兽疫链球菌透明质酸合成途径和相关基因已研究地比较透彻，其中由谷氨酰胺提供氨基合成葡萄糖胺-6-磷酸是透明质酸合成的限速反应。前期通过构建突变体库发现谷氨酰胺合成酶基因glnA影响透明质酸荚膜的合成，但机制尚不清楚。本课题拟开展：（1）通过凝胶迁移、DNase Ⅰ足迹、qPCR、酶活检测等实验，研究全局性氮源调控蛋白GlnR对谷氨酰胺合成酶操纵子glnRA和谷氨酰胺转运蛋白操纵子glnPQ的转录调控作用，以及GlnA协同GlnR执行转录调控作用的机制；（2）构建过表达菌株和通过无痕敲除技术构建相应基因突变菌株，研究GlnR、GlnA和GlnP、GlnQ对胞内谷氨酰胺水平和透明质酸合成及分子量的影响；（3）谷氨酰胺参与其他代谢途径对透明质酸合成的影响。以上研究将揭示谷氨酰胺代谢相关基因功能对透明质酸合成和分子量的影响，为理性改造兽疫链球菌生产高品质透明质酸奠定基础。

Hyaluronic acid (HA) biosynthesis pathway and related genes in Streptococcus equi subsp. zooepidemicus have been thoroughly studied, and the rate-limiting reaction is the synthesis of glucosamine-6-phosphate. The source of amino groups in this reaction is glutamine. A mutant library was constructed and screened for the bacterial capsule defect strain. Through gene knockout and plasmid rescue, the glutamine synthetase gene glnA disruption leads to capsule biosynthesis, but the underlying mechanisms are not clear. In this proposal, we will perform the following work: (1) through EMSA, DNase Ⅰ footprint assay, qPCR, enzyme activity assay and other experiments, the nitrogen regulatory protein GlnR effects on glnA and glutamine transportor gene glnPQ transcriptional regulation and GlnA effects on GlnR activity will be explored; (2) the corresponding overexpression strains and gene knockout strains will be constructed, and GlnR, GlnA, GlnP and GlnQ effects on the regulation of glutamine and HA biosynthesis will be determined; (3) the other glutamine metabolism pathway effects on HA biosynthesis. This work will reveal the functions of the genes in glutamine metabolism pathway and the molecular mechanisms of these genes effects on HA biosynthesis and molecular control, and provide the theoretical basis for rational modification of S. zooepidemicus to produce high quality HA.