平邑甜茶MhVPE基因ihpRNA干扰载体的构建及转拟南芥验证  

Construction of ihpRNA Expression Vector of MhVPE Gene from Malus hupehensis and Verification by Transgenic Arabidopsis thaliana

作　　者：冉昆[1] 杨洪强[1] 孙晓莉[1] 沈伟[1] 姜倩倩[1] 李强[1] 刘智新[2] 

机构地区：[1]山东农业大学园艺科学与工程学院,作物生物学国家重点实验室,国家苹果工程技术研究中心,山东泰安271018 [2]山东农业大学信息科学与工程学院,山东泰安271018

出　　处：《园艺学报》2011年第12期2365-2372,共8页Acta Horticulturae Sinica

基　　金：国家自然科学基金项目(31171923);国家转基因生物新品种培育重大专项(2008ZX08009-3);高校博士学科点专项科研基金项目(20103702110003);山东省自然科学基金项目(ZR2010CM020)

摘　　要：根据平邑甜茶液泡加工酶(vacuolar processing enzyme)基因MhVPE(GenBank登录号为FJ891065)cDNA序列,设计两对含有酶切位点的特异性引物F1/F2和R1/R2,以pMD-MhVPE质粒为模板,分别克隆了用于构建干扰载体的正反义片段pMD-F和pMD-R。将该正反义片段分别插入表达载体pART27的相应位置,构建成了含有内含子发夹结构的ihpRNA表达载体pART-RNAi-MhVPE。通过农杆菌介导,用花序浸泡法转化拟南芥进行验证,经过抗性筛选和PCR检测,得到17株转基因阳性植株;半定量RT-PCR结果显示所获得的转基因拟南芥植株中AtVPE同源基因的表达量明显降低,表明该干扰载体能够有效抑制VPE基因的表达,MhVPE基因的ihpRNA干扰载体构建成功,为进一步鉴定该基因的功能奠定了基础。Based on the cDNA sequence of MhVPE gene（GenBank accession number：FJ891065） from Malus hupehensis（Pamp.）Rehd.,two pairs of specific primers containing different enzyme sites,named F1/F2 and R1/R2 respectively,were designed.With the template of pMD-MhVPE plasmid constructed previously,positive-sense strand pMD-F and antisense strand pMD-R were obtained,which were inserted into specific sites of the expression vector pART27,separately.The RNAi vector of pART-RNAi-MhVPE containing a hairpin structure was confirmed by the digestion of restriction enzymes.pART-RNAi-MhVPE vector was then transformed into Arabidopsis thaliana col-0 using the floral dip method by Agrobacterium-mediated transformation system to verification its efficacy.Seventeen transgenic plants were confirmed by kanamycin resistant selection and PCR testing.Semi RT-PCR results suggested that the expression level of AtVPE homologous gene in transgenic Arabidopsis thaliana was decreased,which proved that the expression of the targeted gene was inhibited efficiently and the ihpRNA vector of the MhVPE gene was constructed successfully,building a solid foundation for further study on the function of vacuolar processing enzyme（VPE）gene.

关 键 词：平邑甜茶 MhVPE RNA干扰 ihpRNA 拟南芥 

分 类 号：Q943.2[生物学—植物学]