枯萎病菌诱导棉花基因表达谱分析及抗病相关基因的挖掘

Xinjiang is the one and only region to produce sea-island cotton in China. In recent years, fusarium wilt of sea-island cotton dramatically affects the yield. Although breeding disease-resistant cultivars is the most economic and effective measure to control fusarium wilt, the lack of resistance source which has stable resistance and the violent segregation of offspring produced by distant hybridization between upland cotton and sea-island cotton result in the difficulty of making use of traditional breeding methods. The development of molecular biology provides new methods to develop new disease-resistant cultivars of sea-island cotton. Because molecular mechanism of the resistance of cotton to fusarium wilt is not fully understood at present and some key genes involved in the resistance of cotton to fusarium wilt are not cloned, there is no significant progress to make use of genetic engineering to enhance the resistance of cotton to fusarium wilt. In this study, making use of upland cotton cultivars with highly resistant to fusarium wilt and upland cotton cultivars with highly susceptible to fusarium wilt as experimental materials, gene expression profiles of different infection times of cotton roots induced by Fusarium oxysporium will be established with the aid of high throughput Solexa sequencing technology to analyse the variation of transcriptome during inducement of Fusarium oxysporium in cotton; the resistance genes of fusarium wilt will be separated and the function of these genes will be identified by rapid amplification of cDNA ends with the aid of bioinformatics. The research results will be helpful to ascertain molecular mechanism of cotton's resistance to fusarium wilt and to make use of genetic engineering to breed new cultivars of sea-island cotton resistant to fusarium wilt.

新疆是我国唯一的长绒棉产区，优越的自然生态条件和集约化生产为新疆长绒棉发展提供了得天独厚的发展优势。近年，长绒棉枯萎病的日益加剧严重影响了长绒棉发展，培育抗病品种是最为经济有效的措施。但长绒棉因缺少抗性强、稳定性好的枯萎病抗源, 加之陆海远缘杂交后代的疯狂分离，传统育种方法难度很大。分子生物学的发展为长绒棉抗病品种选育提供了新的途径。目前由于人们对棉花抗枯萎病的分子调控机制还不是十分清楚，仍有大量抗枯萎病的关键基因未被克隆，利用基因工程技术改良长绒棉的研究进展缓慢。本研究以高抗与高感枯萎病的陆地棉为材料，利用高通量solexa测序技术建立枯萎病菌诱导棉花根部不同侵染时间的基因表达谱，分析棉花在枯萎病菌诱导过程中转录组水平的变化；利用RACE技术结合生物信息学分离棉花抗枯萎病相关基因并分析其功能。该项目的研究将为明确棉花抗枯萎病的分子机制及利用基因工程手段培育抗枯萎病长绒棉新品种奠定基础。

棉花枯萎病是影响长绒棉生产的重要因素，培育抗病品种是最为经济有效的措施。本课题以陆地棉抗枯萎病品种“中棉所12号”和感病品种“新陆早7号”为材料，采用孢子悬浮液浸根法对两叶一心期的水培棉花幼苗进行枯萎病菌诱导处理，利用高通量solexa测序技术建立枯萎病菌诱导不同时期棉花基因的表达谱，分析病原菌侵染前后、侵染不同时间及不同抗、感品种间基因的差异表达。发现枯萎病菌侵染后不同时期，两个品种的显著差异表达基因在各个比对组中下调基因均明显多于上调基因，并且感病品种的显著差异表达基因明显多于抗病品种。基因功能分析表明, 显著差异表达基因可划分为生物学过程、细胞组分和分子功能注释本体三类；利用KEGG数据库对显著差异表达基因进行功能注释和分类,在两个品种中分别鉴定出126条和127条Pathway；各个比对组的Pathway涉及的基因可被划分为碳水化合物代谢，次生代谢产物的生物合成，多糖的生物合成和代谢，环境适应和免疫系统等13类；重点分析了植物激素信号转导代谢Pathway和植物与病原菌相互作用Pathway，获得大量可能与棉花抗枯萎病有关的基因信息。利用表达谱筛选获得的目的基因片段，通过电子克隆结合RT-PCR技术从抗枯萎病棉花品种“中棉所12”中克隆了6个转录因子基因（GhB301、GhERFB101、GhWRKY22、 GhWRKY44、 GhWRKY106-1、GhTGA2.2），对这些基因的表达特性进行分析，发现这6个基因对枯萎病菌和激素诱导均有响应。通过农杆菌介导的转基因技术对GhB301、 GhWRKY22和GhWRKY44 进行功能初步研究，结果表明在过表达目的基因的转基因植株中，其下游PRs基因的表达均有明显的提高，初步证明这些基因可能参与了棉花对枯萎病菌的抗性反应。本项目首次利用基因表达谱系统分析棉花与枯萎菌间的互作关系，研究结果对阐明棉花抗枯萎病的分子调控机制及利用基因工程手段培育抗枯萎病的棉花品种奠定了基础。