利用VIGS技术挖掘谷子十里香抗锈相关基因

Millet leaf rust(Uromyces setariae)is a important fungal pathogen of foxtail millet leaf disease.There were not harvest at all ,when the disease is epidemic due to millet lodging.Resistance cultivars are the most economical and environmentally-friendly way to reduce the damage caused by millet leaf rust,but the breakdown of resistance always occured.A promising measure that could simultaneously speed up millet resistance breeding and prolong working life of resistance variety is to identify the resistance mechanisms and rust resistance related gene. The rust resistance gene, derived from resistant cultivar "Shilixiang" was maped and located in a chromosome based on re-sequencing and RNA-seq﹠digital gene expression were carried out after inoculating with rust.On the one hand rust resistant candidate genes were found based on the bioinformatics analysis of candidate region and genes expression pattern were identified by real-time-PCR. On the other hand digital gene expression profiling analysis was used for estimating gene expression level after inoculating with rust and specific expressed﹠upregultate were screened for further analysis.The molecular basis of resistance mechanisms and regulate network were finaly unraveled by clustering analysis of differential gene expression pattern.Firstly the candidate gene function were identified by BMV induced gene silence and then the accurate gene functoin were studied through the further use of agrobacterium mediated transformation of millet transgenic technology.The study will lay the foundation for molecular breeding of rust resistant variety.

谷子锈病是谷子上重要病害，流行年份不少地块植株倒伏、颗粒无收。生产中利用抗锈品种是最经济有效的防治方法。但抗锈品种应用中易出现抗性丧失现象，为此，克隆抗锈基因、揭示抗锈机理对快速培育抗锈品种，延长品种使用寿命具有重要意义。本课题组以高抗锈病资源十里香为材料，进行了重测序，锈菌诱导的转录组和数字基因表达谱测序，且标记和定位了其抗锈基因，本研究一方面对标记内基因进行生物信息学分析，找到抗锈候选基因，并利用实时荧光PCR对候选基因进行表达分析；另一方面对抗源十里香接种锈菌后不同时间叶片的表达谱中抗锈特异表达、上调表达基因进行分析，获得抗锈相关基因的信息，通过差异基因表达模式聚类分析，从分子水平上揭示谷子抗锈的分子调控途径与机理。然后，利用谷子上病毒诱导的基因沉默平台对抗锈候选基因及其抗锈相关基因进行初筛，进一步利用农杆菌介导的谷子转基因技术最终确定候选基因功能，为开展谷子抗锈分子育种奠定基础。

谷子锈病为谷子全球种植区上重要气传流行性病害，短期流行后可造成10–30%产量损失。抗锈品种的选育与推广是控制该病害的最经济有效的方法，但是随着抗锈品种大面积种植，抗锈品种抗锈性容易丧失，为此，克隆抗锈基因、揭示抗锈机理对快速培育抗锈品种，延长品种使用寿命具有重要意义。本研究对谷子十里香接种谷锈菌不同时间点取样进行3个表达谱测序，对差异表达基因的KEGG pathway显著性富集分析，发现苯丙素类生物合成、苯丙烷生物合成、类黄酮生物合成等防卫反应相关次生物质合成途径可能调控十里香抗锈，利用定量PCR研究表达谱中差异表达的抗锈相关基因，发现这些基因在抗病反应中的表达量明显高于感病反应。构建了十里香基因组的BAC文库，通过抗锈基因共分离标记设计的引物（SLAF75294）对文库进行PCR筛选，鉴定到70-7-G阳性克隆末端测序结果匹配基因组较唯一，并且位于抗锈基因精细定位的区间内，测序结果证实了定位区间DNA序列在抗亲与感亲中存在很大差异，共预测到9个基因，生物信息学分析结果表明，g5与g6二个基因属于“NBS-LRR类抗病基因”，串联重复，推测为谷子抗锈候选基因。摸索优化了谷子基因沉默体系，利用该体系沉默了谷子SGT1、RAR、R11、R13和GL6基因,通过接种鉴定表明这些基因调控十里香抗锈性。对根癌农杆菌介导的谷子愈伤组织及成熟种子的遗传转化技术进行了优化，根据抗锈候选基因G5L、G5S和G6及抗病相关基因RAR、SGT、R11、R13和GL6核苷酸序列，构建了这些基因的转化载体，并利用农杆菌介导的基因转化技术，转化到谷子冀谷11的幼穗愈伤。目前，经过愈伤抗性筛选和成苗诱导，已经获得谷子转基因T0代植株，正在进行阳性鉴定、繁种及抗锈性鉴定。通过本课题的研究，从分子水平上揭示谷子抗锈的分子调控途径与机理，为开展谷子抗锈分子育种及抗锈基因可持续利用奠定基础。