叶酸生物合成途径中调控基因folB表达载体的构建及在大豆中的转化

Construction of the Folate Biosynthetic Pathway Regulatory Gene folB and Transformation of Soybean

叶酸是一种水溶性B族维生素,是维持人体正常活动所必需的营养元素之一,也是植物体内参与一碳单元反应的重要辅酶。folB基因是叶酸调控基因,来源于拟南芥,调控叶酸合成途径中的关键酶DHNA的生成。本研究将人工合成的folB基因转入大豆受体材料Williams82中。共侵染200个大豆外植体,得到26株转化苗。通过对转化苗的草丁膦(浓度150 mg/L)涂抹鉴定,获得16株除草剂抗性转化苗。通过PCR检测,得到12株PCR阳性转化苗。经过bar试纸条检测,其中10株为阳性。Southern杂交结果表明,9株转化苗为Southern杂交阳性。研究结果表明:外源功能基因folB和筛选标记基因bar已经整合到大豆基因组中。相应的转基因后代表现出除草剂抗性和folB基因Southern杂交阳性。本研究通过将folB基因转入大豆,获得了转基因后代,以此为基础可进一步选育高叶酸含量的转基因大豆品种。

Folate is one of the necessary nutrient elements which is water-soluble B-vitamins supported human body normal activities. Also is an important coenzyme involved in one carbon unit reaction in the plant. The folB is a key gene of folate pathway, which regulated production of a key enzyme for DHNA and derived from Arabidopsis thaliana. In this study, we expressed the folB by employing the soybean cotyledonary nodes method. We co-cultivated about 200 explants, and got 26 transgenic plants. Stock of Basta(150 mg/L) was painted on half the upper surface of tested soybean leaves, and we got 16 transgenic plants. PCR analysis showed that 12 of 14 transgenic plants were positive. Strip analysis show that 10 of 12 transgenic plants were positive. Southern blot analysis of PCR-positive plants indicated the integration of the folB gene into the soybean genome, and we also got 9 transgenic lines. Acquired transgenic plants provide the basis for high folate transgenic soybean varieties.