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SDSPAGE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 glucosamine6phosphate 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 overexpressed using Escherichia coli and purified. The results of Gel filtration chromatography and SDSPAGE revealed that it had an estimated molecular mass of 306 kD and consisted of four equalsized subunits of 77 kD. The optimal reaction conditions for the recombinant GlmS were pH 6.4 at 37℃, the halflife 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 endproduct of the hexosamine pathway, UDPGlcNAc.
\[1\]Luo C, Shao W, Li X, et al.Molecular cloning, sequencing, and expression of a Lglutamine Dfructose 6phosphate amidotransferase gene from Volvariella volvacea. Protein J, 2009, 28(1): 3443.
\[2\]Kato N, Dasgupta R, Smartt C T, et al. Glucosamine: fuctose6phosphate aminotransferase: Gene characterization, chitin biosynthesis and peritrophic matrix formation in Aedes aegypti. Insect Mol Biol, 2002, 11(3): 207216.
\[3\]Bearne S L, Blouin C.Inhibition of Escherichia coli glucosamine6phosphate synthase by reactive intermediate analogues: The role of the 2amino function in catalysis. J Biol Chem, 2000, 275(1): 135140.
\[4\]Wojciechowski M, Milewski S, Mazerski J, et al. Glucosamine6phosphate synthase, a novel target for antifungal agents:Molecular modelling studies in drug design. Acta Biochim Pol, 2005, 52(3): 64753.
\[5\]Gary L M, Sherry L M, Shannon L M, et al. Molecular cloning, cDNA sequence, and bacterial expression of human glutamine fructose6phosphate amidotransferase. J Bio Chem, 1992, 267(50), 2520825212.
\[6\]Gabriele W, Widmar T. Cloning of the Glutamine: Fructose6phosphate amidotransferase gene from yeast. J Bio Chem, 1989, 264, 87538758.
\[7\]Smith R J, Milewski S, Brown A J, et al. Isolation and characterization of the GFA1 gene encoding the glutamine:Fructose6phosphate amidotransferase of Candida albicans. J Bacteriol, 1996; 178(8): 23202327.
\[8\]国广泰史， 钱前， 佐藤宏之， 等. 水稻纹枯病抗性QTL分析. 遗传学报， 2002， 9（1）： 5055.
\[9\]鄂志国， 张丽靖， 黄世文， 等. 水稻纹枯病抗性研究进展. 核农学报， 2009， 23（6）： 9971000.
\[10\]Arnold K, Bordoli L, Kopp J, et al. The SWISSMODEL Workspace: A webbased environment for protein structure homology modelling. Bioinformatics, 2006, 22(2):195201.
\[11\]Golinelli P B, Badet B. Possible involvement of lys603 from Escherichia Coli glucosamine6phosphate synthase in the binding of its substrate fructose6phosphate. Eur J Biochem, 1991, 201(1): 175182.
\[12\]Chang Q, Su K, Baker J R, Yang X, et al. Phosphorylation of human glutamine: Fructose6phosphate amidotransferase by cAMPdependent protein kinase at serine 205 blocks the enzyme activity. J Biol Chem, 2000, 275(29): 2198121987.
\[13\]Dorota A B, Mariusz O, Hector A L, et al. Chitin synthesis in saccharomyces cerevisiae in response to supplementation of growth medium with glucosamine and cell wall stress. Eukaryotic Cell, 2003, 2(5): 886900.
