Polymorphism And Molecular Evolution Analysis Of MHC ?A And ?B Genes Of Miiuy Croaker

Compared to higher vertebrates, teleosts as lower vertebrates contain both innate immunity and adaptive immunity, and the immune system is not perfect. Especially teleosts live in the aquatic environment is extremely complex, with a number of microorganism, therefore in the process of antigen invasion, the specific recognition of foreign antigens for the survival of teleosts is significant. Major histocompatibility complex plays an important role in adaptive immunity. MHC is composed of a group of closely linked genes, mainly refers to the MHC class I genes and class II genes. MHC class I genes encode proteins that recognize endogenous antigens and present them to the CD8+ T lymphocytes, while the MHC class II genes encode proteins that are capable of exogenous antigens and present them to CD4+ T lymphocytes.MHC is the highest polymorphism in the genome, in which the polymorphism of MHC class II gene is more studied. The polymorphism of MHC class II gene is expressed in many alleles in each locus, which leads to a lot of antigen binding peptides, and can identify a variety of antigens. MHC polymorphism analysis has become a hot research topic in vertebrate disease resistance. The MHC polymorphism analysis was focus on exon 2, because the code of the amino acid composition of the antigen binding region(PBR). This domain directly determine antigen recognition and presentation, however, few studies focus on the full-length of MHC class II gene. My study has yielded the conclusions as follow:1. As an important economic marine species, miiuy croker(Miichthys miiuy) has become necessary for breeding by molecular markers. The specific primers were designed based on the c DNA sequence of miiuy croker MHC IIA(DAA) and IIB(DAB), respectively. And then, the c DNA of 26 miiuy croker were used to amplify the full-length c DNA sequence of DAA and DAB. 18 positive clones of each individual were selected and sequenced, and 39 novel DAA alleles and 47 novel DAB alleles were identified.2. The 40 different Mimi-DAA sequences can translate into 30 unique amino-acid sequences and 14 alleles have identical amino acid sequences. The result of sequence alignment with complete Mimi-DAA sequences showed deletions in amino-acid position 95, nucleotide position 283-285, and the remaining alleles did not show any gaps. There were 79 variable sites in CDS from 40 Mimi-DAA genes and 56 variable sites is present in exon 2 which encodes alpha-1 domain of Mimi-DAA. When the deduced amino acid sequences from the 40 different alleles were compared it was clear that exon 2 encoded the highest level of diversity as compared to the other exons(exon 1, 3, and 4). The number of variable sites in non-PBR is less compared to the PBR. The codon-based maximum likelihood(ML) approach and Data Monkey ML methods were implemented to test the selective pressure imposed on individual site–domains for the 40 Mimi-DAA sequences. Total 22 sites were singled out for candidates of robust positively selected sites. Thereinto, 19 sites were located on the ?1 domain encoded by the second exon, and the other 3 sites were situated in ?2 domain encoded by the third exon.3. Compared with other teleost, more DAB alleles were identified in miiuy croaker. The 48 different Mimi-DAB sequences can be translated into 34 unique amino-acid sequences. A total of 103 variable sites were present in CDS from 48 Mimi-DAB alleles and 72 variable sites present in exon 2, which encodes the ?1 domain of Mimi-DAB. The average ratio(dN/dS) was 1.531 for the complete exon 2 of the 48 Mimi-DAB alleles, which was significantly higher than the average ratio for other exons. The average ratio of exon 1 and exon 6 was 0, indicating that the high nucleotide diversity middle of the Mimi-DAB alleles, indicated that positive selection only in the middle part of Mimi-DAB. In total, 18 common sites were detected containing significantly positive selection, and seventeen positively selected sites were located on the ?1 domain encoded by the second exon, and the other site was situated in the ?2 domain encoded by the third exon. The polymorphism of MHC class II genes provides an important resource for analyzing the association between the polymorphism of MHC gene and disease susceptibility/resistance, and for molecular selective breeding of miiuy croaker with enhanced disease resistance.