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Abstract: 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, especially 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 detected in all 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.

水稻泛素缀合酶基因OsUbc13的分子特征和蛋白互作研究

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。

关键词：水稻；泛素缀合酶；实时定量聚合酶链式反应；酵母双杂交

[1] Agrawal, G.K., Abe, K., Yamazaki, M., 2005. Conservation of the E-function for floral organ identity in rice revealed by the analysis of tissue culture-induced loss-of-function mutants of the OsMADS1 gene. Plant Mol Biol, 59(1):125-135. 

[2] Akashi, K., Nishimura, N., Ishida, Y., 2004. Potent hydroxyl radical-scavenging activity of drought-induced type-2 metallothionein in wild watermelon. Biochem Biophys Res Commun, 323(1):72-78. 

[3] Al Bitar, F., Roosens, N., Smeyers, M., 2003. Sequence analysis, transcriptional and posttranscriptional regulation of the rice vdac family. Biochem Biophys Acta, 1625(1):43-51. 

[4] Andersen, P.L., Zhou, H., Pastushok, L., 2005. Distinct regulation of Ubc13 functions by the two ubiquitin-conjugating enzyme variants Mms2 and UEV1A. J Cell Biol, 170(5):745-755. 

[5] Apel, K., Hirt, H., 2004. Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu Rev Plant Biol, 55(1):373-399. 

[6] Ashley, C., Pastushok, L., McKenna, S., 2002. Roles of mouse UBC13 in DNA postreplication repair and Lys63-linked ubiquitination. Gene, 285(1-2):183-191. 

[7] Broomfield, S., Chow, B.L., Xiao, W., 1998.  MMS2, encoding a ubiquitin-conjugating-enzyme-like protein, is a member of the yeast error-free postreplication repair pathway. PNAS, 95(10):5678-5683. 

[8] Broomfield, S., Hryciw, T., Xiao, W., 2001. DNA postreplication repair and mutagenesis in Saccharomyces cerevisiae . Mutat Res, 486(3):167-184. 

[9] Brusky, J., Zhu, Y., Xiao, W., 2000.  UBC13, a DNA-damage-inducible gene, is a member of the error-free postreplication repair pathway in Saccharomyces cerevisiae . Curr Genet, 37(3):168-174. 

[10] Chau, V., Tobias, J.W., Bachmair, A., 1989. A multiubiquitin chain is confined to specific lysine in a targeted short-lived protein. Science, 243(4898):1576-1583. 

[11] Chen, Z.J., 2005. Ubiquitin signalling in the NF-κB pathway. Nat Cell Biol, 7(8):758-765. 

[12] Chen, Z.J., 2012. Ubiquitination in signaling to and activation of IKK. Immunol Rev, 246(1):95-106. 

[13] Chen, Z.J., Parent, L., Maniatis, T., 1996. Site-specific phosphorylation of IκBα by a novel ubiquitination-dependent protein kinase activity. Cell, 84(6):853-862. 

[14] Chien, C.T., Bartel, P.L., Sternglanz, R., 1991. The two-hybrid system: a method to identify and clone genes for proteins that interact with a protein of interest. PNAS, 88(21):9578-9582. 

[15] Dai, L., Xu, L., Huang, D., 2002. ASK1 physically interacts with COI1 and is required for male fertility in Arabidopsis . Sci China C Life Sci, 45(6):631-636. 

[16] Ehsani, A., Alluin, J.V., Rossi, J.J., 2013. Cell cycle abnormalities associated with differential perturbations of the human U5 snRNP associated U5-200kD RNA helicase. PLoS ONE, 8(4):e62125

[17] Fields, S., Song, O., 1989. A novel genetic system to detect protein-protein interactions. Nature, 340(6230):245-246. 

[18] Finley, D., Ozkaynak, E., Varshavsky, A., 1987. The yeast poly-ubiquitin gene is essential for resistance to high temperatures, starvation and other stresses. Cell, 48(6):1035-1046. 

[19] Finley, D., Bartel, B., Varshavsky, A., 1989. The tails of ubiquitin precursors are ribosomal proteins whose fusion to ubiquitin facilitates ribosome biogenesis. Nature, 338(6214):394-401. 

[20] Galan, J.M., Haguenauer-Tsapis, R., 1997. Ubiquitin lys63 is involved in ubiquitination of a yeast plasma membrane protein. EMBO J, 16(19):5847-5854. 

[21] Geelen, D.N., Inz, D.G., 2001. A bright future for the bright yellow-2 cell culture. Plant Physiol, 127(4):1375-1379. 

[22] Godbole, A., Varghese, J., Sarin, A., 2003. VDAC is a conserved element of death pathways in plant and animal systems. Biochim Biophys Acta, 1642(1-2):87-96. 

[23] Godbole, A., Mitra, R., Dubey, A.K., 2011. Bacterial expression, purification and characterization of a rice voltage-dependent, anion-selective channel isoform, OsVDAC4. J Membr Biol, 244(2):67-80. 

[24] Grisvard, J., Aubusson-Fleury, A., Baroin-Tourancheau, A., 2010. Multiple uses of Lys63-polyubiquitination in the ciliate Sterkiella histriomuscorum . Protist, 161(3):479-488. 

[25] Guan, X., Diez, T., Prasad, T.K., 1999. Geranoyl-CoA carboxylase: a novel biotin-containing enzyme in plants. Arch Biochem Biophys, 362(1):12-21. 

[26] Hashimoto, M., Komatsu, K., Maejima, K., 2012. Identification of three MAPKKKs forming a linear signaling pathway leading to programmed cell death in Nicotiana benthamiana . BMC Plant Biol, 12(1):103

[27] Hiraishi, H., Mochizuki, M., Takagi, H., 2006. Enhancement of stress tolerance in Saccharomyces cerevisiae by overexpression of ubiquitin ligase Rsp5 and ubiquitin-conjugating enzymes. Biosci Biotechnol Biochem, 70(11):2762-2765. 

[28] Hochstrasser, M., 1996. Ubiquitin-dependent protein degradation. Annu Rev Genet, 30(1):405-439. 

[29] Hoege, C., Pfander, B., Moldovan, G.L., 2002. RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO. Nature, 419(6903):135-141. 

[30] Hofmann, R.M., Pickart, C.M., 1999. Noncanonical MMS2-encoded ubiquitin-conjugating enzyme functions in assembly of novel polyubiquitin chains for DNA repair. Cell, 96(5):645-653. 

[31] Jentsch, S., 1992. The ubiquitin-conjugation system. Annu Rev Genet, 26(1):179-207. 

[32] Jentsch, S., McGrath, J.P., Varshavsky, A., 1987. The yeast DNA repair gene RAD6 encodes a ubiquitin-conjugating enzyme. Nature, 329(6135):131-134. 

[33] Kim, J.A., Cho, K., Singh, R., 2009. Rice OsACDR1 (Oryza sativa accelerated cell death and resistance 1) is a potential positive regulator of fungal disease resistance. Mol Cells, 28(5):431-439. 

[34] Lauber, J., Fabrizio, P., Teigelkamp, S., 1996. The HeLa 200 kDa U5 snRNP-specific protein and its homologue in Saccharomyces cerevisiae are members of the DEXH-box protein family of putative RNA helicases. EMBO J, 15(15):4001-4015. 

[35] Lee, B.H., Kapoor, A., Zhu, J., 2006. STABILIZED1, a stress-upregulated nuclear protein, is required for pre-mRNA splicing, mRNA turnover, and stress tolerance in Arabidopsis . Plant Cell, 18(7):1736-1749. 

[36] Lee, L.Y., Fang, M.J., Kuang, L.Y., 2008. Vectors for multi-color bimolecular fluorescence complementation to investigate protein-protein interactions in living plant cells. Plant Methods, 4(1):24

[37] Li, J., Wen, R., Andersen, P., 2010. Zebrafish Ubc13 is required for Lys63-linked polyubiquitination and DNA damage tolerance. Mol Cell Biochem, 343(1-2):173-182. 

[38] McKenna, S., Spyracopoulos, L., Moraes, T., 2001. Noncovalent interaction between ubiquitin and the human DNA repair protein Mms2 is required for Ubc13-mediated polyubiquitination. J Biol Chem, 276(43):40120-40126. 

[39] McKenna, S., Hu, J., Moraes, T., 2003. Energetics and specificity of interactions within Ub.Uev.Ubc13 human ubiquitin conjugation complexes. Biochemistry, 42(26):7922-7930. 

[40] Muralidhar, M.G., Thomas, J.B., 1993. The Drosophila bendless gene encodes a neural protein related to ubiquitin-conjugating enzymes. Neuron, 11(2):253-266. 

[41] Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco cultures. Physiol Plant, 15(3):473-493. 

[42] Obata, K., Fukuda, T., Morishita, R., 2001. Human biotin-containing subunit of 3-methylcrotonyl-CoA carboxylase gene (MCCA): cDNA sequence, genomic organization, localization to chromosomal band 3q27, and expression. Genomics, 72(2):145-152. 

[43] Oh, C.E., McMahon, R., Benzer, S., 1994.  bendless, a Drosophila gene affecting neuronal connectivity, encodes a ubiquitin-conjugating enzyme homolog. J Neurosci, 14(5):3166-3179. 

[44] Parusel, C.T., Kritikou, E.A., Hengartner, M.O., 2006. URI-1 is required for DNA stability in C. elegans . Development, 133(4):621-629. 

[45] Pastori, G.M., Foyer, C.H., 2002. Common components, networks, and pathways of cross-tolerance to stress. The central role of “redox” and abscisic acid-mediated controls. Plant Physiol, 129(2):460-468. 

[46] Pastushok, L., Xiao, W., 2004. DNA postreplication repair modulated by ubiquitination and sumoylation. Adv Protein Chem, 69:279-306. 

[47] Petersen, A., Suck, R., Lindner, B., 2006. Phl p 3: structural and immunological characterization of a major allergen of timothy grass pollen. Clin Exp Allergy, 36(6):840-849. 

[48] Pickart, C.M., 2001. Ubiquitin enters the new millennium. Mol Cell, 8(3):499-504. 

[49] Prasad, K., Parameswaran, S., Vijayraghavan, U., 2005. OsMADS1, a rice MADS-box factor, controls differentiation of specific cell types in the lemma and palea and is an early-acting regulator of inner floral organs. Plant J, 43(6):915-928. 

[50] Rothofsky, M.L., Lin, S.L., 1997. CROC-1 encodes a protein which mediates transcriptional activation from the human FOS promoter. Gene, 195(2):141-149. 

[51] Sato, S., Sanjo, H., Takeda, K., 2005. Essential function for the kinase TAK1 in innate and adaptive immune responses. Nat Immunol, 6(11):1087-1095. 

[52] Schweimer, K., Petersen, A., Suck, R., 2008. Solution structure of Phl p 3, a major allergen from timothy grass pollen. Biol Chem, 389(7):919-923. 

[53] Sheen, J., 2001. Signal transduction in maize and Arabidopsis mesophyll protoplasts. Plant Physiol, 127(4):1466-1475. 

[54] Silva, A.T., Nguyen, A., Ye, C., 2010. Conjugated polymer nanoparticles for effective siRNA delivery to tobacco BY-2 protoplasts. BMC Plant Biol, 10(1):291

[55] Song, J., Wurtele, E.S., Nikolau, B.J., 1994. Molecular cloning and characterization of the cDNA coding for the biotin-containing subunit of 3-methylcrotonoyl-CoA carboxylase: identification of the biotin carboxylase and biotin-carrier domains. PNAS, 91(13):5779-5783. 

[56] Steller, H., 1995. Mechanisms and genes of cellular suicide. Science, 267(5203):1445-1449. 

[57] Susin, S.A., Zamzami, N., Castedo, M., 1997. The central executioner of apoptosis: multiple connections between protease activation and mitochondria in Fas/APO-1/CD95- and ceramide-induced apoptosis. J Exp Med, 186(1):25-37. 

[58] Takacs, E.M., Suzuki, M., Scanlon, M.J., 2012. Discolored1 (DSC1) is an ADP-ribosylation factor-GTPase activating protein required to maintain differentiation of maize kernel structures. Front Plant Sci, 3:115

[59] Tamura, K., Dudley, J., Nei, M., 2007. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol, 24(8):1596-1599. 

[60] Tokunaga, F., Sakata, S., Saeki, Y., 2009. Involvement of linear polyubiquitylation of NEMO in NF-κB activation. Nat Cell Biol, 11(2):123-132. 

[61] Tripathi, P., Rabara, R.C., Lin, J., 2013. GmWRKY53, a water- and salt-inducible soybean gene for rapid dissection of regulatory elements in BY-2 cell culture. Plant Signal Behav, 8(5):e24097

[62] Ulrich, H.D., Jentsch, S., 2000. Two RING finger proteins mediate cooperation between ubiquitin-conjugating enzymes in DNA repair. EMBO J, 19(13):3388-3397. 

[63] Wang, W.M., Ma, X.F., Zhang, Y., 2012. PAPP2C interacts with the atypical disease resistance protein RPW8.2 and negatively regulates salicylic acid-dependent defense responses in Arabidopsis . Mol Plant, 5(5):1125-1137. 

[64] Wang, Y., Zhang, X.B., Lu, S.J., 2012. Inhibition of a basal transcription factor 3-like gene Osj10gBTF3 in rice results in significant plant miniaturization and typical pollen abortion. Plant Cell Physiol, 53(12):2073-2089. 

[65] Weaver, L.M., Lebrun, L., Franklin, A., 1995. Molecular cloning of the biotinylated subunit of 3-methylcrotonyl-coenzyme A carboxylase of Arabidopsis thaliana . Plant Physiol, 107(3):1013-1014. 

[66] Wei, W., Ayad, N.G., Wan, Y., 2004. Degradation of the SCF component Skp2 in cell-cycle phase G1 by the anaphase-promoting complex. Nature, 428(6979):194-198. 

[67] Weil, R., Israel, A., 2004. T-cell-receptor and B-cell-receptor-mediated activation of NF-κB in lymphocytes. Curr Opin Immunol, 16(3):374-381. 

[68] Wen, R., Newton, L., Li, G., 2006.  Arabidopsis thaliana UBC13: implication of error-free DNA damage tolerance and Lys-63-linked polyubiquitylation in plants. Plant Mol Biol, 61(1-2):241-253. 

[69] Wen, R., Torres-Acosta, J.A., Pastushok, L., 2008.  Arabidopsis UEV1D promotes Lysine-63-linked polyubiquitination and is involved in DNA damage response. Plant Cell, 20(1):213-227. 

[70] Wong, H.L., Sakamoto, T., Kawasaki, T., 2004. Down-regulation of metallothionein, a reactive oxygen scavenger, by the small GTPase OsRac1 in rice. Plant Physiol, 135(3):1447-1456. 

[71] Wu, X., Yamamoto, M., Akira, S., 2009. Regulation of hematopoiesis by the K63-specific ubiquitin-conjugating enzyme Ubc13. PNAS, 106(49):20836-20841. 

[72] Xiao, W., Lin, S.L., Broomfield, S., 1998. The products of the yeast MMS2 and two human homologs (hMMS2 and CROC-1) define a structurally and functionally conserved Ubc-like protein family. Nucl Acids Res, 26(17):3908-3914. 

[73] Yamaguchi, T., Kim, N.S., Sekine, S., 1996. Cloning and expression of cDNA encoding a human ubiquitin-conjugating enzyme similar to the Drosophila bendless gene product. J Biochem, 120(3):494-497. 

[74] Yamamoto, M., Sato, S., Saitoh, T., 2006. Cutting edge: pivotal function of Ubc13 in thymocyte TCR signaling. J Immunol, 177(11):7520-7524. 

[75] Yamamoto, M., Okamoto, T., Takeda, K., 2006. Key function for the Ubc13 E2 ubiquitin-conjugating enzyme in immune receptor signaling. Nat Immunol, 7(9):962-970. 

[76] Yang, R.F., Tang, Q.C., Wang, H.M., 2011. Analyses of two rice (Oryza sativa) cyclin-dependent kinase inhibitors and effects of transgenic expression of OsiICK6 on plant growth and development. Ann Bot, 107(7):1087-1101. 

[77] Yang, Y., Liu, Z.H., Ware, C.F., 1997. A cysteine protease inhibitor prevents activation-induced T-cell apoptosis and death of peripheral blood cells from human immunodeficiency virus-infected individuals by inhibiting upregulation of Fas ligand. Blood, 89(2):550-557. 

[78] Yang, Z., Wu, Y., Li, Y., 2009. OsMT1a, a type 1 metallothionein, plays the pivotal role in zinc homeostasis and drought tolerance in rice. Plant Mol Biol, 70(1-2):219-229. 

[79] Yoshida, S., Forno, D.A., Cock, J.H., 1976. Routine procedures for growing rice plants in culture solution.  Laboratory Manual for Physiological Studies of Rice. IRRI,Los Baños, Philippines :61-66. 

[80] Yuan, J., Chen, D., Ren, Y., 2008. Characteristic and expression analysis of a metallothionein gene, OsMT2b, down-regulated by cytokinin suggests functions in root development and seed embryo germination of rice. Plant Physiol, 146(4):1637-1650. 

[81] Yuan, S., Fu, Y., Wang, X., 2008. Voltage-dependent anion channel 1 is involved in endostatin-induced endothelial cell apoptosis. FASEB J, 22(8):2809-2820. 

[82] Zang, Y., Wang, Q., Xue, C., 2012. Rice UBC13, a candidate housekeeping gene, is required for K63-linked polyubiquitination and tolerance to DNA damage. Rice, 5(1):24-34.