果桑响应桑椹菌核病菌Scleromitrula shiraiana侵染的抗性机制研究

Mulberry popcorn disease is one of the most important diseases for fruits production and become the bottleneck problem of mulberry industry development. Breeding resistant varieties is the most economical and effective means to prevent the disease. However, the research progress on molecular resistant mechanism against the disease was slow. Scleromitula shiraiana was the main isolate of popcorn disease fruits collected from Hubei and Guangdong provinces, and high resistant (Morus macroura) and high susceptible (Big 10) mulberry cultivars were screened in previous study. This research will analyze the differences of histopathology, physiology and biochemistry, and metabolites changes between high resistant and high susceptible cultivars during S.shiraiana infection process, to reveal the mechanism of different mulberry cultivars in response to pathogen infection and the change of metabolites during mulberry fruit decayed. Based on the transcriptomics and metabolomics sequencing datas, the signal pathways and genes closely related to disease resistance reaction will be predicted, and the NBS-LRR gene family associated with popcorn disease resistance will be identified and cloned, and the specific tissue expression patterns of the gene family will be analyzed. This research will provide a basis for deep analysis of mulberry and sclerotinia interactions and molecular mechanism of mulberry resistance, and provide a new way for disease control and disease resistance breeding.

桑椹菌核病是果桑上最重要病害之一，是限制果桑产业发展的瓶颈问题，培育抗病品种是防治该病最经济、有效的手段，然而针对果桑抗桑椹菌核病分子机制研究进展缓慢。申请人前期研究中发现桑椹核地杖菌Scleromitula shiraiana是湖北、广东省桑椹菌核病的主要分离物，长果桑和大10高抗和高感菌核病。本项目拟研究S.shiraiana侵染过程中，不同抗性果桑品种组织结构、生理生化及代谢产物变化，揭示果桑响应菌核病菌的侵染机制及桑椹小核果溃解的代谢产物变化；基于不同时间点转录组和代谢组，全面分析参与果桑抗病相关的信号途径和关键基因；鉴定并克隆与抗病相关的NBS-LRR类基因家族，并对其进行生物信息学及时空表达模式分析。本研究为深入分析果桑与菌核病菌互作及果桑抗病分子机制奠定基础，最终为病害防控及抗病育种提供依据。

桑椹菌核病是果桑最重要病害之一，是制约果桑产业发展的瓶颈问题，培育抗病品种是防治该病最经济、有效的手段，然而针对果桑抗桑椹菌核病分子机制研究进展缓慢。本研究从湖北、重庆、广东、浙江省采集病样，发现Ciboria carunculoides引起的小粒型菌核病在各产区广泛分布。从分离的病菌中选取单孢分离的桑椹缩小型菌核病菌Scleromitrula shiraiana和首次获得的可培养的桑椹小粒型菌核病菌Ciboria carunculoides进行了全基因组测序分析。成功实现了S. shiraiana菌核形成相关基因Mmk1的CRISPR/Cas9系统编辑，并根据其基因组和转录组测序结果分析了菌核形成相关MAPK基因，为后续设计阻断菌核形成的杀菌剂提供了靶标位点，为病害防控提供了新的途径。通过组织染色、石蜡切片和扫描电镜进行侵染观察发现病菌可通过气孔、表皮毛侵入桑果与已报道只通过花序侵染不同。C. carunculoides胁迫下，POD酶活呈显著增加趋势；CAT酶在感病初期变化不明显，但在发病严重时，酶活达到高峰期，在核小果形成菌核后酶活又下降；SOD酶活力呈下降趋势。用生物信息学方法，筛选获得165个桑树NB-ARC蛋白序列，构建ML系统进化树结果显示NBS-LRR类基因主要分为 2个大的聚类组。通过不同发病级别桑椹小粒型菌核病菌C. carunculoides和高感果桑（粤椹大10）的互作转录组数据及高抗果桑品种（长果桑）转录组数据，对664个共表达的差异基因进行分析，有7个抗病相关基因发现显著上调表达，7个发现显著下调表达，其GO功能富集在生物学过程方面，主要富集在有机氮化合物分解代谢过程，离子运输和碳水化合物衍生物分解代谢过程。在分子功能方面，主要富集在主要活性跨膜转运蛋白活性。KEGG富集主要涉及类单萜生物学合成、植物和病菌互作Plant-及植物MAPK信号通路三个方面。结合NBS-LRR类抗病基因分析及RT-qPCR结果，我们筛选出3个与抗病相关基因，为后续抗病基因功能验证提供了依据。