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

摘要

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 inhihited by the end-product of the hexosamine pathway, UDP-GlcNAc.%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2529bp,含有8个内含子；GlmS cDNA序列全长2094bp,推测编码一个含有697个氨基酸残基,分子量约为76.7kD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306kD和77kD,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1h,在pH5.5～7.5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.