旱稻抗旱基因DBL9的功能鉴定及分子机制研究

Drought is a major abiotic stress factor that limits crops growth and production. It has great significance for China and the world food security to clone and apply the key drought-resistance genes in rice. Based on the previous work of rice expression profiling between upland and lowland rice cultivars under water stress using a cDNA microarray, we isolated a candidate drought-resistance gene DBL9 from upland rice IRAT109. Previous works demonstrated that DBL9 respond to various abiotic stresses and phytohormone treatments. Transgenic rice with DBL9 overexpression exhibited significantly improved growth performance under drought stress at the seedling stage. The goal of this project is to determine the drought resistant value of DBL9 at reproductive stage, and to investigate the underlying molecular mechanism of DBL9 regulating rice drought resistance. Mainly includes the evaluation of drought-stress tolerance at reproductive stage, analyses the upstream and downstream genes and detection the protein interaction network of DBL9. Altogether, this project aims to functional characterization of the drought resistant gene DBL9 and elucidating its underlying molecular mechanism of drought resistance. Eventually these works provide genetic resources for drought resistance breeding.

干旱是影响农作物生产的最严重的非生物胁迫因素之一。克隆并应用抗旱基因，提高水稻的抗旱能力，对于我国乃至全世界粮食安全都具有重要意义。本研究基于干旱胁迫处理下的水旱稻芯片表达谱，从旱稻IRAT109中克隆到受水分胁迫诱导表达，并在典型水旱稻间存在序列变异的抗旱候选基因DBL9。前期的工作证明了DBL9基因参与多种非生物逆境和激素的应答过程，并且超量表达DBL9能够提高水稻植株在苗期对干旱胁迫的抗性。本项目的目标是鉴定DBL9转基因植株在成株期的抗旱性，以及对DBL9调控植株抗旱性的分子机制进行深入研究。主要包括：对转基因植株进行成株期的系统抗旱鉴定、分析DBL9提高植株抗旱性的生理生化机制、解析DBL9基因的上下游调控途径及其互作蛋白网络等。通过以上系列的抗旱性鉴定和抗旱机理的研究，明确DBL9基因的抗旱功能，阐明其在旱稻中抗旱的生理机制和分子调控机制，最终为抗旱育种提供基因资源。

干旱是影响农作物生产的最严重的非生物胁迫因素之一。克隆并应用抗旱基因，提高水稻的抗旱能力，对于我国乃至全世界粮食安全都具有重要意义。调节细胞壁组分是植物适应非生物胁迫的主要机制之一。本研究基于水分胁迫处理下的芯片表达谱中筛选出受到水分胁迫诱导表达抗旱候选基因DBL9/OSCOBL4。DBL9/OsCOBL4编码COBRA-like蛋白。DBL9/OsCOBL4基因在典型水稻旱稻材料中存在多处变异，启动子中的3个SNP导致旱稻中DBL9/OsCOBL4的表达水平高于水稻。DBL9/OsCOBL4基因在IRAT109和日本晴中都受到PEG、高盐、低温、H2O2、脱水和ABA胁迫诱导表达，并且在IRAT109中的受诱导表达程度显著高于日本晴。Real-time PCR和GUS组织化学染色结果表明DBL9/OsCOBL4基因在幼苗期的根、叶鞘、叶片和生殖生长期的根、茎、叶鞘、叶和穗中都有表达，并且在IRAT109中的表达量显著高于在日本晴中的表达量；亚细胞定位结果表明DBL9/OsCOBL4在细胞壁中表达。过量表达DBL9/OsCOBL4能够提高转基因植株对高盐、高渗透、PEG模拟干旱胁迫、苗期土壤干旱胁迫和低温胁迫的抗性。在脱水、高盐和低温胁迫处理下，超表达DBL9/OsCOBL4的转基因植株积累的ROS含量显著地低于野生型对照。超表达DBL9/OsCOBL4基因引起植株苗期根部细胞壁的单糖组分发生变化，因此推测，DBL9/OsCOBL4可能通过影响细胞壁单糖组分含量，参与到细胞壁的建设过程中，从而提高转基因植株的抗旱性。DBL9/OsCOBL4的自然变异有助于增强水稻和旱稻的耐旱性，DBL9/OsCOBL4的优异等位基因为耐旱水稻品种选育提供了重要的基因资源。