Half Smooth Tongue Sole Mhc â…¡ Gene Cloning And Correlation With Disease Resistance And Genetic Linkage Analysis

Half-smooth tongue sole (Cynoglossus semilaevis) is an important economic fish, and accounts for a high percentage in fish aquaculture in coastal area of China. Half-smooth tongue sole has to face many kinds of pathogens during its growth in aquiculture. Hence, in order to understand its main immune genes and improve its anti-disease ability, we cloned the major histocompatibility complex (MHC) class II genes from half-smooth tongue sole, and studied the open reading frame (ORF) polymorphism, genomic structure of A gene and also the developmental stages expression of B gene; moreover,200half-smooth tongue sole individuals were injected with Vibrio anguillarum in order to find the potential anti-bacterial or susceptible alleles; meanwhile, three families were used to analyze the linkage relationship of different alleles of class II genes. The main results are listed below:1. Expression study of two B genes in developmental stagesAccording to previous study, there are two B genes in half-smooth tongue sole, named as Cyse-DAB and Cyse-DBB. Diffferent expression patterns were found in19developmental stages using the two genes’ specific primes. Cyse-DAB was undetectable from fertilized eggs to middle gastrula stage. Its expression was observed again from tail-bud stage and increased until2-d larvae, then it decreased in5-d larvae and could not be detected in16-d larvae, but the expression of Cyse-DAB was high just before metamorphosis. Cyse-DBB had a continuous expression from unfertilized eggs to2-d larva. The expression of Cyse-DBB was not detected in5-d larvae, but high expression was detected in16-d larvae. The expression of both genes decreased when metamorphosis began, and had low expression during metamorphosis; then the expression increased and high expression was found in56-d and87-d fingerlings. The different expression patterns in early developmental stages indicated that the two B genes might play different or complementary roles in those stages.2. Cloning, ORF polymorphism and genomic structure of class ⅡA geneA992bp cDNA encoding A gene was cloned from spleen of half-smooth tongue sole, and the ORF is717bp in length, encoding a pepetide of238amino acids. The immunoglobulins and major histocompatibility complex proteins signature and conserved cysteines were identified in the primary structure of the A protein. The secondary structure indicates that A protein is a complex protein. Sequence alignment and phylogenetic analysis indicate that half-smooth tongue sole A protein has a close relationship with that from turbot, Japanese flounder and Atlantic halibut.Nine individuals were used to study the ORF polymorphism and25peptides were identified. Sequence alignment and phylogenetic analysis indicate that those peptides had divergent sequences, clustered into two groups, so they should belong to two A genes and named as Cyse-DAA and Cyse-DBA accordingly. Cyse-DAA and Cyse-DBA has11and14alleles, respectively. The two A genes’specific amino acids in specific positions are Asn91, Ser92, Ser94, Gly95, Lys101and Lys103for Cyse-DAA, and Thr/Ala91, Val/Gly92, Glu/Gln94, Ala95, Leu101and Asn103for Cyse-DBA. Phylogenetic analysis with other teleosts who has at least two A loci indicates that the two A genes’alleles from half-smooth tongue sole clustered into two groups, then joined as a big branch, then with other A genes from other species, which suggests the originating time of two A genes should be after half-smooth tongue sole seperating from other species.The whole genomic sequences indicate that both of the two A genes are comprised of four exons and three introns, and the distribution and length of the exons are same, but the introns are quite different:Cyse-DAA’s intron2is136bp in length, while Cyse-DBA’s intron2is205bp long; but the two genes’intron1and intron3are not quite divergent. Intron3is well conserved, all are90bp long; while in intron1a (GTCA) repeat of about100times was discovered.3. Vibrio anguillarum infection and the search for anti-bacterial/susceptible allelesTwo hundreds half-smooth tongue sole individuals were selected for the V. anguillarum intraperitoneal infection experiment. The first20died individuals and20active ones were screened for the class II genes’alleles. Five clones of each gene were sequenced from each individual. Then60peptides of B genes’were identified, including32Cyse-DAB alleles and28Cyse-DBB alleles. The two B genes have specific nucleotides and amino acids in specific positions. And13Cyse-DAB intron1and11Cyse-DBB intron1sequences were found. Cyse-DAB’s allele Cyse-DAB*0701and Cyse-DAB*1301are potential susceptible alleles with significant difference (P value is0.001and0.01, respectively). Though the difference is not significant in Cyse-DAB’s allele Cyse-DAB*0601, Cyse-DAB*0706, Cyse-DBB’s allele Cyse-DBB*0101and Cyse-DBB*1301, these four alleles are potential anti-bacterial alleles for they were only detected in several surviving individuals.According to the amino acids in exon2and exon3,85peptides of A genes’were identified, including41Cyse-DAA alleles and45Cyse-DBA alleles. The two A genes’ specific nucleotides and amino acids are well conserved. The length of intron2of the two A genes are also well conserved, and three Cyse-DAA intron2and six Cyse-DBA intron2sequences were found because of single nucleotide polymorphism. Though the difference is not significant, Cyse-DAA’s allele Cyse-DAA*0901, Cyse-DBA’s allele Cyse-DBA*1101and Cyse-DBA*1401are potential susceptible alleles with higher frequencies in dead individuals, and Cyse-DAA’s allele Cyse-DAA*0201, Cyse-DAA*1101, Cyse-DBA’s allele Cyse-DBA*0401, Cyse-DBA*1102, Cyse-DBA*1801and Cyse-DBA*2201are potential anti-bacterial alleles for they had higher frequencies in surviving individuals than dead ones.4. Linkage analysis of alleles from class II genesThough only one fragment was detected by Southern blot analysis after PstI digestion using B genes’probe, the different sequences of B genes support the conclusion that two B genes exist in half-smooth tongue sole. Three families were used for linkage analysis. Using poly-PCR and different primer combination, the linkage relationships are as follows:Cyse-DAB’s allele Cyse-DAB*0403and Cyse-DAB*0601are linked with a recombination fraction of8.00%; Cyse-DAB’s allele Cyse-DAB*0601and Cyse-DBB’s allele Cyse-DBB*0101are tightly linked; Cyse-DBB’s allele Cyse-DBB*0101and Cyse-DBB*1601are linked with a recombination fraction of4.17%; Cyse-DAB’s allele Cyse-DAB*2401and Cyse-DBB’s allele Cyse-DBB*2501are tightly linked; Cyse-DAB’s allele Cyse-DAB*0403and Cyse-DBB’s allele Cyse-DBB*0501are linked with a recombination fraction of14.00%. These information indicates that the two B genes repeat at least twice in one chromosome with the unit of a tightly linked Cyse-DAB and Cyse-DBB. Sequencing of B genes from about four offsprings from each family indicates allele’s inheritance from parents to the offsprings.For the nucleotide variations in A genes are quite high, so poly-PCR is not suitable here and only one allele’s specific primer could be used in each PCR. Compared with the B genes’alleles from the same parent indicated that Cyse-DAA’s allele Cyse-DAA*2201are not linked with three of B’s sequences (Cyse-DAB*0601, Cyse-DBB*0101and Cyse-DBB*1601) in family2, and Cyse-DBA’s allele Cyse-DBA*3101are not linked with two of B’s sequences (Cyse-DAB*0403and Cyse-DBB*0501) in family3for the high recombination fraction near50%. Sequencing of A genes in the offsprings indicates alleles have good inheritance and separation from parents to the offsprings.