平邑甜茶MhVPE基因ihpRNA干扰载体的构建及转拟南芥验证

 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。

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

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 confi...