银鲫两个蛋白合成相关基因全长cDNA的克隆及其特征分析
CLONING AND CHARACTERIZATION OF FULL LENGTH cDNAs OF TWO GENES RELATED TO PROTEIN BIOSYNTHESIS IN GYNOGENETIC GIBEL CARP
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摘要: 通过构建雌核发育银鲫心跳期SMART cDNA 质粒文库并从文库中随机挑选克隆测序,克隆得到银鲫翻译起始因子3亚单位2(GTIF3-S2)和翻译延伸因子1亚单位α(GEF-1α)基因全长cDNA.银鲫翻译起始因子3亚单位2基因cDNA全长1280bp,开放阅读框位于117-101bp 之间,编码325个氨基酸.其推断的氨基酸序列存在三个WD结构域.该基因在鱼类中为首次报道.银鲫翻译延伸因子1亚单位alpha基因cDNA全长1784bp,开放阅读框位于82-1467bp之间,编码462个氨基酸.RT-PCR表明,这两个基因在成熟卵母细胞和胚胎发育早期可以检测到少量的转录产物,在胚胎发育期间从原肠期开始转录,并随着发育进程逐渐增强.成鱼组织中除精巢表达较弱外,其他组织都表达较强.同源分析比较表明,TIF3-S2和EF-1α在物种进化过程中具有高度的进化保守性,在物种间的同源性很高.因此,作者认为,这两个基因是研究物种间系统发育的优良对象.Abstract: The SMART cDNA plasmid library was constructed from heartbeat stage embryos of gibel carp(Carassius auratus gibelio). Randomly selected clones from the library were sequenced and their homologies were analyzed. The full length cDNAs of two genes related to protein biosynthesis were obtained from the sequenced clones. One gene,gibel carp translation initiation factor 3 subunit 2,was named GTIF3-S2. It is 1280 bp in length and has an open reading frame of 975 nucleotide acids for encoding 325 amino acids. The deduced amino acid sequence has three WD domains. The gibel carp GTIF3-S2 was first reported fish translation initiation factor 3 subunit 2. Another gene,gibel carp translation elongation factor 1α, was named GEF-1α. It is 1784 bp in length and has an open reading frame of 1386 nucleotide acids encoding 462 amino acids. The open reading frame is between 82—1467 bp. RT-PCR analysis showed that the maternal mRNAs of the two genes were detected in matured eggs and in early developmental embryos. In embryonic development, the two genes began to transcribe from gastrula stage, and their expressions became stronger as the embryos developed. The two genes were detected to express strongly in most of the examined tissues of gibel carp,but weaker expressions were found in testis. Homology analysis indicated that there exists high evolutionary conservation in gibel carp GTIF3-S2 and GEF-1α. among different species, from animals to plants. Therefore, we conclude that gibel carp GTIF3-S2 and GEF-1α. are two excellent target genes for studies on evolutionary genetics and systematics among species.
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[1] Clark B F C, Thirup S,Kjeldgaard M,et al. Structural information for explainlng the molecular mechanism of protein biosynthesis [J]. FEBS Letters,1999, 452: 41-46[2] Zhou L,Gui,J F.Karyotypic diversity in polyploid gibel carp,Carassius auratus gibelio Bloch [J]. Genetica,2002,115:223-232[3] Jiang Y G,Liang S C,Chen B D,et al. Biological effect of heterologous sperm on gynogenetic offspring in Carassius auratus gibelio [J]. Acta Hydrobiol. Sinica,1983,8(1):1-13. [蒋一NB042,梁绍昌,陈本德等.异源精子在银鲫雌核发育子代中的生物学效应.水生生物学报,1983,8(1):1-13][4] Jiang YG,Liang S C,Chen B D,et al. Artificial and natural gynogenesis in carp [J]. Acta Hydrobiol. Sinica,1982,7(4):471-477. [蒋一NB042, 梁绍昌,陈本德等. 鲫鱼的人工和天然雌核发育.水生生物学报,1982,7(4):471-477][5] Gui J F,Liang S C,Zhu L F,et al. Discovery of multiple tetraploids in artificially propagated populations of allogynogenetic silver crucian carp and their breeding potentialities [J]. Chinese Sci. Bull., 1993, 38: 327-331. [桂建芳,梁绍昌,朱蓝菲等.异育银鲫人工繁育群体中复合四倍体的发现及其育种潜力.科学通报,1993,38:327-331][6] Gui J F, Liang S C,Zhu L F,et al.Discovery of two different responsive modes of eggs of artificially propagated multiple tetraploids allogynogenetic silver crucian carp on male parent sperms and maternal sperms [J]. Chinese Sci. Bull., 1992,37(11):1030-1033. [桂建芳,梁绍昌,朱蓝菲等.人工复合四倍体异育银鲫卵子应答父本种精子和母本种精子两种不同发育方式的发现.科学通报,1992,37(11):1030-1033][7] Zhou L,Wang Y,Gui,J F. Genetic evidence for gonochoristic reproduction in gynogenetic silver crucian carp(Carassius auratus gibelio Bloch) [J]. J Mol Evol,2000,51:498-506[8] Gui J F. Retrospect and prospects of studies on the mechanism of natural gynogenesis in silver crucian carp(Carassius auratus gibelio) [J]. Bull Nat Nat Sci Found China,1997, 11(1):11-16. [桂建芳. 天然雌核发育机理研究的回顾与展望. 中国科学基金,1997,11(1):11-16][9] Xin J,Wen J J,Chen B,Gui J F. Differential gene expression in fully-grown oocytes between gynogenetic and gonochoristic crucian carps [J]. Gene, 2001,271: 109-116[10] Wu Q J,Gui J F, Fish genetics and breeding engineering[M]. Shanghai: Shanghai Scientific and Technical Publishers, 1999. [吴清江,桂建芳.鱼类遗传育种工程.上海:上海科学技术出版社,1999][11] Twyman R M. Instant Notes in Developmental Biology. London: BIOS Scientific Publishers Limited, 2002[12] Moriya S,Ohkuma M, Kudo T. phylogenetic position of symbiotic protist Dinenympha [correction of Dinemypha]exilis in the hindgut of the termite Reticulitermes speratus inferred from the protein phylogeny of elongation factor 1 alpha [J]. Gene,1998,210:221-227[13] Kamaishi T,Hashimoto T,Nakamura Y, et al. Protein phylogeny of translation elongation factor EF-1 alpha suggests microsporidians are extremely ancient eukaryotes [J]. J Mol Evol, 1996,42:257-263[14] Shearer G Jr. Cloning and analysis of cDNA encoding an elongation factor 1 alpha from the dimorphic fungus Histoplasma capsulatum [J]. Gene,1995,161:119-123[15] Hovemann B,Richter S,Walldorf U,et al. Two genes encode related cytoplasmic elongation factors 1α(EF1-α) in Drosophila melanogaster with continuous and stage specific expression [J]. Nucleic Acids Res.,1998,16:3175-3194[16] Mita K,Morimyo M,Ito K,et al. Comprehensive cloning of Schizosaccharomyces pombe genes encoding translation elongation factor [J]. Gene,1997,187:259-266[17] Dje M K,Mazabraud A,Viel A, et al. Three genes under different developmental control encode elongation factor 1 alpha in Xenopus laevis [J]. Nucleic Acids Res.,1990,18:3489-3493[18] Shiina N,Gotoh Y and Iwanatsu A. Microtubule severing gy elongation factor 1 alpha [J]. Science,1994,266:282-285[19] Condeelis J. Elongation factor 1 alpha, translation and the cytoskeleton [J]. Trends in Biochem. Sci.,1995,20:169-170[20] Gonen H,Smith C E,Siegel N R,et al. Protein synthesis elongation factor EF1-α is essential for ubiquitin-dependent degradation of certain N-acetylated proteins and may be substituted for by bacterial elongation factor EF-Tu [J]. Proc. Natl. Acad. Sci. USA,1994,91:7648-7652[21] Gao D,L Z F,Murphy T,et al. Structure and transcription of the gene for translation elongation factor 1 subunit alpha of zebrafish (Danio rerio) [J]. Biochimica et Biophysica Acta,1997,1350:1-5[22] Burks E A,Bezerra P P,Le H,et al. Plant initiation factor 3 subunit composition resembles mammalian initiation factor 3 and has a novel subunit [J]. J. Biol. Chem.,2001,276:2122-2131[23] Carninci P and Hayashizaki Y. High-efficiency full-length cDNA clonlig [J]. Meth. Enzymol., 1999,303:19-44[24] Chen R H,Miettinen P J,Maruoka E M,et al. A WD-domain protein that is associated with and phosphorylated by the type Ⅱ TGF-beta receptor [J]. Nature,1995,377:548-552[25] Wood V,Gwilliam R,Rajandream M A,et al. The genome sequence of Schizosaccharomyces pombe [J]. Nature,2002,415:871-880 Clark B F C, Thirup S,Kjeldgaard M,et al. Structural information for explainlng the molecular mechanism of protein biosynthesis [J]. FEBS Letters,1999, 452: 41-46[2] Zhou L,Gui,J F.Karyotypic diversity in polyploid gibel carp,Carassius auratus gibelio Bloch [J]. Genetica,2002,115:223-232[3] Jiang Y G,Liang S C,Chen B D,et al. Biological effect of heterologous sperm on gynogenetic offspring in Carassius auratus gibelio [J]. Acta Hydrobiol. Sinica,1983,8(1):1-13. [蒋一NB042,梁绍昌,陈本德等.异源精子在银鲫雌核发育子代中的生物学效应.水生生物学报,1983,8(1):1-13][4] Jiang YG,Liang S C,Chen B D,et al. Artificial and natural gynogenesis in carp [J]. Acta Hydrobiol. Sinica,1982,7(4):471-477. [蒋一NB042, 梁绍昌,陈本德等. 鲫鱼的人工和天然雌核发育.水生生物学报,1982,7(4):471-477][5] Gui J F,Liang S C,Zhu L F,et al. Discovery of multiple tetraploids in artificially propagated populations of allogynogenetic silver crucian carp and their breeding potentialities [J]. Chinese Sci. Bull., 1993, 38: 327-331. [桂建芳,梁绍昌,朱蓝菲等.异育银鲫人工繁育群体中复合四倍体的发现及其育种潜力.科学通报,1993,38:327-331][6] Gui J F, Liang S C,Zhu L F,et al.Discovery of two different responsive modes of eggs of artificially propagated multiple tetraploids allogynogenetic silver crucian carp on male parent sperms and maternal sperms [J]. Chinese Sci. Bull., 1992,37(11):1030-1033. [桂建芳,梁绍昌,朱蓝菲等.人工复合四倍体异育银鲫卵子应答父本种精子和母本种精子两种不同发育方式的发现.科学通报,1992,37(11):1030-1033][7] Zhou L,Wang Y,Gui,J F. Genetic evidence for gonochoristic reproduction in gynogenetic silver crucian carp(Carassius auratus gibelio Bloch) [J]. J Mol Evol,2000,51:498-506[8] Gui J F. Retrospect and prospects of studies on the mechanism of natural gynogenesis in silver crucian carp(Carassius auratus gibelio) [J]. Bull Nat Nat Sci Found China,1997, 11(1):11-16. [桂建芳. 天然雌核发育机理研究的回顾与展望. 中国科学基金,1997,11(1):11-16][9] Xin J,Wen J J,Chen B,Gui J F. Differential gene expression in fully-grown oocytes between gynogenetic and gonochoristic crucian carps [J]. Gene, 2001,271: 109-116[10] Wu Q J,Gui J F, Fish genetics and breeding engineering[M]. Shanghai: Shanghai Scientific and Technical Publishers, 1999. [吴清江,桂建芳.鱼类遗传育种工程.上海:上海科学技术出版社,1999][11] Twyman R M. Instant Notes in Developmental Biology. London: BIOS Scientific Publishers Limited, 2002[12] Moriya S,Ohkuma M, Kudo T. phylogenetic position of symbiotic protist Dinenympha [correction of Dinemypha]exilis in the hindgut of the termite Reticulitermes speratus inferred from the protein phylogeny of elongation factor 1 alpha [J]. Gene,1998,210:221-227[13] Kamaishi T,Hashimoto T,Nakamura Y, et al. Protein phylogeny of translation elongation factor EF-1 alpha suggests microsporidians are extremely ancient eukaryotes [J]. J Mol Evol, 1996,42:257-263[14] Shearer G Jr. Cloning and analysis of cDNA encoding an elongation factor 1 alpha from the dimorphic fungus Histoplasma capsulatum [J]. Gene,1995,161:119-123[15] Hovemann B,Richter S,Walldorf U,et al. Two genes encode related cytoplasmic elongation factors 1α(EF1-α) in Drosophila melanogaster with continuous and stage specific expression [J]. Nucleic Acids Res.,1998,16:3175-3194[16] Mita K,Morimyo M,Ito K,et al. Comprehensive cloning of Schizosaccharomyces pombe genes encoding translation elongation factor [J]. Gene,1997,187:259-266[17] Dje M K,Mazabraud A,Viel A, et al. Three genes under different developmental control encode elongation factor 1 alpha in Xenopus laevis [J]. Nucleic Acids Res.,1990,18:3489-3493[18] Shiina N,Gotoh Y and Iwanatsu A. Microtubule severing gy elongation factor 1 alpha [J]. Science,1994,266:282-285[19] Condeelis J. Elongation factor 1 alpha, translation and the cytoskeleton [J]. Trends in Biochem. Sci.,1995,20:169-170[20] Gonen H,Smith C E,Siegel N R,et al. Protein synthesis elongation factor EF1-α is essential for ubiquitin-dependent degradation of certain N-acetylated proteins and may be substituted for by bacterial elongation factor EF-Tu [J]. Proc. Natl. Acad. Sci. USA,1994,91:7648-7652[21] Gao D,L Z F,Murphy T,et al. Structure and transcription of the gene for translation elongation factor 1 subunit alpha of zebrafish (Danio rerio) [J]. Biochimica et Biophysica Acta,1997,1350:1-5[22] Burks E A,Bezerra P P,Le H,et al. Plant initiation factor 3 subunit composition resembles mammalian initiation factor 3 and has a novel subunit [J]. J. Biol. Chem.,2001,276:2122-2131[23] Carninci P and Hayashizaki Y. High-efficiency full-length cDNA clonlig [J]. Meth. Enzymol., 1999,303:19-44[24] Chen R H,Miettinen P J,Maruoka E M,et al. A WD-domain protein that is associated with and phosphorylated by the type Ⅱ TGF-beta receptor [J]. Nature,1995,377:548-552[25] Wood V,Gwilliam R,Rajandream M A,et al. The genome sequence of Schizosaccharomyces pombe [J]. Nature,2002,415:871-880
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