Functional Analysis Of OGG1 In Rice

OGG1 is an abbreviation for 8-oxoguanine DNA glycosidase, which can repair the oxidative DNA damage and maintain the genomic stablility. Previous studies have indicated that mutations of human OGG1 (hOGG1) can induce a variety of tumors, and Arabidopsis OGG1 (AtOGG1) can play a role in seed maturation and germination, and may be related to seed longevity. However, it’s not clear whether rice (Oryza ostiva) OGG1 (OsOGG1) acts in the repair of oxidative DNA damage, stress tolerance or seed longevity. To dissect the function of OsOGG1, we made the stable transformation constructs for OsOGG1 overexpression, RNA silencing, protein subcellular localization, prokaryotic expression, promoter fragment activity analysis, and obtained the corresponding transgenic lines. Subsequently, we performed promoter activity analysis, transient expression test for subcellular localization in tobacco (Nicotiana benthamiana) cells, abiotic stress treatment, and detected the activity of OsOGG1 and purified the GST-OsOGGl protein from Escherichia coli. The results are summarized as follows:(1) The analysis of GUS staining experiment indicated that the 1331 bp fragment before ATG of OsOGG1 can initiate the GUS gene expression, suggesting it works as a functional promoter.(2) The confocal fluorescent obervation showed that the OsOGG1-GFP fusion protein was localized in the nucleus of tobacco cells.(3) 38 overexpression T1 lines and 54 RNAi T1 lines were respectively generated by Agrobacterium-mediated transformation, and six overexpression T2 lines and nine RNAi T2 lines were identified through genotyping and gene expression test. We will screen single-copy insertions from OsOGG1 overexpression and RNAi T2 lines for subsequent experimental analysis.(4) Abiotic stress assay of 3-4 leaves stage rice seedlings respective with 30 μM Dimethyl amethyst (MV),38℃,4℃,250 mM NaCl or 20% PEG6000 showed that the OsOGG1 gene expression pattern is similar to the enzyme activity trend of superoxide dismutase (SOD) after induced by MV, OsOGG1 was suppressed at 3 h respective with salt stress (250 mM NaCl), cold stress (4℃) or heat stress (38℃), and osmotic stress (20% PEG6000) treatment could enhance OsOGG1 expression. These results suggested that OsOGG1 could be related to abiotic stresses, and its mechanism needs further study.(5) Expression analysis of OsOGG1 during seed germination showed that the expression level of OsOGG1 was highest at 24 h after imbibition among transgenic lines of OsOGG1 overexpression and RNAi lines together with the control Nipponbare, implying that OsOGG1 may be associated with seed germination in rice.(6) We obtained the fusion protein GST-OsOGG1 by procaryotic expression system and purified it. Meanwhile, we generated the polyclonal antibody against OsOGGl. These works will provide a basis for studying OsOGG1-mediated oxidative DNA damage.