一个全生育期广谱抗白粉病NLR1-V基因的克隆、抗性机制及其进化研究

Wheat powdery mildew is a destructive disease leading to the serious yield loss. Exploration, utilization and cloning resistance genes is the prerequisite for the breeding and mechanism elucidation. The broad-spectrum resistance gene Pm21 from Haynaldia villosa was transferred to the common wheat by development of wheat-H.villosa translocation line of T6VS/6AL, which has been widely used in wheat production. In the previous study of this project, a NBS-LRR gene NLR1-V was cloned from the Pm21 locus by inovative approach combining the mutagenesis, RenSeq, PacBio and cytogenetic stocks development. NLR1-V was found to play improtant role in the broad-spectrum and whole-growth stage powdery midlew resistance by sequence comparison, transient assay and gene silencing analysis. So, cloning the resistance gene in Pm21 locus is valuable for the resistance theory study and wheat production. In this project, the expression pattern and splicing manner, the effect in different genetic background, the trangenic plants creation and the effect on the agronomic traits, gene evolution, interactive proten and signal transduction about NLR1-V will be studied in details. This project will try to make clear the mechanism of broad-spectrm and whole growth stage resistance medicated by NLR1-V, prilimilary contruct the signal tranduction network, and elucidate the relationship between the gene sequence variation and the function determination.

小麦白粉病对小麦生产造成严重危害。发掘、利用和克隆小麦抗白粉病基因是进行抗性育种和抗性机制研究的前提。本实验室通过创制T6VS•6AL易位系将定位于6VS上的簇毛麦广谱抗白粉病基因Pm21导入到普通小麦，在育种上得到广泛应用，克隆Pm21位点抗病基因具有重要理论价值和实践意义。本项目前期将细胞学材料创制、突变体筛选、R基因富集、三代测序技术相结合，克隆出Pm21区间内一个NBS-LRR基因NLR1-V，并通过突变体分析、瞬间表达、基因沉默等技术，明确该基因发挥全生育期广谱抗白粉病功能。本项目拟针对NLR1-V进行表达模式和剪切方式、不同遗传背景中抗性效应、转基因植株的获得和对农艺性状影响、所在位点基因组成和基因进化、互作蛋白和信号传导网络构建等研究。通过本项目研究，明确NLR1-V介导全生育期广谱抗白粉病机制，初步构建信号传导网路；同时解析进化模式，明确基因序列、关键区段与功能之间的关系。

小麦白粉病已成为危害我国小麦安全生产的主要病害。聚焦生产上有重要育种利用价值的抗白粉病基因，对小麦抗白粉病基因进行克隆和特征分析，是进行转基因抗病育种和抗病机制研究的前提和基础。小麦中已经定位了68个抗白粉病基因，但是已经克隆的小麦抗白粉病基因如Pm2、Pm3和Pm8等均是小种专化抗白粉病基因。来自小麦野生种簇毛麦的抗白粉病基因Pm21具有持久广谱抗性，携带Pm21的小麦-簇毛麦易位系T6VS·6AL作为白粉病主要抗源广泛用于中国小麦育种，在生产发挥了巨大作用，克隆Pm21位点抗病基因具有重要理论价值和实践意义。本项目在前期利用细胞学材料创制、突变体筛选、R基因富集、三代测序技术等综合技术，克隆出Pm21区间内一个NBS-LRR基因NLR1-V。本项目针对NLR1-V进行表达模式和启动子活性研究、NLR1-V在不同遗传背景中抗性效应分析、NLR1-V转基因植株的创制、NLR1-V所在位点基因组成和基因进化、NLR1-V互作蛋白和信号传导网络构建等研究。通过本项目研究，明确了NLR1-V的广谱抗白粉病功能，部分阐明了NLR1-V介导全生育期广谱抗白粉病机制，初步构建信号传导网路；解析了NLR1-V进化模式，明确基因序列、关键区段与功能之间的关系。本项目为广谱抗白粉病育种提供了具有较高利用价值的基因资源；将细胞遗传学、分子生物学、现代基因组学等相结合克隆外源抗病基因，为克隆小麦其他外源物种抗病基因提供可参考的技术路线。