SsFoxE1转录因子调控核盘菌子实体形成与发育的机制研究

Sclerotinia sclerotiorum (Lib.) de Bary is a Discomycetes in the class Leotiomycetes necrotrophic fungal pathogen with a broad host range. The causal agent is capable of major infecting soybean, rape, sunflower and so on, causing white mold and stem rot disease, which results in significant yield losses. However, the Sclerotinia sclerotiorum does not produce conidia, but ascospores in the apothecia during growth and development period of its life cycle. And the sexual spores are the primary source of inoculum in susceptible hosts naturally. Our group previously discovered that a typical MADS box transcription factor participates in the sexual developmental process, and probably through regulating a novel transcription factor FoxA1 controls this process in Sclerotinia sclerotiorum. Based on the work above, this project is furthere planning to clarify the SsFoxE1 genes function and deciphering regulating network in fruiting body formation and sexual deevelopment. The research carried out as four respects: (1) to construct SsFoxE1 knockout or knockdown mutant, and identify properties of biology and pathogenicity; (2) to analyze and compare mRNA expression profiles between Ss FoxE1 mutant and wild field type, and clone candidates of the regulated genes; (3) to analyze corresponding relations between the spatial & temporal dynamic and the stages of fruiting body development;(4) to identify and confirm candidates of the regulated genes, and its egulated core sequence of the DNA binding motif of SsFoxE1; (5) to exploit related network of the target genes with fruiting formation and development, and imagine the model of SsFoxE1 transcriptional regulation mechanism. The goal of this project is to reveal the regulation network through SsFoxE1 transcription factor, and to clarify the relationship between regulated genes and sexual process, and hereby provide novel idears for control sclerotinia disease of plant crops in the fields.

核盘菌（Sclerotinia sclerotiorum）是一种不产生无性孢子的植物病原真菌，其中子实体形成与子囊孢子产生是菌核病大发生的前提。项目组前期研究发现：核盘菌SsMADS转录因子参与了核盘菌的有性生殖阶段，并可能调控另一类FoxE1转录因子而起作用。据此，本项目将全面研究核盘菌SsFoxE1的功能，解析其在子实体形成与发育中的调控网络。拟开展工作：（1）构建SsFoxE1突变体，验证SsFoxE1在子实体形成中的生物学特性及对致病性的影响；（2）分析核盘菌野生型与SsFoxE1突变体菌株差异表达谱，克隆SsFoxE1所调控的靶标基因；（3）阐明所调控基因的时空表达动态及与子实体发育之间的关联；（4）针对候选的调控基因及其调控序列，鉴定SsFoxE1所结合的DNA特征序列；（5）阐明SsFoxE1调控机制模型，揭示调控基因与有性生殖之间的关系。为菌核病有效防治提供新思路和新途径。

生物信息学分析发现，核盘菌基因组中含4个编码 Forkhead 家族转录因子的基因，分别命名Ss-FoxE1、 Ss-FoxE2、 Ss-Fox3 和 Ss-Fkh1。.通过基因敲除和基因沉默技术研究表明， Ss-FoxE1 和Ss-FoxE2 对核盘菌子实体发育起重要调控作用，而 Ss-Fkh1 和 Ss-Mcm1 在菌丝生长、菌核形成、致病力等方面具有重要功能。同时，Ss-Fkh1 也影响了菌体对于环境逆境的响应。通过定量 PCR检测，发现 Ss-FoxE2 在子囊盘时期大量表达。同时，利用瞬时表达技术验证了二者均定位在细胞核内。.通过 Ss-FoxE1与野生型在差异蛋白质组分析，发现42个表达差异点，预测对核盘菌细胞结构、翻译过程和子囊盘发育有重要影响。进一步研究核盘菌野生型（WT）与敲除突变体（Ss-FoxE2）转录组差异表达谱，发现 野生型与突变体在菌丝阶段有 12 个差异表达基因，在菌核阶段有 7 个差异表达基因。 KEGG pathway 分析表明，3 个菌丝差异表达基因分别与 N 连接糖链合成（MGAT3）、酵母高甘露糖型 N 连接糖链合成(MNN2)， GPI 锚生物合成途径相关(PIG-M)。另外，在野生型菌丝和菌核阶段的差异基因数量为 1972 个， 突变体中菌丝和菌核阶段的差异基因数量为 2680 个。说明Ss-FoxE2 内菌核阶段的基因表达与野生型相比明显增多。同时，针对Ss-FoxE2 下调靶标基因Ss-PIG-M等，证明其影响菌丝的生长与菌核的形成速度和数量。.酵母双杂交技术初步筛选出 Ss-GAPDH、 Ss-Rpb 等 6 个候选蛋白与Ss-FoxE2 存在互作关系，Ss-Dw1、Ss-Pgm3 等 12 个候选蛋白与 Ss-Mcm1 存在互作关系。通过酵母单杂交技术初步筛选出 Ss-KamA、Ss-Phd 等7个候选蛋白可能调控Ss-FoxE2 的表达。基于最新研究明确， Ss-KamA、Ss-Phd 等调控 Ss-FoxE2的转录表达。Ss-FoxE2 与 Ss-GAPDH、Ss-Rpb 等互作，共同调控下游靶标基因。同时，可能 Ss-Mcm1 与 Ss-Dw1、 Ss-Pgm3 互作调控靶标基因，调控核盘菌子实体形成。.本项目发表 SCI论文3篇、国际会议论文6 篇。培养研究生9人，获吉林省科技进步一等奖 1 项。