突变肠杆菌株NRS-1中5个草甘膦逆境应答基因的克隆与功能研究

Cloning and functional analysis of 5 glyphosate-response-genes in mutant Enterobacter NRS-1 strain

摘要:

【目的】微生物对草甘膦的抗性受复杂的遗传体系调控，涉及靶基因和大量相关调控基因。对肠杆菌属细菌NRS-1突变菌株在高浓度草甘膦逆境下的5个重要差异表达基因进行功能研究，以期深入了解非靶标基因在抗草甘膦微生物的作用特点，为发掘优异基因资源，服务抗草甘膦转基因生物育种提供参考。【方法】NRS-1的差异表达基因可能在蛋白质合成、代谢、细胞膜等水平发挥作用，保护细胞免受高浓度草甘膦逆境，因此分别选取易位酶延伸因子fusA、丁二酸脱氢酶sdhA、胸苷磷酸化酶deoA、鸟氨酸氨甲酰转移酶argF、周质蛋白osmY进行克隆，采用大肠杆菌原核表达、转化拟南芥实验研究其功能，并通过细菌双杂及KEGG pathway分析基因间互作特点。【结果】在明确5个基因结构特点基础上，通过大肠杆菌原核表达及转基因拟南芥鉴定，发现这5个基因及草甘膦的靶基因5-烯醇式丙酮莽草酸-3-磷酸合成酶基因aroA对提高2种生物的草甘膦耐性均有不同程度的作用，其中argF、deoA的抗性较好，与aroA相当，表明在应对草甘膦逆境时，芳香族、含有胸腺嘧啶氨基酸及精氨酸的合成代谢通路可能起重要作用；利用基因互作与KEGG分析发现5个基因与靶基因aroA间形成复杂的调控网络，但无直接的蛋白互作。【结论】NRS-1的5个差异表达基因对草甘膦逆境具有抗性，argF、deoA优于其他3个基因，其与靶基因aroA间表现复杂的基因互作关系。

Abstract:

[Objective] The mechanism of microbe’s tolerance to glyphosate stress is controlled by complex genetic systems including the target gene and many related regulation genes. In the present study, five different expression genes (DEGs) of a mutant Enterobacter strain NRS-1 were identified, the function and interaction were investigated to reveal the role of non-target genes on glyphosate resistance. The work intends to provide potential gene resources for transgenic breeding of glyphosate resistance. [Methods] DEGs of NRS-1 may play a role on the processes of protein biosynthesis, metabolism, cell membrane etc. Accordingly, five DEGs, encoding elongation factor G (fusA), periplasmic protein (osmY), ornithine carbamoyltransferase 2, chain F (argF), thymidine phosphorylase (deoA), succinate dehydrogenase flavoprotein subunit (sdhA), were selected. The sequences of the genes were obtained through T-A cloning, the functions were detected though analyses of prokaryotic expression and transgenic programs. Moreover, the interaction between five genes and the glyphosate-target gene aroA (5-enolpyruvylshikimate-3-phosphate synthase) was also detected using bacteria two-hybrid system and KEGG pathway bioinformatic analysis. [Results] The characteristic of five genes was revealed. According to the performance of prokaryotic expression and transgenic Arabidopsis thaliana, genes were confirmed to help cell to improve the tolerance to glyphosate. The genes argF, deoA have better resistance trait than other genes, similar as the target gene aroA. It seems that the pathways including the synthesis and metabolism of aromatic amino acids, amine acids containing thymine and arginine might be important for NRS-1 to tolerant to glyphosate. Regulation network showed a complicated relationship between the five selected gene and aroA, and no direct interaction was found between them. [Conclusion] The selected five genes had positive effects on the resistance to glyphosate stress, among them, argF, deoA were better than others. Complex gene interaction could be found between genes and aroA.