新型HMW-GS基因1Dy12.1~(*t)植物双元表达载体的构建及其遗传转化

为了建立快速研究高分子量谷蛋白亚基(high molecular weight glutenin subunit,HMW-GS)基因功能活性的植物表达体系,以自主克隆的新型HMW-GS基因1Dy12.1~(*t)为目的基因,利用限制性内切酶Xho Ⅰ和Sal Ⅰ切割后5'端能形成相同粘性末端的特性,成功构建了1Dy12.1~(*t)的植物双元表达载体pBINHP-GluT.该载体选择使用胚乳特异性强启动子,利用已知HMW-GS基因内部均不含有的Xba Ⅰ和Kpn Ⅰ内切酶位点引入1Dy12.1~(*t)编码区,并将1Dy12.1~(*t)的表达调控置于T-DNA区内,从而奠定了其在不同HMW-GS基因功能活性研究上的便利性.通过根癌农杆菌烟草叶盘转化研究目的基因1Dy12.1~(*t)在植物体的表达特性,SDS-PAGE和western blotting鉴定结果表明,1Dy12.1~(*t)在转基因烟草种子胚乳中得到高效表达,进一步证实了所克隆基因的正确性和所构建载体的有效性.

Plant Binary Expression Vector Construction and Genetic Transformation of the New Type HMW-GS Gene 1Dy12.1~(*t)

In order to establish a fast plant expression system for studying the functions and activations of the high molecular weight glutenin subunit (HMW-GS) genes, the plant binary expression vector pBINHP-GluT was successfully constructed by taking advantage of the same 5'-terminus ends of the endonucleases Xho Ⅰ and Sal Ⅰ using the new type HMW-GS gene 1Dy12.1~(*t) as the target gene. The vector pBINHP-GluT, which placed the encoding domain of 1Dy12.1~(*t) between the restriction sites of Xba Ⅰ and Kpn Ⅰ that have not been found in all published HMW-GS genes, and placed 1Dy12.1~(*t) under the regulation of the verified endosperm-specific promoter and placed all operation cassette between the left and right borders of T-DNA, having great predominance in the study of other new HMW-GS gene functions. The T-DNA fragments containing the new type gene 1Dy12.1~(*t) was transferred to tobacco lamina mediated by Agrobacterium tumefaciens. The results of SDS-PAGE and western blotting showed that 1Dy12.1~(*t) expressed an expected protein successfully in the endosperm of transgenic tobacco, which confirmed the authenticities of the new gene 1Dy12.1~(*t) and the validity of the constructed expression vector pBINHP-GluT.