Cloning Of Thymidylate Synthase From Zebrafish, Danio Rerio And Its Expression Regulation

Thymidylate synthase (TS) is a folate-dependent enzyme that catalyzes the reductive methylation of 2-deoxyuridine-5-monophosphate (dUMP) to dihydrofolate and thymidylate (dTMP), an essential precursor for DNA synthesis. TS represents an important target for cancer chemotherapy. Zebrafish has been developed successfully as a screening model for many drugs in recent years. However, the TS gene sequence analysis, mechanism of regulation and the role in zebrafish development have not been reported yet. The aim of the research is to address mechanism of the TS regulation in zebrafish, to document its role in zebrafish development and develop zebrafish as a model for TS inhibitor screening.The complete TS cDNA was amplified by RT-PCR and RACE. The open reading frame (ORF) of zebrafish TS cDNA sequence was 957 nucleotides, encoding a 318-amino acid protein with a calculated molecular weight of 36 kD. The complete ORF encoding the zebrafish TS gene was PCR amplified and inserted into pET-28a to construct an expression vector pET-28/Z-TS. The expression of zebrafish TS protein was induced by IPTG in E. coli BL21 (DE3). High level expression of zebrafish TS was found at 1mM IPTG condition after incubation for 3h. However, increased concentration of IPTG had little effect on protein expression. The zebrafish TS was purified to homogeneity using Ni-NTA spin column from the total cellular proteins. Enzymatic analysis confirmed that the purified zebrafish TS showed the highest activity at 28℃ with similar Km value as human TS.Western blot analysis was used to determine whether the purified zebrafish TS could be recognized by human TS monoclonal antibody. Our results confirmed that zebrafish TS protein interacts with human TS-106 monoclonal antibody with a similar affinity with human TS. So we could use this method to determine the expression profile of zebrafish TS during embryonic development using human TS monoclonalantibody. Careful analysis indicated that zebrafish TS was significantly expressed in embryos at different stages with highest level at 1-cell to 2-cell stages.To study the function of TS in zebrafish development, we generate a short hairpin RNA (shRNA) expression vector, pSilencer 4.1-CMV /TS, and microinjected the plasmid into zebrafish embryos at the 1-cell to 2-cell stage. Abnormal genotype of zebrafish embryos, especially with head and tail, was found after 25 hpf in pSilencer 4.1-CMV /TS injected embryos. The results suggested that TS plays some important roles in zebrafish development. However, the abnormal development of zebrafish embryos was restored if 10 nM thymidine was included in the incubation buffer. Our research indicated a salvage pathway was occurred in zebrafish embryos. To further confirm if the abnormality of the zebrafish development was resulted in by TS gene silencing, western blot analysis was performed to determine the expression level of TS protein in zebrafish embryos. Our results confirmed that the level of TS expression was significantly decreased in pSilencer 4.1-CMV /TS microinjected zebrafish embryos.Our present research confirmed that the gene of zebrafish TS has high similarity with human TS. TS play some key roles in the zebrafish development, and the silencing of TS gene results in abnormal genotype of zebrfish development. The finding contributes to our understanding of TS in zebrafish and provide foundation for developing zebrafish as a new screening model for TS inhibitors.