GlnA基因对刺糖多孢菌生长发育及多杀菌素合成的影响

谷氨酰胺合成酶（GS，glutamine synthetase）是由GlnA基因编码的生物体中最古老也是最广泛存在的酶，它参与许多生物体中的氮和碳代谢，是生物体氮代谢的关键酶之一，对微生物的生命活动起着重要作用。本文研究了GlnA基因对刺糖多孢菌Saccharopolyspora spinosa的生长发育及次级代谢产物合成的影响，从刺糖多孢菌基因组中克隆了glnA 基因的部分片段，将其与穿梭载体pOJ260连接，构建敲除载体pOJ260-glnA，通过接合转移将其导入刺糖多孢菌野生菌中，获得刺糖多孢菌敲除菌株S.spinosa-△glnA；同时，利用重叠延伸PCR将glnA基因置于红霉素强启动子P_ermE下，并将融合片段P_ermE-glnA与穿梭载体pOJ260进行酶切酶连，构建过量表达载体pOJ260-P_ermE-glnA，通过接合转移将其导入刺糖多孢菌中，获得了刺糖多孢菌过量表达菌株S.spinosa-glnA。表型分析发现，glnA基因的敲除对刺糖多孢菌的菌丝形态、孢子萌发等方面都有显著的抑制作用。HPLC检测分析发现，过量表达菌株中多杀菌素产量相比原始菌株提高到170%；该研究表明glnA基因对刺糖多孢菌的生长发育与多杀菌素的合成有一定的影响，为研究其在链霉菌次级代谢过程中的作用奠定了重要基础。

The Effect of glnA Gene on Growth Development and Spinosad Biosynthesis in Saccharopolyspora spinosa

Glutamine synthetase (GS) encoded by glnA is the oldest enzyme and most widely exists in organisms. It participates in nitrogen and carbon metabolism in many organism and is one of the key enzymes for nitrogen metabolism, thus, it has played a crucial role in the life activities of microorganisms. This study explored the effect of glnA gene on the growth development and secondary metabolites of Saccharopolyspora spinosa. First, partial fragment of glnA gene was cloned from the genome of S. spinosa and ligated with shuttle vector pOJ260 to construct knockout vector pOJ260-glnA; Then, the recombinant vector was introduced into S. spinosa by conjugal transfer, and the knockout strain S. spinosa-△glnA was generated. Second, glnA gene was placed under the P_ermE strong promoter by overlap extension PCR, and the fusion fragment P_ermE-glnA was ligated with the shuttle vector pOJ260 to construct the overexpression vector pOJ260-P_ermE-glnA; Then, the recombinant vector was introduced into S. spoinosa by conjugal transfer and generated the over expression strain S. spinosa-glnA. Phenotypic analysis found that the knockout of glnA gene had a significant inhibition effect on the mycelial development and spore germination of S. spinosa. HPLC analysis demonstrated that spinosad A+D yield increased by 170% in S. spinosa-glnA strain compared to the parental strain. Thus, our study revealed that the glnA gene regulated the mycelia development and the spinosad synthesis of S. spinosa, which laid an important foundation on studying the function in the secondary metabolism of Streptomyces.