玉蜀黍赤霉有性生殖发育不同阶段特异性转录因子的调控机制研究

Gibberella zeae (anamorph: Fusarium graminearum) is an important plant pathogen that causes Fusarium head blight (FHB) in wheat throughout the world. Perithecia or associated hyphae are known to be the components of fungi that aid in survival during the winter season. The sexual spores, ascospores, serve as the primary inocula for disease epidemics. Sexual reproduction also helps the G. zeae population maintain genetic diversity and genetic stability. In addition, high genetic diversity of virulence-related genes is thought to provide G. zeae with a great capacity for adaptability toward host plants. Thus, the understanding of sexual development is a prerequisite for integrated control of FHB disease. Studies on transcription factors will enable us to link upstream signaling pathways with downstream target genes. Based on previous studies, we selected five transcription factors that specificlly play regulatory roles in sexual development at different stages (including the regulation of female fertility, the number of perithecia, the size of the perithecia, perithecia mature and ascospores mature). In this project we intend to identify target genes of these transcription factors by performing Chromatin Immunoprecipitation Sequencing（ChIP-Seq）, combining with the results of RNA-sequencing （RNA-Seq）and Electrophoretic Mobility Shift Assay（EMSA）. Finally, we will carry out functional analysis for these target genes. It is first time to clarify regulatory mechanisms of transcription factors that are specific for sexual development in G.zeae. And we will obtain a comprehensive insight of the gene regulatory mechanism in sexual development at different stages. So this project will provide clues for the prevention and treatment of FHB disease.

玉蜀黍赤霉（无性态：禾谷镰孢）引起的赤霉病是世界范围内的小麦流行病害。其有性生殖产生的子囊壳是该菌越冬的主要形式。同时有性孢子（子囊孢子）也是该病流行的初侵染源。有性生殖还保持了居群致病力相关基因的遗传多样性和稳定性，从而提高其对宿主植物的适应能力。因此对有性生殖的理解是综合控制赤霉病的先决条件。研究转录因子能将上游信号途径和下游靶基因连接起来。从前期的研究结果中，我们筛选出5个在有性生殖不同阶段（包括调控雌配子育性、子囊壳数量、子囊壳大小、子囊壳成熟、子囊孢子成熟等）特异发挥调控作用的转录因子。本项目拟以这5个转录因子为研究对象，利用染色质免疫沉淀测序技术并结合表达谱测序结果和体外电泳迁移率实验找出它们的候选靶基因。最后对候选靶基因进行功能分析。该研究将首次阐明特异地作用于玉蜀黍赤霉有性生殖不同发育阶段转录因子的基因调控机制，从整体上认知其有性生殖发育表达调控机制，为赤霉病防治提供线索。

利用分割标记法，构建了5个转录因子MYT1、MYT2、GzZC183、FGSG_08626、FGSG_10286的敲除突变体和回复突变体。实验表明，以上转录因子在有性生殖过程中发挥重要作用。研究发现，光照的强度对子囊壳的数量及成熟起到了关键作用。通过Blast比对，找出了玉蜀黍赤霉中8个编码光敏色素的基因并对其进行敲出及回复。结果显示三个编码吸收蓝光的光感受器基因敲除明显影响子囊壳及子囊孢子的发育,而吸收红光与绿光的光感受器基因敲除对子囊壳形成没有明显的影响。吸收蓝光的光感受器基因敲除突变体中ΔGzWC-1和ΔGzWC-2菌株子囊孢子释放较野生型菌株提前，进一步观测子囊孢子形态，与野生型相比并没有显著差异。ΔGzVVD菌株则无法形成成熟的子囊壳，将GzVVD基因重新回复到突变体菌株后恢复子囊壳发育，说明GzVVD基因的确调控子囊壳的发育。这三个编码蓝光感受器基因的敲除导致子囊壳发育的变化，说明蓝光在子囊壳的发育过程中起到关键作用。. 在研究小麦赤霉病菌相关转录因子功能的同时，结合生产实践，组建了湖北省小麦赤霉病菌对多菌灵、戊唑醇和咪鲜胺的敏感基线，并连续三年就湖北省小麦赤霉病菌对多菌灵、戊唑醇和咪鲜胺的敏感性进行测定，发现湖北省小麦赤霉病菌仍然对上述杀菌剂敏感，可在生产中继续使用。