β-伴大豆球蛋白α′-亚基基因ihp-RNAi表达载体的构建

【目的】构建β-伴大豆球蛋白α′-亚基基因具有功能性间隔序列的发夹结构（Intron-hairpin RNAi，ihp-RNA）的RNA干扰(ihp-RNAi)表达载体并转化大豆，为通过RNA干扰技术改良大豆的营养品质奠定基础。【方法】以大豆总RNA反转录获得的cDNA为模板，通过PCR扩增克隆了β-伴大豆球蛋白α′-亚基基因的核心保守序列(400 bp)，并将该片段的反义和正义片段插入到重组植物表达载体p3301P的种子特异性启动子7αp下游，将功能性间隔序列intron-SSR插入反义片段与正义片段之间，构建α′-亚基基因ihp-RNAi安全型表达载体p3301-PFNZ-α′-BADH，并进行PCR及双酶切鉴定。利用农杆菌介导法将带有p3301-PFNZ-α′-BADH 的菌株转化“吉农27”大豆植株，对转基因植株进行PCR、Southern杂交检测，并对转基因植株α′-亚基基因的表达量进行RT-PCR。【结果】成功构建了β-伴大豆球蛋白α′-亚基基因ihp-RNAi表达载体p3301-PFNZ-α′-BADH，利用农杆菌介导法转化大豆得到7株阳性转化植株；Southern杂交结果显示，外源基因以1～2个拷贝整合于大豆基因组中；RT-PCR检测表明，β-伴大豆球蛋白α′-亚基基因的表达被明显抑制。【结论】成功构建了β-伴大豆球蛋白α′-亚基基因ihp-RNAi表达载体，获得了α′-亚基基因被明显抑制的转基因大豆植株，为应用基因工程技术进行大豆品质改良奠定了基础。

Construction of the ihp-RNAi expression vector of soybean β-conglycininα′-subunits gene

【Objective】 Theα′-subunit gene functional spacer sequence of the hairpin structure(intronhairpin RNAi,ihp-RNAi) RNA interference expression vector was built with transformation of soybean by RNA interference techniques,which could improve the nutritional quality of soybeans.【Method】 The core region ofα′-subunit gene of soybean β-conglycinin was cloned by PCR using soybean cDNA as template and inserted into downstream of seed-specific promoter(7αp) in the vector p3301-P,and the intron-SSR was inserted between anti-sense gene and sense gene and then appraised by PCR and double digestion.Then,the vector p3301-PFNZ-α′-BADH was mobilized into soybeans by Agrobacterium mediation.Transgenic plants were analysed by PCR and Southern blot and the expression ofα′-subunit gene was analysed by RT-PCR.【Result】 Then seven transgenic plants were obtained by the results of PCR.And the results of Southern-blot showed that foreign gene had been integrated into genomic DNA of soybean by one or two copies,and the results of RT-PCR demonstrated that the mRNA ofα′-subunit in transgenic soybeans was significantly inhibited.【Conclusion】 The β-conglycininα′-subunit gene ihp-RNAi expression vector was constructed successfully and theα′-subunit gene was significantly inhibited in transgenic soybean plants.This study is the basis of the application of genetic engineering technology to improve the quality of soybean.