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

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。

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 3CRACE and 5CRACE . Homologous to other reported GlmS in sequence, the GImS 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 GImS were pH 6.4 at 37°C, the half-life period for the recombinant GImS was 1 h at 42°C 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.