c-di-GMP调控荧光假单胞菌MS82抑菌物质生物合成的机制研究

Bacterial strain MS82, isolated from the rhizosphere of a soybean plant, belongs to the species Pseudomonas fluorescens. The most important feature of strain MS82 is the production of antifungal activity against the mushroom pathogenic fungus Trichoderma viride and Mycogone perniciosa but not against the mushroom fungus Agaricus bisporus and Pleurotus ostreatus. During preliminary study, three mutation strains generated with the EZ-Tn5 transposon system completely lost the antifungal activity against T. viride and M. perniciosa. The mutation site of MT19 was related of metabolic pathway of c-di-GMP (PafR gene). Then the mutation MD19 was got by site-specific mutagenesis the GGDEF domain of PafR gene. The results of qPCR showed that the expression quantity of phenazine, pyoverdine and arthrofactin partial synthesis genes of MD19 was reduced distinctly than MS82 wildtype strain. And the phenazine production of MD19 was decreased obviously. The aim of this study is to further define the gene function of c-di-GMP metabolism of MS82. There are four main research contents in this study. (i) The site-specific mutagenesis of all the genes containing GGDEF domain in MS82 will be done. (ii) The target genes will be selected according to the detection results of PDA plate bioassays and qPCR detection technigues, phenazine, pyoverdine and arthrofactin, for example. (iii) Some important phenotypic function of target gene in MS82, such as biofilm formation, swimming motility, swarming motility, twitching motility, colonization ability and biocontrol efficiency, will be analyzed. (iv) The potential receptor transcription factor of c-di-GMP in MS82 will be selected. The specific interaction between target receptor transcription factor and c-di-GMP will be definite. The target receptor transcription factor will be imported into the mutant to verify its function. The molecular mechanism of regulated antifungal substances by target receptor transcription factors will be verified preliminarily. The result of this study not only is the theoretical direction for the mechanism of regulated antifungal substances synthesis by c-di-GMP in Pseudomonas fluorescens MS82, but also is important for developing a new type of biocontrol antifungal pesticide.

荧光假单胞菌Pseudomonas fluorescens MS82对食用菌两个重要致病真菌-绿色木霉Trichoderma viride和疣孢霉Mycogone perniciosa具有较强的抑制能力。前期研究结果发现：突变体MD19完全丧失抑菌能力，突变位点是c-di-GMP代谢通路中PafR基因GGDEF结构域，qPCR结果表明突变体MD19分泌的抑菌物质-吩嗪、嗜铁素、环脂肽合成基因表达量明显下调。本项目拟对MS82中c-di-GMP代谢通路中含GGDEF结构域的基因逐个突变，以室内抑菌试验和抑菌物质基因表达量为检测指标，确定目标基因，探明其生物膜形成、运动、定殖等功能。筛选MS82中c-di-GMP潜在受体转录因子，明确受体转录因子与c-di-GMP是否特异性互作，通过回复突变验证其功能。研究结果对解析荧光假单胞菌MS82中c-di-GMP调控抑菌物质合成的作用机制具有理论指导。

为揭示假单胞菌中c-di-GMP 调控小分子抗菌物质生物合成机制，本研究对荧光假单胞菌MS82中含有GGDEF 结构域的c-di-GMP 代谢相关基因调控抑菌物质的合成以及对菌株生物膜、运动性、抑菌效果的影响进行了研究。通过原生质体双亲接合同源重组的方式，成功构建10个含有GGDEF结构域基因的缺失突变体。以木霉作为病原指示菌，采用抑菌圈法检测对食用菌病害木霉的抑菌效果变化，发现突变株 MT0189、 MT2587、MT5092、MT4701 对木霉的抑菌活性皆明显低于野生菌株，其他突变体的抑菌活性变化不明显。通过荧光定量PCR技术检测9个抑菌物质相关合成基因在4个突变体中表达情况，发现突变体中表达量减少的基因数量为MT4701>MT2587=MT5092>MT0189。构建四个突变体的回复突变体，对木霉的抑制作用明显提升，且与野生菌株差异不明显。4个基因的缺失突变体对菌体生物膜形成、多糖含量、运动性及离体防效均有不同程度的降低，而4个回复突变体对菌体生物膜形成、多糖含量、运动性和离体防效的影响与其突变体相比都有显著恢复，且与野生菌株MS82差异不显著。MS82中含有11个c-di-GMP的受体效应蛋白。2587基因为MucR蛋白的编码基因，该基因含有MHYT-GGDEF-EAL结构域，长度为2082bp，编码693个氨基酸，同时具有DGC活性和PDE活性。MucR蛋白通过控制抑菌物质合成基因表达、菌体生物膜形成、运动性等功能影响对木霉的抑菌活性。本项目结果认为荧光假单胞菌的c-di-GMP调控不仅可以直接通过调控抑菌物质合成影响抑菌作用，而且可以通过调控生物膜形成、多糖合成及运动性影响菌体定殖能力达到间接影响抑菌功能的效果。通过本项目的开展，发表文章2篇，其中1篇SCI；培养研究生2名，完成毕业论文1篇；获得科研奖励1项。通过学术会议交流对本项目的成果进行介绍和宣传。