeneticengineeringtotranscriptahairpinstructureofdsRNA，interferephospholipaseCgeneexpressionandincreaseth
5、etotalcontentofCastor0ii．Wechosecastorastheresearchobject，andextractedtotalRNAofthedevelopingseed，thenmadeRNAreversedtranscriptionintocDNA．ByusingTD—PCR．Weobtainedtwogenefragmentsofphosph01ipaseC，thelengthsofwhichwere803bpand458bprespectively．WhensequencingC1andC2，w
6、eusedSangerdideoxy—mediatedchain—terminationmethod，Afteranalysingtheresultsof‘sequencingandthehomologywiththemRNAofphosph01ipaseCgenefromOenBank，wefoundthatthehomologyW&Supto99．88％and99．13％respectivelyandatlastwesucceededincloningthecastorphospholipaseCgene．Thenwesu
7、ccessfullybuiittheRNAiexpressionvectorofCastorphospholipaseCgene，andthiStechnologylaidafoundationforanewCastormaterial．Keywords：Castor,"PhospholipaseC：R^Ai：ExpressionVector目錄摘要?．?．．??????????????．j?????．IAbstract??????????????????????．．II錄?．．．??．?????．．．??．．????????
8、．．．．III1引言?．???．．???．．?．-．?．．???????????．12試驗(yàn)材料??????????????????．????62．1植物材料???????????一????．．????72．2菌種與載體???????????????????．．72．3試劑??
