甘蓝型油菜BnKCR2基因cDNA的克隆和真核表达载体的构建  

cDNA Cloning and Eukaryotic Expression Vector Construction of BnKCR2 Gene in Brassica napus L.

作　　者：王慧[1] 王茂华[1] 刘绵学[1] 杨毅[1] 李旭锋[1] 

机构地区：[1]四川大学生命科学学院生物资源与生态环境教育部重点实验室,四川成都610064

出　　处：《安徽农业科学》2007年第23期7105-7107,共3页Journal of Anhui Agricultural Sciences

基　　金：国家自然科学基金资助项目(30570986)

摘　　要：[目的]为了克隆甘蓝型油菜的BnKCR2基因,并构建其真核表达载体,以期得到高芥酸含量的油菜。[方法]根据GenBank中甘蓝型油菜3-酮酯酰-CoA还原酶(BnKCR2)的cDNA序列设计引物,提取蜀杂九号油菜叶片的总RNA,进行RT-PCR扩增,将产物与T-A克隆载体pMD18-T连接,通过中间载体pMD18-T和pBluescript将BnKCR2分别正向、反向导入pBI121质粒载体,构建BnKCR2基因的正向和反向表达质粒。[结果]通过甘蓝型油菜总RNA的RT-PCR扩增,成功克隆到一个960 bp的BnKCR2基因;重组质粒pMD18-BnKCR2测序结果与NCBI中甘蓝型油菜cDNA(AY196197)序列的相似性达99%;而蛋白质序列仅有1个氨基酸之差,且蛋白质天然构象未改变,BnKCR2是反向连接克隆。[结论]通过冻融法将重组质粒导入根癌农杆菌,为通过根癌农杆菌介导法将BnKCR2基因导入油菜奠定了基础。[Objective] The aim of the research was to clone BnKCR2 gene and construct its eukaryotic expression vector in order to find the rape with high erucic acid. [Method] Designing the primers based on the cDNA sequence of 3-ketoacyl-CoA reducase （BnKCR2） in Brass ica napus L. from GenBank, total RNA was extracted from the rape leaves of Shuza 9 to amplify by RT-PCR and connect the amplification products with T-A cloning vector pMD18-T. Through the intermediate vectors pMD18-T and pBluescript, the BnKCR2 gene was introduced to plasmid vector pBI121 in forward and reverse directions to construct the forward and reverse expression plasmids of BnKCR2 gene, [Result] Through the RT-PCR amplification of total RNA in Brassica napus L., BnKCR2 gene of 960 bp was cloned successfully. The similarity between the sequencing results of recombinant plasmid and the cDNA sequence （AY196197） in Brassica napus L. from NCBI website was over 99 %, The sequences of protein had difference with one amino acid, the natural conformation of protein didn＇t change and BnKCR2 was the reverse connection cloning. [Conclusion] Using freezing-thawing method, the recombinant plasmid was introduced to Agrobacterium tumefaciens, which laid the foundation for introducing the BnKCR 2 gene to rape by Agrobacrerium tumefacie ns -mediated transformation.

关 键 词：BnKCR2基因 真核表达载体 载体构建 

分 类 号：Q78[生物学—分子生物学]