Des和GlnR调控链霉菌Snea253杀线虫活性氨基寡糖代谢研究

Plant pathogenic nematodes are important pathogens in worldwide agriculture and forestry production. A few effective nematicides can control these diseases,such as soybean cyst nematode,root knot nemtode and so on. So,the researches are becoming focus on nematicides with high efficiency and low toxicity and low residue in the international pesticide fields. Nematicidal activity of actinomycetes Streptomyces venezuelae Snea253 as research materials of this project had independent intellectual property rights and patent in our lab. In the preliminary researches we separated and purified the bioactive metabolite of Snea253 and initially identified as amino oligosaccharides. Bioinformatics,Proteomics,Genomic Minning method and microbial secondery metabolites databases will be used for analysis of biological synthesis gene clusters and regulators of Snea253 secondary metabolites in this project, and determined the key regulation factors and genes in metabolic pathway. Then, two regulators will be researched in here，which Des with six or seven enzymes involved in dTDP-desosamine production, and GlnR provided global nitrogen regulation in S.venezuelae. The Plackett-Burman method will be screened glycosylated precursor and amine precursor, then RT-PCR will be monitored the Des and GlnR key gene expression in Snea253 metabolic process,and detected amino oligosaccharides yields by HPLC. Meanwhile, we will operate Des and GlnR key gene overexpression and knockout to clarify metabolic regulation mechanism of Snea253 producting efficient amino oligosaccharins. The research results will proven the metabolitic regulation mechanism and gene clusters and key regulators producing nematicidal amino oligosaccharins in S.venezuelae Snea253 at the molecular level, and increase a now biological nematicides to control plant nematode diseases and provide technical support for developing high efficient pesticides.

植物病原线虫是世界范围内农林业生产中重要病原物，有效药剂很少，因此研发高效低毒杀线剂是当前国际研究热点。本项目以拥有自主知识产权杀线虫活性委内瑞拉链霉菌Snea253为研究试材，前期研究确定活性代谢产物为氨基寡糖化合物，拟利用微生物代谢数据库，通过生物信息学和蛋白质组学方法分析Snea253产氨基寡糖生物合成基因簇和代谢调控途径；进而从糖代谢Des和氮代谢GlnR调控子入手，利用Plackett-Burman设计法筛选糖基和氨基前体，RT-PCR监测Des和GlnR中关键调控基因的表达，HPLC检测氨基寡糖的产率；同时过表达和基因敲除Des和GlnR中关键调控基因，以明确关键调控子对Snea253产氨基寡糖的代谢调控机理，获得目的代谢产物高效表达。研究结果将从分子水平探明链霉菌产氨基寡糖的基因簇及糖代谢和氮代谢中关键代谢调控子的作用，为防控植物线虫病害的新型生物农药的研发奠定理论研究基础。

植物病原线虫是世界范围内农林业生产中重要病原物，有效药剂很少，因此研发高效低毒杀线剂是当前国际研究热点。本项目以拥有自主知识产权杀线虫活性委内瑞拉链霉菌Snea253为研究试材，从分子水平探明链霉菌杀线虫活性基因簇及糖代谢和氮代谢中关键代谢调控子的作用，取得如下重要研究结果：.1.通过紫外线复合氯化锂诱变筛选获得遗传稳定性高的强毒菌株UV-L-16和弱毒菌株UV-L-8。.2.Snea253强毒株和弱毒株的转录组测序及分析，结果显示糖代谢的基因包括DesⅠ基因17个，DesⅡ基因7个，DesⅢ基因10个，DesⅣ基因2个，DesⅤ基因13个，DesⅥ基因2个。氮代谢的基因包括GlnR基因1个，Amt基因4个，OmpR基因17个。本研究主要筛选了8个在碳氮代谢通路上的重要基因，即5个上调基因和3个下调基因，为下一步选择Snea253杀线虫活性产物的代谢调控基因进行功能研究奠定基础。.3. Snea253菌株的营养代谢调控研究结果表明，利用Plackeet-Burman试验设计方法研究确定出初始pH值和发酵时间的显著影响，再利用最陡爬坡试验法确定最大响应区域，应用Central composite design试验设计和响应面分析方法确定最优发酵条件，为种龄（72 h）、接种量（5%）、初始pH值（6.6）、发酵温度（28℃）、发酵时间（5.25 d）、摇床转速（175 r/min）和装液量（20%）。优化后的发酵液的线虫校正死亡率与原始发酵液相比，提高了29.7%。.4. 通过对碳代谢基因gabT调控生防菌Snea253的GABA代谢途径对杀线虫活性的研究发现，在生防菌Snea253中 gabT基因表达量越高，GABA含量越低，琥珀酸含量越高，杀线活性越强；培养基碳源可改变gabT基因的表达，不同培养基碳源培养的菌株中gabT基因相对表达量越高，GABA含量越低，杀线活性越强。研究结果证实了gabT基因具有调控生防菌Snea253代谢以增强其杀线活性的作用。. 本研究通过对Snea253菌株中杀线虫活性相关基因的分析和功能验证研究，明确了碳氮代谢中部分相关基因的调控作用，为杀线虫生防菌的代谢调控研究开拓了新思路，同时也为新型生物农药研发奠定理论基础，在未来生防制剂的生产实践中具有重要意义。