甘蓝型油菜PEPC基因ihpRNA表达载体的构建与遗传转化研究

Construction of Seed-specific ihpRNA Expression Vector Targeting PEPC Gene and Transformation of Rapeseed

为提高油菜含油量,构建了基于油菜磷酸烯醇式丙酮酸羧化酶基因(PEPC)的ihpRNA植物表达载体。分别从甘蓝型油菜基因组中克隆了种子储藏蛋白napin基因启动子(315 bp)、PEPC基因的外显子片段(246 bp)及PEPC基因的内含子(754 bp),包含了其剪切位点之前4 bp和之后18 bp片段。通过中间载体pBluescriptⅡSK(+)再构建到pBI121中,构成种子特异表达的内含子剪接的PEPC基因的RNA干扰载体p BI121.NP+IP-。利用农杆菌介导法对湘油15号进行遗传转化,获得卡那霉素抗性转化植株9株,PCR检测证实外源片段成功导入其中7株油菜基因组中。对T2种子中含油量检测发现比对照提高10%左右。

To study the relationship between rapeseed oil content and phosphoenolpyruvate carboxylase activity,we constructed the seed-specific ihpRNA expression vector targeting PEPC gene in Brassica napus L..A fragment of 315 bp napin promoter and 246 bp exon fragment of PEPC gene were amplified from the genomic DNA of Xiangyou15.At the same time,a intron（754 bp） of PEPC gene including 4 bp and 18 bp DNA fragment before and after the splice site respectively was cloned into corresponding enzyme sites.According to the structural principle of expression vector,these fragments were cloned into p Bluescript ⅡSK（＋）,respectively.Then we inserted the ihpRNA structure into plant expression vector p BI121,designated p BI121.NP＋IP-.Transformed Xiangyou 15 by Agrobacterium tumefaciens containing p BI121.NP＋IP-,seven transgenic plants from nine anti-kanamycin plants were confirmed by PCR detection.The oil content of transgenic plants was about 10% higher than that of the controls.