金钱鱼MHC II β 基因结构、多态性与组织表达分析

为探究金钱鱼(Scatophagus argus)MHC Ⅱβ基因的结构和特性,采用同源克隆和RACE等技术,在获得cDNA全序列的基础上,分析其内含子序列、基因多态性和组织表达情况。结果表明,金钱鱼MHC IⅡβ基因cDNA序列全长1172 bp,其中5′UTR长34 bp,3′UTR长388 bp,开放阅读框(ORF)长750 bp,编码249个氨基酸,包含信号肽、β1结构域、β2结构域、连接肽(CP)、跨膜区(TM)和胞质区(CYT);MHC Ⅱβ基因由6个外显子和5个内含子组成,其中内含子3将β2结构域分开。从43尾金钱鱼的209个有效克隆中,获得209条不同的核苷酸序列,可归为48个等位基因主型,分别命名为Scar-DXB*0101~Scar-DXB*4801,揭示金钱鱼MHC Ⅱβ基因的多态性很丰富。RT-PCR检测发现,金钱鱼MHC Ⅱβ基因在所检测的11种组织中均有表达,其中在脾、鳃、肠和皮肤中表达量较高,在肾、胃、心脏表达量中等,而在眼、脑、肝、肌肉中表达量较低。对健康金钱鱼人工感染嗜水气单胞菌后,发现其MHC Ⅱβ基因在肝、脾、鳃、肾等组织中的表达量均发生了不同程度的变化,证明该基因在金钱鱼免疫反应中有重要作用。在NJ法构建的系统树中,金钱鱼与舌齿鲈、大西洋鲑等硬骨鱼类的亲缘关系相对较近,而与铰口鲨、原鸡、小鼠、人等的亲缘关系则依次渐远。

Genomic structure,polymorphism, and expression analysis of the spotted scat (Scatophagus argus) MHC-Ⅱ β gene

The major histocompatibility complex (MHC) is a gene family common to vertebrates and is encoded by a group of genes closely related to immunological functions. Many studies have indicated that MHC occurs on the surface of all karyocytes and that the MHC II genes of fish differ by species in structure, character, and polymorphisms. The spotted scat (Scatophagus argus) is eurythermic and euryhaline and is widely distributed in brackish water and marine habitats of the Indo-Pacific region. Spotted scat has become an economically important species in southern China in recent years owing to its strong tolerance to a variety of environmental conditions. However, most spotted scat studies in the last two decades have focused on artificial breeding, biological characteristics, gonadal development, reproductive characteristics, and genes related to osmotic adjustment and gonadal development, but not immune system function. Furthermore, farmers have recently found that spotted scat are susceptible to infections by parasites, bacteria, and viruses, which could cause serious economic losses. Thus, we determined the complete cDNA sequence of the spotted scat MHC-IIβ gene for the first time using homology-based cloning and rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR) and analyzed the introns, gene polymorphisms, and tissue expression. As results, the cDNA sequence of the spotted scat MHC-IIβ gene was 1172 bp in length, including a 34 bp 5''-untranslated region (UTR), a 388 bp 3''-UTR, and a 750 bp open reading frame (ORF). The ORF contained the signal peptide, the β1 domain, the β2 domain, a connecting peptide, a transmembrane region, and a cytoplasmic tail. The total length of the gene was 2920 bp. The MHC-IIβ gene contained six exons and five introns (85-1010 bp), and the third intron separated the β2 domain into two parts. This structure differs from some other teleosts and provides new evidence for the lack of conservation of MHC-IIβ gene structure. The molecular formula was C₂₂₃₉H₃₇₃₀N₇₅₀O₉₃₄S₁₉₈, and relative molecular mass was 62.45 kD. Some classic functional sites were found in the amino acid sequence, such as a protein kinase C phosphorylation site, an N-glycosylation site, a casein kinase II phosphorylation site, a tyrosine kinase phosphorylation site, and an N-nutmeg acetoxylation site. Interestingly, the number of protein functional sites in the spotted scat MHC-IIβ gene was very similar to that in other fish, such as common carp, yellow croaker, and Japanese flounder, indicating conservation of MHC in fish. We identified 48 alleles from 209 clones in 43 individuals to examine polymorphisms in the spotted scat MHC-IIβ gene. These alleles were named Scar-DXB^*0101-Scar-DXB^*4801, indicating high polymorphism of the MHC-IIβ gene in S. argus. However, allelic polymorphisms of the MHC-IIβ gene are quite different in fish, which may be an inherent characteristic of the species or it may be related to other factors, such as the founder effect, gene flow, or gene selection during evolution. The RT-PCR analysis showed that the MHC-IIβ gene was expressed in all 11 tissues examined, with the highest levels in spleen, gill, intestine and skin; moderate levels in kidney, stomach, and heart; and low levels in the eyes, brain, liver, and muscle. MHC-IIβ gene mRNA expression levels varied in liver, spleen, gill, and kidney after infection with Aeromonas hydrophila, indicating that the gene has important effects on the immune response. A neighbor-joining tree showed that the S. argus MHC-IIβ gene had a fairly close genetic relationship with those of Paralichthys olivaceus, Dicentrarchus labrax, and Salmo salar and more distant genetic relationships with Ginglymostoma cirratum, Gallus gallus, Mus musculus, and Homo sapiens. These results lay the foundation for screening resistance genes and breeding S. argus for disease resistance.