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  

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

作　　者：张艳贞[1,2] 王轲[2] 张静[1] 晏月明[2] 

机构地区：[1]北京联合大学应用文理学院,北京100083 [2]首都师范大学生命科学学院,北京100037

出　　处：《沈阳农业大学学报》2009年第6期683-687,共5页Journal of Shenyang Agricultural University

基　　金：国家自然科学基金项目(30571154);"863"计划资助项目(2006AA10Z186)

摘　　要：为了建立快速研究高分子量谷蛋白亚基(high molecular weightglutenin 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XhoⅠ和SalⅠ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XbaⅠ和KpnⅠ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。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 XhoⅠand SalⅠusing 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 XbaⅠand KpnⅠthat 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.

关 键 词：HMW-GS基因 双元表达载体 遗传转化 植物体 高分子量谷蛋白亚基 SDS-PAGE 限制性内切酶 基因功能 

分 类 号：S512.1[农业科学—作物学] Q943.2[生物学—植物学]