大豆BBi基因ihpRNA种子特异性表达载体的构建

Construction of ihpRNA seed-specific expressed vector of Bowman-Birk inhibitor gene from soybean

【目的】构建具有大豆蛋白酶抑制剂BBi基因反向重复结构的ihpRNA种子特异性表达载体,并将其转入根癌农杆菌中,为大豆品质改良奠定基础。【方法】采用PCR技术克隆大豆蛋白酶抑制剂BBi基因的正义和反义片段、大豆种子特异性启动子7αP和作为内含子的GFP基因片段,分别连入克隆载体pMD18-T Vector中。然后根据植物中ihpRNA原理,以植物表达载体pCAMBIA1301为基础,将组成RNAi载体的4个目的片段分别连入其中,然后利用冻融法转化根癌农杆菌,并进行PCR鉴定。【结果】PCR扩增得到组成RNAi载体的4个目的片段,分别构建成重组克隆载体和ihpRNA种子特异性表达载体p7αP-GFP-BBiS,并转化得到含有p7αP-GFP-BBiS的农杆菌EHA101和EHA105。【结论】成功构建了具有BBi基因反向重复结构的ihpRNA种子特异性表达载体p7αP-GFP-BBiS,BBi基因的ihpRNA表达框架成功转入到农杆菌中。

【Objective】 The ihpRNA seed-specific expressed vector of Bowman-Birk inhibitor gene from soybean was constructed,and then introduced into Agrobacterium tumefaciens to lay a foundation for quality improvement of soybean.【Method】 In this paper,the BBi gene,soybean seed-specific promotor 7αP and the fragment of GFP gene which were used as intron were isolated by PCR method,and then inserted into pMD18-T Vector respectively.The ihpRNA seed-specific expressed vector p7αP-GFP-BBiS with inverted repeats of BBi gen...