土壤宏基因组中抗草甘膦新基因的克隆与转化水稻的研究

Cloning of New Herbicide Resistant Gene in Soil Metagenomics and Generation of Transgenic Rice Plants

EPSPS(5-烯醇丙酮莽草酸-3-磷酸合酶,EC 2.5.1.19)是植物芳香族氨基酸和植物次生代谢产物生物合成中莽草酸途径的关键酶;同时也是广谱性除草剂草甘膦的作用目标。本实验通过对草甘膦污染土壤宏基因组文库的建立及筛选,成功克隆了一个新的草甘膦抗性的EPSPS基因(命名为soilEPSPS)。序列分析表明,soilEPSPS基因全长1404 bp,其编码的467个氨基酸中未涉及已公布专利中保护的氨基酸序列。原核功能验证表明,该基因对草甘膦的耐受能力优于EPSPS CP4基因。将该基因与水稻Rubisco SSU引导肽相融合构建由actin启动子驱动的植物表达载体,用农杆菌介导法实现了水稻的遗传转化。抗性再生植株的PCR和Southern杂交结果表明所获得的26株再生植株均为转基因阳性植株,其中共有3个单拷贝转化事件。草甘膦抗性鉴定证明纯合体T2代植株能够耐受高达500 mmol L–1的草甘膦。本研究为转基因抗除草剂水稻新品种的培育奠定了基础。

The EPSPS （5-enolpyruvylshikimate-3-phosphate synthase, EC 2.5.1.19） is considered as one of the crucial enzymes in the shikimate pathway for the biosynthesis of essential aromatic amino acids and secondary metabolites in plant. It is also proved as a specific target of broad spectrum herbicide glyphosate. In this research, a new glyphosate resistant EPSPS gene named soilEPSPS was isolated by screening the metagenomic library. The sequence analysis suggested that soilEPSPS consists of 1404 bp and encodes a polypeptide of 467 amino acids that are not involved in the amino acid sequence protected by the published patent- sions. The result of prokaryotic expression confirmed that soilEPSPS had a better ability of glyphosate resistance than EPSPS CP4 gene. Fusing the gene and the Rubisco SSU signal peptide constructed the plant expressing vector with the actin promoter driven, and transformation of rice mediated by Agrobacterium tumefaciens. PCR and Southern analyses showed that a total of 26 trans genic plants were obtained and three of them were single copy insertion events. Resistant experiment showed that the homozy gous T2 plants in these events could tolerate the spray of 500 mmol L glyphosate solution. This study laid a foundation for breeding transgenic herbicide resistant rice varieties.