为探究青海湖裸鲤中主要组织相容性复合体 (MHC) 基因在免疫功能调节过程中可能发挥的作用，实验利用cDNA末端快速扩增技术(RACE技术)克隆获得了青海湖裸鲤MHCⅡα/β基因和伴侣基因Ii全长cDNA序列以及花斑裸鲤MHCⅡα/β和Ii基因的编码区序列。将获得的基因序列进行对比分析，结果显示，两种鱼中该基因的序列特性基本一致。系统发生树结果显示，两种鱼的MHCⅡ与鲤科鱼类在进化地位上更接近，聚为一支。两种鱼中MHCⅡα/β氨基酸序列均包括信号肽、两个功能区、跨膜区和胞质区，且均在跨膜区发现1个在不同物种中的保守结构。实时荧光定量PCR (qRT-PCR)结果显示，青海湖裸鲤MHCⅡ主要在肾脏、脑、鳃、肝脏、臀皮中表达较高，而花斑裸鲤主要在脑、肌肉、眼、鳃中高表达。对比正常状态和感染水霉后青海湖裸鲤鳃、肾脏、肝脏、肠组织中MHCⅡ 基因的表达变化，发现MHCⅡ 3个基因在肾脏组织中的表达水平显著下调，而MHCⅡα/β基因在鳃、肝脏、肠组织中均显著上调。对青海湖裸鲤进行不同盐碱胁迫处理后，检测到鳃、肾脏组织中MHCⅡ 基因的mRNA水平均显著降低。对两种鱼中MHCⅡα/β基因的多态性进行分析，分别获得青海湖裸鲤和花斑裸鲤MHCⅡα 8种、12种等位基因型，分别编码23和24种氨基酸序列，且这种序列多态性主要集中在α1功能区；MHCⅡβ 分别获得12、14种等位基因型，编码22、23种氨基酸序列。青海湖裸鲤MHCⅡα/β 的多态性均低于花斑裸鲤，暗示其与青海湖裸鲤的盐碱耐受过程联系不紧密。遗传多样性分析结果表明群体多样性在两个物种中均较高，其单倍型多样性接近1，且核苷酸多样性之间的差异较小。花斑裸鲤MHCⅡ 基因的单倍型多样性和核苷酸多样性均略高于青海湖裸鲤，反映了青海湖裸鲤种群稳定性略低于花斑裸鲤。研究表明，MHCⅡ 分子不仅在青海湖裸鲤和花斑裸鲤的免疫防御应答中发挥着重要作用，还可能参与了青海湖裸鲤的盐碱耐受过程，而基因多态性与盐碱耐受的关联度不高。

The major histocompatibility complex (MHC) is crucial in immune function regulation and is a primary factor in immune responses within organisms. To explore the characteristics and roles of this gene in Gymnocypris przewalskii, the full-length cDNA sequences of MHCⅡ and chaperone gene Ii were obtained by cloning using rapid-amplification of cDNA ends (RACE) technique. Meanwhile, the sequences of the coding regions of MHCⅡ and Ii in G. eckloni were also obtained and compared. Sequence alignment and analysis revealed that the characteristics of respective genes were largely identical in both species. The construction of phylogenetic tree indicated the closer evolutionary position of MHCⅡ in both fish, clustered to one family, Cyprinidae. The amino acid sequences of MHCⅡα/β from two species both contained a signal peptide, two functional domains, a transmembrane domain, in which a conserved structure found in many different species located, and a cytoplasmic region. Quantitative real-time PCR (qRT-PCR) results showed that MHCⅡ was highly expressed in the kidney, brain, gill, liver, and skin tissue around the anal fin of G. przewalskii, while the expression of which displayed a higher level in the brain, muscle, eye, and gill of G. eckloni. The expression of MHCⅡ in gill, kidney, liver, and intestinal tissues was also compared between normal and Saprolegnia infected individuals of G. przewalskii, results demonstrated that the expression level of 3 genes in MHCⅡ was significantly down-regulated in the kidney tissues, while α, β genes were significantly up-regulated in gill, liver and intestinal tissues. MHCⅡ mRNA levels were significantly reduced in gill and kidney tissues after different saline stress treatments on G. przewalskii. Gene polymorphism analysis indicated that MHCⅡα has 8 and 12 allelic genotypes, encoding 23 and 24 amino acid sequences in two species, respectively, with sequence polymorphisms primarily in the α1 functional region. MHCⅡβ had 12 and 14 allelic genotypes, encoding 22 and 23 amino acid sequences in two species, respectively. The lower polymorphisms in MHCⅡ α/β of G. przewalskii suggested a weaker association with salinity tolerance compared to G. eckloni. Genetic diversity analysis showed high population diversity in both species, with haplotype diversity approaching 1 and minimal differences in nucleotide diversity. The slightly higher haplotype and nucleotide diversity of the MHCⅡ in G. eckloni indicated a somewhat greater population stability compared to G. przewalskii. Our research verified that MHCⅡ not only plays an important role in immunologic defence of two kinds of fish but also involved in the salinity tolerance process of G. przewalskii, while gene polymorphisms were not highly associated with salinity tolerance.

Q 786；S 917.4

国家自然科学基金 (31960741, 31660745)；青海省科技项目 (2016-ZJ-940Q)