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漆树TvGST7基因克隆及其功能分析
何学高, 赵爱国, 谢冬冬,等
西北农林科技大学 林学院
摘要:
【目的】克隆漆树(Toxicodendron vernicifluum (Stokes) F. A. Barkl.)叶片中的谷胱甘肽S-转移酶(glutathione S-transferase,GST)基因并分析其结构特征,明确该基因编码蛋白的酶活性及该基因的抗逆性,为其功能研究提供理论依据。【方法】根据已有的漆树叶片转录组数据,利用RT-PCR克隆漆树GST基因,应用生物学网站对其所编码蛋白的理化性质进行分析,并构建系统进化树和三维结构模型;利用原核表达系统和Ni-NTA亲和层析技术表达、纯化漆树GST重组蛋白,并利用不同底物测定重组蛋白的酶活性;将原核表达载体转入大肠杆菌BL21(DE3),验证该基因对低温、高温、高盐、高渗透压等非生物逆境胁迫的耐受性。【结果】获得了漆树谷胱甘肽S-转移酶基因TvGST7(GenBank登录号为MK956145)的完整编码序列,开放阅读框为711 bp,编码236个氨基酸,分子质量为27.17 ku,理论等电点为5.25;进化树分析和三维结构模拟表明,TvGST7蛋白属于Lambda(L)亚家族。酶活性测定结果显示,重组蛋白TvGST7具有巯基转移酶活性;胁迫试验表明,TvGST的表达能增强大肠杆菌对低温、高温、高盐、高渗透压等非生物胁迫的抗性。【结论】根据TvGST7重组蛋白具有巯基转移酶活性及TvGST7的表达提高了大肠杆菌对非生物胁迫的抗性,推测TvGST7基因在漆树中的表达对于维持漆树体内的正常代谢和清除非生物胁迫产生的氧化损伤起着重要作用。
关键词:  漆树  谷胱甘肽S-转移酶  酶比活力  胁迫耐受性
DOI:
分类号:
基金项目:国家重点研发计划项目“漆树丰产经营与精细产品开发技术研究”(2017YFD0600705)
Cloning and characterization analysis of glutathione S-transferase TvGST7 in Toxicodendron vernicifluum
HE Xuegao, ZHAO Aiguo,, XIE Dongdong,et al
Abstract:
【Objective】This research cloned the glutathione S-transferase (GST) gene (TvGST7) from the leaves of lacquer tree,Toxicodendron vernicifluum,analyzed its structure properties,and clarified the resistance and enzyme specific activity of protein coded by TvGST7,so as to provide basis for fundamental function research of TvGST7.【Method】Based on the transcriptome data of leaves from T. vernicifluum,the complete coding sequence of TvGST7 was cloned using RT-PCR.The biological website was used to analyze the physicochemical properties of the encoded protein,and a phylogenetic tree and a three-dimensional structural model were constructed.The recombinant protein was prokaryotically expressed and purified by Ni-NTA affinity chromatography.Simultaneously,enzyme specific activity of recombinant protein was determined by using different substrates.The prokaryotic expression vector was transferred into E. coli BL21 (DE3) to verify the tolerance of TvGST7 to abiotic stresses including low temperature,high temperature,high salt and high permeability.【Result】The complete coding region sequence of TvGST7 (GenBank accession No.:MK956145) was obtained,and its ORF was 711 bp in length,encoding 236 amino acids with the predicted molecular mass of 27.17 ku and theoretical isoelectric point of 5.25.The phylogenetic tree and the three-dimensional structure analysis indicated that TvGST7 belonged to Lambda(L) subfamily of GSTs.The specific activity showed that the recombinant protein TvGST7 had thiol transferase activity.The expression of TvGST7 enhanced the resistance of DE3 to low temperature,high temperature,high salt and high permeability.【Conclusion】According to the fact that TvGST7 recombinant protein had thiol transferase activity and its expression enhanced the resistance of E.coli to abiotic stress,it is speculated that the expression of TvGST7plays an important role in maintaining normal metabolism and eliminating oxidative damage caused by abiotic stress.
Key words:  Toxicodendron vernicifluum  glutathione S-transferase  enzymatic specific activity  stress tolerance