Research On Goldfish Tgf2 Transposon And Its Amplification In Transgenesis

Transposable elements are mobile genetic elements capable of moving from one site to another by―cut-paste‖or―replicate-paste‖mechanism. For example, Tol2, (transposable element of Oryzias latipes, number 2) and SB (Sleeping Beauty transposon) are elements move in a―cut-paste‖fashion. Research demonstrated that terminal inverted repeats (TIR) and sub-terminal repeats of Tol2 transposon are essential for its transposition, for mutation in these regions leads to a notable decrease in transfer activity. As for SB, 230 bp IRs at its terminal and directly repeated DNA sequence motifs (DRs) at the ends of each IR are recognized by its transposase. According to that, transgenesis system have been constructed, and showed no host specificity. Up to now, Tol2 and SB have been extensively used in transgenesis, gene trap and gene therapy exploring.Most recently, Tgf2, a new hAT family transposon was found from goldfish genome and further study was carried out in this study. First, the complete cDNA of transposase gene was cloned and RT-PCR analysis and in-situ hybridization was carried out to explore its expression; Second, PCR analysis revealed that Tgf2 distributes in all strains of goldfish studied, and both complete and incomplete Tgf2 transposons were detected; Finally, donor vector plasmid and helper plasmid were constructed using the terminal sequence of Tgf2 transposon and 1734bp coding sequence of Tgf2 transposase gene separately and transgenesis research was carried out in zebrafish, grass carp, goldfish and johnny carp.1. The complete cDNA of Tgf2 transposase gene was cloned using rapid application of cDNA ends method (RACE), and the result indicate that there are at least 7 different transcripts, named gfTP-1, gfTP-2, gfTP-3, gfTP-4, gfTP-5, gfTP-6, gfTP-7 separately, encoding 3 kind of transposase proteins which are 686AA (gfTP-1), 650AA (gf TP-2, gfTP-3) and 577AA (gfTP-4, gfTP-5, gfTP-6, gfTP-7) in length; The result of homology comparison between deduced amino acid sequence of Tgf2 transposase and that of Tol2 showed a sequence similarity of 99%, with only 7 amino acid distinction and amino acid difference was found among different copies of Tgf2 transposase;2. RT-PCR using primers that shared by 3 kind of transposase in adult fish revealed that Tgf2 transposase expresses in all tissues studied but especially in ovum and liver, while RT-PCR and in-situ hybridization analysis showed a big difference in expression pattern among embryos;3. PCR analysis revealed that Tgf2 transposon distributes in all strains of goldfish studied, and Tgf2 transposon with large deletions was also detected in goldfish genome by PCR analysis;4. The vector plasmid pTgf2-ef1α-eGFP, pTgf2-ef1α-eGFP and pTgf2-ef1α-RFP were constructed using the 220 base pairs of the left and 185 base pairs of the right terminal sequence of the Tgf2 and the 1734bp coding sequence of Tgf2 transposase was subcloned into a helper plasmid, from which transposase mRNA could be transcribed in vitro; Transgenesis research was carried out by coinjection of the vector plasmid and transposase mRNA at a concentration of 50 ng/μL and 50 ng/μL separately in zebrafish, grass carp, johnny carp and goldfish; PCR analysis revealed that the GFP reporter gene was transferred from the vector plasmid into grass carp and goldfish genomes, and the incorporation efficiency was 96% and 37% in separate.