番茄广谱抗性microRNA发掘及其分子机制

Tomato powdery mildew and late blight are important worldwide plant disease caused by Oidium neolycopersici and Phytophthora infestans, respectively, which can cause decrease of production and quality. microRNAs (miRNA) play important roles in disease-resistance and are often induced by pathogens. The mining and mechanism decoding of miRNAs with broad-spectrum resistance against pathogens will be helpful for elucidating the molecular mechanisms of disease resistance and broad-spectrum resistance breeding in tomato. Oidium neolycopersici and Phytophthora infestans are biotrophy and semi-biotrophy, respectively. O. neolycopersici infect tomato only with biotrophic manner, and P. infestans infect tomato with both biotrophic and nectrophic manners. In this project, we will focus on finding changed miRNAs induced by the above two pathogens with different infection manners through comparative transcriptome method, which are specifically involved in biotrophic infection stages, these key miRNAs should show broad-spectrum resistance against the pathogens. The molecular mechanism of the key miRNAs will be deciphered to enrich the theory of tomato response to pathogens. The key miRNAs and their target genes are important resources of genes showing broad-spectrum resistance against pathogens with biotrophic infection manners. Targeting the obtained broad-spectrum resistance miRNA genes, tomato new germplasm with broad-spectrum resistance will be created using genome editing.

由番茄白粉菌和致病疫霉引起的白粉病和晚疫病都是重要的世界性番茄病害，能够造成番茄的减产和品质下降。microRNA(miRNA)在植物与病原物互作中起着重要作用，病原物可诱导其表达变化，发掘具广谱抗性miRNA并解析其分子机制，对阐释番茄抗病反应机理和广谱抗性育种具有重要意义。白粉菌和致病疫霉分属活体寄生和半活体寄生真菌，白粉菌仅以活体寄生方式侵染番茄，而致病疫霉侵染番茄兼具活体寄生和腐生两个方式，本项目拟通过比较分析这两种病原物诱导的番茄miRNA转录组，旨在发掘病原物活体寄生侵染阶段特异诱导番茄信号途径中的关键miRNA，并通过确定其下游基因调控网络和靶基因解析其分子机制，可以丰富番茄抗病反应机理理论，同时这些关键miRNA及其靶基因是对采用活体寄生侵染方式的病原物具有广谱抗性的重要基因资源。利用基因组编辑对具有广谱抗性的关键miRNA及其靶基因进行编辑，可以创制广谱抗性番茄新种质。

番茄晚疫病和白粉病都是重要的世界性真菌病害。miRNA在植物与病原菌互作中发挥着重要作用，利用RNA-seq技术对致病疫霉菌和白粉菌侵染番茄前后miRNA的比较转录组分析获得重要的候选抗病相关miRNA，并利用过量表达、干扰表达以及基因编辑等技术对候选miRNA进行功能分析，得出以下结果和结论：（1）获得522个番茄抗病反应的重要候选miRNA，为番茄抗病育种提供了丰富的基因资源；（2）从转录组数据中，挑选7个候选miRNA （miR397、miR1919c、miR156、miR162、miR6022、miR171b和miR395），构建其过表达载体、STTM干扰载体以及Crispr/Cas9敲除载体，目前部分转基因已获得T3代转基因植株，正在对其接种病原菌进行功能验证；（3）对候选miRNA的靶基因进行预测，并利用RACE技术进一步确认靶基因；（4）解析了番茄miR397、miR1919c以及miR156在番茄抗病中的功能，且这些miRNA除影响番茄抗病性外，也影响番茄植株的发育；（5）miR397负调控番茄对晚疫病和灰霉病的抗性，正调控侧枝数目的发育；（6）miR156c负调控番茄对晚疫病的抗性，并调控叶、侧枝以及番茄株高的发育；（7）miR162正调控番茄对晚疫病的抗性；（8）miR171b负调控番茄对晚疫病的抗性；（9）miR1919c负调控番茄对致病疫霉菌的抗性，正对灰霉菌的抗性增强；过量表达miR1919c的转基因株植株侧枝数目增多；（10）采用RNA-seq技术对接种致病疫霉菌前后过量表达miR1919c的转基因植株和野生型植株的基因表达差异情况进行了分析，发现595个差异表达基因，对这些基因进行GO、COG和KEGG分析，推断出miR1919c可能通过激素信号途径影响番茄对病原菌的抗性，同时为进一步研究番茄与病原菌互作机制提供基因来源。本项目初步阐释miRNA在番茄与不同病原菌互作中的机制，为挖掘具有持久、广谱抗性的番茄miRNA提供资源。基于以上研究成果，本项目发表SCI论文7篇，中文核心论文2篇，申请和授权专利5项。