苹果腐烂病菌转录调控因子VmSom1互作蛋白的鉴定及功能分析

Apple is the largest fruit in China. The canker disease caused by Valsa mali is the most widely distributed and the most destructive biological disaster to the apple tree of production areas in China. Mapping the mechanism of the development and pathogenesis of the pathogen will be helpful to explore new strategy for disease control. Transcription regulation factor plays an important role in regulating the development and pathogenesis in plant fungi, VmSom1 is a newly discovered transcription regulatory factors in apple rot pathogen, the results before showed that deletion VmSom1 gene could significantly reduce growth, fruit body morphology formation and pathogenicity, the function and mechanism of interaction protein of VmSom1 remains largely unknown, it is necessary to understanding the types and acting mechanism of interaction proteins in metabolic process. This research mainly screening and identification new proteins partner of VmSom1, which participate in growth, development and virulence. This research mainly using RNA-seq transcriptomics and biological information, yeast two hybrid, BiFC, Immuno-co-precipitation technology and real-time fluorescence quantitative PCR technology, and clarify its protein expression types, the dynamic, subcellular localization and signal transduction pathway. To elucidate the mechanism of molecular regulation in the growth and development of pathogen. The research results will provide molecular mechanism for growth and pathogenic of apple rot pathogen and exploring novel targets for control disease.

苹果是我国第一大水果。由苹果黑腐皮壳菌（Valsa mali）引起的苹果腐烂病是我国苹果产区分布最广、危害最为严重的生物灾害。明确腐烂病菌生长发育及致病分子机制有助于研发新的病害防控策略。转录调控因子及其互作蛋白在调控植物病原真菌生长发育与致病机制方面发挥着重要作用。VmSom1是新发现的苹果腐烂病菌转录调控因子，VmSom1基因突变可显著降低腐烂病菌生长、子实体形态建成及致病力，但目前尚无关于其互作蛋白的鉴定及作用机制方面研究。本项目应用RNA-seq转录组学、酵母双杂交、双分子荧光互补、免疫共沉淀和实时荧光定量qRT-PCR等技术筛选和鉴定新的与VmSom1互作、参与病菌生长发育及致病过程的蛋白，明确其表达动态、亚细胞定位、信号传导途径，阐明VmSom1调控苹果腐烂病菌生长发育和致病的分子机制，研究结果将为病害防控提供新的靶标。

由Valsa mali引起的腐烂病是危害我国苹果树最为严重的生物灾害。本研究围绕参与苹果腐烂病菌生长发育及致病分子机制的转录调控因子VmSom1的表达调控及互作蛋白进行了筛选、鉴定及功能分析，应用RNA-seq转录组学测序技术和荧光定量技术证明VmSom1基因在病菌侵染过程中的表达呈现升高趋势，病菌侵染第6d比12d表达量分别上调1.18和1.08倍，同时正调控 VM1G_06657 和 VM1G_11970 基因表达，而负调控VM1G_07354 基因表达；应用酵母双杂交、双分子荧光互补和实时荧光定量PCR等技术筛选和鉴定了与VmSom1互作、参与病菌生长发育及致病作用等功能基因13个，其中直接互作的基因有VM1G_09838（VmStuA）、VM1G_00359（VmSFL1）、VM1G_06057、VM1G_00917（VmCdf1）、VM1G_01499 和VM1G_00266（VmPKA2），这些基因均对病菌的生长发育及致病性有显著影响；间接互作的基因有VmMkh1、VmMkk1、VmSpm1、VmWis4、VmWis1、VmHog1、VmHMG7。VmMkh1和VmMkk1基因调控病菌细胞壁完整性、渗透胁迫；VmSpm1和VmHog1基因调控病菌生长、细胞壁完整性、渗透胁迫以及致病性；VmWis4和VmWis1基因调控病菌细胞壁完整性、渗透胁迫以及致病性；VmHMG7基因的缺失可抑制病菌的生长，增强病菌致病性。VM1G_09838（VmStuA）和VM1G_00266（VmPkA2）主要位于cAMP/PKA途径上，而VmMkh1、VmMkk1、VmSpm1、VmWis4、VmWis1、VmHog1、基因主要位于MAPK通路上, VM1G_09838（VmStuA）、som1都和VM1G_00266（VmPKA2）互作，VmPkA2 和VmstuA也与MAPK途径上的VmSpm1互作，存在两个代谢通路的交叉，说明VmSom1及其互作蛋白基因分子调控网复杂，研究结果对于进一步明确病菌的分子互作机制提供了理论依据。