MADS-box转录因子Bcmb调控灰霉菌致病力的分子机制

Botrytis cinerea is one of the necrotrophic plant pathogen with a wide host range. Grey mould disease caused by B. cinerea has brought serious economic losses to agricultural production. Studying the function of transcription factor has become a research foucs to understand the biological phenomenon in the international field of plant pathology. Supporting by the technology of gene knockout of our lab we generated the mutants of MADS-box transcription factor Bcmb in B. cinerea. The results of preliminary experiments showed that the pathogenicity of mutants were significantly depressed. This project mainly study the target genes regulated by Bcmb and the interaction proteins of Bcmb. We will analyse the differentially expressed genes between the mutant and wild type strain throuth the proteomics and transcriptomics method, use the technique such as chromatin immunoprecipitation assay (ChIP) and electrophoretic mobility shift assay (EMSA) to find out the downstream target genes regulated by Bcmb. On the other hand, we perform the mathods of co-immunoprecipitation (Co-IP) and mass spectrometry (MS) to explore the interaction proteins of Bcmb, combining the technology of yeast two-hybrid and bimolecular fluorescence complementation (BiFC) to check the interaction between Bcmb and the indentified proteins. This study will not only provide the new evidence for the molecular regulating mechanism of Bcmb on the pathogenicity of B. cinerea, but also provide the technical guidance for exploring the new antifungal agents.

灰霉菌（Botrytis cinerea）是一种寄主范围极其广泛的腐生型病原真菌。该病原菌所引发的灰霉病在农业生产中造成了巨大的经济损失并且较难防治。通过对转录因子的研究来解析生物学现象逐渐成为了当前国际植物病理学领域的研究热点。课题组利用基因敲除技术筛选到灰霉菌MADS-box转录因子Bcmb的突变株，初步研究发现该转录因子的缺失导致灰霉菌致病力显著下降。本项目主要研究转录因子Bcmb的下游靶基因和与之互作的蛋白，通过蛋白质组学和转录组学的方法分析突变株中差异表达的基因，利用染色质免疫共沉淀和凝胶阻滞等方法探明受Bcmb调控的靶基因；另外，通过免疫共沉淀结合质谱鉴定的方法确定Bcmb的互作蛋白，结合酵母双杂交和双荧光分子互补的方法对蛋白互作进行验证。该研究不但为深入解析转录因子Bcmb对B. cinerea致病力的分子网络调控机制提供新证据，而且为研制新型抑菌剂提供技术指导。

MADS-box家族转录因子在真核生物中高度保守并参与了多种重要的生命过程。灰霉菌Botrytis cinerea是一种寄主非常广泛的植物病原真菌。B. cinerea中的MADS-box转录因子的功能之前尚未见报道。本研究中我们分析了B. cinerea中的MADS-box转录因子编码基因Bcmads1在发育、致病力和菌核产生中的作用。B. cinerea的菌核只在黑暗的培养条件下产生，并可以作为越冬的组织和有性繁殖中的母本。本研究发现Bcmads1对菌核的产生必不可少。RT-qPCR分析结果表明Bcmads1可以通过影响光响应基因的表达模式来调节菌核的产生。本研究同时发现Bcmads1在致病力调控中发挥重要作用，Bcmads1的敲除导致B. cinerea在苹果果实上的的致病力显著降低。我们通过比较蛋白质组学分析，在Bcmads1突变株和野生型菌株之间鉴定出55个差异蛋白，其中包括囊泡转运相关的蛋白Bcsec14和Bcsec31。遗传学分析表明Bcsec14和Bcsec31基因的敲除可导致B. cinerea致病力和胞外蛋白的分泌能力明显下降。以上研究结果表明Bcmads1可以通过影响光响应基因的表达模式来调节菌核的产生，同时可以通过对蛋白分泌相关蛋白的调节来进一步对致病力产生影响。