兴国红鲤MHCⅡ类α基因多态性、表达及其与鱼体抗病力关系的分析

THE ANALYSIS OF THE POLYMORPHISM AND EXPRESSION OF MHC CLASS IIα GENE AND ITS ASSOCIATION WITH RESISTANCE/SUSCEPTIBILITY TO AEROMONAS HYDROPHILA IN XINGGUO RED COMMON CARP

对41尾感病和22尾抗病兴国红鲤的308个有效克隆进行测序,获得171条不同的MHCⅡ类α基因编码序列,分属26个不同的等位基因,其中Cyca-DXA24—Cyca-DXA36为新发现的13个等位基因。MHCⅡ类α基因片段的长度为624 bp,包括第1—4个外显子,分别编码信号肽、α1和α2结构域及连接肽/跨膜区。α1结构域的变异明显大于α2结构域,表现在α1结构域中核苷酸和氨基酸变异位点比例(55.16%和79.76%)明显高于α2结构域的变异位点比例(45.96%和68.42%)。α1结构域的PBR区的非同义碱基替换率(dN)与同义碱基替换率(dS)的比值ω(ω=dN/dS)为5.742,远远高于非抗原结合位点(non-PBR)及α2结构域的0.755、0.592,揭示兴国红鲤MHCⅡ类α基因的α1结构域在进化过程中受到正向选择作用。等位基因Cyca-DXA24(P<0.01)与兴国红鲤对嗜水气单胞菌的抗性相关,等位基因Cyca-DXA3(P<0.05)、Cyca-DXA4(P<0.01)、Cyca-DXA6(P<0.05)、Cyca-DXA33(P<0.05)与兴国红鲤对嗜水气单胞菌的易感性相关。荧光定量PCR结果表明,MHCⅡ类α基因在健康兴国红鲤的肾、肝、鳃等10个组织均能普遍表达。人工感染嗜水气单胞菌后,肾、肝、脾3个组织中的MHCⅡ类α基因的表达量均发生了不同程度的变化,表明MHCⅡ类α分子在兴国红鲤的免疫反应中起到重要作用。

We cloned 171 sequences of MHC class IIαgene were determined from 41 susceptible and 22 resistant indi-viduals of Cyprinus carpio var. singguoensis. These sequences belonged to 26 alleles, thirteen of which were novel （Cyca-DXA24-Cyca-DXA36）. The total length of the sequence was 624 bp, consisting of a 33 bp exon1, a 252 bp exon2, a 285 bp exon3, and a 54 bp exon4. These exons encoded a partial signal peptide, aα1 domain, aα2 domain and a con-necting peptide （CP）/partial transmembrane （TM）, respectively. The mutation-Prone positions of nucleotide or amino acid sites in α1 domain （55.16%, 79.76%） significantly out number than those in α2 domain （45.96%, 68.42%）. The ωvalues （ω=dN/dS） were 5.742 for peptide-binding region （PBR）, 0.755 for non-PBR in theα1 domain and 0.592 for theα2 domain, implying that the positive selection pressure probably had occurred in the PBR region of the α1 domain. Cyca-DXA24 （P〈0.01） was significantly correlated with the carp’s resistance to Aeromonas hydrophila, while Cyca-DXA3 （P〈0.05）, Cyca-DXA4 （P〈0.01）, Cyca-DXA6 （P〈0.05） and Cyca-DXA33 （P〈0.05） alleles were signifi-cantly associated with the fish’s susceptibility to the disease. Real-time quantitative PCR demonstrated that MHC IIαgenes were ubiquitously expressed in ten tissues. The expression was up-regulated in kidney, down-regulated in liver and spleen during infection. Our results indicated that the MHC class IIα gene might play an important role in the im-mune reactions of C. carpio var. singguoensis.