过表达谷胱甘肽合成酶基因增强烟草对镉的耐受性

doi:10.13523/j.cb.20141013

中国生物工程杂志

2014, Vol. 34

Issue (10): 79-86 DOI: 10.13523/j.cb.20141013

技术与方法

过表达谷胱甘肽合成酶基因增强烟草对镉的耐受性

贾翠翠¹, 季静¹, 王罡¹, 田小卫^2,3, 杜希龙², 关春峰¹, 金超¹, 吴电云²

1. 天津大学环境科学与工程学院天津 300072;
2. 天津大学化工学院天津 300072;
3. 天津农学院园艺园林学院天津 300384

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

JIA Cui-cui¹, JI Jing¹, WANG Gang¹, TIAN Xiao-wei^2,3, DU Xi-long², GUAN Chun-feng¹, JIN Chao¹, WU Dian-yun²

1. School of Environmental Science and Engineering, Tianjin University, Tianjin 300072, China;
2. School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China;
3. College of Horticulture and Landscape, Tianjin Agricultural University, Tianjin 300384, China

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摘要：

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.

关键词： 枸杞; LcGS; CdCl₂胁迫; PCs

Abstract:

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 24h 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 24h. 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.

Key words: Lycium chinense Glutathione synthetase CdCl₂ Stress PCs

收稿日期: 2014-07-16 出版日期: 2014-10-25

ZTFLH:

Q789

基金资助:

国家自然科学基金(31271793,31271419)、国家转基因生物新品种培育重大专项(2014ZX08003-002B, 2014ZX07203-009)资助项目

通讯作者: 季静 E-mail: jijingtjdx@163.com;wanggangtjdx@126.com

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引用本文:

贾翠翠, 季静, 王罡, 田小卫, 杜希龙, 关春峰, 金超, 吴电云. 过表达谷胱甘肽合成酶基因增强烟草对镉的耐受性[J]. 中国生物工程杂志, 2014, 34(10): 79-86.

JIA Cui-cui, JI Jing, WANG Gang, TIAN Xiao-wei, DU Xi-long, GUAN Chun-feng, JIN Chao, WU Dian-yun. Over-expression of Glutathione Synthetase Gene Enhances Cadmium Tolerance in Transgenic Tobacco Plant. China Biotechnology, 2014, 34(10): 79-86.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20141013 或 https://manu60.magtech.com.cn/biotech/CN/Y2014/V34/I10/79

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