团头鲂MHCⅡα基因的SNP位点开发、鉴定及与抗病性状关联分析
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摘要
用嗜水气单胞菌(Aeromonas hydrophila)感染团头鲂(Megalobrama amblycephala)区分抗病和易感组,通过PCR扩增及测序在MHCⅡα基因上筛选单核苷酸多态位点(single nucleotide polymorphisms,SNP),利用高分辨率熔解曲线法和限制性内切酶酶切法对SNP进行分型,分析其多态性及与抗病性状之间的关系。在MHCⅡa基因上共筛选出35个候选SNP位点,占MHCⅡa基因总碱基的1.45%,包含30个转换位点、5个颠换位点。其中外显子部分有16个,内含子部分7个,5′非编码区有1个,3′非编码区有11个。有13个SNP位点位于编码区,占总氨基酸位点的5.56%,位于839bp的T/A颠换和1 663bp的A/G转换是无义突变,其余11个SNP位点为有义突变。α1结构域的SNP位点分别占总碱基和总氨基酸位点的4.88%和10.98%,明显高于α2结构域的1.08%和3.22%。MHCⅡα基因的21个抗原结合位点(peptide binding region,PBR)中,有4个位点变异,变异率为19.05%;而non-PBR的变异率仅为8.20%(5/61)。用SPSS软件对分型成功的5个SNP位点在易感和抗病组各100尾中的基因型频率和等位基因频率进行了统计。通过卡方检验分析发现位于1 395bp(T/A)位点的基因型频率和等位基因频率在易感和抗病组中差异极显著,位于221bp(G/T)和1 859bp(G/T)位点的基因型频率和等位基因频率在易感组和抗病组中差异显著。本研究结果表明团头鲂MHCⅡα基因的多态性和抗细菌性败血症性状显著关联。
Blunt snout bream(Megalobrama amblycephala)were infected with Aeromonas hydrophilato distinguish susceptible and disease resistant individuals.PCR and DNA sequencing were used to screen single nucleotide polymorphisms(SNP)in the MHCⅡαgene of blunt snout bream.Highresolution melting and restriction enzyme digestion were used to analyze the relationship between gene polymorphism and disease resistance with SNP genotyping.A total of 35 SNP sites(1.45% of total nucleotide)out of the MHCⅡα gene were screened,including 30 transition sites and 5transition sites.Among these 35 SNPs,1was found to be located in the 5′-untranslated region(UTR),16 located in exon,7located in intron,and 11 located in 3′UTR.Coding sequence region contained 13SNPs(5.56% of total amino acid).Among them,TA transversion located at 839 bp and AG transition located at 1 663 bp were nonsense mutations,and the other 11 SNPs were sense mutations.The percentage of SNPs of nucleotide and coding region inα1domains were 4.88% and 10.98%,respectively,significantly higher than that inα2domain(1.08% and 3.22%).There were 4variable sites in the 21 peptide binding region(PBR),with the mutation rate of 19.05%.However,the mutation rate of non-PBR was just 8.20%(5/61).The genotype and allele frequencies of the 5successfully genotyped SNP sites in the 100 bacterial septicemia susceptible and 100 resistant individuals were statistically different.Result of Chi-square tests showed that the differences of genotype and allele frequencies of SNP(T/A)located at 1 395 bp were extremely significant(P<0.01),SNPs located in 221bp(G/T)and 1 859bp(G/T)were significant different between susceptible and resistant groups(P<0.05).It is indicated that the MHCⅡagene polymorphism is significant correlated with bacterial septicemia resistance.
Blunt snout bream(Megalobrama amblycephala)were infected with Aeromonas hydrophilato distinguish susceptible and disease resistant individuals.PCR and DNA sequencing were used to screen single nucleotide polymorphisms(SNP)in the MHCⅡαgene of blunt snout bream.Highresolution melting and restriction enzyme digestion were used to analyze the relationship between gene polymorphism and disease resistance with SNP genotyping.A total of 35 SNP sites(1.45% of total nucleotide)out of the MHCⅡα gene were screened,including 30 transition sites and 5transition sites.Among these 35 SNPs,1was found to be located in the 5′-untranslated region(UTR),16 located in exon,7located in intron,and 11 located in 3′UTR.Coding sequence region contained 13SNPs(5.56% of total amino acid).Among them,TA transversion located at 839 bp and AG transition located at 1 663 bp were nonsense mutations,and the other 11 SNPs were sense mutations.The percentage of SNPs of nucleotide and coding region inα1domains were 4.88% and 10.98%,respectively,significantly higher than that inα2domain(1.08% and 3.22%).There were 4variable sites in the 21 peptide binding region(PBR),with the mutation rate of 19.05%.However,the mutation rate of non-PBR was just 8.20%(5/61).The genotype and allele frequencies of the 5successfully genotyped SNP sites in the 100 bacterial septicemia susceptible and 100 resistant individuals were statistically different.Result of Chi-square tests showed that the differences of genotype and allele frequencies of SNP(T/A)located at 1 395 bp were extremely significant(P<0.01),SNPs located in 221bp(G/T)and 1 859bp(G/T)were significant different between susceptible and resistant groups(P<0.05).It is indicated that the MHCⅡagene polymorphism is significant correlated with bacterial septicemia resistance.
引文
[1]EDWARDS S V,HEDRICK P W.Evolution and ecology of MHC molecules from genomics to sexual selection[J].Trends Ecol Evol,1998,13(8):305-311.
[2]李雪松,刘至治,赵雪锦,等.“全红”体色瓯江彩鲤MHC-DAB基因多态性及其与鱼体抗病力关系的分析[J].水产学报,2011,35(9):1293-1301.
[3]AOYAGI K,DIJKSTRA J M,XIA C,et al.Classical MHC classⅠgenes composed of highly divergent sequence lineages share a single locus in rainbow trout(Oncorhynchus mykiss)[J].J Immunol,2002,168(1):260-273.
[4]XU S X,REN W H,LI S Z,et al.Sequence polymorphism and evolution of three cetacean MHC genes[J].J Mol Evol,2009,69(3):260-275.
[5]庞纪彩,高凤英,卢迈新,等.广东养殖尼罗罗非鱼3个不同群体MHCⅡB基因序列多态性和遗传分化[J].基因组学与应用生物学,2014,33(2):299-306.
[6]SHEILA C,PRISCILLA S B A,ROLF B,et al.Strong and weak histocompatibility gene differences in mice and their role in the rejection of homografts of tumors and skin[J].Bar Harb Maine,1956,144(2):198-204.
[7]HASHIMOTO K,NAKANISHI T,KUROSAWA Y.Isolation of carp genes encoding major histocompatibility complex antigens[J].Proce Nat Acad Sci,1990,87:6863-6867.
[8]STRAUSBERG R L,FEINGOLD E A,GROUSE L H,et al.Generation and initial analysis of more than 15 000fulllength human and mouse cDNA sequences[J].Proce Nat Acad Sci,2002,99(26):16899-16903.
[9]陈松林.海水养殖鱼类抗病分子育种研究进展及前景展望[J].科技导报,2004(9):10-13.
[10]扶晓琴,丁祝进,饶友亮,等.团头鲂白细胞介素6基因启动子报告载体的构建及活性分析[J].华中农业大学学报,2016,35(1):86-91.
[11]姚伦,吴小平,欧阳珊,等.团头鲂对嗜水气单胞菌的免疫应答[J].湖北农业科学,2009,48(2):416-417.
[12]周凤娟.团头鲂NLRC3-like基因的克隆、表达及其SNP的开发和鉴定[D].武汉:华中农业大学,2015.
[13]PAABO S,WILSON A C.Polymerase chain reaction reveals cloning artifacts[J].Nature,1988,334(6181):387-388.
[14]胡丹丹,刘哲.MHC基因在鱼类遗传育种中德研究与应用[J].水产杂志,2013,26(3):64-67.
[15]STET R J M,VRIES B D,MUDDE K,et al.Unique haplotypes of co-segregating major histocompatibility classⅡA and classⅡB alleles in Atlantic salmon(Salmo salar)give rise to diverse classⅡgenotypes[J].Immunogenetics,2002,54(5):320-331.
[16]CUESTA A,ESTEBAN M A,MESEGUER J.Cloning distribution and up-regulation of the teleost fish MHC classⅡalpha suggests a role for granulocytes as antigen-presenting cells[J].Mol Immunol,2006,43(8):1275-1285.
[17]KRUISWIJK C P,HERMSEN T,FUJIKI K,et al.Analysis of genomic and expressed major histocompatibility class I a and classⅡgenes in a hexaploid Lake Tana African‘large’barb individual(Barbus intermedius)[J].Immunogenetics,2004,55(11):770-781.
[18]XU T J,SUN Y N,SHI G,et al.Characterization of the major histocompatibility complex classⅡgenes in Miiuy Croaker[J].PLoS ONE,2011,6(8):e23823.
[19]PAITI Y K M,NICHOLS K,WALLER J E,et al.Association between DNA polymorphisms tightly linked to MHC classⅡgenes and IHN virus resistance in backcrosses of rainbow and cutthroat trout[J].Aquaculture,2001,94(3/4):283-289.
[20]逄锦菲.“黄海2号”中国对虾高通量SNP筛选及其与抗WSSV性状的关联分析[D].上海:中国海洋大学,2013.
[21]KJGLUM S,LARSEN S,BAKKE H G,et al.How specific MHC class I and classⅡcombinations affect disease resistance against infectious salmon anaemia in Atlantic salmon(Salmo salar)[J].Fish Shellfish Immunol,2006,21:431-441.
[2]李雪松,刘至治,赵雪锦,等.“全红”体色瓯江彩鲤MHC-DAB基因多态性及其与鱼体抗病力关系的分析[J].水产学报,2011,35(9):1293-1301.
[3]AOYAGI K,DIJKSTRA J M,XIA C,et al.Classical MHC classⅠgenes composed of highly divergent sequence lineages share a single locus in rainbow trout(Oncorhynchus mykiss)[J].J Immunol,2002,168(1):260-273.
[4]XU S X,REN W H,LI S Z,et al.Sequence polymorphism and evolution of three cetacean MHC genes[J].J Mol Evol,2009,69(3):260-275.
[5]庞纪彩,高凤英,卢迈新,等.广东养殖尼罗罗非鱼3个不同群体MHCⅡB基因序列多态性和遗传分化[J].基因组学与应用生物学,2014,33(2):299-306.
[6]SHEILA C,PRISCILLA S B A,ROLF B,et al.Strong and weak histocompatibility gene differences in mice and their role in the rejection of homografts of tumors and skin[J].Bar Harb Maine,1956,144(2):198-204.
[7]HASHIMOTO K,NAKANISHI T,KUROSAWA Y.Isolation of carp genes encoding major histocompatibility complex antigens[J].Proce Nat Acad Sci,1990,87:6863-6867.
[8]STRAUSBERG R L,FEINGOLD E A,GROUSE L H,et al.Generation and initial analysis of more than 15 000fulllength human and mouse cDNA sequences[J].Proce Nat Acad Sci,2002,99(26):16899-16903.
[9]陈松林.海水养殖鱼类抗病分子育种研究进展及前景展望[J].科技导报,2004(9):10-13.
[10]扶晓琴,丁祝进,饶友亮,等.团头鲂白细胞介素6基因启动子报告载体的构建及活性分析[J].华中农业大学学报,2016,35(1):86-91.
[11]姚伦,吴小平,欧阳珊,等.团头鲂对嗜水气单胞菌的免疫应答[J].湖北农业科学,2009,48(2):416-417.
[12]周凤娟.团头鲂NLRC3-like基因的克隆、表达及其SNP的开发和鉴定[D].武汉:华中农业大学,2015.
[13]PAABO S,WILSON A C.Polymerase chain reaction reveals cloning artifacts[J].Nature,1988,334(6181):387-388.
[14]胡丹丹,刘哲.MHC基因在鱼类遗传育种中德研究与应用[J].水产杂志,2013,26(3):64-67.
[15]STET R J M,VRIES B D,MUDDE K,et al.Unique haplotypes of co-segregating major histocompatibility classⅡA and classⅡB alleles in Atlantic salmon(Salmo salar)give rise to diverse classⅡgenotypes[J].Immunogenetics,2002,54(5):320-331.
[16]CUESTA A,ESTEBAN M A,MESEGUER J.Cloning distribution and up-regulation of the teleost fish MHC classⅡalpha suggests a role for granulocytes as antigen-presenting cells[J].Mol Immunol,2006,43(8):1275-1285.
[17]KRUISWIJK C P,HERMSEN T,FUJIKI K,et al.Analysis of genomic and expressed major histocompatibility class I a and classⅡgenes in a hexaploid Lake Tana African‘large’barb individual(Barbus intermedius)[J].Immunogenetics,2004,55(11):770-781.
[18]XU T J,SUN Y N,SHI G,et al.Characterization of the major histocompatibility complex classⅡgenes in Miiuy Croaker[J].PLoS ONE,2011,6(8):e23823.
[19]PAITI Y K M,NICHOLS K,WALLER J E,et al.Association between DNA polymorphisms tightly linked to MHC classⅡgenes and IHN virus resistance in backcrosses of rainbow and cutthroat trout[J].Aquaculture,2001,94(3/4):283-289.
[20]逄锦菲.“黄海2号”中国对虾高通量SNP筛选及其与抗WSSV性状的关联分析[D].上海:中国海洋大学,2013.
[21]KJGLUM S,LARSEN S,BAKKE H G,et al.How specific MHC class I and classⅡcombinations affect disease resistance against infectious salmon anaemia in Atlantic salmon(Salmo salar)[J].Fish Shellfish Immunol,2006,21:431-441.