Molecular Cloning And Expression Of Translation Elongation Factors (eEF1As) In The Nile Tilapia

Eukaryotic translation elongation factor 1A (eEFIA) is a ubiquitous protein family present in abundance in many types of cells, promoting guanosine triphosphate (GTP)-dependent binding of aminoacyl-transfer ribonucleic acid (tRNA) to ribosomes during peptide chain elongation as part of the translational elongation complex.42Sp50 is a special member of eEFIA family which has been found only in the previtellogenic oocytes of teleosts and amphibians so far. Many studies of eEFIA revealed that it has several isoforms that are expressed in a tissue- and stage-specific manner during development. For example, two eEFIA isoforms exist in birds and mammals, designated as eEF1A1 and eEF1A2, while three were found in amphibians, i.e. eEFIA1, eEF1A2 and 42Sp50. There are few reports on phylogenetic analysis of eEFIA family, and there is no report on their expressions and roles in gonads. In order to investigate eEF1As expression patterns and functions in gonads,42Sp50 was cloned from the Nile tilapia using RT-PCR and RACE technology, and other three eEF1As were identified using sequences from the 3-month Nile tilapia gonads transcriptomes. Moreover, a complete phylogenetic analysis was performed, and the expression patterns of 42Sp50 and eEF1A1b were investigated by RT-PCR, Realtime-PCR and ISH. The results are as follows, The full-length cDNA of 42Sp50 was 1718 bp, with an open reading frame (ORF) of 1371 bp, encoding a protein of 456 amino acids; and all the ORFs of three eEF1As had 1389bp, encoding proteins of 462 amino acids. Based on the alignment of eEF1A sequences isolated from GenBank, a phylogenetic analysis was performed. The phylogenetic tree showed that eEF1A sequences from various vertebrates are clustered into five separate clades:eEF1A1, eEFlA2, eEF1A3, eEF1A4 and 42Sp50. eEF1A1 and eEF1A2 existed in vertebrates, while eEF1A3 and eEF1A4 existed only in teleosts. 42Sp50 which was found in teleosts and amphibians clustered into one clade different from the other eEF1 As clades.By RT-PCR, eEF1A1b was found to be expressed in both testis and ovary with higher expression in the testis, whereas 42Sp50 was only detected in the ovary. Moreover,42Sp50 was found to be strongly expressed in 17(3-estradiol induced XY ovaries, but completely inhibited in Fadrozole induced XX testes. The expression of eEF1Alb was higher in XX testes than that in XY ovaries. By Real-time PCR, ontogenic expression of 42Sp50 was detected from 10 dah (days after hatching) in the ovary, peaked at 60 dah, and then decreased onwards, while only faint signals were detected in all stages of testicular development. As for eEF1Alb only faint expression was detected from 10 to 70 dah in the testis, increased sharply from 90 dah onwards, peaked at 120 dah, and decreased gradually, then peaked again at 240 dah, while in the ovary, it remained low during the whole period. ISH (in situ hybridization) was performed to determine the cell types which express 42Sp50. eEF1Alb and eEF1A4. In previtellogenic oocytes,42Sp50 signal was found to be uniformly distributed, while eEF1A4 appeared to be concentrated in the small area. Both eEF1Alb and eEF1A4 were expressed in primary and secondary spermatocytes. Moreover, promoter analysis of 42Sp50 was performed using three estrogen receptors (ERa, ERβ1 and ERβ2) in HEK293 and CHO cells. The results showed that all three ERs suppressed the promoter activity of 42Sp50 in both cell lines. Recombinant protein with His-Tag at the N-terminal (expressed using pCold I vector in E. coli) of 42Sp50 was obtained and purified, and then its polyclonal antibody was produced in mouse. Western blot analysis was performed to check the quality of the antibody. Our antibody could recognize the same recombinant protein as recognized by His-Tag antibody.In summary, five eEF1As named as 42Sp50, eEF1A1a. eEF1A1b, eEF1A3 and eEF1A4 were identified and cloned in tilapia by transcriptome analysis and gene cloning (eEF1A4 had been reported before). A thorough phylogenetic analysis of eEFlAs from animal kingdom revealed that eEF1A1b is a new member of eEF1A family. Sexual dimorphic expression of 42Sp50 and eEF1A1b were detected in gonads. 42Sp50 is specifically expressed in the previtellogenic oocytes while eEF1A1b mainly in primary and secondary spermatocytes. Moreover,42Sp50 promoter activity was found to be suppressed by all three ERs in the luciferase assay. Our findings suggest for the first time that sex specific protein synthesis in male and female gonad might be undertaken by different translation elongation factors,...