芸薹属TT19基因家族RNA干扰载体的构建

摘要

为构建芸薹属TT19基因家族的RNAi载体,为芸薹属植物的种皮色素、观赏色彩等性状的研究和修饰打基础.利用PCR克隆芸薹属TT19基因家族的RNAi片段,采用双酶切将其反义片段和正义片段分别从T-载体亚克隆到平台载体pFGC5941M的启动子与间隔区之间和间隔区与终止子之间,形成RNAi载体,转化大肠杆菌并完成PCR鉴定,再转化根癌农杆菌LBA4404得到工程菌株.结果表明:克隆得到200 bp(含人工酶切位点)的芸薹属TT19基因家族RNAi片段BTT19I,其对应于甘蓝型油菜BnTT19-1 mRNA的363～540 bp,其反义和正义片段分别被NcoI+AatII和BamHI+XbaI双酶切亚克隆到pFGC5941M中,得到10552 bp的RNAi载体pFGC5941M-BTT19I(简称pBTT19I).成功构建了芸薹属TT19基因家族的RNAi载体.%The RNAi fragment of Brassica TT19 gene family is cloned by PCR first, and then its sense and antisense fragments are respectively sub-cloned into between promoter and spacer, and between spacer and terminator in platform vector pFGC5941M for forming RNAi vector and finally the RNAi vector is transformed into Agrobacterium tumefaciens strain LBA4404 after PCR identification to lay a foundation for research and modification of testa pigment and ornamental colo characters. The sequence results showed that the RNAi fragment (BTT19I) with 200 bp of Brassica TT19 gene family was cloned, which corresponds to 363～540 bp of BnTT19-1 mRNA, its sense and antisense fragments were respectively subcloned into pFGC5941M by NcoI+AatII and BamHI+ XbaI1 double digestion and pFGC5941M-BTT19 I,a RNAi vector with 10 552 bp was obtained.