抗除草剂Bar基因的优化及PAT蛋白的表达

Opitimization of Bar Gene Sequence and Expression of Phosphinothricin Acetyltransferase Protein

利用生物信息学方法优化了适用于原核表达的Bar基因序列密码子,克隆了Bar基因序列并构建了p ET28a-Bar融合表达载体,并转入大肠杆菌宿主菌BL21(DE3)。结果表明,融合表达载体在大肠杆菌BL21(DE3)菌株中以包涵体形式表达膦丝菌素乙酰转移酶(PAT蛋白质),并通过镍离子亲和层析纯化后获得了PAT蛋白质,该纯化蛋白质的获得为利用酶联免疫法定量检测和筛选转基因植物提供了必要前提。

In this thesis,the Bar gene sequence was optimized by using the method of bioinformatics to make it more suitable. The full gene fragment with restriction sites was inserted into the E. coli expression vector pET28a （＋） to construct a fusion expression vector pET28a-Bar. Then the fusion expression vector was transformed into the E. coli host strain BL21 （DE3）. The results showed that phosphinothricin acetyhransferase was expressed in the form of an inclusion body protein in the strain,Through the metal chelate chromatography the PAT protein was purified. The obtainment of the protein could provide a necessary prerequisite for the quantitatively testing and screening of the transgenie plants using the method of enzyme-linked immunosorbent assay.