Cloning, Polymorphism And Expression Study Of Two Subtypes MHC Class Ⅱ B From Half-smooth Tongue Sole (Cynoglossus Semilaevis)

Half-smooth tongue sole (Cynoglossus semilaevis) is an important economic fish, and has many strongpoints such as grow fast in aquaculture. Recently, there are many studies focus on its aquaculture, development and population genetics; but little is known about its important genes such as immune-related genes. Major histocompatibility complex(MHC)encodes immunoglobulin-like receptors in nearly all vertebrate and is highly polymorphic gene family. It is given great attention for its important roles in immune systems. At present, many MHC genes are studied in several kinds of fishes. This paper discussed the cDNA of MHC class II B got from half-smooth tongue sole, studied its polymorphism, discovered the two subtypes, and studied the two subtypes'specific genomic organization and expression pattern.First of all, the Open Reading Frame (ORF) of MHC class II B was got from the cDNA library of half-smooth tongue sole constructed by the lab, and then the full cDNA sequence was got by using RACE (Rapid amplification of cDNA ends, RACE). The cDNA comprises a 28bp 5'UTR (Untranslated region, UTR), a 750bp ORF and a 437bp 3'UTR. The first 21 putative amino acids form the signal peptide, and the 215th-237th amino acids constitute the transmembrane region. There are four conserved cysteines in theβ1 andβ2 domain; and also one N-glycosylation site, four protein kinase C phosphorylation site, six casein kinase II phosphorylation site, two tyrosine kinase phosphorylation site and four N-myristoylation site in the putative amino acid. The secondary structure shows that class II B protein is a mixed protein, and its tertiary structure is very similar to HLA-DRB'. Its putative amino acid identity is quite high with the same gene from three-spined stickleback, Japanese flounder and turbot; and it clusters first with that from three-spined stickleback, then with Japanese flounder, turbot and large yellow croaker in phylogenetic tree, which demonstrate half-smooth tongue sole's closer relationship with those fishes.Then ORF polymorphism was studied by using primers in 5'UTR and 3'UTR and high fidelity polymerase from 9 individuals. Positive clones were sequenced from both directions, which resulted in 24 nonredundant sequences. There are apparent divergence among those sequences, which cluster into two subgroups, and named as MhcCyse-DAB1 and MhcCyse-DAB2, respectively. MhcCyse-DAB1 includes 14 alleles, while MhcCyse-DAB2 includes 8 alleles; and the most variable region isβ1 domain. Most of the substitutions among alleles are nonsynonymous, which lead to 22 alleles from the 24 sequences. There are also two sequences, which differ from all the other sequences in nucleotides, but have the same putative amino acids as Cyse-DAB1*0101 and Cyse-DAB2*0201, respectively, so they are named as Cyse-DAB1*0102 and Cyse-DAB2*0202, respectively. The intragroup alleles'divergence in Cyse-DAB1 and Cyse-DAB2 is 0.4-6.7 and 0.4-8.5, respectively; and the intergroup alleles between Cyse-DAB1 and Cyse-DAB2 is 8.5-16.6. Still, there are another two sequences which has a deletion mutation of 2bp and 7bp, respectively; and are presumed as pseudogenes. For they have similar nucleotide sequence as from Cyse-DAB1 and Cyse-DAB2, so they are named as Cyse-DAB1-pse1 and Cyse-DAB2-pse1, respectively. And most of the individuals expresse both of the two subtypes, except the S1 individual which cannot be detected the Cyse-DAB2's expression..At the same time, 3'UTR was studied and two kinds of 3'UTR sequences were discovered. It turns out that each subgroup own one 3'UTR sequence, which indicates that each subgroup has at least one locus. However, the divergence between the two 3'UTR sequence is small, which includes only two basepair variations and 1bp difference in length.The study of intron 1 from 5 individuals indicates similar results: intron 1 from the same subgroup cluster first and distinct difference was discovered between the two subgroups. Five kinds of Cyse-DAB1 intron1 and five kinds of Cyse-DAB2 intron1 sequences were got, which indicate that each subgroup has at least five loci and individual study shows that each individual has at least one to two loci of each subgroup.The genomic whole length was got by primers in 5'UTR and 3'UTR and Extaq. It turns out that both the two subtypes consist of six exons and five introns, both of which span about 2.4kb. The two subtypes have the same exon length and distribution, but have great divergence in the intron length and sequence. It shows that only intron 1 can be the parameter to distinguish the subgroups, all others cannot for the similarity and crossover in the nucleotide sequences. There are also microsatellites in intron 1, 2 and 4. The genomic sequence of Cyse-DAB2 from S1 individual was also got, and no mutation in RNA splicing and no deletion\insertion mutation in ORF was discovered, which show that the expression of Cyse-DAB2 may be relevant to the body's physiological environment.The expression profile of different tissues and developmental stages were studied by using RT-PCR. The two subtypes have similar expression pattern in different tissues, and express in most tissues (liver, spleen, kidney, gill, brain, heart, intestine, ovary and testis), and have high expression in spleen, kidney and heart; while the expression in muscle and fin cannot be detected and Cyse-DAB2 has a lower expression in gill, intestine, ovary and testis than Cyse-DAB1. In developmental stages, Cyse-DAB1 can be detected in fertilized egg, gastrula stage, eye bud stage and 5-22 day after hatching, and has a drop from 5 day after hatching; while Cyse-DAB2 cannot be detected under the same condition, which indicate that the two subtypes may have different functions.