水稻纹枯病菌6磷酸葡萄糖胺合成酶基因的克隆、测序及表达分析

doi:10.3969/j.issn.10017216.2012.02.002

中国水稻科学 ›› 2012, Vol. 26 ›› Issue (2): 137-143.DOI: 10.3969/j.issn.10017216.2012.02.002

水稻纹枯病菌6磷酸葡萄糖胺合成酶基因的克隆、测序及表达分析

罗楚平^1，3,刘永锋¹,陈志谊^1,3,* ,王晓宇¹,方先文²,陈忠明²,刘邮洲¹ ,聂亚锋¹ ,张荣胜¹

¹江苏省农业科学院植物保护研究所, 江苏南京 210014； ²江苏省农业科学院粮食作物研究所, 江苏南京 210014；³南京农业大学植物保护学院, 江苏南京 210095

收稿日期:2011-01-25 修回日期:2011-09-19 出版日期:2012-03-10 发布日期:2012-03-10
通讯作者: 陈志谊1,3,*
基金资助:
国家自然科学基金资助项目（30900929，31071754）

Molecular Cloning, Sequencing, and Expression of a Glucosamine6phosphate Synthetase Gene from Rice Pathogen Rhizoctonia solani

LUO Chuping ^1,3, LIU Yongfeng ¹, CHEN Zhiyi ^1,3，* , WANG Xiaoyu ¹, FANG Xianwen ², CHEN Zhongming², LIU Youzhou ¹, NIE Yafeng ¹ , ZHANG Rongsheng¹

¹ Institute of Plant Protection， Jiangsu Academy of Agricultural Sciences， Nanjing 210014， China； ²Institute of food crops, Jiangsu Academy of Agricultural Sciences， Nanjing 210014， China; ³Plant Protection College, Nanjing Agricultural University, Nanjing 210095, China;

Received:2011-01-25 Revised:2011-09-19 Online:2012-03-10 Published:2012-03-10
Contact: CHEN Zhiyi1,3，*

摘要/Abstract

摘要： 为筛选新型水稻纹枯病菌GlmS活性抑制物质，采用3′RACE和5′RACE克隆了水稻纹枯病菌GlmS的基因组DNA序列和完整的cDNA序列。GlmS基因组DNA序列全长2 529 bp，含有8个内含子；GlmS cDNA序列全长2094 bp，推测编码一个含有697个氨基酸残基，分子量约为76.7 kD的蛋白质。生物信息学分析表明水稻纹枯病菌GlmS含有1个谷氨酰胺氨基转移酶结构域和2个葡萄糖异构酶结构域。采用大肠杆菌重组融合表达GlmS，重组蛋白的分子量经过葡聚糖凝胶层析和SDSPAGE电泳测得分别为306 kD和77 kD，表明GlmS是由4个相同大小亚基组成的多聚酶复合体。重组蛋白的酶学性质研究表明其最适反应温度为37℃，最适pH为6.4，42℃下的半衰期为1 h，在pH 5.5～7.5时比较稳定。GlmS催化反应能被己糖胺通路末端产物鸟苷氮乙酰葡萄糖胺反馈抑制。

关键词: 水稻纹枯病菌, 6磷酸葡萄糖胺合成酶, 活性物质, 克隆表达

Abstract: To develop novel antimicrobial agents to Rhizoctonia solani, the genomic gene and complete cDNA encoding glucosamine6phosphate synthetase were cloned and sequenced from the rice pathogen Rhizoctonia solani by 3′RACE and 5′RACE . Homologous to other reported GlmS in sequence, the GlmS contains eight introns, and encodes a predicted protein of 697 amino acids. Domain structure analysis revealed that R.solani GlmS contained a glutamine transferase motif and two sugar isomerase motifs. Recombinant native R.solani GlmS enzyme was overexpressed using Escherichia coli and purified. The results of Gel filtration chromatography and SDSPAGE revealed that it had an estimated molecular mass of 306 kD and consisted of four equalsized subunits of 77 kD. The optimal reaction conditions for the recombinant GlmS were pH 6.4 at 37℃, the halflife period for the recombinant GlmS was 1 h at 42℃ and the enzyme was stable at pH 5.5-7.5. R.solani GlmS activity was inhibited by the endproduct of the hexosamine pathway, UDPGlcNAc.

Key words: Rhizoctonia solani, glucosamine6phosphate synthetase, antimicrobial agents, cloning and expression

中图分类号:

罗楚平1，3刘永锋1陈志谊1,3,*王晓宇1方先文2陈忠明2刘邮洲1聂亚锋1张荣胜1. 水稻纹枯病菌6磷酸葡萄糖胺合成酶基因的克隆、测序及表达分析[J]. 中国水稻科学, 2012, 26(2): 137-143.

LUO Chuping1,3, LIU Yongfeng1, CHEN Zhiyi1,3，*, WANG Xiaoyu1, FANG Xianwen2, CHEN Zhongming2, LIU Youzhou1, NIE Yafeng1 , ZHANG Rongsheng1 . Molecular Cloning, Sequencing, and Expression of a Glucosamine6phosphate Synthetase Gene from Rice Pathogen Rhizoctonia solani [J]. Chinese Journal of Rice Science, 2012, 26(2): 137-143.

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水稻纹枯病菌6磷酸葡萄糖胺合成酶基因的克隆、测序及表达分析

Molecular Cloning, Sequencing, and Expression of a Glucosamine6phosphate Synthetase Gene from Rice Pathogen Rhizoctonia solani

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