抗草甘膦基因的克隆及在原核表达系统中的功能分析

摘要

This study was aimed to clone a glyphosate-resistant gene and analyze its glyphosate-resistance in Exhe-richia coli. A glyphosate-resistant strain G6PP was isolated from extremely glyphosate-polluted soil, which could grow on medium with 200 wnol/L glyphosate. It was identified as Pseudomonas putida by scanning electron microscopy and 16S rDNA. Total DNA was isolated from G6PP. The EPSPS was cloned by PCR, which encoded 440 amino acid residues. EPSPS was then subcloned into the expression vector pET-28a and the recombinant prokary-otic expression vector (pET-G6) was transformated into E. Coli BL21 (DE3). A fusion protein of about 46 ku was obtained with IPTG induction. E. Coli BL21 (DE3) harboring pET-G6 plasmid could tolerate 150 wnol/L glyphosate in M63 liquid medium. The EPSPS G6 had glyphosate-resistant activities and might be used to develop glyphosant-resistant transgenic crops.%为了克隆抗草甘膦基因并在原核表达系统中分析其抗性水平,利用200 μmol/L草甘膦平板从草甘膦严重污染的土壤中筛选到1株抗草甘膦菌株G6PP,经电镜和16S rDNA鉴定为恶臭假单胞菌；以该菌株基因组DNA为模板,通过PCR方法扩增出5-烯醇丙酮莽草酸-3-磷酸酯合成酶(EPSPS)基因,该基因编码440个氨基酸,亚克隆到原核表达载体pEET-28a中构建原核表达载体pET-G6;将重组表达载体转化大肠杆菌BL21 (DE3)中,经IPTG诱导,表达出46 ku的融合蛋白；携带pET-G6质粒的大肠杆菌BL21 (DE3)在液体M63培养基中能耐受150 μmol/L草甘膦的抑制.本研究结果表明克隆到的G6基因具有一定的抗草甘膦活性,对抗草甘膦作物的培育具有实践意义.