过表达谷胱甘肽合成酶基因增强烟草对镉的耐受性

Over-expression of Glutathione Synthetase Gene Enhances Cadmium Tolerance in Transgenic Tobacco Plant

随着工业的发展,土壤污染问题愈发严重,利用基因工程修复土壤技术备受青睐,因此,开发重金属应答中的限速酶基因,将为植物修复重金属污染的土壤提供可应用的基因资源。通过RT-PCR及末端克隆方法获得枸杞谷胱甘肽合成酶(Lycium chinense,Glutathione synthetase,LcGS)基因,采用半定量RT-PCR分析了枸杞LcGS在不同时间镉(Cd)胁迫下表达量的变化,LcGS表达量随着胁迫时间的延长而增强,胁迫9h、12h和24h后LcGS表达量维持在较高水平。同时构建了植物双元表达载体pCAMBIA2300-LcGS,通过农杆菌介导的方法将LcGS基因转入烟草,PCR证明了LcGS基因成功整合到烟草基因组中,在Cd处理条件下,转基因植株谷胱甘肽(glutathione,GSH)、植物螯合肽(phytochelatins,PCs)和叶绿素含量比对照组明显高,即转基因植株对重金属的耐逆性比对照组更强,因此,过表达GS植物将是植物修复重金属污染的一个有效策略。

Soil pollution,as a result of industrial development,is becoming a serious problem in China and raising increasing concerns from the public. Soil remediation through plant genetic engineering is a widely used method to tackle this problem. The theory is to provide a new genetic resource for heavy metal phytoremediation through the study and manipulation of heavy metal responsive rate-limiting enzymes in plants. The gene LcGS,coding glutathion synthetase（GS） of Lycium chinense was cloned using reverse transcription polymerase chain reaction（RT-PCR） and rapid amplification of cDNA ends（RACE）. Expression of LcGS in L. chinense under CdCl2 stress over a 24 h time period was monitored and analyzed using semi-quantitative RT-PCR. The expression of LcGS gene up-regulated initially following the increase of Cd stress time,then remained at a relatively high level from 9h,till the end of the monitoring period at 24 h. The expression vector containing this gene was also constructed and named pCAMBIA2300-LcGS. Then the LcGS expression cassette was transformed into tobacco via Agrobacterium-mediated transformation.Integration of this foreign gene was confirmed using PCR. Under CdCl2 stress,transgenic plants carrying LcGS gene contain higher contents of glutathione（GSH）,phytochelatins（PCs） and chlorophyll than wild-type plants,confirming higher tolerance to CdCl2. Therefore,over-expression of glutathion synthetase genes through genetic engineering can be a promising strategy for heavy metal phytoremediation.