番茄与致病疫霉互作中的miRNA解析

Late blight caused by Phytophthora infestans is a kind of devastating disease which is common and epidemic in tomato production. Currently, there are no tomato varieties immune to the disease while the pesticide residue caused by chemical control and the drug resistance of the pathogens are increasingly prominent. This situation is largely due to lack or insufficient of related basic research. We have mastered the induced resistance and the induction condition of tomato inoculated with P. infestans. We have already identified the resistance-related tomato miRNAs and predicted the existence of P. infestans miRNAs. On these bases, small RNA library construction, high throughput sequencing, bioinformatics analysis, molecular hybridization and real-time quantitative PCR are to be used in this project to explore the crucial miRNAs in both tomato and P. infestans during the interactions between the two. The resistance of transgenic tomato plants to P. infestans will be measured to decipher the functions of the key miRNAs by over expression and miRNA sponge. Then, we will construct the regulatory networks model of these miRNAs and reveal the molecular defense mechanism of tomato. Findings of this work will fill the gap for the absence of any P. infestans miRNAs in the current miRBase. In the meantime, both new disease-resistant tomato germplasm and the targets for the prevention of late blight could be obtained. This project is expected to lay a foundation for molecular breeding of late blight resistance in tomato via targeted control of the pathogen, and hence to improve both quality and quantity of tomato production.

致病疫霉侵染番茄造成的晚疫病是生产中最严重的常发性、流行性病害，目前尚无对其完全免疫的品种，而化学防治导致的农药残留和病原菌的耐药性等问题日益突出，相关的基础研究相当薄弱。本项目在掌握了致病疫霉接种番茄过程中的诱导抗性和发病条件，并挖掘鉴定了番茄中与抗性相关的miRNA以及预测到致病疫霉中存在miRNA的基础上，拟采用小RNA文库构建、高通量测序、生物信息学分析、分子杂交、实时荧光定量PCR等手段，探究番茄与致病疫霉互作中双方体内起关键作用的miRNA；利用过表达及miRNA海绵技术，通过对转基因植株的抗性检测，解析各miRNA的功能；建立它们相互之间的调控网络模型，揭示番茄抗病防御的分子机制；填补miRNA数据库中尚无致病疫霉miRNA的空白；创制出新的番茄资源材料；找到防治晚疫病的靶点。为抗病分子育种和病原菌的生物靶向治理奠定基础；对番茄无公害生产、提高产量和品质具有重要的意义。

为了揭示番茄与致病疫霉互作的分子机制，给晚疫病的防治提供理论和材料基础，本项目利用miRBase中所有物种的miRNA成熟体及前体序列、NCBI中的番茄和致病疫霉的EST序列以及构建的番茄与致病疫霉互作过程中的miRNA文库，发现了二者体内起关键作用的miRNA；结合降解组及5’ RACE 技术鉴定了这些miRNA的靶基因；利用过表达及人工miRNA、TM和STTM沉默技术，明确了番茄miR172、miR396、miR1916以及致病疫霉miR1918在二者互作中的功能，发现番茄miR172、致病疫霉miR1918分别与晚疫病的抗性呈正、负相关关系；这些miRNA及靶基因之间构成了巨大的调控网络，在该网络中，番茄NBS-LRR基因Solyc06g008070.2.1可同时受番茄miR482和miR6024的调控，而Solyc11g069020.1.1同时受番茄miR6024和miR6027的调控；番茄miR396既可以靶向GRF基因，也可以靶向TRAF-like等基因；致病疫霉的miR1918可以跨界调控番茄中的RING finger基因。在逐渐探明番茄与致病疫霉互作中各主要miRNA及其功能的过程中，还创制出了21个抗、感病新材料。与此同时，我们利用获取的大量序列信息，借助数据挖掘技术手段，开发出一系列预测miRNA、靶基因等的算法工具。四年来共发表期刊论文31篇，其中有23篇被SCI收录，影响因子合计为56。该项目的研究结果将极大地推动番茄抗病分子育种的进程，也将促进其他植物分子生物学的研究。