The Cloning, Expression And Polymorphism Of MHC Class â…¡ B Gene Of Grass Carp (Ctenopharyngodon Idella) Populations Of Yangtze River

The major histocompatibility complex(MHC) is a group of closely linked loci presented in remarkably similar form in all mammals and perhaps in all vertebrates.It is hetero-dimer gene encoding glycoprotein which presents peptides to T4 cell, and plays a central role in immune system.This research studied the cloning, expression, polymorphism and the associations between some genotypes and resistance ability of major histocompatibility complex(MHC)using the methods of PCR, RT-PCR from the important freshwater fish in our country, Ctenopharyngodon idella.Our aim was to screen some MHCⅡB alleles associated with resistance in order to realize application of molecular markers in fish breeding.(1) To investigate the evolutionary patterns and population genetic variation of grass carp (Ctenopharyngodon idella) major histocompatibility complex (MHC), the DNA fragments of exon 2 and exon 3, encodingβ1 domain and P2 domain of MHC ClassⅡP respectively, and the intron between them from three grass carp populations of collected from Yangtze River were cloned and sequenced. A total of 34 alleles was defined from 572 clones, each including an exon 2 of 130-136bp, an exon3 of 218bp and an intron of 81-371bp in length. Analysis of alignments of nucleotide acid and putative amino acid sequences revealed that there were 106 variable nucleotide acid (78%) and 40 amino acid (88%) sites in exon 2,100 variable nucleotide acid (45%) and 41 amino acid (56%) sites in exon 3, suggesting more variability inβ1 than inβ2. Two phylogenetic trees constructed separately from sequences ofβ1 andβ2 domains showed 5 distinct lineages with high bootstrap values, considering the different introns of alleles in lineages, which indicated it should harbor 5 loci at least in grass carp. The numbers of synonymous substitutions per synonymous site (dS) and of nonsynonymous substitutions per nonsynonymous site (dN) were calculated for peptide binding residues (PBR) and non-PBR of theβ1 domain andβ2 domain. The results showed the ratio of dN to dS in PBR was 2.03 (P<0.05), whereas ratios in non-PBR andβ2 domain were lower than 1, implying divergent selection impacted at MHC of grass carp. Analysis of molecular variance (AMOVA) based on allele frequency, FST=0.37%, revealed little genetic differentiation among populations of grass carp in Yangtze River. (2) Semiquantitative RT-PCR demonstrated that high expression was detected in kidney, intestine and heart, low expression in muscle and the brain was not expressed. Challenge of Grass Carp with pathogenic bacteria, Aeromonas hydrophila,resulted in a significant decrease and rise in the expression of MHC classⅡB mRNA from 5 h to 88h after infection in liver,kidney,heart,intestine and gill except spleen in which the expression maintained the upward trend,followed by a recovery to normal level after 112h,and the expression still kept up a high level in intestine at 112h.