利用Cre/loxp定位重组系统构建雄性不育基因和恢复基因表达载体  

Constructions of Male Sterility Gene and Fertility Restoring Gene Expression Vectors by Cre/loxp Site-specific Recombination System

作　　者：宋洪元[1] 曹必好[2] 丁建刚[1] 雷建军[2] 宋明[1] 

机构地区：[1]西南农业大学园艺园林学院,重庆400716 [2]华南农业大学园艺系,广州510642

出　　处：《农业生物技术学报》2004年第4期396-400,共5页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(No.30200185);重庆市应用基础研究项目(No. 200217)资助。

摘　　要：结合Cre/lox定位重组系统和杂种一代的育种特点,构建了反义肌动蛋白雄性不育基因表达载体pCABARTAAct和相应的恢复基因表达载体pBINPLUSCre。将位于质粒pSTAAct中的反义肌动蛋白雄性不育基因表达盒(含TA29启动子、反向插入TA29启动子下游的Actin基因及Nos终止子)切下插入中间克隆载体pGloxp两个同向lox位点之间,再将上述表达盒连同lox位点切下插入pCAMBar,获得以除草剂Basta为筛选剂的反义肌动蛋白雄性不育基因表达载体pCABARTAAct。PCR方法从溶原菌BM25.8基因组DNA扩增出Cre基因,克隆测序验证无误后插入PBI525 CaMV35S-35S双启动子和Nos终止子之间,再将表达盒切下插入pBINPLUS质粒相应位点,得到恢复基因植物表达载体pBINPLUSCre。The antisense actin sterile gene expression vector pCABARTAAct and the fertility-restoring gene expression vector pBINPLUSCre were constructed by combining the features of Cre/loxp site-specific recombination system and F1 hybrid breeding. The antisense actin sterile gene expression box in pSTAAct vector which contained the TA29 promtor antisense actin gene and Nos terminator, was cut down and then inserted in between the two directly oriented loxp sites of pGloxp. The final expression vector pCABARTAAct was obtained by inserting the antisense actin sterile gene expression box with the two directly oriented lox sites into the plasmid pCAMBar , which contained the Bar gene. The Cre gene was cloned from lysogen BM25.8 by PCR. The Cre gene was then inserted in between the CaMV35S-35S double promtor and Nos terminator of PBI525 plasmid after sequencing. The expression box was cut down and inserted into plasmid pBINPLUS to obtain the final fertility-restoring expression vector pBINPLUSCre.

关 键 词：基因表达载体 雄性不育基因 系统构建 定位 除草剂Basta Nos终止子 CRE/LOX 肌动蛋白 植物表达载体 基因表达盒 CRE基因 DNA扩增 PCR方法 启动子 杂种一代 重组系统 克隆载体 克隆测序 恢复基因 插入 反义 位点 n基因 筛选剂 

分 类 号：Q782[生物学—分子生物学] S565.4[农业科学—作物学]