土壤宏基因组中抗草甘膦新基因的克隆及其功能验证

Cloning and functional analysis of a novel glyphosate resistant gene soilA from soil metagenome

【目的】从土壤宏基因组中克隆新的抗草甘膦新基因,并对其功能进行验证,为培育抗除草剂转基因新品种奠定基础。【方法】利用被草甘膦污染的土壤建立宏基因组文库,经筛选从中成功克隆了一个新的具有草甘膦抗性的5-烯醇丙酮莽草酸-3-磷酸合成酶(EPSPS)基因(命名为soilA)。构建了Prrn启动子驱动soilA基因的原核表达载体pSYTH-soilA,将其导入大肠杆菌进行原核草甘膦抗性试验,构建了35S启动子驱动soilA基因的植物表达载体pC330E,利用农杆菌介导法转化烟草,并对经PCR和胶体金试纸条鉴定的转基因烟草植株进行草甘膦耐受能力检测。【结果】序列分析表明,所获得的soilA基因长1 347bp,编码448个氨基酸,经BLAST分析,结果表明其属于ClassⅡ型EPSPS基因家族。原核表达soilA可使受体菌具有耐受1 200mmol/L草甘膦。构建soilA植物表达载体pC330E,通过农杆菌介导法将其导入烟草,经PCR和胶体金试纸条检测共获得107株阳性烟草植株,经过喷洒试验获得了3株高耐受草甘膦植株,这3株转基因烟草最高可耐受200mmol/L草甘膦。【结论】新克隆的编码EPSPS的soilA基因能够提高受体材料的草甘膦耐受能力。

[Objective] This paper aimed to clone and identify a novel glyphosate resistant gene from the metagenome derived from glyphosate-contaminated soil,which would lay foundation for cultivating new herbicide resistant and genetically modified breeds. [Method] In the present study, a novel glyphosate resistant 5-enolpyruvylshikimate-3-phosphate synthase （EPSPS） gene, named soilA, was isolated from the metagenomic library derived from glyphosate-contaminated soil. The cloned soilA gene was driven by Prrn promoters to construct prokaryotic expression vector pSYTH-soilA,which was introduced into E. coli for resistance analysis against glyphosate, and driven by 35S promoters to construct plant expression vector pC330E. Then,vector pC330E was introduced into tobacco for resistance analysis against glyphosate. [Re sult] The sequence analysis suggested that soilA consisted of 1 347 bp and encoded a polypeptide of 448 amino acids. BLAST analysis showed that it belonged to the Class Ⅱ family of EPSPS genes. Expression of soilA in prokaryotic system conferred the host cell could tolerate 1 200 mmol/L glyphosate solution. A plant expression vector pC330E harboring soilA gene was constructed and transferred into tobacco genome via agrobacterium-mediated transformation. A total of 107 transgenic plants were obtained by molecular i- dentification,3 of which were selected by the spray of glyphosate solution. Resistance experiment showed that the 3 transgenic plants could tolerate the spray of 200 mmol/L glyphosate solution. [Conclusion] The soilA gene enhanced the glyphosate tolerance of receptor materials.