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（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 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.
出处
《西北农林科技大学学报（自然科学版）》
CSCD
北大核心
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)
