SEQUENCE AND EXPRESSION ANALYSIS OF THE GENE ENCODING PROGRANULIN IN TILAPIA

Progranulin (PGRN) is an epithelial tissue growth factor (also known as proepithelin, acrogranin and PC-cell-derived growth factor) which plays an important role in innate immunity regulation and growth development. The full-length cDNA of Pgrn (GenBank Accession number: GQ241348) was obtained by screening the full-length cDNA library of the peripheral blood leucocytes from Oreochromis niloticus. The full size of the cDNA was 843 bp, containing one complete open reading frame encoding a peptide of 206 amino acid residues (aa) that contains a presumptive signal peptide (20 aa) and two repeat units of granulin (GRN, both 56 aa). The two predicted GRN peptides were arranged in tandem repeats interrupted by a GAP peptide. The deduced amino acid sequences of the two predicted GRN peptides were consistent with the motif of X2-3CX5-6CX5CCX8CCX6CCX5CCX4CX5-6CX2. The positions of the cysteine residues were highly conserved in the GRN consensus sequence of tilapia, carp, zebra fish and human. respectively. GRN1 and GRN2 of O. niloticus and O. mossambicus had two and one amino acid variation sites respectively. Furthermore, phylogenetic analysis of GRN1 and GRN2 from tilapia, carp, zebra fish and human revealed that while tilapia GRN2 was closer to carp and zebra fish GRNs, tilapia GRN1 was the closest to human GRN2 and GRN3. In the present study, four species of tilapia (O. niloticus×O. aureus, O. niloticus, O. aureus and GIFT) were divided into infection and control groups with 50 tilapias in each group. The infection dose for each infection group (2.0×107 CFU) was the median lethal dose (LD50) for O. niloticus×O. aureus based on our previous results. The tilapias in the control group were intraperitoneally injected with equal volume of normal saline. Occurrence of disease and mortality was observed and recorded. Mortality in all the tilapia species was observed within 25 days of infection; more specifically, the mortality appeared to reached peak during day 4-6, and completely ceased 13 days after infection. The mortality rate for GIFT, O. aureus, O. niloticus and O. niloticus×O. aureus was 64%, 52%, 32% and 20%, respectively. Expression of Pgrn was determined by real-time RT-PCR (qRT-PCR) in four tissues (brain, liver, spleen and head kidney) of those four species. Specificity of PCR primers was assessed by melting curve analysis of PCR reaction products. Only one peak was observed in the melting curve for both genes indicating that there were no non-specific amplifications which met the requirement of quantitative analysis. Standard curve for the internal control β-actin gene and Pgrn gene was generated by using the concentration of cDNA as the X-axis and Ct value as the Y-axis. The correlation coefficient between the concentration of cDNA and Ct value was 1.000 and 0.999 for β-actin and Pgrn genes, respectively. The PCR efficiency for the quantitation of β-actin and Pgrn gene was 89.3% and 91.3%, respectively. To determine relative fold differences for each sample, the Ct value for Pgrn was normalized to the Ct value for β-actin gene and was calculated relatively to a calibrator using the formula 2-Ct. Pgrn was significantly up-regulated (P 0.05), most prominently in head kidney and spleen. In particular, spleen expression was more than 100 fold higher than that of brain, which may suggest PGRN plays an important role in innate immunity regulation of fish. Moreover, Pgrn mRNA was down-regulated on 6h in brain and liver, 6h and 12h in spleen and head kidney of O. niloticus×O. aureus, which was not observed in the other three species. This may be one of the reasons why O. niloticus×O. aureus had high resistance. The results provided data for studying on innate immunity regulation of fish and provided reference molecular marker for breeding disease resistant tilapia.