AP2/ERFs转录因子和油菜素内酯互作调控黄瓜疫病抗性机制研究

Cucumber blight, caused by Phytophthora melonis Leonian, is a destructive soil-borne fungal disease in South China. No fully immune or highly resistant varieties are found. It is also difficult to control the disease infection using chemical fungicide and agronomic measures in production. Thus, it is a noteworthy scientific topic to explore the regulatory mechanisms of cucumber blight. Our research found that brassinosteroids had good control effect on cucumber blight, and also showed significant interaction with APETALA2/ethylene-responsive factor (AP2/ERFs) transcription factor. However, the mechanism is not clear. Based on the results of RNA-seq analysis, we aimed to screen and get candidate AP2/ERFs genes related to disease resistance by qRT-PCR and Arabidopsis thaliana transgenetic methods. Furthermore, biological function of candidate AP2/ERF genes was verified in transgenic cucumber plants. Afterwards, the direct targets, interaction proteins and biological functions of candidate AP2/ERFs genes will be further confirmed through DNA-protein and protein-protein approaches. Subsequently, the molecular mechanism of AP2/ERFs transcription factors and BRs in induced resistance of cucumber plants to P. melonis will be comprehensively explored. The results obtained by this project can provide the theory basis for cucumber blight control in production and resistance breeding of cucumber plants.

黄瓜疫病是危害华南地区黄瓜生产的毁灭性土传病害，不仅缺乏完全免疫或高抗的品种类型，同时生产中也缺乏有效的杀菌剂和农业措施。因此，研究黄瓜疫病的调控机制是一个值得关注的科学问题。BRs在黄瓜疫病防治中具有良好的防效，且与AP2/ERFs转录因子表现出明显的相互作用，但是互作机制不明确。本项目在转录组测序分析结果的基础上，通过基因表达分析和拟南芥转基因等手段获得最有可能参与黄瓜疫病抗性反应的AP2/ERFs基因，并在黄瓜转基因植株中验证其生物学功能。在此基础上，进一步通过DNA-蛋白质、蛋白质-蛋白质互作等手段，明确候选AP2/ERFs转录因子的下游靶基因、互作蛋白以及生物学功能，进而对BRs和AP2/ERFs转录因子互作调控黄瓜疫病抗性的分子机制进行全面的解析，为黄瓜疫病的调控以及抗病育种提供扎实的理论基础。

黄瓜疫病是危害华南地区黄瓜生产的毁灭性土传病害，不仅缺乏完全免疫或高抗的品种类型，同时生产中也缺乏有效的防控措施。本项目主要研究结果如下：（1）下胚轴“短而粗”的黄瓜品种抗病性高于下胚轴“细而长”的品种；外源BRs施用可促进黄瓜下胚轴加粗生长，同时可诱导木质素在黄瓜下胚轴维管束的外韧皮部和木质部导管细胞壁的沉积，以及胼胝质在外韧皮部筛板的积累，激活细胞壁防御反应，进而增强黄瓜疫病抗性。（2）内源BRs生物合成末端氧化酶基因CYP85A1缺失突变体抗病性显著降低，分析其突变诱导的黄瓜转录本变化，发现58个AP2/ERFs转录因子受CYP85A1缺失或者疫霉菌接种诱导。运用VIGS、遗传转化技术以及生物信息学分析，筛选出ERF亚家族DREB1（CsaV3_3G016760）负调控、RAP2-3-like（CsaV3_6G022740）正调控黄瓜疫病抗性，RAP2-3-like靶向调控GCC盒和CYP85A1启动子。（3）酵母双杂筛库共筛选到6个可能的互作蛋白，一对一验证表明，仅E3泛素连接酶MARCH6（CsaV3_7G008470）与CYP85A1互作。本项目研究成果可为黄瓜疫病抗性育种以及外源激素BRs的应用提供理论依据。