根结线虫miRmc1跨界调控番茄类隐花色素蛋白基因的作用机制研究

植物病原物会产生一系列效应分子来调控寄主的防卫反应，协助病原物的侵染，小RNA（small RNA）是其中的一类重要的非蛋白类效应分子，目前在植物线虫上这类研究还鲜有报道。申请人前期通过测序发现源自南方根结线虫的miRNA（miRmc1），可能通过跨界抑制番茄类隐花色素蛋白基因SlCRY1的表达，调控番茄对线虫的防御反应。本课题拟采用RACE-PCR方法验证miRmc1切割靶基因的位点；构建miRmc1转基因番茄验证该小分子RNA的生物学功能；构建靶基因SlCRY1敲除和过表达转基因番茄验证其对线虫的抗性；通过Co-IP筛选和鉴定SlCRY1信号途径互作蛋白，从而解析miRmc1跨界调控的作用机理，揭示线虫致病新机制，为该病害的防控提供理论基础。

Small RNAs (sRNA) produced by pathogenic fungi are recently defined as non-proteinous effectors and regulate trans-kingdom plant immunity. However, it is rarely known about the function of sRNA from the root-knot nematode (Meloidogyne incognita, RKN) during infection and parasitism. In current study, we found 45 nematode miRNAs in RKN-infected tomato roots with Illumina deep sequencing. One of the miRNA (miRmc1) showed high abundance in RKN- infected sites. Silencing the miRmc1 precursor of the second stage of juvenile of M. incognita resulted in weak infection in tomato seedlings. Bioinformatics analysis and experimental evidences showed that miRmc1targets the cryptochrome-like protein 1 gene (SlCRY1) in tomato. Given that CRYs play vital role in plant resistance to pathogens, we speculate that RKN-produced miRmc1 may manipulate SlCRY1 gene expression in a trans-kingdom manner. Based on these results, this project will identify the cleave sites of target gene by RACE-PCR; the miRmc1 overexpression tomato plants and the SlCRY1 knock-out mutants will be generated to assess the transgenic plant response to RKN infection;. Further, mass spectrometry-based immuno-precipitation proteomics were carried out for identify the interacting protein of SlCRY1 in the transgenic tomato plants. This project will elucidate the role of RKN miRmc1 in trans-kingdom regulating plant immunity and the knowledge gained from this study will be instrumental in improving the control of RKN disease.