中国水稻科学

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

• 研究报告 • 上一篇    下一篇

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. 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ming 2, LIU Youzhou 1, NIE Yafeng 1 , ZHANG Rongsheng1   

  1. 1 Institute of Plant Protection， Jiangsu Academy of Agricultural Sciences， Nanjing 210014， China； 2  Institute of food crops, Jiangsu Academy of Agricultural Sciences， Nanjing 210014， China;  3 Plant 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，*

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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

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