基于高通量测序平台解析小桐子抗旱性提高过程中的重要通路和关键基因

Research and development of the energy plant Jatropha curcas L. is a strategical demand for Yunnan province and our country. Drought is a key limiting factor for development and production of J. curcas. We found that there was obvious variation of drought resistance in J. curcas from different provenances, and drought-hardening could significantly enhance drought resistance of J. curcas seedlings. Based on our previous work and Illumina Hiseq 2000 high-throughput sequencing platform, using two populations of J. curcas with different drought resistance as experimental materials, the present proposal is going to study: 1. changes of transcriptomes and their analysis and comparison in J. curcas seedlings during drought hardening-enhanced drought resistance, and construction of drought-responsive EST libaries; 2. analysis and verification of important signal tranduction and metabolic pathways related to the enhancement of drought resistance of J. curcas, and construction of drought-responsive gene network; 3. based on the pathway analysis, screening and cloning key drought resistance-related genes, identifying their function, and carrying out genetic improvement of J. curcas using the above-mentioned key drought resistance-related genes to enhance their drought resistance. The research will elucidate the machanisms of drought hardening-enhanced drought resistance in J. curcas seedlings and molecular basis for the difference of drought resistance among various J. curcas populations to some extent, obtain some key drought resistance-related genes, and improve drought resistance of J. curcas by genetic engineering. These results will be of scientific importance and great potential for further application.

研究开发能源植物小桐子是云南省和国家的能源战略需求，干旱是影响小桐子产量的主要环境因素。我们前期研究表明不同生境的小桐子居群抗旱性存在明显差异，干旱锻炼可显著提高小桐子幼苗的抗旱性。基于前期工作基础和已有的Hiseq 2000高通量测序系统，以两个抗旱性有显著差异的小桐子居群为材料，本项目拟研究：①干旱锻炼提高小桐子抗旱性过程中转录组的变化与分析比较，构建干旱响应EST文库；②解析并验证与小桐子抗旱性提高相关的重要信号转导与代谢通路，构建干旱应答基因网络；③基于通路分析结果，筛选与小桐子抗旱性提高相关的关键基因进行克隆与功能解析，并对小桐子进行基因工程改良。本研究成果将初步揭示干旱锻炼提高小桐子抗旱性的分子机制及不同居群小桐子抗旱性差异的分子基础，获得一批与抗旱性密切相关的关键基因，并通过基因工程手段改良小桐子的抗旱性。研究成果具有重要的科学意义和潜力巨大的应用前景。

研究开发和利用能源植物是国家和云南省产业发展战略的需求，小桐子是具有巨大开发潜力的能源植物树种，干旱是限制小桐子产业发展的重要环境因素。因此，弄清小桐子抗旱性形成的代谢途径及其分子机制，克隆抗旱性密切相关的关键基因，并通过基因工程手段进行改良已成为急需解决的问题。. 本项目研究了干旱锻炼对小桐子幼苗的抗旱性的效应，分析比较了干旱锻炼提高小桐子抗旱性过程中转录组的变化，构建了干旱响应的EST文库，解析并验证与小桐子抗旱性提高相关的重要代谢通路，分离克隆了与小桐子抗旱性提高相关关键基因并进行了功能解析与验证。此外，还进行了小桐子干旱锻炼及干旱胁迫过程中microRNA组分析及部分重要miRNA的功能解析。. 研究结果表明，干旱锻炼能显著提高小桐子幼苗的抗旱性，渗透调节过程参与了其抗旱性的提高，部分是通过激活甜菜碱和脯氨酸的生物合成途径。通过RNA-seq测序，我们对小桐子干旱锻炼和随后的干旱胁迫过程中的转录组和microRNA组的全景变化进行了解析，鉴定干旱响应的差异表达基因与miRNAs，通过数字基因表达谱及qPCR分析揭示了小桐子抗旱相关基因与miRNAs的表达模式，构建了干旱锻炼提高小桐子抗旱性过程中一些重要的代谢通路，克隆了8个与抗旱性相关的基因并进行了功能验证，发现了一些新的与抗旱性相关的miRNAs。这些结果有助于揭示小桐子干旱锻炼诱导的抗旱性形成的分子机制，并为后续通过基因工程手段改良小桐子提供了理论和实验基础。. 这些研究结果目前发表论文17篇，其中SCI引用论文6篇。1名博士生和6名硕士生获得了相应的学位。