柑橘青霉菌Dicer基因的功能及其诱导的sRNA对夏橙致病机制的研究

Dicer is one of the most important genes associated with molecular mechanism of RNA interference, which is be functional for regulating small RNA formation in varied organisms. In addition, it has been known that small RNA deriving from plant pathogen could be a new type of virulence factors, but we still poorly understand about this new field of plant diseases. On the basis of data from a previous youth fund, this project is based on the model of interaction between Penicillium italicum and Citrus sinensis. Using gene knockout, small RNA sequencing and so on, it is firstly aimed to illuminate the function of Dicer associated with the pathogenicity of P. italicum, and further to reveal the types and features of its guided small RNA in P. italicum. Secondly, we plan to confirm the interaction between the small RNA in P. italicum and target genes in C. sinensis by using the transgenic tobacco system containing fluorescent reporter gene and the degradome sequencing. According to the physiological function and biological effect of Dicer gene in P. italicum, we aim to prepare dsRNA of Dicer gene used to control postharvest pathogens from Penicillium spices, and further to illustrate its mechanism of action. These are based on the comprehensive application of gene silencing techniques and routine analytical methods from molecular biology and pesticide science. Through the implementation of this project, it will help us to understand the pathogenic mechanism of P. italicum against C.sinensis in small RNA level, and the expected results are possible to be used in the field of fruit fresh-keeping and citrus breeding.

Dicer是RNA干扰分子机理的关键基因，其功能之一在于调控生物体小分子RNA的形成。然而植物病原真菌分泌的小分子RNA可能是一类新型的致病因子，但我们对这一植物病害研究新领域还知之甚少。在前期青年基金资助的基础上，本项目以柑橘青霉菌与夏橙的相互作用为研究模型，首先利用基因敲除技术、小分子RNA测序技术等阐明柑橘青霉菌Dicer基因与其致病性的相关性、及其调控小分子RNA的种类和特征；其次利用含荧光报告基因的转基因烟草体系、降解组测序等揭示柑橘青霉菌小分子RNA靶向沉默的夏橙目标基因；在阐明柑橘青霉菌Dicer基因的生理功能和生物学效应的基础上，结合基因沉默技术和分子生物学、农药学常规分析方法，阐明Dicer基因dsRNA对多种采后青霉属病原菌的防治效果和作用机理。通过本项目的实施，将有助于我们从小分子RNA水平理解柑橘青霉菌侵染夏橙的致病机制，预期成果有望应该在水果保鲜及柑橘育种等领域。

Dicer是RNA干扰分子机理的关键基因，其功能之一在于调控生物体小分子RNA的形成。然而植物病原真菌分泌的小分子RNA可能是一类新型的致病因子，但我们对这一植物病害研究新领域还知之甚少。我们利用基因沉默技术揭示了柑橘青霉菌 Dicer 基因的功能，通过小分子RNA测序鉴定了表达的sRNA，研究发现柑橘青霉菌 Dicer 2基因沉默后对其致病性有显著影响，也对其sRNA 的种类、丰度、长度有较大影响。我们用qPCR和Northern blot技术验证了柑橘青霉菌 Dicer 2基因沉默显著降低了Pit-novel7的表达，预测结果表明Pit-novel7靶向夏橙免疫相关基因Cs7g19510（AP2/B3-like transcriptional factor），进一步利用烟草体外转染实验，验证了Pit-novel7确实靶向Cs7g19510。此外，通过测定柑橘青霉菌基因组及比较基因组分析，阐明了柑橘青霉菌的进化地位；通过双重转录组分析揭示了柑橘青霉菌与夏橙互作的分子基础以及寄主专一性的分子机制;我们的研究阐明了柑橘青霉菌、柑橘绿霉菌和扩展青霉三者之间的进化关系和物种形成的机理，结果表明扩展青霉是通过蛋白家族扩张、基因平行转移、正向选择的物种进化机制实现了寄主范围扩大和寄主跳跃。针对柑橘青霉菌抗药性严重，目前防治药剂药效降低的问题，我们研发了基于纳米木质素的柑橘青霉菌防治增效助剂。采用溶解度参数原理，制备木质素Pickering乳液，再通过滴涂工艺和透析技术制备木质素纳米颗粒，最终发现纳米木质素对植物精油有增溶和增效的作用，使其对柑橘青霉菌的防治能力提高30%，该增效技术不使用乙醇等有机溶剂、全过程采用食品级材料，是一种环境友好和食品安全的柑橘保鲜剂增效方法。