拮抗酵母菌诱导的猕猴桃果实lncRNA与抗性相关基因挖掘及功能分析

Kiwifruit is one of important economic fruits in China. It is called as “the king of fruit”, due to the high content of vitamin C. However, the fungal pathogen, Botrytis cinerea, causes gray mold, and results in significant economic losses of kiwifruit industry. The previous study of our research group has indicated that the antagonistic yeast, Pichia cecembensis XF-1, could manage postharvest gray mold safely and effectively. Moreover, long noncoding RNA (lncRNA) of kiwifruit plays a regulatory role in fruit development and stress response. In this project, we plan to utilize kiwifruit cv. ‘Hongyang’ and the antagonistic yeast XF-1 as materials to investigate the mode of action of lncRNA and the regulated genes in the disease resistance of kiwifruit induced by antagonistic yeast against gray mold. This study includes transcriptomic analysis of the expression profile of lncRNA and related genes in kiwifruit; characterization of their function via transient expression and RNAi techniques; utilization of chromatin immunoprecipitation assay, RNA pull-down and RNA binding protein immunoprecipitation to find out the target genes regulated by lncRNA and the interactive proteins. Collectively, unveiling the regulatory network of lncRNA will provide a new theoretical basis for biological control of postharvest diseases in kiwifruit.

猕猴桃是我国重要的经济林果之一，因富含维生素C被誉为“水果之王”。灰霉菌Botrytis cinerea引起的采后灰霉病是导致果实腐烂的主要病害，给猕猴桃产业带来巨大的经济损失。申请者前期研究发现，拮抗酵母菌Pichia cecembensis XF-1可安全高效地防治猕猴桃采后灰霉病；并且发现，长链非编码RNA（lncRNA）在猕猴桃果实发育和抗逆应答中发挥调控作用。本项目拟以红阳猕猴桃果实为材料，采用拮抗酵母菌XF-1处理，从转录调控网络分析拮抗菌诱导猕猴桃lncRNA和抗性相关基因的表达谱，通过瞬时表达和RNAi技术解析lncRNA功能，利用染色质免疫共沉淀、RNA pull-down和RNA结合蛋白免疫沉淀等方法挖掘lncRNA的靶基因及互作蛋白，进而明确lncRNA及其调控基因在拮抗酵母菌诱导果实抵抗灰霉病菌侵染中的作用机制，为猕猴桃采后病害的生物防治提供新的理论依据。

猕猴桃在采后贮藏加工过程中极易受到真菌侵染而发生腐烂变质，会给果农造成巨大的经济损失。本项目分别以红心、绿心和黄心猕猴桃为研究材料，利用生防效力较好的拮抗酵母菌Candida oleophila和Pichia cecembensis，检测其对猕猴桃果实寄主的抗性基因和lncRNA的诱导情况，分析了lncRNA的靶基因及互作蛋白。在此基础上，比较了C. oleophila和P. cecembensis对猕猴桃果实采后病害的生防效力，结果表明两种酵母菌对于猕猴桃果实采后灰霉病、青霉病和黑斑病均有明显的防治效果。发现果实防御相关酶和物质对拮抗酵母菌有应答变化；C. oleophila中转录因子rlm参与了对逆境耐受性调控。此外，还发现拮抗酵母菌Wickerhamomyces anomalus对猕猴桃果实的青霉病和灰霉病具有较好的生防效果。采用lncRNA高通量测序和生物信息学分析W. anomalus处理后的猕猴桃果实lncRNA，并根据靶基因预测了其功能；热处理在采后生物防治系统中起着重要作用，项目深入研究了拮抗酵母的热应激分子反应机制，发现热处理降低了拮抗酵母菌Debaryomyces hansenii对猕猴桃灰霉病的防治效果，并通过RNA-seq鉴定到了与应激反应、生物调控和发育过程相关的差异表达基因；Alternaria alternata是一种广泛存在于农作物中的病原菌，可以造成猕猴桃采后黑斑病。项目组从“红阳”猕猴桃中分离鉴定到A. alternata菌株（Y784-BC03），证实其能引起果实的黑斑病，因此，绘制了该菌株首张全基因图谱。综述所述，本项目明确了拮抗酵母菌可以通过诱导激活寄主猕猴桃果实lncRNA与抗性基因的表达，挖掘了lncRNA的靶基因及互作蛋白，阐明了拮抗酵母菌对寄主诱导抗性的新机制，为猕猴桃采后重要病害的绿色防治研究提供了理论基础和科技支撑。