NO诱导采后番茄果实抗病中NPR1与MAPK的协同互作机制研究

As NO induced disease resistance of fruits and vegetables is closely related to MAPK and NPR1, it is of significant importance for exploring a green, broad-spectrum, efficient and convenient disease control strategy by analysis of the "NO-NPR1-MAPK" network to elucidate their crosstalk in disease resistance. Tomato fruit of antisense ACS2 and LeMAPK1/2/3, and over-expression LeMAPK1/2/3 have been obtained by the applicant and will be used in this research to construct two kinds of double silence tomato fruit of "LeACS2-LeMAPK1/2/3" and "LeACS2-NPR1", and also to establish two comparative research systems of "regulary-silence- overexpression" of LeMAPK1/2/3 and "regulary-silence" of NPR1. More over, tomato materials which have been skillfully applied by our lab, will be used to scientificly avoid the interference from endogenous ethylene to hierarchically define the roles, interaction and cross-regulation mode of NPR1 and MAPK in NO induced disease resistance of tomato fruit. And then, relevant new technologies of protein conformation, site mutagenesis, immunoprecipitation assay, gel autophosphorylation assay, nuclear localization, and molecular modelling & structure prediction, will be brought in to gradually and precisely reveal the interaction mechanism of NPR1 and MAPK in NO induced disease resistance in tomato fruit for the purpose of laying a necessary theoretical basis in realizing a green and simply regulatory method in postharvest storage and processing of fruits.

NO诱导采后果蔬抗病与MAPK和NPR1密切相关，解析其中的"NO-NPR1-MAPK"网络关系，对探索绿色、广谱、高效、简便的果蔬采后病害防控策略具有重要意义。本项目拟采用申请人已获得的转反义LeACS2、LeMAPK1/2/3转基因沉默和LeMAPK1/2/3超表达番茄材料，构建"LeACS2-LeMAPK1/2/3"和"LeACS2-NPR1"双价缺失番茄材料，建立LeMAPK1/2/3"正常-缺失-超表达"对应体系和NPR1"缺失-正常"对应体系，并利用已成熟多年的乙烯缺陷型材料，科学避开内源乙烯，分层次确定NO调控番茄果实抗病中MAPK、NPR1的作用及互相调控模式；进而引入蛋白构象、定点突变、免疫共沉淀、磷酸化位点分析、核内定位、分子预测与建模等相关学科成熟新技术，逐步精准解析NO诱导番茄果实抗病中NPR1与MAPK的交叉互作机制，为实现果蔬采后病害的绿色简易防控奠定理论基础。

NO 诱导采后果蔬抗病与MAPK 和NPR1 密切相关，解析其中的“NO-NPR1-MAPK”网络关系，对探索绿色、广谱、高效、简便的果蔬采后病害防控策略具有重要意义。本项目拟采用申请人已获得的转反义SlACS2、SlMAPK3基因沉默番茄材料、SlMAPK1过表达番茄材料，结合MAPK激酶抑制剂U0126和NPR1抑制剂，构建“SlACS2-MAPK级联”和“SlACS2-SlNPR1”双价缺失番茄材料，科学避开内源乙烯，分层次确定NO 调控番茄果实抗病中MAPK、NPR1 的作用及互相调控模式；进而引入生物信息学、实时定量PCR、亚细胞定位、CRISPR/Cas9基因编辑等相关学科成熟新技术，逐步精准解析NO 诱导番茄果实抗病中NPR1 与MAPK的交叉互作机制，为实现果蔬采后病害的绿色简易防控奠定理论基础。