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1 10 个半滑舌鳎家系 MHC IIB 基因多态性初步研究牛宝珍 1, 杜民 1 2, 陈松林 (1., ; 2., ) 摘要 : 为检测 10 个半滑舌鳎 (Cynoglossus semilaevis) 家系 MHCⅡB(Cyse-DAB) 基因的多态性水平 MHCⅡ 类 B 位点数目以及平衡选择的作用, 作者利用多聚酶链式反应 (PCR) 和直接测序的方法对 10 个半滑舌鳎家系 MHCⅡB 基因位点遗传变异和平衡选择进行了研究用特异性引物和 PCR 扩增的半滑舌鳎 MHCⅡB 基因片段大约 397 bp, 包含一部分第一外显子, 全部第一内含子和全部第二外显子 10 个半滑舌鳎家系中, 每家系选取 5 个体, 每个体 5 个克隆序列分析发现 60 个不同序列, 代表 60 个等位基因, 其中有 28 个是新发现的, 已提交到 GenBank 同源分析表明 60 个等位基因相似性为 89.36% 共 50 个个体中, 有 6 个存在 5 个不同等位基因, 表明在半滑舌鳎至少存在 3 个座位有 9 个家系的 MHCⅡB 序列多肽结合区 (PBR) 的非同义替换 (d N ) 显著高于同义替换 (d S ) 关键词 : 半滑舌鳎 (Cynoglossus semilaevis); 多态性 ; MHCⅡB; 平衡选择中图分类号 : S917.4 文献标识码 : A 文章编号 : (2015) doi: //hykx (major histocompatibility complex, MHC), [1-2], T (TCR) [3-4] MHC, T MHC [5] MHC Hashimoto [6] 1990 (Cyprinus carpio), MHC, (Carcharodon carcharias) [7-8] (Alticorpus macrocleithrum) [9] (Scophthalmus maximus) [10] (Pseudosciaena crocea) [11] [12] (Cynoglossus semilaevis) (Pleuronectiformes) (Cynoglossidae) (Cynoglossus),,, [13],,, [14] ; MHC [15] β2m [16] A B [17] MHC B β1 [18] ; β1 MHC A α1 MHC, [19] MHC, [5], MHC, (Salmo salar)mhc [20-21], Du [22] (Paralichthys olivaceus)7 MHC MHC (Edwardsiella tarda) MHC [23] [24] 10 MHC B(Cyse-DAB) MHC B, : ; : : ( ); (ZD ); (2015HB059); (2014HB0203); (14bs11) : (1979-),,,, ;,,, du2005min@126.com 70 / 2015 / 39 / 12

2 1 材料和方法 1.1 实验鱼 ,, [24] 1.2 取样及 DNA 提取 10 5, 7~8 g, MS222(1g/kg) - [25], DNA 1%, DNA, 100 ng/µl, 引物设计和多聚酶链式反应 (PCR) Xu [15] cdna : hmpn12 (5 -CTCTCTTCTCTTCCTCCTCAC-3 ) hmpc-12 (5 -ACACTCACCTGATTTAGCCA-3 ), MHC B, 25 μl PCR 1 μl DNA 2.5 μl 10 TaqDNA (TransGen Biotech) 1.5 mmol/l MgCl mmol/l dntps 0.2 μmol/l ( ) 1 UTaq PCR 94 5 min 30 (94 40 s s s), min PCR Peltier (PTC-200) (Molecular Imager Gel Doc XR Biorad ) 1.4 克隆和 DNA 序列测定 PCR QIAEXII (QIAGEN) PBS-T, TOP10 M13 PCR ; 5 ABI3730 M13+/ 1.5 基因型序列分析和统计检验分析 DNAMAN (d S )(d N ) MEGA4.0 [26] Nei Gojobori - [27] DAMBE DnaSP5.0 [28] SPSS13.0 Davies [29] [30-31] 2 结果与分析个半滑舌鳎家系中 MHC II 第二外显子序列的多态性 10 50, 5, 250 MHC B cdna [17] - GT-AG, 397 bp, 35 bp (84 bp, 12bp CA ) (270 bp) MHC B β1, PCR, Taq DNA [32-33] 60, 32 [34], 28, 28, GeneBank ( 1) 10 5, 1 2, 2 6, 3 21, 4 15, 5 6 2, 10 MHC B, 4% ( ), 4# 19# ( ) 1# 3# 4# 6# 16# 19# 24# 28# 33# 34# 89.89% 94.26% 88.24% 92.35% 94.67% 93.7% 90.37% 90.16% 93.22% 88.84% 89.36% 60 MHC B, 60, Pi (p) Theta-W ; Marine Sciences / Vol. 39, No. 12 /

3 表 1 等位基因和 Genbank 登录号 Tab.1 Alleles and their GenBank accession number GenBank GenBank GenBank Cyse-DBB*0502 GU Cyse-DBB*3303 GU Cyse-DBB*4901 GU Cyse-DBB*0601 GU Cyse-DBB*3304 GU Cyse-DBB*5001 GU Cyse-DBB*0802 GU Cyse-DBB*3601 GU Cyse-DBB*5102 GU Cyse-DBB*1101 GU Cyse-DBB*3602 GU Cyse-DBB*5201 GU Cyse-DBB*1401 GU Cyse-DBB*3603 GU Cyse-DBB*5301 GU Cyse-DBB*1603 GU Cyse-DBB*3604 GU Cyse-DBB*5603 GU Cyse-DBB*1901 GU Cyse-DBB*3801 GU Cyse-DBB*6101 GU Cyse-DBB*2001 GU Cyse-DBB*4004 GU Cyse-DBB*6701 GU Cyse-DBB*2804 GU Cyse-DBB*4401 GU Cyse-DBB*3001 GU Cyse-DBB*4603 GU 表 2 10 个半滑舌鳎家系中每尾鱼的 Scma-DAB 等位基因数目和 5 种等位基因情况下的个体数目 Tab.2 The allele number per individual in 10 half-smooth tongue sole families and the individual number in five different allele models 5 ( ) : : 91 (H) (k) 半滑舌鳎 MHCⅡB 基因选择作用 MHC B (PBR), 23 20(86.96%), 69 40(57.97%) 60, (d N ) (d S ) , (d N ) (d S ) ;, (d N ) (d S ) , ( 3) 3, 28#, 9, MHC B Brown [19] HLA-DRB 3 讨论 MHC B β, PCR 10 MHC class B ; : Xu [35] ; Rakus [36] 9 7 MHC class B ; Stet [37] 84 7 Sasa-DAA 7 72 / 2015 / 39 / 12

4 表 3 10 个半滑舌鳎家系中 MHCⅡB 基因第二外显子中多肽结合区 (PBR) 非多肽结合区(non-PBR) 的同义替代率 (ds) 和非同义替代率的比值及显著性分析 Tab.3 Synonymous(dS) and non-synonymous(dn) substitution rates and significant levels in the putative peptides binding region(pbr) and non-peptides binding region(non-pbr) among half-smooth tongue sole alleles d N (SE) d S (SE) d N /d S z(p) PBR ± ± p=0.001 Non-PBR ± ± p= ± ± p=0.251 PBR ± ± p=0.012 Non-PBR ± ± p= ± ± p=0.172 PBR ± ± p=0.004 Non-PBR ± ± p= ± ± p=0.374 PBR ± ± p=0.045 Non-PBR ± ± p= ± ± p=1.000 PBR ± ± p=0.041 Non-PBR ± ± p= ± ± p=1.0 PBR ± ± p=0.000 Non-PBR ± ± p= ± ± p=0.069 PBR ± ± p=0.000 Non-PBR ± ± p= ± ± p=0.219 PBR ± ± p=0.054 Non-PBR 67 14± ± p= ± ± p=1 PBR ± ± p=0.000 Non-PBR ± ± p= ± ± p=0.11 PBR ± ± p=0.004 Non-PBR ± ± p= ± ± p=0.336 PBR ± ± p=0.001 Non-PBR ± ± p= ± ± p=0.001 Sasa-DAB ; Langefors [38] MHC class B MHC B, 6 5, MHC B 3, Xu [17] : [39] PCR-SSCP SLA-DQB 5, 3 DQA ; [40] Marine Sciences / Vol. 39, No. 12 /

5 , 5, 3 5, MHC B : [41] [42-43], (dn) (ds) dn/ds 1, 10 MHC B, 28# MHC B (PBR) (Z P=0.054>0.05), 9 MHC B (PBR) ( 3) MHC B, MHC B, MHC B HLA-DRB [19], [43] 60, 32 [34], 28,, , MHC B, MHC B / : [1] Nikolich-Žugich J, Fremont D H, Miley M J, et al. The role of mhc polymorphism in anti-microbial resistance[j]. Microbes and Infection, 2004, 6: [2] Klein J. Natural history of the major histocompatibilitycomplex[m]. New York: John Wiley& Sons, 1986: [3] Rothbard, J B, Gefter M L. Interactions betweenimmunogenic peptides and MHC proteins[j]. Annu Rev Immunol, 1991, 9: 527. [4] Srisapoome P, Ohira T, Hirono I, et al. Cloning, characterization and expression of cdna containing major histocompatibility complex classi, IIa and IIb genes of Japanese flounder. Paralichthys olivaceus[j]. Fish Sci, 2004, 70: [5] Rakus K Ł, Wiegertjes G F, Stet R J M, et al. Polymorphism of major histocompatibility complex class II B genesin different lines of the common carp (Cyprinus carpio)[j]. Aquat Living Resour, 2003, 16: [6] Hashimoto K, Nakanishi T, KurosawaY. Isolation of carp genes encoding major histocompatibility complex antigens[j]. Proc Natl AcadSci USA, 1990, 87: [7] BartlS. What sharks can tell us about the evolution of MHC genes[j]. Immunol Rev, 1998, 166: [8] Ohta Y, Okamura K, McKinney E C, et al. Primitive synteny of vertebrate major histocompatibility complex class I and class II genes[j]. Proc Natl Acad Sci USA, 2000, 97: [9] Figueroa F, Mayer W E, Sültmann H, et al. Mhc classii B gene evolution in East African cichlid fishes[j]. Immunogenetics, 2000, 51(7): [10] Zhang Y X, Chen S L. Molecular identification, polymorphism and expressio nanalysis of major histocompatibility complex class II A and B genes of turbot Scophthalmus maximus[j]. Mar Biotechnol, 2006, 8: [11] Yu S H, Ao J Q, Chen X H. Molecular characterization and expression analysis of MHC class II a and b genes in large yellow croaker (Pseudosciaena crocea)[j].mol Biol Rep, 2010, 37: [12] Stet R J M, Kruiswijk C P, Saeij J P, et al. Majorhistocompatibility genes in cyprinid fishes: theory and practice[j]. Immunol Rev, 1998, 166: [13]. [M]. :, 2005: [14] Chen S L, Tian Y S, Yang J F, et al. Artificial gynogenesis and sex determination in half-smooth tongue sole (Cynoglossus semilaevis) [J]. Mar Biotechnol, 2009, 11: [15] Xu T J, Chen S L. Molecular cloning, genomic structure and expressionanalysis of major histocompatibility complex class I a gene of half-smooth tongue sole (Cynoglossus semilaevis)[j]. Fish Physiol Biochem, 2011, 37: [16] Xu T J, Sha Z X, Chen S L. Unexpected variations of b2-microglobulin gene in the half-smooth tongue sole[j]. Fish & Shellfish Immunology, 2010, 28: [17] Xu T J, Chen S L. Molecular cloning, genomic structure, polymorphism and expression analysis of major histocompatibility complex class IIA and IIB genes of half-smooth tongue sole (Cynoglossus semilaevis) [J]. Fish Physiol Biochem, 2009, 27(2): [18] Klein D, Ono H, O huigin C, et al. Extensive Mhc variability in cichlid fishes of Lake Malawi[J]. Nature, 1993, 364: [19] Brown J H, Jardetzky T S, Gorga J C, et al. Three-dimensional structure of the human class II histocompatibility antigen HLA-DR1[J]. Nature, 1993, 364: [20] Grimholt U, Olsaker I, de Vries Lindstrøm C, et al. A study of variability in the MHC class II b1 and class I 74 / 2015 / 39 / 12

6 a2 domain exons of Atlantic salmon, Salmo salar L[J]. Anim Genet, 1994, 25: [21] Langefors Å, Lohm J, von Schantz T. Allelic polymorphism in MHC class II B in four populations of Atlantic salmon (Salmo salar)[j]. Immunogenetics, 2001, 53: [22] Du M, Chen S L, Liang Y, et al. Polymorphism and balancing selection of MHC class II DAB gene in 7 selective flounder (Paralichthys olivaceus) families[j]. Evidence-Based Complementary and Alternative Medicine, 2011, [23] Du M, Chen S L, Liu Y H, et al. MHC polymorphism and disease-resistance to Edwardsiella tarda in six turbot (Scophthalmus maximus) families[j]. Chin Sci Bull, 2012, 57(25): [24],,,. [J]., 2010, 34(12): [25] Chen S L, Li J, Deng S P, et al. Isolation of female-specific AFLP markers and molecularidentification of genetic sex in half-smooth tongue sole (Cynoglossus semilaevis) [J]. Marine Biotechnology, 2007, 9: [26] Tamura K, Dudley J, Nei M, et al. MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0[J]. Molecular Biology and Evolution, 2007, 24: [27] Nei M, Gojobori T. Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions[j]. Mol Biol Evol, 1986, 3(5): [28] Librado P, Rozas J. DnaSP v5: A software for comprehensive analysis of DNA polymorphism data[j]. Bioinformatics, 2009, 25: [29] Davies C J, Andersson L, Ellis S A, et al. Nomenclature for factors of the BoL Asystem, report of the ISAG BoLA nomenclature committee[j]. Anim Genet, 1997, 28: [30] Xu R F, Li K, Chen G H, et al. Characterization of geneticpolymorphism of novel MHC B-LBII alleles in Chinese indigneous chickens[j]. J Genet Genomics, 2007, 34(2): [31] Xu R F, Li K, Chen G H, et al. Genetic variation within exon 2 of the MHC B-LBII genein Tibetan chicken[j]. Acta Genet Sin, 2005, 32(11): [32] Longeri M, Zanotti M, Damiani G. Recombinant DRB sequences produced by mismatch repair of heterodu plexes during cloning in Escherichia coli[j]. Euro J Immunogenet, 2002, 29: [33] Zorn A M, Krieg P A. PCR analysis of alternative splicingpathways: identification of artifacts generated by heteroduplexformation[j]. Biotechniques, 1991, 11: [34] Du M, Chen S L, Liu Y H, et al. MHC polymorphism and disease resistance to Vibrio anguillarum in 8 families of half-smooth tongue sole (Cynoglossus semilaevis) [J]. BMC Genetics, 2011, 12: 78. [35] Xu T J, Chen S L, Ji X S, et al. MHC polymorphism and disease resistance tovibrio anguillarum in 12 selective Japanese flounder Paralichthys olivaceus families[j]. Fish Shellfish Immunol, 2008, 25, [36] Rakus K.Ł, Wiegertjes G F, Stet R J M, et al. Polymorphism of major histocompatibility complex class II B genes in different lines of the common carp (Cyprinus carpio) [J]. Aquat Living Resour, 2003, 16: [37] Stet R J M, Vries B, Mudde K, et al.unique haplotypes of co-segregating major histocompatibility class II A and class II B alleles in Atlantic salmon (Salmo salar) [J]. Immunogenetics, 2002, 54: [38] Langefors Å, Lohm J, von Schantz T. Allelic polymorphism inmhc class II B in four populations of Atlantic salmon (Salmo salar)[j]. Immunogenetics, 2001, 53: [39],,. SLA-DQB 2 [J]., 2005, 27(2): [40],. MHC IIB [J]., 2006, 30(5): [41] Parham P, Ohta T. Population biology of antigen presentation by MHC class I molecules[j]. Science, 1996, 272(5258): [42],,. DAB [J]., 2008, 54(5): [43],. MHC-DAA [J]., 2009, 31(10): Marine Sciences / Vol. 39, No. 12 /

7 Polymorphisms and balancing selection in the half-smooth tongue sole, Cynoglossus semilaevis NIU Bao-zhen 1, DU Min 1, CHEN Song-lin 2 (1. Key Lab for Quality, Efficient cultivation and Security Control of Crops in Colleges and University of Yunnan province, Honghe University, Mengzi , China; 2. Key Lab for Sustainable Utilization of Marine Fisheries Resources, Ministry of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao , China) Received: Aug., 3, 2014 Key words: half-smooth tongue sole Cynoglossus semilaevis; polymorphism; MHC IIB; balancing selection Abstract: In order to detect the polymorphism, number of alleles, and equilibrium selection of the MHC II B (Cyse-DAB) gene ofthe half smooth tongue sole, Cynoglossus semilaevis, we measured the genetic variation and balancing selection at MHC IIB loci in 10 half-smooth tongue sole by polymorphism chain reaction and the direct sequence method. We cloned and sequenced a 397bp fragment of the MHC class IIB exon2. An amplified fragment spanned the partial exon1, complete intron1, and exon2. Five clones and five fry per individual (n = 50, 10 groups) were selected for MHC IIB sequence analysis. A total of 60 sequences were discovered which revealed 60 alleles, 28 of which were first found in this study and have been submitted to Genbank. The homology similarity was 89.36% among the sixty sequences. Five distinct sequences were presented in each of six individuals which denoted that there were at least three loci in half-smooth tongue sole MHC class IIB genes. Non-synonymous (d N ) substitution was significantly higher than synonymous (d S ) in the peptide-binding region of the MHC IIB gene in nine of the ten half-smooth tongue sole groups. ( 本文编辑 : 谭雪静 ) 76 / 2015 / 39 / 12

48 5 ( Pinctada) ( P. fucata ), 3 [ 7 ] ( P. maxima ) ( P. albina), ( P. margaritifera) ( P. nigra) RAPD, ( P. chemnitzi), ( Pteria penguin) [ 1-2 ] [

48 5 ( Pinctada) ( P. fucata ), 3 [ 7 ] ( P. maxima ) ( P. albina), ( P. margaritifera) ( P. nigra) RAPD, ( P. chemnitzi), ( Pteria penguin) [ 1-2 ] [ 5 6 2 00 9 1 2 South China Fisheries Science Vol. 5, No. 6 Dec., 2009 doi: 10. 3969/ j. issn. 1673-2227. 2009. 06. 009 16S rrna,,, (, 510300) : PCR ( Pinctada fucata) ( P. chemnitzi) ( Pteria penguin)