水稻抗旱基因网络重建及关键节点基因OsHSF01的功能鉴定

Drought tolerance is a very complex trait, controlled by many genes, and its molecular mechanism is still unclear. Therefore, we propose to adopt the network biology research strategy to facilitate gene discovery and to better understand the molecular mechanism underlying drought tolerance in rice. In this study, we will combine bioinformatic data and resources to reconstruct a drought-tolerant gene network and identify gene modules and key node genes controlling the trait of drought tolerance in rice. In order to verify the assumption that the key node gene OsHSF01 as a new regulator involved in ABA dependent pathway to response to drought stress by regulating root development and control the opening and closing of the stomata, we will characterize OsHSF01 function through gain-of-function and loss-of-function approaches. The physiological, biochemical and morphological characteristics of transgenic rice will be further analyzed to investigate the pathway of OsHSF01 in the resistance of rice plant to drought stress. RNA-Seq analysis will be used to survey transcriptome changes of transgenic rice and examine the impact of OsHSF01 silencing or overexpression on gene expression in the network. Furthermore, we also use the ChIP-Seq experiments to identify the OsHSF01 binding sites (TFBSs) and its regulated genes in rice genome. These results will help us better understand the OsHSF01 regulatory network and its biological roles in drought tolerance in rice, which will provide new insight into plant tolerance to drought stress.

水稻抗旱机制十分复杂，而缺乏对抗旱基因相互协作关系的全面认识，严重制约了水稻抗旱品种的分子设计育种。故我们采用网络生物学研究策略来促进抗旱新基因发现、加深对抗旱分子机制的认识。本研究将综合多种生物信息数据与资源，构建水稻抗旱基因网络模型，结合水稻抗旱性状GWAS数据，探明控制水稻抗旱性的基因模块及关键基因。结合前期网络解析提出"水稻中存在一个依赖ABA信号调控、以OsHSF01为核心连接根部发育和气孔运动的抗旱基因网络"的假设，进一步通过过表达与抑制表达等实验，评价OsHSF01基因在水稻抗旱中的功能；并对转基因水稻根系发育特征与叶片生理性状进行研究，明确其作用途径。通过ChIP-seq分析，结合转基因水稻转录组变化，探明受OsHSF01直接调控的下游基因及参与的网络通路，力争在OsHSF01抗旱基因网络及调控机制方面获得有创新意义的进展或突破，为较全面的认识水稻抗旱分子机制提供新的视角。

蛋白质-蛋白质相互作用研究能够从分子水平上揭示蛋白质功能，帮助揭示水稻响应干旱胁迫的分子机制。本研究提出了一种基于机器学习的水稻蛋白质与蛋白质互作预测新方法，该方法结合了蛋白质空间结构与功能信息，有效提高了蛋白质相互作用预测的准确性，其阳性率达到70.62%，同时假阳性率仍维持在0.07%的极低水平，各项可比指标均优于现有预测方法。利用该方法在全基因组尺度重建了水稻蛋白质互作网络，该互作网络包含16,895节点蛋白质，涉及708,819对相互作用关系，由1,356个相对独立的功能模块组成。基于水稻互作网络，发展了抗旱基因发掘新策略，系统评估表明基于蛋白质互作网络进行关联推断可有效发掘水稻抗旱基因。在此基础上，筛选获得一批重要抗旱候选基因。通过转录组测序分析表明OsHSF1的表达水平明显受干旱及ABA诱导，超过43%的OsHSF1互作基因的表达水平受干旱调控。水稻种子萌发实验表明抑制OsHSF1表达能提高水稻对外源ABA敏感性。生化指标分析测定发现在干旱胁迫条件下过表达OsHSF1能提高转基因水稻细胞体内超氧化物歧化酶活性，从而降低对植株细胞的伤害。抗旱性实验表明抑制OsHSF1表达能增强水稻抗旱性，耐热性实验显示过表达OsHSF1提高转基因植株的耐热性，同时增加转基因水稻的株高。此外，研究还发现在干旱胁迫条件下过表达OsHSF1促进植株叶片热耗散，增强光系统II的效率。