兴国红鲤MHCⅡ类基因多态性、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 Ⅱ GENE AND ITS ASSOCIATION WITH RESISTANCE/SUSCEPTIBILITY TO AEROMONAS HYDROPHILA IN XINGGUO RED COMMON CARP
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摘要: 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MHC Ⅱ类基因编码序列，分属26个不同的等位基因，其中Cyca-DXA24Cyca-DXA36为新发现的13个等位基因。MHCⅡ类基因片段的长度为624 bp，包括第14个外显子，分别编码信号肽、1和2结构域及连接肽/跨膜区。1结构域的变异明显大于2结构域，表现在1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2结构域的变异位点比例（45.96%和68.42%）。1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 （=dN/dS）为5.742，远远高于非抗原结合位点（non-PBR）及2结构域的0.755、0.592，揭示兴国红鲤MHC Ⅱ类基因的1结构域在进化过程中受到正向选择作用。等位基因Cyca-DXA24 （P0.01）与兴国红鲤对嗜水气单胞菌的抗性相关，等位基因Cyca-DXA3 （P0.05）、Cyca-DXA4 （P0.01）、Cyca-DXA6 （P0.05）、Cyca-DXA33 （P0.05）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类基因的表达量均发生了不同程度的变化，表明MHCⅡ类分子在兴国红鲤的免疫反应中起到重要作用。Abstract: We cloned 171 sequences of MHC class Ⅱ gene were determined from 41 susceptible and 22 resistant individuals 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, a 1 domain, a 2 domain and a connecting peptide (CP)/partial transmembrane (TM), respectively. The mutation-Prone positions of nucleotide or amino acid sites in 1 domain (55.16%, 79.76%) significantly out number than those in 2 domain (45.96%, 68.42%). The values (= dN/dS) were 5.742 for peptide-binding region (PBR), 0.755 for non-PBR in the 1 domain and 0.592 for the 2 domain, implying that the positive selection pressure probably had occurred in the PBR region of the 1 domain. Cyca-DXA24 (P0.01) was significantly correlated with the carp's resistance to Aeromonas hydrophila, while Cyca-DXA3 (P0.05), Cyca-DXA4 (P0.01), Cyca-DXA6 (P0.05) and Cyca-DXA33 (P0.05) alleles were significantly associated with the fish's susceptibility to the disease. Real-time quantitative PCR demonstrated that MHC II genes 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 II gene might play an important role in the immune reactions of C. carpio var. singguoensis.
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Keywords:
- Cyprinus carpio var. singguoensis /
- MHC /
- Polymorphism /
- Expression /
- Disease resistance
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