基于蛋白质互作网络的水稻抗旱基因发掘及关键基因OsMYC7功能解析

Drought tolerance is a very complex trait, controlled by many genes, and its molecular mechanism is still unclear. Therefore, We propose to adopt the biological big data driven network biology research strategy to facilitate gene discovery and to advances our understanding of molecular mechanism underlying drought tolerance in rice. In this study, we will develop a new machine learning method for discovering rice protein-protein interactions (PPIs) based on the three-dimensional structures and functional evidences, and then construct a direct PPI network in rice. In order to verify the assumption that OsMYC7 as a new regulator involved in crosstalk between JA (Jasmonic acid) and ABA (Abscisic acid) signaling pathways by interacting with OsbZIP23 under drought stress, we will characterize OsMYC7 function through gain-of-function and loss-of-function approaches. The physiological, biochemical and morphological characteristics of transgenic rice will be analyzed to investigate the role of OsMYC7 under drought stress. Sensitivity of transgenic rice plants to exogenous hormones will be also analyzed to validate that OsMYC7 is involved in JA signaling pathway in response to drought stress. RNA-Seq analysis will be further used to survey transcriptome changes between 35S:OsMYC7/osbzip23 and 35S:OsMYC7/wt transgenic rice plants and examine the impact of OsbZIP23 on OsMYC7 to regulate gene expression. All these results will help us better understand the molecular crosstalk between JA and ABA signaling pathways under drought stress, which would facilitate the identification of important genes for genetic improvement to generate the drought tolerant rice.

水稻抗旱机制十分复杂，而缺乏对抗旱基因互作关系的全面认识，严重制约着抗旱品种的分子设计育种。故我们采用生物大数据研究策略促进水稻抗旱基因互作关系解析，加深对抗旱分子机制的认识。本研究拟采用机器学习方法对生物大数据进行深度挖掘，构建水稻抗旱基因互作网络，剖析抗旱通路交叉互作关系，探明调控抗旱通路的关键节点基因。根据前期基础提出“OsMYC7作为JA与ABA信号通路调控水稻抗旱性的一个重要分子连接点”的假设为研究突破口，通过过表达与抑制表达转基因水稻抗旱性鉴定，评价OsMYC7的抗旱功能，并分析转基因水稻对外源激素的敏感性，明确其作用通路。通过不同背景过表达转基因植株的耐旱性比较，阐明OsMYC7作为信号通路连接点的抗旱意义，并结合转录组变化，探明OsMYC7调控的抗旱网络，力争在水稻抗旱基因发掘及抗旱通路互作方面获得有创新意义的进展，为抗旱水稻品种的分子设计育种提供重要基因资源和理论支撑。

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。本研究通过同源建模构建了水稻蛋白质空间结构组，结构组共包含2,132个高分辨率的水稻蛋白质空间结构，占水稻整个蛋白组的5.5%，其中94.5%同源模型与结构模板之间的RMSD小于1Å。在高分辨率蛋白质结构组基础上发展了一种基于分子对接的水稻蛋白质互作预测方法。系统评估表明该方法可以根据蛋白质同源模型的对接结果有效区分其互作与非互作关系。利用该方法构建了水稻蛋白质空间结构互作组，互作组包含2,131个节点蛋白质，涉及24,653对互作关系。借助水稻蛋白质空间结构互作组，利用已知水稻抗旱基因进行关联推断，筛选获得486个抗旱候选基因。项目进一步系统分析了干旱胁迫、外源激素以及干旱胁迫叠加外源激素处理后的水稻转录组变化，767个干旱差异表达基因能响应一种或多种外源激素，其中24个干旱胁迫差异表达基因同时受4种激素调控，表明水稻基因在干旱胁迫与不同激素间存在广泛的重叠转录应答。水稻蛋白质互作网络和水稻转录组数据挖掘显示OsMYC7作为重要候选基因参与调控水稻对干旱胁迫的响应。酵母双杂和BiFC实验证明OsMYC7与ABA信号通路的核心基因OsbZIP23相互作用，其作为JA和ABA信号通路的分子连接点协同调控水稻响应干旱胁迫。