甘蓝型油菜丙酮酸激酶基因RNA 干扰载体的构建

Construction of a RNA interference vector of Brassica napus L.pyruvate kinase gene

采用基因芯片技术,从油菜中鉴定了一个油分积累优势表达基因即丙酮酸激酶(Pyruvate kinase,PK)基因。为构建甘蓝型油菜PK基因的RNA干扰(RNAi)载体,通过设计引物克隆了PK基因长498 bp的siRNA靶序列,连接到pEASY-T1载体上,采用Not I和Xho I酶切,酶切产物连接到RNAi平台载体pHurricane的Not I和Xho I位点,通过多重PCR鉴定证实载体构建成功,然后将上述PK基因反向重复框克隆到加入napin启动子的pCAMBIA1390表达载体相应的酶切位点上,构建具有种子特异性表达的PK基因的RNAi载体。限制性内切酶酶切证实载体构建成功,为进一步研究PK基因参与决定油菜油分积累的分子机理和代谢调控奠定了基础。

A predominantly expressed gene, pyruvate kinease（PK） gene, controlling oil accumulation, had been identified in the previous study. To construct a PK RNAi vector, a 498鄄bp target PK gene sequence was amplified and transferred into the pEASY鄄T1 vector. Subsequently, the target DNA fragments were cut off by enzymes Not I and Xho I and directionally inserted into plant RNAi platform vector pHurricane, a newly developed RNAi vector in our lab,to form the PK inverse repeats. The PK inverse repeats fragment was then cloned into a modified vector pCAMBIA1390 under the driven of a rapeseed seed鄄specific promoter napin. Restriction enzyme digestion verified the successful construction of RNA interference vector. The PK RNAi construction will lay a foundation for study in the function of PK in oil accumulation and metabolism in rapeseeds.