甘蓝型油菜丙酮酸激酶基因RNA干扰载体的构建

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 NotI和XhoI酶切，酶切产物连接到本课题组新近改造的 RNAi 平台载体 pHurricane 的 NotI 和XhoI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。

Construction of a RNA Interference Vector of Brassica napus L. Pyruvate Kinase Gene

A predominantly expressed gene, pyruvate kinease （PK） gene, control ing oil accumulation, had been identified in previous study. To construct a PK RNAi vector, a 498-bp target PK gene sequence was amplified and transferred into the pEASY-T1vector. Subsequently, the target DNA fragments were cut off by enzymes Not I and Xho I and directional y inserted into plant RNAi platform vector pHurricane, a newly developed RNAi vector in our lab, to form the PK inverse repeats. The PK inverted repeats fragment was then cloned into a modified vector pCAMBIA1390, driven by a rapeseed seed-specific promoter napin. Restriction enzyme digestion verified the successful construction of RNA interference vector. The PK RNAi con-struction wil lay a foundation for study in the function of PK in oil accumulation and metabolism in rapeseeds.