38 4 Vol. 38, No.4 2014 7 ACTA HYDROBIOLOGICA SINICA Jul., 2 0 1 4 doi: 10.7541/2014.108 Cytb 1, 2 吴旭东 2 肖伟 1, 2 连总强 3 侯玉霞 (1., 730070; 2., 750001; 3., 100193) : ( lanzhouensis) Cytb, ( glanis) Cytb PCR, 1138 bp Cytb 13 Cytb,, 61.38% 91.12%, 76.62% 95.52% ( meridionalis)( asotus)( cochinchinensis) Cytb, Cytb, 8.87%, / 3.21;, 14.41%, / 1.83 Cytb Cytb, 11.16%, 127, / 4.08, 0.1230 NJ 13 Cytb, : ; Cytb; 中图分类号 : S917 文献标识码 : A 文章编号 : 1000-3207(2014)04-0772-08 ( lanzhouensis), ()(Siluridea)(),,,,, () [1], [2] ;,, [3 6] ; [7, 8] ; PCR [9] (Mitochondria), [10],, [11] b(mitochondria Cytochrome b), ATP, [12] Meyer, b, 13, [13], Cytb, [14] Cytb [15] Cytb, GenBank 收稿日期 : 2013-07-05; 修订日期 : 2014-02-13 基金项目 : (30760191) 通信作者 : (1967 ),,, ; E-mail: amy95@163.com
4 : Cytb 773 Cytb, 1 1.1 60 [(10±1.5) cm, (120±8) g] EcoRⅠSalⅠ T4 DNA Taq DNA ; IPTGX-gal Sigma ; (Escherichia Coli)JM109, ; pmd18-t Promga 1.2 DNA 0.5 g, [16] Spectrophotometer DNA, 50 ng/µl 1.3 Cytb (GenBank : AM398435.2) Cytb : HD: 5-CCA AAGCGGCAAAATAAG-3; LD: 5-GGAGTTAGGG GCGGGAGT-3 PCR PCR 25 µl, 10 buffer( Mg 2+ )3 µl, dntp 2 µl, 0.5 µl, Taq 0.5 µl(2.5 U/µL), ddh 2 O 25 µlpcr : 94 5min; 94 30s, 54 30s, 72 30s, 30 ; 72 5minPCR 1%,, pmd18-t 16, JM109, LB 37 16h,, [17] 1.4 Sal I EcoR I, (2 ) DNAClub Clustal x ; Mega4.0 [18] Cytb, Cytb, /; Mega4.0 Kimura s 2 Parameter, NJ Cytb 2 2.1 Cytb PCR PCR, 1100 bp ( 1A) T, JM109,, 2000 bp, 1100 bp ( 1B), 1 Cytb PCR Fig. 1 The whole mitochondria Cytb PCR amplification product and enzyme digestion of S. lanzhouensis M DNA Marker; ; A Cytb PCR ; B DNA M is marker; Number are lanes; A is mitochondria Cytb PCR product of S. lanzhouensis; B is mitochondria Cytb enzyme digestion of pmd18-t 2.2 Cytb DNAClub,, Clustal x, 1138 bp, Mega4.0 Cytb, Cytb 1138, 379, ATG, T GenBank, HQ890503 2.3 13 Cytb 13 Cytb 1137 1141, 378 380 ( 1), TAA
774 38 Tab. 1 表 1 兰州鲇与其他 13 种鱼类线粒体 Cytb 基因碱基组成及密码子比较 Comparison of base composition and codons of mitochondria Cytb genes between S. lanzhouensis and other 13 species Taxonomy Genera Species Length (bp) Number of AA Initiator Terminator (A+T) (%) S. lanzhouensis 1138 379 ATG T 57.7 S. asotus 1138 379 ATG T 55.8 S. meridionalis 1138 379 ATG T 56.1 S. glanis S. cochinchinensis 1138 379 ATG T 56.0 1138 379 ATG T 56.0 Tetraodontiformes Takifuku T. fasciatus 1137 378 ATG TAA 55.0 Perciformes Cypriniformes Thunnus Cyprinus T. thynnus C. carpio 1141 380 ATG T 54.0 1140 380 ATG T 56.0 Anguilliformes Anguilla A. japonica 1140 379 ATG TAA 58.0 Clupeiformes Pleuronectiformes 鳀 Engraulis Paralichthys 鳀 E. japonicas P. olivaceus 1141 380 ATG T 56.0 1141 380 ATG T 55.0 Salmoniformes Salmo S. salar 1141 380 ATG T 55.0 Acipenseriformes Squaliformes Acipenser Squalus A. stellatus S. acanthias 1141 380 ATG T 53.0 1146 381 ATG TAA 60.0, T, Perdices, et al. Rhamdia [19] Cytb,,,,, Cytb, Cytb [20] Mega4.0 Cytb, 13 Cytb ( 2),, Cytb, 91.12% 95.52% Mega4.0 Kimura s 2 Parameter, NJ 5 Cytb, bootstrap1000 ( 2) 2.4 Cytb Mega4.0 Cytb, Cytb ( 3), Cytb, 8.87%, / 3.21;, 14.41%, / 1.83 2.5 Cytb PCR, [9] (Gen- Bank : JF895472) Mega4.0
4 : Cytb 775 Tab. 2 Taxonomy 表 2 兰州鲇与其他 13 种鱼类线粒体 Cytb 基因序列和氨基酸序列同源性比较 Comparoson of homology of nucleotide and amino acid of mitochondria Cytb between S. lanzhouensis and other 13 species Genera Species GenBank GenBank accession Nucleotide homology (%) Amino acid homology (%) S. asotus AF416898 91.12 95.52 S. meridionalis AF416892 89.37 95.51 S. glanis AM398435 88.18 94.12 S. cochinchinensis AF416881 85.59 94.20 Takifuku T. fasciatus AJ421455 74.31 83.42 Thunnus T. thynnus AP003091 75.81 85.53 Cyprinus C. carpio X61010 78.88 87.11 Anguilla A. japonica AB038556 75.90 84.47 鳀 Engraulis 鳀 E. japonicas AB040676 74.78 83.68 Paralichthys P. olivaceus AB028664 74.06 83.95 Salmo S. salar U12143 75.11 84.47 Acipenser A. stellatus AJ585050 74.93 82.89 Squalus S. acanthias NC002012 64.38 76.62 2 13 Cytb Fig. 2 Phylogenetic trees derived from mitochondria Cytb nucleotide sequences of S. lanzhouensis and other thirteen species of fishes based on Neighbor-Joining method Cytb, Cytb ( 4) Cytb, 2.63%, / 4.00; 18.45%, / 2.00; Cytb 11.16%, 127, 102, 25, / 4.08 Mega4.0 Kimura s 2-Parameter Cytb Cytb ( 5 3 W-fishes ) Cytb ( 5) Cytb, ; Cytb, ; Cytb Cytb 0.1230 5 NJ ( 3), bootstrap1000 100%,,,
776 38 表 3 兰州鲇与四种鲇属鱼类线粒体 Cytb 基因的碱基替换分析 Tab. 3 Number and frequencies of nucleotides substitution of mitochondria Cytb genes between S. lanzhouensis and other four species Nucleotide substitution S. glanis S. meridionalis Number of base S. asotus S. cochinchinensis (TS) A G 25 22 23 24 C T 77 71 54 82 Total of TS 102 93 77 106 (TV) A C 12 14 11 39 A T 10 11 6 14 G C 3 2 4 4 G T 1 1 3 1 Total of TV 26 28 24 58 Total of nucleotides substitution 128 121 101 164 Total of bases (bp) 1138 1138 1138 1138 Frequencies of nucleotides substitution (%) 11.25 10.63 8.87 14.41 / TS/TV 3.92 3.32 3.31 1.83 表 4 王庆容等测定的线粒体 Cytb 基因与五种鲇属鱼类线粒体 Cytb 基因之间的碱基替代分析 Tab. 4 Number and frequencies of nucleotides substitution of mitochondria Cytb genes between which was measured by Wang Qing-Rong and other five species Number of base Nucleotide substitution S. glanis S. asotus S. cochinchinensis S. meridionalis S. lanzhouensis (TS) A G 4 31 36 27 25 C T 20 71 104 49 77 Total of TS 24 102 140 76 102 (TV) A C 4 15 45 11 12 A T 2 3 15 4 9 G C 0 3 6 1 3 G T 0 1 4 2 1 Total of TV 6 22 70 18 25 Total of nucleotides substitution 30 124 210 94 127 Total of bases (bp) 1138 1138 1138 1138 1138 Frequencies of nucleotides substitution (%) 2.63 10.90 18.45 8.26 11.16 / TS/TV 4.00 4.64 2.00 4.22 4.08 Tab. 5 表 5 兰州鲇等几种鲇形目鱼类基于线粒体 Cytb 基因的遗传距离 Genetic distance of S. lanzhouensis and other species based on mitochondria Cytb Species S. lanzhouensis S. lanzhouensis S. glanis S. asotus S. cochinchinensis S. meridionalis W-fishes S. glanis 0.1230 S. asotus 0.0962 0.1284 S. cochinchinensis 0.1687 0.2172 0.2185 S. meridionalis 0.1169 0.1200 0.0946 0.2198 W-fishes 0.1230 0.0269 0.1198 0.2242 0.0889
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4 : Cytb 779 THE CLONING AND SEQUENCE ANALYSIS OF THE MITOCHONDRIAL CYTOCHROME b GENE OF SILURUS LANZHOUENSIS WU Xu-Dong 1, 2, XIAO Wei 2, LIAN Zong-Qiang 1, 2 and HOU Yu-Xia 3 (1. College of Animal Science and Technology, Gansu Agricultural University, Lanzhou 730070, China; 2. Ningxia Fisheries Research Institute, Yinchuan 750001, China; 3. College of Science, China Agricultural University, Beijing 100193, China) Abstract: To clone the sequence of the mitochondrial cytochrome b (Cytb) gene of 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. Key words: lanzhouensis; Mitochondria Cytochrome b; Sequence analysis