Molecular Cloning And Analysis Of Differentially Expressed Genes From Japanese Flounder (Paralichthys Olivaceus) Injected With Vibrio Anguillarum

The mRNA Differential Display technique (DD)was performed to identify and isolate differentially expressed cDNAs representing transcripts from Japanese flounder( Paralichthys olivaceus ) injected with Vibrio anguillarum. Based on the technicques of DD and RACE,three full length cDNAs of Japanese flounder wre cloned,and their bioinformatics were analyzed by softwares. In addition, a detailed investigation was made on the expression patterns of these differentially expressed cDNAs. The main results are reported as follows.1. Isolation of differentially expressed cDNAs from Japanese flounder injected with Vibrio anguillarum by DDTotal 20 differentially expressed cDNAs were isolated, recoveried, cloned and sequenced, of which 9 were confirmed to be positive fragments, after elimination of false-positives by RT-PCR expression studies. Among these 9 positive fragments, BLAST analysis indicated that two of them shared high homology with known genes, one was homologous with complement component C3(98% identity),another showed 98% identity with TEGT(testis-enhanced gene transcript), the others were new cDNA fragments and no high homologous genes were found in Genebank database.The tempral expressions of C3 was measured by semi-quantitative RT-PCR. In the controlled fish,the mRNA expression of C3 could only be detected in liver. After 6 hours stimulated by Vibrio anguillarum,the C3 expression could be detected in intestine, and the longer after stimulation,the higher expression could be observed in liver and intestine of challenged fish. The result indicated that C3 might play a critical role in the fish immune response.Analysis of the semi-quantitative RT-PCR showed that the mRNA expression of TEGT could be detected in all the studied tissues of controlled fish, and was up-regulated after 6 hours stimulated in challenged fish, especially in spleen. The results suggested that TEGT might play a critical role in the host-pathogen interaction.2. Cloning and expression analysis of IL-1RII from Japanese flounderThe IL-1RII gene was cloned from Japanese flounder by using the techniques of DD-PCR and RACE.The full length cDNA of IL-1RII contained a 5' untranslated region (UTR) of 100 bp, an open readingframe(ORF) of 1263bp by encoding a polypeptide of 420 amino acids with an estimated molecular mass of 46.65 kDa and a 3' UTR of 430bp. In addition, three repeated AU sequences(ATTTA) were found in the 3' UTR and a potential 16-amino acid signal peptide in the polypeptide. Two Ig-like domains and a transmembrane region were detected in the extracellular portion of the flounder IL-1RII. Morover, bioinformatic analysis showed that the protein of the flounder IL-1RII belonged to a mixed protein and its tetiary structure was quite similar to the crystal structure of human IL-1RI. The deduced amino acid sequence of flounder IL-1RII had 60.0% and 47.4% identity with that of seabream seabream and rainbow trout respectively. The mRNA expression of IL-1RII could be observed in all the studied tissues of controlled fish,and increased after stilulated for 6 hours in challenged fish,especially in kidney(1.9-fold)and spleen(2.0-fold) . These results suggested that IL-1RII of flounder might mediate the signal transduction and made an important role in the host-pathogen interaction.3. Cloning and expression analysis of TM4SF4 from Japanese flounderBased on the techniques of DD-PCR and RACE, the TM4SF4 gene was obtained from Japanese flounder. The full length cDNA of contained a 5'UTR of 174bp, followed by an ORF of 594bp, and a 3' UTR of 489bp. The ORF was capable of encoding a polypeptide of 197 amino acids with an estimated molecular mass of 20.58 kDa. The deduced amino acid sequence had four transmembrane domains and the Cys-Cys-Gly sequence that were the typical characteristics of Transmembrane 4 Superfamily. Morever, a conserved sequence (53GSGVLMIFPALVFL GLKNNDCCGCCGN79) among the compared species except mouse was found. The deduced amino acid sequence of flounder TM4SF4 had 80.2%,68.5%,64.5%,68.0%,65.5% ,66.5%,66.5% and 66.5% identity with those of Tetraodon nigroviridis,Danio rerio,Xenopus tropicalis,Mus musculus,Macaca mulatta,Homo sapiens,Canis familiaris and Bos taurus, respectively. The mRNA expression level of flounder TM4SF4 was very low and could only be found in spleen and kidney of controlled fish. Infection drastically increased the mRNA levels of TM4SF4 in spleen(5.2-fold)after 6 hours,and in kidney(4.2-fold)after 24 hours in challenged fish by V.anguillarum.In addition, the longer after infection,the higher mRNA expression levels of TM4SF4 could be found in liver of treated fish.. These results indicated that TM4SF4 of flounder might play a critical role in the host-pathogen interaction.