MAPK级联途径元件响应生防酵母/细胞壁组分诱导调控番茄果实抗性反应的机制研究

Induction of fruit resistance is a hot topic of controlling postharvest rot diseases in recent years. MAPK cascade as the early signal to control the resistance response has been widely concerned. Different of MAPKs, MAPKKs and MAPKKKs are activated in responses to different biological / abiotic stresses and have expression profilings in different tissues and organs..Biocontrol yeast / cell wall can induce disease resistance of fruit with the increase of the expression of resistant protein and related transcription factors in postharvest fruits. However, there is no systematic report on the mechanism of MAPK cascade in response to this induction and regulation of fruit resistance. Based on this, high-throughput transcriptome sequencing would be used to screening the target MAPK cascade effect element, resistance protein gene and its transcription factor, resistance related hormone synthesis genes of tomato fruit in response to yeast / cell wall induced conditions. RT-qPCR technology would be used to analysis the MAPKs (MAPKKs and MAPKKKs) gene, antagonism related genes and transcription factors, resistance related hormone synthesis genes in transgenic tomato (Lycopersicum esculentum) through ‘knocking-down’ their expression with methods of RNAi and wild type tomato. The resistance related hormones would be analyzed by targeted metabolomics technology also. The function of target MAPK (MAPKK or MAPKKK) and MAPK cascade pathway in the resistance regulatory network system of fruit response to yeast/ cell wall component would be explored through this study. The research results have important theoretical and practical significance for using yeast/ cell wall component to induce fruit disease resistance in more efficient and accurate way and promoting postharvest preservation technology of fruits in China.

诱导抗性是近年来控制果实采后腐烂病害的研究热点。MAPK级联途径作为调控抗性反应的早期信号，其表达及调控抗性的作用机制受到广泛关注。生防酵母细胞壁组分能激活抗性蛋白及其转录因子的表达以提高果实抗性，而MAPK级联途径在此诱导条件下调控果实抗性的机制尚无系统报道。基于此本项目拟采用高通量转录组测序以筛选番茄果实响应此诱导条件的MAPK级联途径元件、抗性蛋白及其转录因子基因、抗性相关激素合成基因及激素调控路径关键基因；利用RNA干扰技术构建目标MAPK级联途径元件缺陷型转基因番茄，运用RT-qPCR、靶向代谢组学技术分析目标MAPK级联途径对抗性相关基因及转录因子、激素调控路径关键基因、激素合成基因及其含量影响，系统分析目标MAPK级联途径元件响应诱导、调控番茄果实抗性反应的网络和机制，研究结果对于更加高效利用酵母细胞壁组分诱导提高果实抗病性、促进我国水果采后防腐保鲜技术具有重要意义。

诱导采后果蔬抗性以减少化学杀菌剂的使用是近年来采后果蔬病害防控研究的热点，其中生防酵母及细胞壁组分做为激发子受到关注。目前MAPK 级联途径元件作为植物响应抗性诱导的初级传导信号，其对酵母细胞壁组分的响应及其对抗性调控的作用机制及调控网络缺乏系统研究。本研究通过酵母及细胞壁组分处理樱桃番茄果实，筛选有效的抗性诱导处理方法，利用转录组测序分析诱导抗性早期响应的差异表达基因及功能富集和通路，筛选关键MAPK激酶，分析MAPK激酶对下游转录因子、抗性蛋白的调控作用，进一步利用MAPK激酶抑制剂和构建基因缺陷型植株，结合靶向代谢组学和RT-qPCR技术分析MAPK路径与植物激素之间互作所形成的果实抗性反应的调控网络。结果表明：①1%酵母细胞壁组分可有效诱导果实抗性提升；转录组测序结果表明处理组差异基因富集在植物与病原菌互作、MAPK信号通路、植物激素信号转导等方面，筛选MAPK3、JA、BRs、SA为后续分析目标 ②酵母细胞壁组分能够激发SlMAPK3快速响应上调表达，转录因子SlPti5/SlERF1及抗性蛋白SlPR1/SlPR5/SlCHI9也显著上调表达且与SlMAPK3具有时间顺序性，抑制剂处理显著降低SlMAPK3及转录因子和抗性蛋白基因的表达，表明MAPK3对下游转录因子、抗性蛋白具有直接调控作用 ③靶向代谢组结果表明JA、BL在诱导处理早期含量显著上升，SA无显著变化，转录水平分析表明BR、JA合成及信号转导相关基因显著上调表达，而SA信号转导基因显著下调表达，表明JA、BRs作为主要激素调控抗性反应，RNA沉默技术构建缺陷型番茄植株体系研究表明，JA/ET路径是植株抗性免疫响应主要路径；抑制剂处理表明JA、SA信号转导受到抑制，MAPK3与JA、SA形成互作网络共同参与果实抗性反应，研究结果为诱导抗性机制分析提供理论依据。