甘蓝型油菜PEPC基因保守序列的克隆和种子特异性ihpRNA表达载体的构建

Cloning of the PEPC Gene and Construction of a Seed-specific ihpRNA Expression Vector in Brassica napus L.

为了通过转基因途径获得油菜种子中PEPC基因发生转录后基因沉默,通过PCR扩增分别分离得到了甘蓝型油菜种子特异性表达的Napin启动子序列(1138bp)和磷酸烯醇式丙酮酸羧化酶(PEPC)基因保守序列(181bp),将它们分别克隆到pMD18-T载体中,利用中间载体pBS-NEI构建了PEPC基因的反向重复框。再将PEPC基因片段以正向的方式连接到一个可剪切的内含子5'末端,以反向的方式连接到该内含子的3'末端;然后将Napin启动子序列克隆到植物表达载体pCAMBIA1391的pUC18多克隆位点上,获得了种子特异表达的载体pCAMNapin;再将PEPase基因的反向重复序列克隆到pCAMNapin载体Napin启动子的3'末端,构建了具有种子特异性表达的PEPC基因的ihpRNA表达载体pCAMNapin-B2-NEI-B1。通过限制性内切酶酶切对载体作了鉴定分析。

A full-length napin promoter sequence1 （138 bp） and a conserved region （181 bp） of PEPC gene were amplified from the genomic DNA of Brassica napus L.and recombined into the pMD18-T vector respectively. To develop an ihpRNA vector for seed-specific expression targeting the PEPC gene in Brassica napus L., an inverted repeat unit of the 181 bp segment of PEPC gene was first cloned into a mediating vector, pBS-NEI vector, with a spliceable PEPC intron sequence in middle. An ihpRNA expression vector pCAMNapin was constructed through inserting the seed-specific Napin promoter into the multiple clonal sites in the binary vector pCAMBIA1391. Then the whole inverted repeat cassette was inserted into the 3＇-end of the Napin promoter in the vector pCAMNapin. It was finally confirmed by the digestion of restriction enzymes, that a seed-specific expression ihpRNA vector, pCAMNapin-B2-NEI-B1, targeting the PEPC gene in Brassica napus L. was constructed.