Expression Characterization And Functional Analysis Of Hypoxia Responsive Gene TGF-?1 In Zebrafish

Hypoxia is a natural and seasonal phenomenon in ponds, reservoirs, lakes and open oceans. In recent years, severe hypoxia has been caused by eutrophication and become one of the main elements that restrict the continuable development of aquiculture. So, it is theoretically and practically significant to elucidate the molecular mechanism of fish responsing to hypoxia.Transforming growth factor-?1 (TGF-?1) is a pleiotropic cytokine and participates in the regulation of key events of development, disease and tissue repair. Our previous work showed that the expression of TGF-?1 gene increased after hypoxia exposure in CAB cells. Based on the result in vitro, we will analyzed the expression pattern and the function of TGF-?1 gene in zebrafish. The results are as follows:Firstyly, we got TGF-?1 DNA sequence of zebrafish from NCBI. The full length cDNA of zebrafish TGF-?1 is 1571bp, which encodes 377 aa. Phylogenetic analysis revealed that TGF-?s clustered strictly according to different types. Zebrafish TGF-?1 gathered with other fish on a branch.Secondly,we analyzed the embryos of different development stages by RT-PCR. It showed that TGF-?1 existed in unfertilized eggs of zebrafish. Its expression increased from 24hpf and remained a high level until 72hpf. Whole-mount in situ hybridization (WISH) analysis showed that TGF-?1 transcripts could be first detected in embryos from 24hpf and increased apparently in subsequent stages. TGF-?1 transcripts were expressed mainly in gill arches, lateral line primordium, otic vesicle, olfactory placode, heart and pronephros. The expression pattern of TGF-?1 gene in different tissues of healthy zebra fish showed that TGF-?l gene was expressed ubiquitously and the expression in eyes and gill was highest, Its obvious expression in the liver, heart, brain in the gonads, low expression level in the kidney, intestine, of muscle. It indicated that TGF-?1 might play an essential role in immunoregulation, lateral line formation and vasculogenesis.Then, we studied the the effect of hypoxia on the expression of TGF-?1 in zebrafish embryos and adult zebrafish by RT-PCR, Real-Time PCR and WISH. It was found that the development of zebrafish embryos were retarded and the expression level of TGF-?1 in embryos decreased dramatically after hypoxia treatment. The TGF-?1 mRNA exsited ubiquitously in adult zebrafish and was induced significantly by hypoxia in eyes, brain, gill, heart, liver, spermary and ovary. We cloned TGF-?1 ORF into pEGFPN1 and constructed pEGFPNl-TGF-?1 plasmid. pEGFPNl-TGF-?1 and pEGFPNl plasmids were respectively transfected into HEK293 cells and were treated with hypoxia. It was found that the viability of cells transfected with pEGFPN1-TGF-?1 was significantly higher than that of the cells trasfected with pEGFPNl. The results suggested that TGF?1 gene may play an important role in hypoxia adaptation of zebrafish.