| Major histocompatibility complex (MHC) is a gene group of inmense polymorphism in vertebrate genomes, and MHC class Ⅰ molecules are composed by α chain and β2m microglobulin. The α chain showed high polymorphism, including extracellular domain (α1, α2, α3), transmenbrane region (TM) and cytoplasmic (CY), in which the peptide binding region (PBR) of α1 and α2 domains is capable for combining antigen peptide and inducing specific immune response. The molecular structure and functional diversity is caused by polymorphism of MHC class Ⅰ, and leading to differences of specific pathogen resistance. Therefore, the investigation of polymorphic characteristics of MHC classⅠα chain is help to reveal the molecular evolution and immune mechanism. In this study, the genomic structure and polymorphic characteristics of MHC classⅠα chain of low vertebrate Ctenopharyngodon idellus (Ctid) were investigated, and compared with other three vertebrates including mouse, cattle and chicken.Firstly, total blood RNA of three individuals was extracted, the Ctid-MHCⅠα gene was amplified by RT-PCR method, then cloned into pMD18-T vector and transformed into E.coli DH5α competent cells, thus the 20 positive clones of each individual were sequenced. Secondly, the molecular characteristics of polymorphic Ctid-MHCⅠα gene was analysed by bioinformatics software, and compared to other vertebrate including mouse, cattle and chicken. The results showed that 1027bp specific band was identified and 54 Ctid-MHC Ⅰα cDNA sequences were obtained.1-3 MHCⅠα gene types were existed in each individual,5 Ctid-MHC Ⅰα gene sequences were picked and uploaded to GeneBank database, then the accession number of Ctid-MHC Ⅰα gene were obtained (KF831073-KF831077). At the same time, MHC class Ⅰαchain accession number of mouse (KF831063-KF831067), cattle (KF831068-KF831072) and chicken (KF274027-KF274031) were obtained as well. Sequence analysis results showed that Ctid-MRC Ⅰα gene encoded 332 amino acids (aa), including signal peptide region (21aa), α1 domain (88aa), α2 domain (90aa), α3 domain (91aa) and TM/CY domain (42aa). Ctid-MHCⅠα gene was rich of genetic diversity, a total of 150 variable amino acid residues located in all five domains,102 of them located in α1 and α2 domains. There were 16 polymorphic sites (Wu-Kabat score≥4) in PBR,9 and 7 sites in α1 and α2 domains respectively, in which the variability with high scores (Wu-Kabat score≥8) was found at four amino acid residues (55,77,80 and 125). A total of 11 polymorphic sites located in a helices or β sheets corresponding to the key position of antigen presentation from PBR spatial structure model. In addition, the ratio (R) of nonsynonymous (dN) and synonymous substitutions (dS) of the Ctid-MHC I a gene was less than 1, indicating its evolution process was influenced by nagative selection, while it was positive selection of other vertebrates. Phylogenetic analysis suggested that Ctid-MHC I a molecules grouped into a single branch and shared distantly genetic relationship with other vertebrates, individuals continued to differentiate into sub-branch with no significant differences.In conclusion, highly polymorphic characteristics existed in both Ctid and mouse, cattle, chicken MHC I a genes, and mostly concentrated in al and a2 domains. PBR spatial structure of four species was highly similar, and polymorphic amino acid residues were mainly distributed in the key position of antigen-presention a helix or p sheet. While Ctid-MHC I a molecules shared distantly genetic relationship and different evolution process compared with other vertebrates. This study not only clarifies the polymorphic characteristics and molecular evolution of Ctid-MHC I a genes from the point of genetics, but also lays the theoretical foundation to further explore the biological characteristics of MHC molecules of lower vertebrates. |
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