Molecular Cloning And Analysis Of Genes Involved In Immune Response In Japanese Flounder(Paralichthys Olivaceus)

We have identified by differential display a number of novel genes that are expressed in Japanese flounder (Paralichthys olivaceus) injected with Vibrio anguillarum. Three of these novel genes were reported here. Based on the technicques of DD and RACE,their full length cDNAs were cloned, and their bioinformatics were analyzed by softwares. In addition, a detailed investigation was made on the expression patterns of these differentially expressed cDNAs. Systems for high-level expression of the two new immune-related genes in Escherichia toll were also constructed, and expression regulation were investigated. The main results are reported as follows.1. Cloning and expression analysis of fibrinogenβfrom Japanese flounderFibrinogenβof flounder was obtained using the techniques of DD-PCR and RACE. The full length cDNA sequence of the flounder fibrinogenβcontained a 5' UTR of 60 bp, a 3'UTR of 316bp, and a putative ORF of 1479bp by encoding a polypeptide of 492 amino acids with an estimated molecular mass of 55.72kDa. Sequence analysis revealed that there were a C-terminal domain signature and a potential 16-amino acid signal peptide in the deduced amino acids. In addition, bioinformatic analysis indicted that the protein of the flounder fibrinogenβbelonged to a kind of beta protein and its tetiary structure was quite similar to the crystal structure of chicken. The deduced amino acid sequence of flounder fibrinogenβshowed 81.4%, 68.0%, 55.5% and 53.8% identity with those of Paralichthys olivaceus, Danio rerio, Mus musculus and Homo sapiens respectively. The tempral expression of fibrinogenβwas measured by semi-quantitative RT-PCR. In the controlled fish, the mRNA expression of fibrinogenβcould only be detected in liver and kidney. After 6 hours stimulated by Vibrio anguillarum, the fibrinogenβexpression could be detected in spleen, heart and intestine, and 9 hours later expression could also be found in gill. Moreover, the longer after stimulation, the higher mRNA expression levels of fibrinogenβcould be detected in liver of challenged fish. These results indicated that on one hand fibrinogenβhelped stop bleeding by assisting blood clots to form, on the other hand it might be associated with other cytokines and play a critical role in the host-pathogen.2. Cloning and expression analysis of PoIR1 (Paralichthys olivaceus immune-related gene 1)PoIR1 (P. olivaceus immune-related gene 1, GenBank accession number: EU224373) was obtained using the techniques of DD-PCR and RACE. The complete cDNA contains a 523 bp 5'UTR, a 300 bp open reading frame (ORF) encoding 99 amino acids and a 174 bp 3'UTR. Three exons and two introns were identified in the PoIR1 gene. Blastp similarity comparisons showed PoIR1 had no significant similarity to known protein sequences in the nr database. The primary structure analysis indicated that the gene product contained a typical prokaryotic membrane lipoprotein attachment site, suggesting that it could be lipid modified and anchored to the bacterial membrane. RT-PCR demonstrated that the expression of PoIR1 was quickly induced and drastically increased in liver, kidney, spleen, gills, intestine, heart, skeletal muscle after challenge with V. anguillarum. These results indicated that the PoIR1 played an important role in Japanese flounder immune response. The mature PoIR1 peptide was expressed in the form of fusion protein using pET32a as expression vectors and BL21(DE3) pLysS as expression host. 15% SDS-PAGE and Western Blotting analysis showed an expression protein band of 29KD corresponding to the recombinant mature peptide. The further analysis proved that a great part of the recombinant mature peptide existed as soluble type.3. Cloning and expression analysis of PoIR1 (Paralichthys olivaceus immune-related gene 2)PoIR2 (P. olivaceus immune-related gene 2, GenBank accession number: (EU224372) was obtained using the techniques of DD-PCR and RACE. The complete cDNA contains a 169 bp 5'UTR, a 336 bp open reading frame (ORF) encoding 111 amino acids and a 556 bp 3'UTR. Six exons and five introns were identified in the PoIR2 gene. Blastp similarity comparisons showed PoIR2 had no significant similarity to known protein sequences in the nr database (cutoff E-value=0.0001). RT-PCR demonstrated that the expression of PoIR2 was quickly induced and drastically increased in liver, kidney, spleen, gills, intestine, heart, skeletal muscle after challenged with V. anguillarum. These results indicated that the PoIR2 played an important role in Japanese flounder immune response. The mature PoIR2 peptide was expressed in the form of fusion protein using pET32a as expression vectors and BL21(DE3) pLysS as expression host. 15% SDS-PAGE and Western Blotting analysis showed an expression protein band of 31KD corresponding to the recombinant mature peptide. The further analysis proved that a great part of the recombinant mature peptide existed as soluble type.4. Phylogenetic relationship of 16 pleuronectiformes fish speciesThe utility of the seventh intron of single-copy fibrinogen gene (β-chain;β-fibrinogen intron 7, fibint7) has been successfully explored at different taxonomic levels in studies of birds,reptiles and mammals, however, still lack in those of fishes. Our work is the first to explore the potential of fibint7 as a genetic maker in pleuronectiformes systematics. We also sequenced two complete NADH dehydrogenase (ND6) and Cytochrome Oxidase subunits II (COII) genes from mt genome, given the general thought that analysis of multiple independently inherited genes is especially effective in testing for congruence and estimating organismal phylogeny. The fibint7 sequences of Pleuronichthys cornutus had a high polymorphism because of the minisatellite sequence. We obtained a single objective band in the amplification in the other species. The length of fibint7 of 16 Pleuronectiformes fish species ranged from 81bp to 1087bp, thus maked fibint7 as an inappropriate maker for phylogenetic analysis. We also got homologous complete sequence of ND6 and COII in 16 fishes except Cynoglossus semilaevis, 522bp and 691bp respectively. The genetic distances and phylogenetic trees conducted by ND6 and COII were consistent with the traditional relationship of the 15 flatfishes in the rough. In both trees, Zebrias zebra and Pleuronichthys cornutus clustered firstly, which worth further study.