油菜PEPase基因的克隆及其对应RNAi载体的构建

Molecular cloning of the PEPase gene from Brassica napus and the construction of its corresponding RNAi vectors

磷酸烯醇式丙酮酸羧化酶(PEPase)是控制油菜蛋白质/油脂含量比例的一个关键酶。采用RT-PCR方法从甘蓝型油菜中双4号中扩增出PEPase基因cDNA片段,与已发表的PEPase基因(登录号为D13987)序列同源性为98%。根据RNA干扰(RNAi)目标序列的选取原则选取两段长度约为400bp的DNA序列,分别克隆到RNAi载体pHBM1301中,构建了油菜中对应于PEPase基因的RNAi载体pHBM1301-PEP1和pHBM1301-PEP2。

Phosphoenolpyruvate carboxylase(PEPase) plays an important role in the control of the ratio of the protein/lipid content in rape seed (Brassica napus). The cDNA fragment of PEPase gene was cloned from B.napus zhongshuang No.4 by RT-PCR. Sequence analysis of the fragment suggested that it possessed 98% homology with the PEPase gene(D14985). In order to down-regulate the expression of PEPase gene, two 400bp fragments were cloned into the Sap I site of RNAi vector pHBM1301 and two recombinant plasmids pHBM1301-PEP1 and pHBM1301-PEP2 were obtained.