β-伴大豆球蛋白α′-亚基基因ihp-RNAi表达载体的构建  

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

作　　者：李夏[1] 戴佳乐[1] 陈子奇[1] 付永平[1] 马建[1] 曲静[1] 王丕武[1] 

机构地区：[1]吉林农业大学生物技术中心,吉林长春130118

出　　处：《西北农林科技大学学报（自然科学版）》2012年第10期191-198,共8页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家自然科学基金项目(30971805)

摘　　要：【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(400bp),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。【Objective】The α′-subunit gene functional spacer sequence of the hairpin structure（intronhairpin 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 of α′-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 of α′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 of α′-subunit in transgenic soybeans was significantly inhibited.【Conclusion】The β-conglycinin α′-subunit gene ihp-RNAi expression vector was constructed successfully and the α′-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.

关 键 词：α′-亚基基因 ihp-RNAi表达载体 农杆菌介导法 大豆 

分 类 号：S565[农业科学—作物学]