| In this paper, the S. prenati as the research objiect with cloning the MHCⅡA gene. Preliminarily exploring the gene’s molecular polymorphism, specificity of the tissues expression, the differences in expression parrerns in embryos and larvae. Looking forward to provide certain theoretical basis for the study of disease-resistant breeding in the fish. Major Histocompatibility Complex(MHC) Ⅱclass moleculars which encode the cell-surface transmembrane proteins are higly polymorphic genes in vertebrates, and a part of the genotype strong correlation with fish’s resistance. Firstly, in present research, using homology-based cloning and RACE technology to isolate the complete sequence of MHCⅡgene from S. prenati. Secondly, by means of the gene sequencing method, analyzing its polymorphism of the open reading frame(ORF). Finally, applying the real-time fluorescence quantitative PCR to detect it tissue distribution and expression in embryos and larvae of the same parents. The main research results as follows.(1) The full-length MHCⅡDNA (Genebank accession No. KM670437) fragment of 1143-bp, which included a 64-bp 5’-untranslated region(UTR), a 711-bp coding region, as well as a 135-bp 3’-UTR with a canonical polyadenylation signal(AATAAA) and a 27-bp poly(A) tail. The coded protein had 236 amino acid residues, with a putative molecular weight of 25.94 Kda and a theoretical isoelectric point of 4.60. The secondary structure of the predicted protein, all the characteristic domains present in the MHCⅡprotein of other species could be found in its sequence, including a signal peptide, two extracellular domains(a-1 and a-2), a transmembrane region, and a cytoplasmic domain. Four conseved cysteine residues and rich phosphorylation sites were found in the coded protein sequence. The transmembrane region of MHC class ⅡA molecule contains a classic a amino acides of GXXXGXXGXXXG motif. Its highest homology with Cyprinus carpiovar Jian and the similarity is 83.03%.(2) We got 17 different cDNA sequences of MHCⅡA gene from 8 healthy adults, including four disparate sequences from one fish at most. The analysis showed that it coded at least 15 different amino acid sequences, contained at least 2 loci. One of the 15 sequences with the others’homology is 89.6%-94.2%.50 variation points were found in the coding amino acid sequence and 35 mutation locis focused on a-1 domain with 70% of the total number of mutation sites. It showed that the gene is highly polymorphism.(3) Quantitative real-time RT-PCR demonstrated that MHC ⅡA gene was ubiquitously expressed in 19 tissues of the S. prenati, but the expression level was significant difference, showing the expression of tissue specificity. The high levels of MHC ⅡA transcripts were detected in midkidney, head-kidney, midgut, foregut, hindgut, spleen, red muscle and heart, moderate levels in brain, ovary, liver, fin-ray and white muscle.The highest expression quantity was found in the kidney, it was 15.869-fold that in blood(P<0.05).Low expression in the blood, eye, testis, skin, gill and bladder, the minimum expression level of the bladder accounted for only 38.5% of the blood. It suggests that the gene expression level is closely related to immune organ or tissue.(4) The RT-PCR demonstrated that the expression level of the MHC ⅡA gene during development in 28 embryonic period. The gene was all detected in unfertilized eggs and fertilezation to archenteron-stage embryos. But the expression level from fertilezation one hour to 8 cell-stage embryos all were lower than unfertilized eggs. It shows that its the expression level is primary controled by the maternal factor mRNA in the early embryos of S. prenati. The highest expression quantity was found in 16 cell-stage embryo, it was 2.259-fold that in unfertilized eggs(P<0.05), showing that the expression level was controled by zygotic gene. After embryo into blastocyst stage, However, the gene expression of quantity significantly reduced and the expression of early-blastula stage amount was only 8.6% of the unfertilized eggs(P<0.05). Especially, the gene was almost undetectable expression while the embryos getting into organ formation stage. It showed that the MHC ⅡA expression level of synthetic of embryo itself significantly reduced or not in this stage, may be caused by the increasing of MHC ⅡB gene expression.(5) After detecting 9 period’s larvae, we found that the MHC ⅡA gene expression level was in differernt period of the larcae of a significant difference. The exprssion in the newly hatched larvae was only 2% of the unfertilized egg(P<0.05). However, the gene expression quantity increased significantly in eye pigment and cycle larvae, its was 7.296-fold and 24.794-fold that in unfertilized eggs, respectively(P<0.05). And the expression Quantity which was from the digestive tract through to the yolk sac disappeared larvae were significant higher than before. The larvae of dorasl fin formation period’s expression was reaching peak, and its was 968.412-fold in unfertilized eggs(P<0.05). The different expression pattern may be related to immune organ or tissue gradually formed and accomplished, as well as endogenous exchanged exogenous nutrition. |
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