Molecular identification and interaction assay of the gene(OsUbc13) encoding a ubiquitin-conjugating enzyme in rice

水稻泛素缀合酶基因OsUbc13的分子特征和蛋白互作研究(英文)

The ubiquitin （Ub）-conjugating enzyme, Ubc13, has been known to be involved in error-free DNA damage tolerance （or post-replication repair） via catalyzing Lys63-linked polyubiquitin chains formation together with a Ubc variant. However, its functions remain largely unknown in plant species, especial y in monocotyledons. In this study, we cloned a Ub-conjugating enzyme, OsUbc13, that shares the conserved domain of Ubc with AtUBC13B in Oryza sativa L., which encodes a protein of 153 amino acids; the deduced sequence shares high similarities with other homologs. Real-time quantitative polymerase chain reaction （PCR） indicated that OsUbc13 transcripts could be de-tected in al tissues examined, and the expression level was higher in palea, pistil, stamen, and leaf, and lower in root, stem, and lemma;the expression of OsUbc13 was induced by low temperature, methylmethane sulfate （MMS）, and H2O2, but repressed by mannitol, abscisic acid （ABA）, and NaCl. OsUbc13 was probably localized in the plasma and nuclear membranes. About 20 proteins, which are responsible for the positive yeast two-hybrid interaction of OsUbc13, were identified. These include the confirmed OsVDAC （correlated with apoptosis）, OsMADS1 （important for development of floral organs）, OsB22EL8 （related to reactive oxygen species （ROS） scavenging and DNA protection）, and OsCROC-1 （required for formation of Lys63 polyubiquitylation and error-free DNA damage tolerance）. The molecular characterization provides a foundation for the functional study of OsUbc13.

研究目的:通过研究水稻泛素缀合酶基因OsUbc13的序列特征、表达模式、亚细胞定位模式及其互作分子,为深入研究该基因的生物学功能和分子作用机理奠定基础。创新要点:首次对植物Ubc13进行了亚细胞定位研究及蛋白互作研究。研究方法:通过序列比对及聚类分析进行OsUbc13的序列特征研究;通过实时荧光定量聚合酶链式反应(PCR)进行OsUbc13的表达模式分析;通过聚乙二醇(PEG)介导转化烟草BY-2原生质体进行OsUbc13亚细胞定位研究(见图4);通过酵母双杂交进行OsUbc13的蛋白质互作分析(见图5和表1)。重要结论:OsUbc13编码具有153个氨基酸的蛋白质,其推断的氨基酸序列与其它同源序列具有很高的相似性;该基因在水稻各组织中均有表达,其中内稃、雌蕊、雄蕊和叶片中的表达量较高,而根、茎和外稃中的表达量较低;低温、甲基磺酸甲酯(MMS)和过氧化氢(H2O2)胁迫处理使胚性愈伤中OsUbc13的表达量显著上调,甘露醇、脱落酸(ABA)和氯化钠(NaCl)胁迫则使愈伤组织中该基因的表达量降低;OsUbc13与绿色荧光蛋白(GFP)的融合蛋白表达于质膜和核膜处;酵母双杂交结果表明约有20个蛋白可能与OsUbc13存在相互作用,其中OsVDAC(与细胞凋亡有关)、OsMADS1(与花器官发育有关)、OsB22EL8(与活性氧清除及DNA保护有关)和OsCROC-1(为Lys63聚合泛素链形成及运行无误性DNA损伤耐受机制所必需)四个蛋白经验证确与OsUbc13互作。