| 甜瓜细菌性果斑病菌MH21中γ-谷氨酰转移酶AcGGT3的异源表达与功能分析 |
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| 引用本文： | 董雅容,杨静,母军霞,赵晓燕,任争光.甜瓜细菌性果斑病菌MH21中γ-谷氨酰转移酶AcGGT3的异源表达与功能分析[J].北京农学院学报,2018,33(2):27-31. |
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| 作者姓名： | 董雅容 杨静 母军霞 赵晓燕 任争光 |
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| 作者单位： | 北京农学院植物科学技术学院/植物生产国家级实验教学示范中心,北京,102206;北京农学院植物科学技术学院/植物生产国家级实验教学示范中心,北京,102206;北京农学院植物科学技术学院/植物生产国家级实验教学示范中心,北京,102206;北京农学院植物科学技术学院/植物生产国家级实验教学示范中心,北京,102206;北京农学院植物科学技术学院/植物生产国家级实验教学示范中心,北京,102206 |
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| 基金项目： | 北京农学院青年科学基金(QZK2015002)，北京农学院高级别科研项目培育计划(GJB2015001) |
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| 摘 要： | 【目的】为了探索γ-谷氨酰转移酶(Gamma-glutamyltransferase,GGT)在甜瓜细菌性果斑病菌(Acidovorax citrulli)中的作用。【方法】以A.citrulli MH21菌株的DNA为模板,设计引物对Acggt3基因进行PCR扩增,克隆到蛋白表达载体pET22b(+)上,并进行测序和生物信息学分析。将重组质粒转化大肠杆菌BL21(DE3)中异源表达并纯化AcGGT3蛋白,用分光光度计法测定了不同温度和pH值条件下AcGGT3的酶活性。【结果】氨基酸序列分析结果显示,AcGGT3在Acidovorax属细菌中保守,并具有N末端信号肽。用IPTG诱导的方法成功表达并纯化出AcGGT3蛋白。酶活检测发现该蛋白在37℃时酶活性最高,最适pH为8.0。【结论】首次在A.citrulli菌株中克隆了γ-谷氨酰转移酶基因,并探索了AcGGT3蛋白的生物学特性,为下一步研究其致病功能奠定基础。
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| 关 键 词： | 细菌性果斑病 西瓜食酸菌 γ-谷氨酰转移酶 异源表达 酶活性 |
Heterologous expression and function analysis of gamma-glutamyltransferase AcGGT3 from Acidovorax citrulli MH21 |
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| DONG Yarong,YANG Jing,MU Junxia,ZHAO Xiaoyan,REN Zhengguang.Heterologous expression and function analysis of gamma-glutamyltransferase AcGGT3 from Acidovorax citrulli MH21[J].Journal of Beijing Agricultural College,2018,33(2):27-31. |
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| Authors: | DONG Yarong YANG Jing MU Junxia ZHAO Xiaoyan REN Zhengguang |
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| Abstract: | Objective]Gamma-glutamyltransferase (GGT) is a key enzyme in maintaining the intracellular redox balance in a number of organisms.In order to explore the function of the GGT in plant pathogen Acidovorax citrulli,a GGT gene Acggt3 was cloned and heterogeneous expressed from A.citrulli MH21,and the conditions of the AcGGT3 enzymatic activity were also detected.Methods]The Acggt3 gene was cloned in plasmid pET22b(+) and the AcGGT3 protein was heterogeneous expressed in Escherichia coli BL21(DE3).The cloned Acggt3 gene was also sequenced and analyzed,and the enzymatic activity of the purification AcGGT3 in different temperature and pH were measured by spectrophotometer method.Results]The amino acid sequence of AcGGT3 was conserved in Acidovorax strains and it had an N-terminal signal peptide.The recombinant protein of AcGGT3 was successfully expressed with the IPTG inducer,and the results of enzyme assay revealed that the temperature suited for the AcGGT3 activity best demonstrating was 37 ℃,the appropriate pH was 8.0.Conclusion]In this study,the GGT gene Acggt3 from A.citrulliwas first cloned and the AcGGT3 was successfully expressed in E.coli.The biological characteristics of this protein were determined.All the results provided references for exploring the pathogenic features of AcGGT3 in A.citrulliin future. |
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