Insights Into Trx1, TRP14 And Prx1 Homologs Of Paralichthys Olivaceus:Molecular Profiles And Transcriptional Responses To Immune Stimulations

Japanese flounder (Paralichthys olivaceus) is an economically important fish. However, to resist the outbreaks of infectious diseases, it’s urgent to know more about fish immune mechanisms. Thioredoxin (Trx) proteins are involved in multiple celluar processes such as anti-oxidative stress and celluar redox homeostasis. In the present study, full length of PoTrxl and PoTRP14 cDNA from Japanese flounder are isolated via the technicque of RACE (Rapid amplification of cDNA ends, RACE) and the sequence bioinformatics are analyzed via softwares. Then the investigation of the expression level of PoTrxl and PoTRP14 mRNA in eight different tissues and fourteen developmental stages are performed. An in vitro hepatocyte primary culture system from Japanese flounder is established to evaluate the PoTrxl, PoTRP14 and PoPrxl gene expression change in response to LPS, CUSO4, and H2O2, The results demonstrate that the three immunostimulants could induce significant expression in time-dependent expression pattern compared with the control (0h).1、Cloning and expression analysis of PoTrxlThe cloned PoTrxl cDNA is 723bp in length and contains a 366 bp open reading frame (ORF) that predicts a translation products of 121 amino acids, preceded by a 5’untranslated region (UTR) of 33 bp, and followed by a 3’-UTR of 324bp containing a polyadenylation signal (AATAAA) and a poly(A) site. The molecular weight and theoretical isoelectric point (pI) of the deduced protein are 15.90 kDa and 5.39 respectively. Like all known Trxl, PoTrxl contains no apparent signal peptide sequence. Complete cDNA sequences of Trx1 genes from six species are obtained from GenBank to perform the homologous comparison with PoTrxl, BLASTP analysis show that PoTrx1 has similarities with Trx1s of other species. The deduced amino acid sequence of PoTrxl shares significant homolog with Cynoglossus semilaevis, Oplegnathus fasciatus and Trachidermus fasciatus of 60%、67% and 61% identity. Multiple alignments reveal that the CGPC motif is highly conserved in all analyzed Trxls. The homologous of Trx1 are further confirmed by the phylogenetic analysis. It’s indicated that mammalian and teleost Trx1s, which includes PoTrxl, form a group separated from that formed by Trx2. Nevertheless, within the Trx1s subgroup, PoTrxl constitute a branch with Cynoglossus semilaevis Trxl that fall outside the cluster formed by other Trx1s except the Epinephelus coioides Trxl. This grouping is well supported by bootstrapping. PoTrxl is expressed in eight tested tissues, with the high level in gill, intestine and heart, followed by liver, kidney, spleen, and the low level in muscle and brain. PoTrxl transcripts are expressed in small amount at the stage of unfertilized, then followed by hardly any transcripts from 1-cell to gastrula. A rapidly rise appeares at the neurula stage, reaching the maximum level at the somatic stage. Although the expression level is decreased at before-hatching stage, it is still relatively high comparing with the stages of hatching and 1dph.2、Cloning and expression analysis of PoTRP14The cloned PoTRP14 consists of a sequence of 909 bp comprising a 58 bp 5’-UTR, a 372bp ORF encoding a putative peptides of 123 amino acids residuces, and a 479bp 3’-UTR including a polyadenylation consensus signal (AATAAA) and a poly(A) tail. The deduced protein of PoTRP14 has a molecular mass of 14.01 kDa and an isoelectric point (pI) of 5.70. No signal peptide can be predicted and similar to most TRP14, PoTRP14 contains the conserved motif CPDC. BLASTP analysis show that PoTRP14 shares high identity with Anoplopoma fimbria、Atlantic salmon and Epinephelus coioides of 87% identity. Multiple alignments reveal that CPDC motif is highly conserved in all analyzed TRP14s. In the phylogenetic analysis, PoTRP14 is grouped with other teleost TRP14 peptides, which are clustered into a branch separated from the mammals and birds. The PoTRP14 transcripts are mainly detected in the tissue of intestine, liver, kidney, brain and heart, slightly lower in spleen, gill and muscle. The highest level is found in intestine. The PoTRP14 mRNA is found a high expression level at the unfertilized eggs stage, suggesting the maternal deposit of PoTRP14. The expression level remains relatively low after fertilization, with a slight increase from gastrula to somatic stage, and then an obvious maximum level appeares at the before hatching stage, then drop in sudden.3、Expression profiles of PoTrxl, PoTRP14 and PoPrxl in response to LPS, CUSO4, and H2O2To examine whether PoTrxl, PoTRP14 and PoPrxl mRNA expression are affected by immunostimulants such as LPS, CuSO4, and H2O2, cultured primary hepatocytes of Japanese flounder are exposed to LPS, CUSO4 and H2O2 treatment at different concentrations respectively, and the cells are analyzed for genes expression by qRT-PCR at 0,2,6,12,24 and 48h post-treatment. The results demonstrate that the three immunostimulants can induce significant expression in time-dependent expression pattern compared with the control (0h).As for LPS challenge, PoTrx1, PoTRP14 and PoPrx1 mRNA expression are enhanced to significant levels at 2,6,12,24 and 48h post-treatment, after a prominent upregulation from 2h to 6h (the first expression peak), the expression levels are decreased at 12h, followed by the peak expression at 24h by 10 fold compared with the control. In brief, the whole variation trend of PoPrxl and PoTRP14 gene expression in response to LPS are consistent to that of PoTrxl, PoPrxl expression peak is at 24h by 32 fold, however, PoTRP14 expression peak occurs at 48h by 14 fold, which is delayed compared with PoTrxl and PoPrxl.In case of CUSO4 stimulation, the PoTrxl, PoTRP14 and PoPrxl transcripts are induced at all tested time points. A sharp increase is monitored at 2h post-treatment by 13,17, and 25 fold respectively, followed by a stable decline, reaching the second peak expression at 48h by the fold of 7,13 and 44 respectively.In the presence of H2O2 treatment, significant elevation of PoTrxl, PoTRP14 and PoPrxl expression are observed at all the tested time points. After an increase from 2h to 6h, the maximum induction occur at 12h post-treatment by 20,21 and 53 fold respectively. The expression amount fall steeply at 24h, but for PoTrxl and PoTRP14, a slight rise occurs at 48h again.