核盘菌转录因子Ss-Nsd1对其小孢子产生与子实体发育的调控机制研究

Sclerotinia sclerotiorum (Lib.) de Bary is a Discomycetes in the class Leotiomycetes necrotrophic fungal plant 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 produce microconidia and ascospores in the life cycle. The microconidia，spermatia, is reported very little about production and function. And the sexual spores are the primary source of inoculum in susceptible hosts naturally. We have found that the Ss-Nsd1 is a novel kind of transcriptional factor to regulate microconidia reproduction and fruiting body davelopment passway as molecular switching on the base of our group previously discovery in Sclerotinia sclerotiorum. Based on the work above, this project is furthere planning to clarify the Ss-nsd1 gene function and deciphering regulating network in microconidia formation and fruiting body formation. The research carried out as four respects: (1) to construct Ss-Nsd1 knockout or knockdown mutant, and identify properties of biology and pathogenicity of the Ss-nsd1 mutants; (2) to analyze and compare mRNA expression profiles between Ss-nsd1 mutant and wild field type, and clone candidates of the regulated genes; (3) to analyze corresponding relations between the spatial and temporal dynamic and the stages of microconidia formation and fruiting body development;(4) to identify and confirm candidates of the regulated genes, and its egulated core sequence of the DNA binding motif of the Ss-Nsd1; (5) to exploit related network of the target genes with microconidium reproduction and fruiting formation development, and imagine the model of Ss-Nsd1 transcriptional regulation mechanism. The goal of this project is to reveal the regulation network through Ss-Nsd1 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 (lib.) de Bary）是一种死体营养型植物病原真菌。在生活史中产生小孢子与子囊孢子。子囊孢子是该菌主要初侵染源，而小孢子的产生与功能鲜见报道。前期研究发现，Ss-Nsd1是调控核盘菌小孢子产生与子实体发育的关键转录因子。其突变株产生小孢子、不形成产囊体和产囊丝及子囊孢子。推断Ss-Nsd1是小孢子产生与子实体发育的分子开关。为此，项目拟：（1）验证Ss-Nsd1与小孢子及子实体形成的关系，明确其生物学功能；（2）通过差异表达谱、CLIP-seq与互作分析，阐明Ss-Nsd1所调控的靶标基因，及其调控元件等功能；（3）解析Ss-Nsd1在小孢子和子实体发育中，与靶标基因及相关因子关联，明确其调控途径与调控网络；（4）揭示Ss-Nsd1转录因子对核盘菌小孢子与子实体发育的调控机制。项目旨为深入解析核盘菌无性繁殖与有性生殖奠定基础

核盘菌（Sclerotinia sclerotiorum (lib.) de Bary）子实体形成与子囊孢子产生影响菌核病的初侵染及其流行程度。本课题组利用基因敲除的方法分析了SsNsd1的生物学功能。首次发现SsNsd1缺失突变体在营养生长阶段产生精孢子(Spermatia)，并大量存在于菌核的髓质内。 SsNsd1功能的缺失将影响菌核组织结构的完整性，阻碍产囊体与子实体发育，最终不形成子囊盘与子囊孢子。揭示了SsNsd1正调控子囊盘形成、负调控精孢子产生的一个重要转录因子和分子开关。同时，Ssnsd1缺失突变体菌丝不能形成附着胞，致使其致病性丧失。.差异表达分析发现，SsNsd1缺失突变体与野生型之间存在2470个表达量差异基因。KEGG分析显示，下调基因显著富集在核糖体代谢、氨基酸生物合成与苯丙氨酸代谢等通路中，上调基因显著富集在淀粉与蔗糖代谢、氨基糖与核苷酸糖代谢以及其他聚糖降解等通路中。蛋白质组学研究表明，SsNsd1缺失突变体与野生型之间存在43个差异蛋白。通过多肽质谱发现差异蛋白中有17个已知功能的蛋白，主要作用于能量代谢，蛋白质组装，应激反应以及细胞生长与分裂。.酵母单杂实验和筛选出SS1G_07183、SS1G_07944、SS1G_03736、SS1G_03803、SS1G_08945、SS1G_05913、SS1G_12468、SS1G_03527、SS1G_10512等9个基因所编码的蛋白与Nsd1启动子区域特异性结合。酵母双杂与BiFC筛选发现SS1G_00872、SS1G_10135、SS1G_14138、SS1G_04744四个基因所编码的蛋白与转录因子SsNSd1存在互作关系。其中，基因SS1G_10135编码的蛋白（SsFDH1）属于乙醇脱氢酶ADH3家族。并揭示了SsNsd1与SsFDH1通过半胱氨酸残基之间形成二硫键，以及GATA锌指结构域中的锌离子进行互作，形成二聚体对该菌生长发育起调控作用。.综上，本项研究明确SsNsd1在核盘菌生长发育中重要生物学功能，揭示了SsNsd1对核盘菌子实体形成与精孢子产生的调控途径与调控网络，对核盘菌等丝状真菌生长发育调控具有普遍科学意义与学术价值。该项目研究结果在《mBio》等杂志上发表论文6篇。在国际会议上发表论文3篇，国内会议上发表论文2篇。培养硕士研究生6人、博士研究生3人。

内容获取失败，请点击重试