Preliminary Studies On Expression Patterns And Functional Analysis Of GATA Factors In Nile Tilapia

GATA factors are important transcription factors, which have two highly conserved zinc finger domains, C-X2-C-X17-C-X2-C (C, cysteine residues; X, variable amino acid; numbers, the numbers of amino acid residues). These two zinc fingers were designated as N-terminal (N) and C-terminal (C) fingers. The GATA family was named from zinc finger binding to the consensus nucleotide sequence [T/A(GATA)A/G] in the promoter and enhancer regions of the target genes. In mammals, the GATA family consists of six members, and can be divided into two subfamily families, GATA1/2/3and GATA4/5/6, based on the structure and function. Recent studies showed that the members of the GATA factors are emerging as mediators of steroidogenesis, sex determination and differenationation during development in mammals. Therefore, this study intends to investigate the expression patterns and biological functions of GATA factors during tilapia gonadal development by transcriptome analysis, Real-time PCR, in situ hybridization and the luciferase assay.In the present study, we isolated7members of GATA family in the Nile tilapia by bioinformatic and transcriptome analysis. Noticeablely, GATA2a and GATA2b were probably derived from the additional genome duplication (the fish-specific genome duplication, FGSD) that occurred at the emergence of actinopterigians. The length cDNA of GATA1,2a,2b,3,4,5,6are1275,1748,1569,1841,1691,850and3260bp, with open reading frames (ORFs) encoding425,463,456,444,393,384and495putative amino acids (aa), respectively. The GATA1/2/3subfamily members comprise five exons and four introns, while GATA4/5/6subfamily members comprise six exons and five introns. Only one GATA123gene and one GATA456gene were isolated in each published invertebrate deuterostome genome. The phylogenetic analysis showed that GATA123s from invertebrate deuterostome were clustered with GATA1/2/3subfamily, while GATA456s were clustered with GATA4/5/6subfamily. These data suggested that the last common ancestor of deuterostome contained at least two GATA genes, with both a GATA123and a GATA456ortholog. The GATA family may have expanded in vertebrates accompanied with whole genome duplication events. Furthermore, a closer relationship between GATA2and GATA3was observerd, and so was between GATA5and GATA6by phylogenetic analysis..Syntenic analysis of vertebrate GATA2,3,5and6demonstrated that GATA2and GATA3might have arisen from a common ancestral gene, while GATA5and GATA6might be derived from aonther ancestral gene.Through the analysis of5,30,90and180dah XX and XY gonad transcriptomes of the Nile tilapia, we found that all the GATA family members except GATA5were highly expressed in5dah gonad. It was also found that the expressions of GATA2b, GATA4and GATA6in were obviously sexual dimorphic, with higher expression in XX gonads than in XY gonads. However, the expressions of all GATA family members except GATA6decreased gradually at30,90and180dah. These results suggested that the GATA family may play important roles in the early development of Nile tilapia gonad. As GATA4played critical role in sex determination and differentiation, and verifying the authenticity of the transcriptome data, Real-time PCR was performed to investigate the expression of GATA4in5,10,20,30,50,70dah gonad. The results were consistent with the transcriptome data. In this study, GATA6was detected in interstitial cells of both male and female gonads by in situ hybridization. Recent studies had shown that GATA2a, GATA2b and GATA4were also expressed in interstitial cells of both male and female gonads, suggesting that GATA family members may have similar cellular expression pattern in the Nile tilapia. Interestingly, the AMH which thought to be associated with gonadal sex differentiation and the steroidogenic genes, such as CYP19ala, the STAR1, and STAR2were expressed in interstitial cells of both male and female gonads, which implies that GATA4may be associated with the expression of AMH, CYP19ala, STAR1and STAR2. In mammal, GATA4and SF1synergistically regulated the CYP19a1a, STAR and AMH, Therefore, luciferase assay were performed to confirm whether GATA4could regulae the CYP19ala, STAR1and STAR2. Our results show that GATA4can increase AMH and STAR1but supress the transcription of CYP19a1a. However GATA4has no effect on the transcription of STAR2. The regulation of GATA4to AMH and CYP19a1a was further illustrated that GATA4may play an important role in the male pathway. In summary,7GATA factors were isolated from Nile tilapia. Through bioinformatics, transcriptome analysis, in situ hybridization and luciferase assay, we got the following conclusions:1) the GATA genes of vertebrates might be derived from the two GATA genes of the last common ancestor of deuterostome;2) GATA2and GATA3might have arisen from a common ancestral gene, while GATA5and GATA6might be derived from aonther ancestral gene;3) GATA4could increase AMH and supress the CYP19a1a transcription which indicated that GATA4may play an important role in the male pathway;4) GATA family might play an important roles in the early development of Nile tilapia gonad by transcriptome analysis.