抗草甘膦的新型II类EPSPs基因的体外分子进化及转基因植物的功能分析

Glyphosate-resistant crop currently takes the biggest share of GM cultivation area all around the world. Thus, the cloning, expression and function of EPSPs become main focuses of modern bio-molecular breeding. AroA gene encoded EPSP synthase was successfully cloned from Bacillus cereus in our laboratory and was proved to be highly resistant to glyphosate. In this program,aroA gene resistant to high glyphosate will be obtained by using complementary genomic library screening technology and gene synthesis and transformation will be carried out by PTDS gene synthesis and POEP many sites mutations methods on the basis of the plant preference codon for these genes better application in production practice. Excellent mutant gene with independent intellectual property rights and high resistance to glyphosate will be obtained by DNA shuffling used synthetical aroA gene as a template, combined with high-throughput screening system. The three structure of protein encoded by mutation gene will be predicted and analyzed using bioinformatics methods. The relationship between mutation sites and the increase of glyphosate-resistant will be discussed. The mutant genes will be introduced into the model plant Arabidopsis and rice and the glyphosate-resistant will be analyzed, which will provide the theory basis for transgenic plant with independent intellectual property rights and high resistance to glyphosate.

抗草甘膦转基因作物是目前全球播种面积最大的转基因作物，抗草甘膦基因(aroA)的克隆、表达及功能验证等也因此成为现代分子生物育种研究的重点。本实验室已从蜡样芽胞杆菌（Bacillus cereus）中成功克隆了编码EPSPS合酶的aroA基因，并证明了其具有较高的抗草甘膦能力。为使该基因能够应用于生产实践，本项目利用基因组文库互补筛选技术获得高抗草甘膦aroA基因，并根据植物偏爱密码，采取PTDS基因合成和POEP多位点突变等方法进行基因合成和改造；以合成的aroA基因为模板进行DNA改组，结合高通量的筛选体系，以期获得优良的具有自主知识产权的高抗草苷膦的突变基因；同时采用生物信息学方法对突变基因编码蛋白质的三级结构进行预测和分析，探讨突变位点与草苷膦抗性增加的关系；并将突变基因转入模式植物拟南芥和水稻，分析其草苷膦耐受性，为培育具有自主知识产权的抗草甘膦转基因植物奠定理论基础。

抗草甘膦转基因作物是目前全球播种面积最大的转基因作物，抗草甘膦基因(aroA)的克隆、表达及功能验证等也因此成为现代分子生物育种研究的重点。本项目利用基因组文库互补筛选技术获得高抗草甘膦aroA基因，并根据植物偏爱密码，采取PTDS基因合成和POEP多位点突变等方法进行基因合成和改造；以合成的aroA基因为模板进行DNA改组，将改组片段构建成原核表达库，转化大肠杆菌草甘膦敏感菌株，在高浓度的草甘膦选择压存在条件下筛选抗性菌株。经过5轮循环建库，最后筛选获得2个高耐受草甘膦突变体。同时采用生物信息学方法对突变基因编码蛋白质的三级结构进行了分析。已经完成蜡样芽胞杆菌的新型的II类EPSP合酶突变基因的植物表达载体构建，分别将表达载体电击转化农杆菌GV3101和EHA105。通过蘸花法遗传转化拟南芥，分别获得20株表达突变体和野生型的aroAB.cereus转基因拟南芥，通过农杆菌介导转化水稻，分别获得15株突变体和野生型的aroAB.cereus转基因水稻，对转基因植物进行了草甘膦耐受性试验，验证突变体转基因植物对草甘膦高度耐受性。利用草甘膦平板实验及喷洒实验，检测草甘膦对植物幼苗生长及根生长等方面的影响，并进行了叶绿素及脯氨酸和POD活性的检测，结果表明无论转基因拟南芥还是转基因水稻，转突变体aroAB.cereus植株都比转野生型植株和对照植株更强的草甘膦抗性。本项目申报并授权3项国家基因发明专利，发表SCI论文5篇，培养硕士和博士研究生2名。