转录因子StMsn2调控玉米大斑病菌高渗胁迫反应及致病性的分子机制研究

In our previous study, we found that HOG-MAPK cascade pathway in Setosphaeria turcica was involved in regulating growth and development, hypertonic stress reaction and pathogenicity, and the probable regulation mechanism was that the pathway activated the key transcription factor named StMsn2, which located in the downstream of this pathway. In this project, research from four aspects will be put into effect if we can obtain the funding. Real-time PCR and western blot will be used to identify the expression pattern in different development stages and under hypertonic stress in Setosphaeria turcica. GFP labeling technology will be done to confirm the subcellular localization of StMsn2. The function of StMSN2 related to growth and development, hypertonic stress reaction, infection and pathogenicity will be explored by virtue of target gene mutagenesis (TGM), RNA interference, and gene overexpression technology. Gene expression differences between TGM mutants and wild-type strain will be analyzed using RNA-sequence technology. The target genes which StMsn2 may bind will be screened using chromatin co-precipitation (ChIP) technique and the functions of these genes will be made sure by employed gene function analysis technology mentioned above. The interaction between StMsn2 and StHog1 will be verified by applying yeast two hybrid and co-immunoprecipitation (CoIP) technique. The results will not only illuminate the regulation molecular mechanism of HOG-MAPK cascade pathway, but also lay the foundation for developing new fungicides in the future.

本课题组在前期研究中发现，玉米大斑病菌的HOG-MAPK级联途径参与调控生长发育、高渗胁迫反应及致病过程，其机制可能是激活了下游的核心转录因子StMsn2。本研究拟利用Real-time PCR、Western blot及GFP标记技术，明确StMSN2在病菌不同发育时期及高渗胁迫下的表达规律及亚细胞定位；利用靶基因敲除技术、RNAi、基因过表达技术明确StMSN2对病菌生长发育、侵染、致病性及高渗胁迫的调控作用；利用RNA-Seq技术筛选StMSN2基因敲除突变体与野生型菌株差异表达基因，并利用ChIP-Seq技术筛选StMsn2直接作用的靶基因，进而利用基因功能分析技术明确靶基因的功能；利用酵母双杂交、免疫共沉淀技术明确StMsn2与StHog1的互作关系。研究结果不仅将深入诠释植物病原真菌HOG-MAPK级联途径调控作用的分子机制，也为新型杀真菌制剂的研制奠定基础。

本课题组在前期研究发现，玉米大斑病菌中转录因子StMsn2很可能受HOG-MAPK级联途径关键因子StHog1所调控，并参与调控病菌生长发育、致病性及高渗胁迫反应，但其调控的分子机制尚不清晰。本研究围绕高渗胁迫对病菌生长发育及致病性的影响、StHog1对StMsn2的调控作用以及StMsn2调控作用的分子机制等开展研究，获得的主要研究结果如下：①利用RNA-seq技术明确了StMSN2在病菌不同发育时期及高渗胁迫条件下的表达规律；利用Western blot技术验证高渗条件下StMsn2蛋白的表达水平变化。②利用基因敲除技术明确了转录因子StMSN2基因对病菌生长发育、致病性及高渗胁迫的调控作用。③利用RNA-seq技术筛选StMSN2 RNAi突变体与WT菌株差异表达基因，初步明确了该基因的调控机制。④利用ChIP-seq技术筛选到9个StMsn2直接作用的靶基因，进而利用EMSA、荧光素酶系统证明了StMsn2对2个重要下游靶基因（StATA1和StODA1）的调控作用。⑤利用RNAi、过表达技术明确了StMsn2下游靶基因StODA1和StATA1的功能。⑥利用qRT-PCR和Western blot技术初步明确了StHog1对StMsn2的调控作用。⑦筛选得到了一个新的转录因子StRTG3，体外试验初步证明转录因子参与调控StMSN2表达。⑧系统研究了高渗胁迫条件对病菌菌丝形态、细胞壁发育、分生孢子的形态、侵入丝形成及致病性等的影响，探索了HOG-MAPK途径及其下游基因参与调控病菌高渗胁迫反应的分子机制。该研究不仅揭示了HOG-MAPK通过其下游转录因子StMsn2调控病菌生长发育、高渗胁迫反应及致病的分子机制，也为可为研制新型杀真菌制剂奠定基础。