水稻OGG1介导的抗逆机制研究

Active oxygen free radicals may cause DNA to form 8-oxoguanine，resulting mutations on DNA. 8-oxoguanine DNA glycosylase (OGG1) is responsible for special recognition and excision of 8-oxoguanine (8-oxoG) in organisms. The homologs of OGG1 from Arabidopsis and Medicago truncatula Gaertn (barrel medic) are induced by abiotic stress factors, and their overexpression enhances the life of seed vigor in Arabidopsis and Medicago respectively. At present, research on rice OGG1 (Oryza sativa OGG1, OsOGG1) has not been reported. Based on rice genomics information, we have cloned OsOGG1 and its promoter sequence, and obtained transgenic lines for OsOGG1 overexpression, RNA silencing, subcellular localization, and its promoter driving GFP. In this project, we will analyze these transgenic lines, dissect the relationship between OsOGG1 and biotic and abiotic stress factors, identify the subcellular localization of OsOGG1, and verify whether OsOGG1 can extend the life of rice seeds. In addition, we will isolate and purify OsOGG1, and test its ability to repair 8-oxoG in vitro. This project will provide evidence and guidance for OsOGG1-mediated abiotic and biotic stress tolerance in rice breeding and germplasm preservation.

活性氧自由基可导致DNA形成8-羟基鸟膘呤(8-oxoG)而引起突变，机体内8-羟基鸟膘呤DNA糖苷酶（OGG1）特异识别并切除修复8-oxoG。已知拟南芥和蒺藜苜蓿OGG1受非生物胁迫因子诱导，其超表达可增强种子寿命活力。目前，尚无水稻OGG1（OsOGG1）的研究报道。 本项目组利用水稻基因组学信息，已克隆了水稻OGG1及其启动子序利，获得了OsOGG1超表达、RNA沉默、亚细胞定位及其启动子操控GFP表达的转基因株系。我们将分析上述转基因株系，研究OsOGG1与生物和非生物胁迫因子的关联性，对OsOGG1进行亚细胞定位，验证OsOGG1能否延长水稻种子寿命，并分离、纯化OsOGG1，检验OsOGG1的体外修复能力，为OsOGG1介导的水稻抗逆育种及优良种质保存提供依据和指导。

8-羟基鸟嘌呤DNA糖苷酶1（8-oxoguanine DNA glycosylase 1），简称OGG1，在生物体内保守存在，其主要作用是修复由活性氧引起的DNA 8-氧鸟嘌呤（8-oxoG）突变。有研究表明，人OGG1（hOGG1）突变易诱发多种肿瘤；拟南芥OGG1（AtOGG1）与种子的成熟和萌发机制有关，对种子寿命有重要影响。本项目首次在水稻中克隆了OsOGG1基因，并通过超表达、RNAi及CRISPR/Cas9基因编辑技术创制了一系列OsOGG1转基因材料，对转基因材料进行抗病性鉴定及种子老化处理实验，结果表明，OsOGG1能够促进水稻种子抗老化，提高种子萌发率，并且负调控水稻的抗病性。项目研究结果还表明，氧化（MV）、高温、低温、高盐和高渗胁迫处理能够诱导或抑制水稻OsOGG1的转录表达；氧化胁迫（MV）处理时，OsOGG1的转录表达水平随着MV浓度的改变与超氧化物歧化酶（SOD）活性变化趋势有较好的相关性，这说明OsOGG1表达量可能与SOD酶活性相关联，OsOGG1可能通过修复胁迫引起的DNA氧化损伤从而影响SOD酶的活性来参与水稻抗逆性的调控。水稻种子萌发试验表明，无论是日本晴、OsOGG1超表达还是RNAi种子，OsOGG1基因表达量都在种子吸涨24 h后达到最高值，这说明OsOGG1在种子萌发过程中的重要作用。本项目首次在水稻中研究了OsOGG1的功能，并对其抗逆调控机理进行了初步探索，了解了其在水稻种子萌发过程中的动态转录水平，这为水稻抗逆机制研究及高萌发率、抗老化种子种质创制提供了新思路。