26957.一個(gè)水稻花藥特異表達(dá)基因的克隆及初步分析

1、摘要摘要利用水稻TDNA插入雄配子不育突變體分離得到一個(gè)花藥特異表達(dá)基因MGA01。突變體mga01自交后代群體潮霉素磷酸轉(zhuǎn)移酶標(biāo)記基因保持穩(wěn)定1：1分離，連續(xù)6代自交未分離出hpt抗性純合株系。突變體mga01花粉育性接近50％，不育花粉淀粉充實(shí)過(guò)程受阻。遺傳分析表明，TDNA插入與雄配子不育性狀連鎖。Southernblot分析發(fā)現(xiàn)突變體mga01ToDNA以單拷貝方式整合到水稻中，TDNA插入位點(diǎn)位于水稻第3號(hào)染色體AC09207

2、6BAC克隆上。mga01突變表型與TDNA插入共分離檢測(cè)發(fā)現(xiàn)：花粉半不育與MGA01／mga01雜合基因型共分離。以上結(jié)果充分說(shuō)明，突變體mga01為一個(gè)TDNA插入導(dǎo)致的配子體花粉不育突變體。MGAOI基因半定量RTPCR分析及MGA01啟動(dòng)子與GUS基因融合表達(dá)轉(zhuǎn)基因水稻植株To、Tl代組織GUS活性檢測(cè)結(jié)果顯示，MGA01在花藥中特異表達(dá)。根據(jù)NCBI數(shù)據(jù)庫(kù)中的基因信息設(shè)計(jì)引物擴(kuò)增MGA01基因CDS，以pCAMBIAl300為

3、基本骨架構(gòu)建基因表達(dá)載體pCAMBIAl300MGA01()及pCAMBIAl300MGA01()，通過(guò)農(nóng)桿菌介導(dǎo)轉(zhuǎn)化水稻，獲得了轉(zhuǎn)基因植株。綜上初步結(jié)果，MGA01可能是調(diào)控水稻雄配子發(fā)育的重要基因，是花粉成熟所必需的。關(guān)鍵詞：水稻雄配子不育ToDNA花藥特異表達(dá)MGA017^‘●‘AbstractAbstractWehaveisolatedallantherpreferentialgene，MGAOl(malegametogenes

4、isadoption01)，fromaTDNAinsertionalpopulationofindicariceTransgenicplantscarryingaTDNAinsertionintheMGA01genedisplayedthephenotypeofsegregationdistortionofthemutatedmga01geneMoreovermga01／mga01homozygousprogenywerenotpres

5、entPollenfertilityofmga01mutantisabout50％，andstarchsynthesisinmga01pollenwasblockedReciprocalcrossesbetweenMGA01／mga01heterozygousplantsandthewildtypeshowedthatthemga01allelecouldnotbetransmittedthroughthemaleSouthernblo

6、tandanalysisoftheDNAsequenceflankingTDNAshowedthatmga01mutantcarryoneTDNAinsertionat5’UTRofMGAOI，whichislocatedonchromosome3Aboveresultsfullyexplainthatmga01mutantisagametophytepollensterilemutantcausedbyTDNAinsertionCo—

7、segregationanalysisrevealedthatMGA01isainsertionalmutant，andtheinfertilityphenotypewasduetoTDNAinsertionTheconsequenceofsemiquantitativereversetranscriptionPCRandGUSactivitydetectionofthetransgenicrice(T0，TI)transformedw

8、ithMGA01PfusedwithGUSgeneindicatedthatMGA01preferentiallyexpressinriceantherMGA01genewereisolatedtoconstructexpressionvectorscombinedwithpCAMBIAl300Transgenicriceplantsoftheconstructsweretransformtoricemediatedbyagrobact