番茄SlMLO4参与白粉菌胁迫应答的机制研究

MLO (Mildew Locus O) belong to a recessive disease resistance gene family, as .susceptible factors, loss of function mutations of these genes confer plants broad-spectrum resistance to all known isolates of the powdery mildew. The previous study results found that the tomato MLOs family member SlMLO4 expressed significantly up-regulated at the early stage of powdery mildew infection, which shows it is closely involved in the stress response of powdery mildew, but the function and mechanism of the stress response are not clear. So this project is proposed. Molecular biology function of SlMLO4 will be revealed by CRISPR/Cas9 system and over expression techniques. The interaction proteins with SlMLO4 will be screened by yeast two hybrid technique. The interaction between SlMLO4 and the candidate proteins will be verified by yeast two-hybrid, bimolecular fluorescence complementation assay and co-immunoprecipitation techniques. The gene expression characteristics of the encoding genes of the interaction proteins will be analyzed by the transgenic lines with SlMLO4 knockout above, and the regulation relationship between the two genes will be determined. Through the expansion of powdery mildew under the target protein gene knockdown, the role of it involved in SlMLO4 to powdery mildew stress will also be determined. The results of this research project will reveal the functions of SlMLO4 in tomato response to powdery mildew fungi stress, explore the response mechanism involved SlMLO4 and genes of its interaction proteins in the interactions of tomato and powdery mildew, enrich the resources of broad spectrum resistance genes to powdery mildew and provide theory basis for studying the mlo resistance mechanism to powdery mildew and tomato disease resistance breeding.

MLO（Mildew Locus O）是抗病负调控基因家族，其作为感病因子功能缺失突变后，可使植物获得广谱抗病性。申请人前期研究结果发现番茄MLOs家族成员SlMLO4在白粉菌侵染初期极显著上调表达，表明其密切参与白粉菌胁迫应答，但其在胁迫应答中的功能与机制尚不明晰。因此本项目拟利用CRISPR/Cas9基因编辑和过表达技术对番茄SlMLO4进行功能分析；利用酵母双杂交筛选SlMLO4互作蛋白；通过酵母回复验证、双分子荧光互补和免疫共沉淀技术验证两者互作关系；通过上述敲除SlMLO4转基因株系的靶蛋白基因表达特征确定两者调控关系；敲除靶蛋白基因，通过白粉菌扩展情况确定其在SlMLO4参与白粉菌胁迫中的作用，以期揭示SlMLO4参与白粉菌胁迫应答的分子生物学功能，探索其与靶蛋白基因在番茄与白粉菌互作中的响应机制，丰富广谱抗性基因资源，为研究mlo对白粉病的抗性机制和番茄抗病育种提供理论依据。

本项目以感病番茄Moneymaker（MM）和抗病番茄62579为材料，利用过表达和基因编辑技术对番茄SlMLO4进行功能分析；利用酵母双杂交筛选SlMLO4互作蛋白，并验证其互作关系，主要研究内容和结论如下：（1）SlMLO4、SlMLO2相对表达量在接种白粉菌3-12 h呈上升趋势，12h形成吸器时达到最高值，24h后显著下调，表明其积极响应白粉菌胁迫；亚细胞定位发现SlMLO4定位在细胞核和细胞膜上，SlMLO2定位在细胞膜和过氧化物酶体上。（2）过表达SlMLO4、SlMLO2转基因株系病情指数均高于野生型感病番茄MM和抗病番茄62579，基因编辑SlMLO4株系病情指数显著低于野生型MM；SlMLO4、SlMLO2过表达株系中受到白粉菌胁迫后不同时间点SlMLO4、SlMLO2的相对表达量，坏死细胞的位点数量，H2O2积累位点数，SOD、POD、CAT活性和MDA含量，以及Glu、PR1b1、NPR1、EDS1、PAD4和PR1b1的相对表达量均上调，表明SlMLO4和SlMLO2为感病基因参与番茄与白粉菌胁迫应答，揭示了番茄感病基因SlMLO4、SlMLO2参与白粉菌胁迫应答分子生物学功能。（3）构建核体系及膜体系番茄酵母cDNA文库；通过酵母回转验证和酵母点对点验证明确有6个候选靶蛋白均与SlMLO4存在互作。（4）利用 geNorm、NormFinder和BestKeeper 3个软件，从8 个番茄候选内参基因种筛选出L33 基因是在番茄与白粉菌互作过程中ABA、JA和 SA这3 种外源激素诱导下表达最稳定的内参基因；抗、感番茄品种在白粉菌胁迫下最稳定的内参基因是Act。（5）抗、感番茄接种白粉菌后，通过转录组测序鉴定出9855个差异表达基因（DEG），KEGG富集结果表明，氨基酸代谢、信号转导、能量代谢和其他次级代谢产物显著富集。广泛靶向代谢组检测到差异累积代谢物（DAMs）1058个，与转录组联合分析表明，番茄对白粉菌具有不同抗性的关键候选基因、代谢产物和代谢途径，并系统、全面地分析了番茄抗白粉菌的分子机制。本研究这为不同抗性番茄对PM感染叶片的抗性反应提供了有利的分子信息，丰富广谱抗性基因资源，为培育抗PM的番茄品种提供了依据。项目开展期间共发表SCI论文1篇，中文核心期刊7篇，共培养毕业硕士研究生4名。