携带芋兰属LATERAL SUPPRESSOR(LS)-EGFP融合基因植物表达载体的构建

Construction of Plant Expression Vector Containing the LATERAL SUPPRESSOR(LS) and EGFP Fusion Gene from Nervilia Genus

LATERAL SUPPRESSOR(LS)是GRAS家族参与植物分生组织发育的转录因子,是控制植物分枝的关键基因。增强型绿色荧光蛋白基因(EGFP)是目前应用广泛的分子生物学报告基因之一,为筛选转基因植株提供了简便有效的方法。为了探讨LS基因在青天葵及其同属植物广布芋兰中的功能,本研究利用降落-重叠PCR(Touchdown-Overlap Extension PCR,TD-OE PCR)方法,分别构建了两种芋兰属植物LS基因和EGFP融合基因。通过酶切和连接的方法,将融合基因构建到植物双元表达载体p RI 101-AN DNA中,成功获得了两个携带芋兰属植物LS-EGFP融合基因的植物表达载体p RI 101-LS-EGFP,并成功转化至农杆菌,下一步拟用于转化拟南芥,为深入研究该基因功能提供基础。

LATERAL SUPPRESSOR(LS)- which regulates the branching and development in plants, is a transcription factor belonging to GRAS family. EGFP(enhanced green fluorescent protein) gene is one of the widely used reporter genes, providing effective and easy approach to screen transgenic plants. In order to elucidate the function of the LS genes in Nervilia fordii and its close relative species Nervilia aragoana, here the EGFP gene, the Nf LS gene from N.fordii and the Na LS from N.aragoana, respectively, were fused by touchdown-overlap extension PCR(TD-OE PCR), and were then cloned into plant binary expression vector p RI 101-AN DNA, resulted in two recombinant expression vectors p RI 101-Nf LS/Na LS-EGFP by restriction and ligation. The recombinant plasmids were successfully transferred into Agrobacterium strain EHA105.Functional of Nf LS/Na LS genes will be carried out using transgenic Arabidopsis thaliana transfected by Agrobacterium tumefaciens carrying recombinant plasmids.