温度诱导的小麦抗条锈病基因表达特征研究

Wheat stripe rust, which treatens wheat production all over the world, is one of important epidemic diseases. Currently, main problem in wheat production is no durable resistance cultivar among resistant cultivars. High temperature resistance of plant is a kind of durable resistance of low infection type, which is controlled by major gene, easy to be screened, identified, and used.The cultivars with high temperature resistance are very rich in China. It is still unclear on the molecular mechanism of high temperature resistance of wheat cultivars to wheat stripe rust. In this research, the transcriptome of Xiaoyan 6 and Lantian 1 treated under high and normal temperature will be sequenced and analyzed using RNA-seq and bioinformatic technologies based on our previous results of temperature transformation method, expression characteristics, and biological and chemical mechanisms of high temperature resistance to stripe rust. The key genes related to high temperature resistance to stripe rust will be cloned. The functioins of these genes will be verified by virus-induced gene-silencing system. The defense responses, signaling, and gene regulation during high temperture resistance expressed to wheat stripe rust will be understood. The molecular mechanism of high temperature resistance to stripe rust will be revealed. This research will make a base for gene cloning and usage of high temperature durable resistance to wheat stripe rust.

小麦条锈病是小麦生产中最重要的流行性病害,严重威胁着我国乃至全世界的小麦安全生产。现阶段生产中存在的主要问题是抗条锈病品种缺乏持久抗病性。研究发现高温抗条锈性是一种低反应型的持久抗病性，由主效基因控制，且易于鉴选和利用。我国具有丰富的高温抗条锈性小麦品种资源，这些品种具有重要的利用价值，但关于高温抗条锈性的分子机制研究迄今仍为空白。本项目拟在前期建立的小麦温度转换培养法及高温抗条锈性的表达规律和生化机制的基础上，以小偃6号和兰天1号小麦品种与条锈菌CYR32为对象，采用RNA-seq技术，分析小麦在高温处理前后的转录组变化，结合生物信息学分析、qRT-PCR分析及病毒诱导的基因沉默技术进行基因功能验证，了解高温抗病性表达过程中的防卫反应激活、信号传递和基因调控规律，揭示小麦品种高温抗条锈性的分子机制，以期为最终实现高温持久抗条锈性基因的克隆和利用奠定基础。

小麦品种的高温抗条锈性是只在相对较高环境温度下表达的一种低反应型抗病性，是一种非小种专化的持久抗病性，由主效基因控制，易于鉴选和利用。小偃6号自1975年推广以来，至今仍保持较好的抗条锈病性，是典型的持久抗病品种。本研究以小偃6号-条锈菌CYR32为模式体系，采用RNA-seq转录组测序技术、实时荧光定量PCR、RACE、RT-PCR及病毒诱导的基因沉默等技术，研究了小偃6号小麦品种与条锈菌在温度调控下从亲和性转变为非亲和性过程中转录组水平变化规律，共获得30个样本的转录本，且Q30水平达到91.23%以上。组装共获得445226个序列，且N50长度达到1849 bp。共有1395个差异基因参与到24 h高温诱导的苗期抗病性中。经过染色体定位、KEGG富集、R基因、转录因子、蛋白互作及实时定量PCR分析，揭示了小偃6号的HTSP是由双因素（20℃温度和条锈菌）诱导产生。同时，基于转录组测序分析结果，筛选并克隆出了参与小麦高温抗条锈病过程的关键基因WRKY49、WRKY62和WRKY70，通过病毒诱导的基因沉默技术验证的这些基因的生物学功能，通过组织病理学和实时荧光定量PCR技术研究了这些基因参与小麦高温抗条锈病过程中的分子调控机理。该项研究为进一步利用这种高温抗病性奠定了理论基础。