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| Effects of T20N Site-directed Mutation on GTPase Activities of OsRacD from Oryza sativa |
| LIU Xiao-fei,LIANG Wei-hong
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| College of Life Sciences , Henan Normal University , Xinxiang 453007, China |
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Abstract OsRacD belonging to rice Rho family of the small GTP binding proteins, is a pivotal gene involved in rice photoperiod fertility conversion of photoperiod sensitive genic male sterile rice, which influences rice fertility via controlling the pollen tube growth.T20N site-directed mutation was introduced into its highly conserved G1 motif by PCR-mediated method to mimic its GDP-binding state based on the sequences alignment and conserved domains analysis. The prokaryotic expression vector of OsRacD and T20N-OsRacD were constructed , and the His6 tag fused proteins were expressed and purified from E.coli. After identified by Western blot,the GTP hydrolysis activities were detected. The results showed that the GTPase activities of T20N-OsRacD were significantly reduced comparing with that of OsRacD, suggested that OsRacD and T20N-OsRacD have different biochemical characteristics.
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Received: 23 June 2009
Published: 27 January 2010
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Corresponding Authors:
Liang Wei－Hong
E-mail: liangwh@henannu.edu.cn
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| ［1］ Bourne H R, Sanders D A, McCormick F. The GTPase superfamily: conserved structure and molecular mechanism. Nature, 1991, 349(6305): 117127.
［2］ Hall A. Small GTPbinding proteins and the regulation of the actin cytoskeleton. Annu Rev Cell Biol, 1994, 10(1):3154.
［3］ Yang Z B, Waston J C. Molecular cloning and characterization of rho, a rasrelated small GTPbinding protein from the garden pea. Proc Natl Acad Sci USA, 1993, 90(18): 87328736.
［4］ Jones M A. The Arabidopsis RopGTPase is a positive regulator of both root hair inhtiation and tip growth. Plant Cell, 2002, 14(4): 763776.
［5］ Fu Y, Yang Z. Rop GTPase controls plant cell morphogenesis via a novel form of cortical Factin. Plant Cell, 2002, 14(4):777794.
［6］ Deborah P D, Julie R, Andrawis A, et al. Genes encoding small GTPbinding analogous to mammalian rac are preferentially expressed in developing cotton fibers. Mol Geb Genet, 1995, 248(1): 4351.
［7］ Trotochaud A E, Hao T, Wu G, etal. The CLAVATA1 receptorlike kinase requires CLAVATA3 for its assembly into a signaling complex that include KAPP and a Rhorelated protein. Plant Cell, 1999, 11(3): 393406.
［8］ Yang Z. Cell Polarity Signaling in Arabidopsis. Annu Rev Cell Dev Biol,2008, 24(1):551575.
［9］ 米志勇, 王树声，吴乃虎. 水稻低分子量GTP结合蛋白基因OsRacD的分离.科学通报,2000,45（19）:20472054. Mi Z Y, Wang S S, Wu N H.Chinese Science Bulletin,2000, 45（19）:20472054.
［10］ 叶建荣,黄美娟,吴乃虎.转水稻OsRacD 反义基因拟南芥植株的育性分析.自然科学进展,2003,13(3):424428. Ye J R，Huang M J, Wu N H.Progress in Natural Science,2003,13(3):424428.
［11］ 叶建荣，黄美娟，吴乃虎.OsRacD基因表达与光敏核不育水稻光周期育性转换的相关性.自然科学进展,2004, 14(2),166172. Ye J R，Huang M J, Wu N H.Progress in Natural Science,2004, 14(2):166172.
［12］ 梁卫红，吴乃虎. G15V点突变对水稻OsRacD基因蛋白产物效应的影响.中国生物化学与分子生物学报,2006,22(4):338342. Liang W H, Wu N H.Chinese Journal of Biochemistry and Molecular Biology, 2006,22(4):338342.
［13］ Aiyar A, Xiang Y, Leis J. Sitedirected mutagenesis using overlap extension PCR.Methods Mol Biol,1996,57:177191.
［14］ Li H,Shen J G,Yang Z B，et al.The Rop GTPase Switch Controls Multiple Developmental Processes in Arabidopsis.Plant Physiology, 2001, 126(2):670684.
［15］ Cool R H, Gudular S, Christian U, et al. The Ras mutant D119N is both dominant negative and active. Mol Cell Biol, 1999, 19(9): 62976305.
［16］ Olofsson B. Rho guanine dissociation inhibitors: pivotal molecular incellular signaling. Cell Signal, 1999, 11(8): 545554.
［17］ Yang Z B, Waston J C. Molecular cloning and characterization of rho, a rasrelated small GTPbinding protein from the garden pea. Proc Natl Acad Sci USA, 1993, 90(8): 87328376.
［18］ Berken A. ROPs in the spotlight of plant signal transduction. Cell Mol Life Sci, 2006, 63(21): 24462459.
［19］ 梁卫红,刘肖飞,毕佳佳.G15V和T20N定点突变对水稻OsRacD胞内定位和蛋白互作特性的影响.中国生物化学与分子生物学学报,2009,25(9):828833. Liang W H, Liu X F, Bi J J.Chinese Journal of Biochemistry and Molecular Biology,2009,25(9):828833. |
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