甘蓝型油菜三种种子储藏蛋白和丙酮酸羧化酶基因片段的克隆及hpRNA表达载体的构建

利用PCR从甘蓝型油菜(B rassica napusL. )华双4号基因组DNA中扩增出种子储藏蛋白cruciferin、nap2 in、oleosin和丙酮酸羧化酶( PEPC) 基因片段,再以扩增出的片段为模板设计引物从一端扩增出4个相应的小片 段,然后将同一基因的大小两个片段反向连接,插入到种子特异表达载体2300 - nap多克隆位点的nap in启动子和 nos终止子之间,构建成可以在油菜种子中转录表达发夹RNA (Hairp in RNA, hpRNA)结构的植物表达载体。

Cloning and hpRNA vector construction for genes of cruciferin, napin, oleosin and PEPC in Brassica napus L.

Three major storage proteins,i.e.,cruciferin,oleosin and napin amount to 80%~90% of the total seed protein in Brassica napus.Phosphoenolpyruvate carboxylase(PEPC) plays an important role in controlling the ratio of protein/lipid content in rapeseed.Gene fragments for cruciferin,napin, oleosin and PEPC were PCR-amplified from B.napus genomic DNA and used as templates to amplify four smaller fragments.The larger and the smaller fragments of the same gene were ligated in opposite orientations into a seed specific expression vector 2300-nap to produce hpRNAi vectors.Sequence analyses indicated that the fragments cloned were indeed genes for cruciferin,napin,oleosin and PEPC.These hpRNA vectors will be useful in the attempt to increase oil content in rapeseed by inhibiting the seed protein synthesis.