团头鲂MHCⅡα基因的SNP位点开发、鉴定及与抗病性状关联分析

SNP screening,identification and association with disease resistance of MHCⅡα gene in blunt snout bream(Megalobrama amblycephala)

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 hydrophila to distinguish susceptible and disease resistant individuals.PCR and DNA sequencing were used to screen single nucleotide polymorphisms （SNP） in the MHC Ⅱα gene of blunt snout bream.High-resolution melting and restriction enzyme digestion were used to analyze the relationship between gene pol ymorphism and disease resistance with SNP genotyping.A total of 35 SNP sites （1.45% of total nucleotide） out of the MHC II a gene were screened, including 30 transition sites and 5 transition sites. Among these 35 SNPs, 1 was found to be located in the 5r-untranslated region （UTR）, 16 located in exon,7 located in intron,and 11 located in 3r UTR.Coding sequence region contained 13 SNPs （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 nucle- otide and coding region in 1 domains were 4.88% and 10.98%, respectively, significantly higher than that in α2 domain （1.08% and 3.22%）.There were 4 variable 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 5 successfully 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 221 bp （G/T） and 1 859 bp （G/T） were significant different between susceptible and resistant groups （P〈0.05）.It is indicated that the MHC Ⅱα gene polymorphism is significant correlated with bacterial septicemia resistance.