小RNA药物对番茄灰霉病菌的防治及其抑菌机制研究

Gray mold is one of the most important diseases in tomato. A large number of chemical pesticides were used to control Botrytis cinerea have caused serious pollution as well as Drug resistance. sRNA drug is a hot research topic in animal and medical field, but it is relatively backward in plant. The sRNA drugs were designed based on the sequences of Botrytis-responsive miRNA in tomato can inhibit the growth of the Botrytis cinerea in the preliminary study. In this project, these sRNA drugs would be furtherly study from two aspects. The inhibition toxicity, drug residues and environmental safety would be detected and analyzed for the application of sRNA drugs in tomato production. Then the targets of these sRNAs would be identified using degradation sequencing and 5’-end RNA linker media rapid amplification cDNA end. In addition, the gene expression profiles in whole genome levels would be investigated through high throughput sequencing to reveal the molecular mechanism of sRNA drugs in growth inhibition of Botrytis cinerea. This project is the first attempt to prevent and control plant pathogens using sRNA drugs. The result is the basis for the development and design of sRNA drug in agricultural production in future. Project research is conducive to the development of a series of sRNA drugs with independent intellectual property rights, to provide new measures for prevention and control of plant diseases.

灰霉病是近年来危害番茄的主要病害之一。目前大量使用的化学农药对生态环境造成了严重的污染；此外，抗药性的加剧也导致防治效果逐年降低。小RNA药物是目前研究的热点，已在动物及人类疾病治疗方面取得巨大进展，但在植物病害防治的应用研究相对滞后，迄今未有相关的文献报道。项目申请人在前期研究中发现，体外施用小RNA药物，能有效抑制灰霉病菌的生长。本项目以这些小RNA药物为研究对象，通过对小RNA药物的抑制毒力及环境毒理学分析，确定小RNA药物的环境安全性；利用降解组及数字表达谱等高通量测序技术，对小RNA药物作用的靶基因进行识别、并从全基因组水平全面分析小RNA药物对灰霉病菌基因表达的影响，揭示小RNA药物抑制灰霉病菌生长的分子机制；本项目是小RNA药物在植物病害防治领域的首次尝试，为开发小RNA药物提供理论依据，也为植物病害的可持续治理提供新措施。

有证据表明，microRNAs作为一种效应分子，可以从一个物种转移到另一个物种的，并且在受体物种中表现出生物活性。但目前对于植物源miRNAs跨物质调控病原真菌毒力的研究却鲜有报道。在本项目中，我们重点研究了番茄编码的miRNA，在灰霉病菌生长发育及感染植物过程中的跨界调控作用。我们发现miRn14对灰霉病菌孢子的萌发有明显的抑制作用、对感染宿主植物的致病性也有显著的抑制作用，这种抑制作用不受受体物种的限制，而且双链miRn14比单链miRn14对病菌的抑制效果更好。通过环境安全性分析表明，miRn14对蝌蚪、蜜蜂等小动物没有毒性；并且，化学合成的sRNA在自然界：包括植物叶片、水体和土壤中基本上几个小时就会完全降解，具有很高的环境安全性。5′-RLM-RACE实验表明，miRn14可直接靶向两个编码番茄灰霉病杆菌ATP依赖性金属肽酶和半胱氨酸型内肽酶的基因。实时RT-PCR分析表明，miRn14处理的番茄灰霉病菌与对照相比，两个靶基因的表达明显下调。我们的发现为病原体和寄主植物的共同进化提供了新的证据，也为利用植物源miRNAs控制病原体提供了新的方向。