解淀粉芽胞杆菌PG12中 c-di-GMP的功能鉴定及其调控防病作用的途径研究

Cyclic dimeric GMP (c-di-GMP) is a small molecule that regulates a lot of multicellular behaviors in many bacteria. Bacillus amyloliquefaciens PG12 is a patented strain which showed high control efficacy against apple ring rot disease. It showed outstanding biocontrol charateristics including forming robust biofilm, efficiently and stably colonizing the surface and within the tissues of apples and producing antifungal antibiotics iturin A and fengycins. c-di-GMP metabolizing protein encoding genes have been preditced in the genome of PG12. Previous results showed that deletion of the c-di-GMP synthetase encoding genes resulted in significant change of biofilm, swarming and antagonism. Based on these results, this project will combine functional genomic techniques, such as gene knockout, complementation, overexpression, transcriptome analysis, qRT-PCR, with the detection the c-di-GMP concentration, biocontrol related phenotye and key gene expression to reveal the function of c-di-GMP in biocontrol activity of PG12 against apple ring rot disease and its regulatory pathways. This project will contribute to not noly exploring the molecular mechanisms of c-di-GMP in PG12 against plant disease, but also searching for new ways to increase the stability of the biocontrol efficacy.

c-di-GMP是细菌中广泛存在的第二信使，调控细菌多种生理功能。解淀粉芽胞杆菌PG12是一株对苹果轮纹病防效显著的野生型菌株，已获得国家授权发明专利，具有很强的生物膜形成能力，能在苹果表面及体内稳定定殖，产生脂肽类化合物Iturin A和Fengycins是其重要的生防机制。基因组序列分析显示，菌株PG12基因组上存在c-di-GMP代谢相关蛋白的编码基因。前期结果显示，c-di-GMP合成酶的突变显著影响菌株PG12生物膜、游动性及拮抗活性。本项目拟通过基因敲除、功能互补验证、基因过表达分析、转录组分析、qRT-PCR等功能基因组学研究方法，结合c-di-GMP浓度检测、防病相关性状及关键基因的检测，探究c-di-GMP在菌株PG12防治苹果轮纹病中的作用及其主要调控途径。本项目的开展不仅有利于揭示菌株PG12防病的分子机制，而且有助于探寻提高生防菌效果稳定性的新途径。

解淀粉芽胞杆菌(Bacillusamyloliquefaciens)PG12菌株是本实验室从苹果中分离获得的对苹果轮纹病表现出良好防效的有益细菌。通过生物信息学分析发现解淀粉芽胞杆菌PG12基因组编码蛋白中含有4个GGDEF结构域蛋白和2个EAL结构域蛋白。利用活性位点分析、GGDEF结构域蛋白的体外表达、构建GGDEF/EAL结构域蛋白编码基因的敲除突变体和互补菌株、并结合LC-MS/MS技术检测各菌株胞内c-di-GMP水平，最终明确了PG12的c-di-GMP合成酶为YhcK和YtrP，降解酶为YuxH，明确了菌株PG12中c-di-GMP的代谢机制。通过检测各菌株游动性、生物膜形成、对轮纹病菌的拮抗活性和防病效果等表型，发现：c-di-GMP负调控菌株的运动能力，正调控PG12薄皮型生物膜的形成，不影响其对苹果轮纹病病原菌(Botryosphaeriadothidea)和西瓜食酸菌(Acidovoraxcitrulli)的拮抗能力，正调控PG12对苹果轮纹病的防病效果，但不影响其对甜瓜细菌性果斑病的防效。通过保守位点预测、单突变和双缺失突变体及互补菌株的构建、及表型检测，发现YdaK是退化的GGDEF结构域蛋白，没有合成c-di-GMP的功能，但其可能作为受体影响运动性。综上所述，本项目明确了菌株PG12中c-di-GMP的代谢机制和c-di-GMP在菌株PG12防病过程中的作用，初步探讨了c-di-GMP调控防病作用的信号通路。