大豆凝集素和脂肪氧化酶双价RNAi表达载体的构建及其遗传转化
Construction of RNAi expression vector of soybean agglutinin and lipoxygenase inhibitor genes and its transformation

【目的】应用RNA干扰技术，同时抑制大豆凝集素（Soybean agglutinin，SBA）和脂肪氧化酶（Lipoxygenase，Lox）基因在种子中的表达，改良大豆营养品质，为培育优质大豆材料奠定基础。【方法】根据RNAi原理，酶切获得Lox目的片段，构建以除草剂Bar基因为筛选标记、种子特异性启动子P7αP启动SBA和Lox双干扰的pCAMBIA3301－SBA－Lox(pSBA－Lox)干扰表达载体，并通过农杆菌介导法转化大豆（品种为吉农28），采用PCR、Southern杂交和实时荧光定量PCR对转基因植株进行检测。【结果】质粒PCR和酶切鉴定结果表明，双价RNAi植物表达载体pSBA-Lox构建成功。将其转入到大豆中，对转化植株进行PCR、Southern 杂交检测，结果显示，外源基因以单拷贝形式整合到植物基因组中，并能遗传给后代。T1代转基因植株的实时荧光定量PCR分析显示，转基因植株中SBA基因和Lox基因在籽粒中的表达量比未转化受体植株均明显降低，SBA基因表达量降低了35.9%～47.2%，Lox基因表达量降低了32.8%～56.1%，而在幼嫩叶片中的表达量相比对照植株变化不大。【结论】获得了大豆凝集素和脂肪氧化酶表达量均明显降低的T1代转基因大豆。
【Objective】The aim of this study was to improve the nutritional quality of soybean and create new elite soybean germplasm by using RNA interference to prohibit the expression of agglutinin and lipoxygenase inhibitor genes in soybean seed.【Method】RNAi expression vector of soybean agglutinin and lipoxygenase inhibitor genes promoted by P7αP and selected using herbicide,was constructed by digestion and cloning.Then the obtained expression vector was introduced into to soybean cultivar Jinong 28 via agrobacterium-mediated transformation and detected by PCR,Southern blot,and Real-time quantitative PCR.【Result】RNAi expression vector of soybean agglutinin and lipoxygenase inhibitor genes was successfully obtained.PCR and Southern blot results showed that the transgenic plants were also successfully established.Real-time quantitative PCR in T1 generation demonstrated that the expression of soybean agglutinin and lipoxygenase in transgenic soybeans was significantly inhibited.The expression of soybean agglutinin decreased by 35.9%-47.2% and that of lipoxygenase decreased by 32.8%-56.1%.【Conclusion】Transgenic plant lines of T1 generation with prohibited expression of soybean agglutinin and lipoxygenase inhibitor genes in seed were obtained