兰州鲇线粒体Cytb基因的克隆与序列分析
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摘要
为克隆兰州鲇(Silurus lanzhouensis)线粒体Cytb基因全序列,根据欧洲鲇(Silurus glanis)线粒体基因全序列设计特异引物进行兰州鲇线粒体Cytb基因的PCR扩增,得到1138 bp兰州鲇线粒体Cytb基因序列。对兰州鲇和其他13种鱼的线粒体Cytb基因核苷酸和氨基酸序列之间进行同源性比较,结果显示具有较高的同源性,核苷酸同源性介于61.38%—91.12%,氨基酸同源性介于76.62%—95.52%。对兰州鲇、欧洲鲇、大口鲇(Silurus meridionalis)、鲇(Silurus asotus)、越南鲇(Silurus cochinchinensis)的Cytb基因之间进行碱基替代分析,结果显示兰州鲇Cytb基因与鲇之间替换率最低,值为8.87%,转换/颠换值为3.21;与越南鲇之间替换率最高,值为14.41%,转换/颠换值为1.83。对本文克隆的兰州鲇Cytb基因与王庆容等测定的兰州鲇线粒体Cytb序列进行序列差异分析,结果显示两者之间替换率为11.16%,存在127个变异位点,转换/颠换比为4.08,遗传距离为0.1230。由NJ法基于兰州鲇和其他13种鱼的Cytb基因序列构建的系统进化树,结果显示与传统的分类地位基本吻合。
To clone the sequence of the mitochondrial cytochrome b(Cytb) gene of Silurus lanzhouensis, we designed a specific pair of primers from the mitochondrial genome of S. glanis. The 1138 bp S. lanzhouensis Cytb gene was obtained after PCR. Then we compared the homology of the mitochondrial Cytb gene between S. lanzhouensis and 13 other species. The results showed that these species have high homology in Cytb gene. The homology value of the nucleotides was between 61.38% and 91.12%, and the value of the amino acids was between 76.62% and 95.52%. We also analyzed the nucleotides substitution of the mitochondrial Cytb gene among S. lanzhouensis, S. glanis, S. meridionalis, S. asotus, and S. cochinchinensis, and found that the lowest frequency of nucleotides substitution occurred between S. lanzhouensis and S. asotus with the value of 8.87%, and TS/TV was 3.21. The highest frequency was between S. lanzhouensis and S. cochinchinensis with the value of 14.41%, and TS/TV was 1.83. Qing-Rong Wang, et al. previously reported the sequence of the mitochondrial Cytb gene of S. lanzhouensis. Here we conducted the sequence difference analysis between our results and theirs. The results showed that the frequency of nucleotides substitution was 11.16%, there were 127 variable sites, TS/TV was 4.08, genetic distance was 0.1230. The phylogenetic tree of S. lanzhouensis and 13 other species constructed by NJ method was consistent with the traditional taxonomic results.
To clone the sequence of the mitochondrial cytochrome b(Cytb) gene of Silurus lanzhouensis, we designed a specific pair of primers from the mitochondrial genome of S. glanis. The 1138 bp S. lanzhouensis Cytb gene was obtained after PCR. Then we compared the homology of the mitochondrial Cytb gene between S. lanzhouensis and 13 other species. The results showed that these species have high homology in Cytb gene. The homology value of the nucleotides was between 61.38% and 91.12%, and the value of the amino acids was between 76.62% and 95.52%. We also analyzed the nucleotides substitution of the mitochondrial Cytb gene among S. lanzhouensis, S. glanis, S. meridionalis, S. asotus, and S. cochinchinensis, and found that the lowest frequency of nucleotides substitution occurred between S. lanzhouensis and S. asotus with the value of 8.87%, and TS/TV was 3.21. The highest frequency was between S. lanzhouensis and S. cochinchinensis with the value of 14.41%, and TS/TV was 1.83. Qing-Rong Wang, et al. previously reported the sequence of the mitochondrial Cytb gene of S. lanzhouensis. Here we conducted the sequence difference analysis between our results and theirs. The results showed that the frequency of nucleotides substitution was 11.16%, there were 127 variable sites, TS/TV was 4.08, genetic distance was 0.1230. The phylogenetic tree of S. lanzhouensis and 13 other species constructed by NJ method was consistent with the traditional taxonomic results.
引文
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[20]Peng Z,He S,Zhang Y.Phylogenetic relationships of glyptostemoid fishes(Siluriformes:Sisoridae)inferred from mitochondrial cytochrome b gene sequence[J].Molecular Phylogenetics and Evolution,2005,31(3):979—987
[21]Avise J C.Molecular Markers,Natural History and Evolution[M].New York:Chapman and Hall.1994,120—144
[22]Cheng Q Q,Ma C Y,Zhuang P,et al.Genetic structure and evolution characters in three populations of Coilia mystus based on cytochrome b gene segment sequence of mitochondrial DNA[J].Journal of Fisheries of China,2008,32(1):2—6[程起群,马春艳,庄平,等.基于线粒体细胞色素b标记探讨凤鲚3群体遗传结构和进化特征.水产学报,2008,32(1):2—6]
[23]Yang X G,Wang Y Q,Zhou K Y,et al.The authentication of Oviductus ranae and their original animals by using molecular marker[J].Biopharm Bull,2002,25(8):1035—1039
[24]Ward R D,Tyler S Z,Bronwyn H I,et al.DNA barcoding Australia’s fish species[J].Philosophical Transactions Royal Society B,2005,360:1847–1857
[25]Kartavtsev Y P,Lee J S.Analysis of nucleotide diversity at the cytochrome b and cytochrome oxidase 1 genes at the population,species,and genus levels[J].Russian Journal of Genetics,2006,42(4):437–461
[26]Chu X L,Zheng B S,Dai D Y,et al.Fauna Sinica-Osteich-thyes Siluriformes[M].Beijing:Science Press.1999,87—88[褚新洛,郑葆珊,戴定远,等.中国动物志·硬骨鱼纲·鲇形目.北京:科学出版社.1999,87—88]
[27]Meng Q W,Su J X,Miao X Z.Taxonomy of Fishes[M].Beijing:China Agriculture Press.1995,995—998[孟庆闻,苏锦祥,缪学祖.鱼类分类学.北京:中国农业出版社.1995,995—998]
[2]Cheng X L.Division of catfish fish overview of China[J].Acta Hydrobiologica Sinica,1977,6(2):197—216[陈湘遴.我国鲇科鱼类的总述.水生生物学集刊,1977,6(2):197—216]
[3]Wu X D,Zhang Q,Zhao H X,et al.A new species of catfish in Ningxia—Silurus lanzhouensis and it’s intensive morphological description[J].Freshwater Fisheries,2006,36(3):26—29[吴旭东,张奇,赵红雪,等.宁夏鲇属鱼类一新纪录种—兰州鲇形态学特征描述.淡水渔业,2006,36(3):26—29]
[4]Shi L N,Zhang Q,Wu X D,et al.Individual fertility of the Silurus lanzhouensis[J].Journal of Gansu Agricultural University,2008,43(1):67—70[史丽娜,张奇,吴旭东,等.兰州鲇个体繁殖力的研究.甘肃农业大学学报,2008,43(1):67—70]
[5]Wu X D,Lian Z Q,Hou Y X,et al.Application analysis of microsatellite markers from Silurus meriaionalis on three species of siluriformes[J].Acta Hydrobiologica Sinica,2011,35(4):638—644[吴旭东,连总强,侯玉霞,等.大口鲇微卫星标记在三个鲇形目鱼类种群间适用性研究.水生生物学报,2011,35(4):638—644]
[6]Wu X D,Lian Z Q,Hou Y X,et al.Genetic structure analyses between wild population and artificial breeding ones of Silurus lanzhouensis[J].Freshwater Fisheries,2011,41(3):34—38[吴旭东,侯玉霞,连总强,等.兰州鲇野生群体和人工繁育群体遗传结构的比较研究.淡水渔业,2011,41(3):34—38]
[7]Qiu X C,Zhao H X,Wang Y J,et al.Nutrient analysis and nutritive evaluation in muscle of catfish Silurus lanzhouensis[J].Fisheries Science,2008,27(8):407—410[邱小琮,赵红雪,王远吉,等.兰州鲇肌肉营养成分分析及营养价值评价.水产科学,2008,27(8):407—410]
[8]Qiu X C,Zhao H X,Wang Y J,et al.In vitro digestibility coefficient and enzymolysis rate of Silurus lanzhouensis to four common feed ingredients[J].Freshwater Fisheries,2008,38(8):31—35[邱小琮,赵红雪,王远吉,等.兰州鲇对四种饲料原料的离体消化率和酶解能力.淡水渔业,2008,38(2):31—35]
[9]Wang Q R,Xu C,Xu C R,et al.Mitochondrial genome structure of Yellow River catfish(Silurus lanzhouensis)and phylogenetic analysis[J].Acta Scientiarum Naturalium Universitatis Pekinensis,2012,48(3):376—380[王庆容,许昌,许崇任,等.兰州鲇线粒体基因组序列结构及系统发育分析.北京大学学报,2012,48(3):376—380]
[10]Gray M W.Origin and evolution of mitochondrial DNA[J].Annual Review of Cell Biology,1989,5:25—50
[11]Xiao W H,Zhang Y P.Genetics and evolution of mitocchondrial DNA in fish[J].Acta Hydrobiologica Sinica,2000,24(4):384—391[肖武汉,张亚平.鱼类线粒体DNA的遗传与进化.水生生物学报,2000,24(4):384—391]
[12]Schoepp B,Breton J,Parot P,et al.Relative orientation of the hemes of the cytochrome bc1 complexes from Rhodobacter Sphaeroides,Rhodospirillum rubrum,and beef heart mitochondria,A linear dichroism study[J].Biological Chemistry,2000,275(8):5284—5290
[13]Meyer A.Shortcoming of the cytochrome b gene as a molecular marker[J].Trends in Ecology and Evolution,1994,9:278—289
[14]Katriina L I,Erie B T.Molecular resolution of the systematics of a problematic group of fishes(Teleostei:Osmeridae)and evidence for morphological homoplasy[J].Molecular Phylogenetics and Evolution,2009,50(1):163—175
[15]Song C B,Near T J,Page L M.Phylogenetic relations among percid fishes as inferred from mitochondrial cytochrome b DNA sequence data[J].Molecular Phylogenetics and Evolution,1998,10(3):343—353
[16]Wu X D,Zhang Q,Zhao H X,et al.The method of extracting and purifing genome DNA of a catfish in the Yellow River[J].Freshwater Fisheries,2006,36(1):19—21[吴旭东,张奇,侯玉霞,等.黄河鲶基因组DNA提取与纯化方法的研究.淡水渔业,2006,36(1):19—21]
[17]Sambrook J,Russell D W.Molecular Clonging:a Laboratory Manual[M].New York:Cold Spring Harbor Laboratory Press.2001,611—627
[18]Tamura K,Dudley J,Nei M,et al.Mega4:Molecular evolutionary genetics analysis(MEGA)software version 4.0[J].Molecular Biology and Evolution,2007,(10):1092—1093
[19]Perdices A,Bermingham E,Montilla A,et al.Evolutionary history of the genus Rhamdia(Teleostei:Pimelodidae)in central America[J].Molecular Phylogenetics and Evolution,2002,25:172—189
[20]Peng Z,He S,Zhang Y.Phylogenetic relationships of glyptostemoid fishes(Siluriformes:Sisoridae)inferred from mitochondrial cytochrome b gene sequence[J].Molecular Phylogenetics and Evolution,2005,31(3):979—987
[21]Avise J C.Molecular Markers,Natural History and Evolution[M].New York:Chapman and Hall.1994,120—144
[22]Cheng Q Q,Ma C Y,Zhuang P,et al.Genetic structure and evolution characters in three populations of Coilia mystus based on cytochrome b gene segment sequence of mitochondrial DNA[J].Journal of Fisheries of China,2008,32(1):2—6[程起群,马春艳,庄平,等.基于线粒体细胞色素b标记探讨凤鲚3群体遗传结构和进化特征.水产学报,2008,32(1):2—6]
[23]Yang X G,Wang Y Q,Zhou K Y,et al.The authentication of Oviductus ranae and their original animals by using molecular marker[J].Biopharm Bull,2002,25(8):1035—1039
[24]Ward R D,Tyler S Z,Bronwyn H I,et al.DNA barcoding Australia’s fish species[J].Philosophical Transactions Royal Society B,2005,360:1847–1857
[25]Kartavtsev Y P,Lee J S.Analysis of nucleotide diversity at the cytochrome b and cytochrome oxidase 1 genes at the population,species,and genus levels[J].Russian Journal of Genetics,2006,42(4):437–461
[26]Chu X L,Zheng B S,Dai D Y,et al.Fauna Sinica-Osteich-thyes Siluriformes[M].Beijing:Science Press.1999,87—88[褚新洛,郑葆珊,戴定远,等.中国动物志·硬骨鱼纲·鲇形目.北京:科学出版社.1999,87—88]
[27]Meng Q W,Su J X,Miao X Z.Taxonomy of Fishes[M].Beijing:China Agriculture Press.1995,995—998[孟庆闻,苏锦祥,缪学祖.鱼类分类学.北京:中国农业出版社.1995,995—998]