| Transposons is widely distributed in bacteria, fungi, insects and other organisms, and is mobile element in the genome, and thought to be used in the area of transgenic and gene therapy with a good prospect. Recently, international development and application of transposon have made significant progress, transposons like PB, SB and Tol2 constitute complementary research tools for gene transfer in mammalian cells with important implication for fundamental and translational research. However, domestic development of transposon with intellectual property rights is lagging behind.This study mainly focused on mining zebrafish genomic DNA transposons, and verified the activity of the new transposon on vertebrate cell and embryonic level, and then obtained a high activity transposon (ZB) to provide independent knowledge property right genetic manipulation tool for transgenis, functional genomics (gene trapping, etc.) and gene therapy research. The main contents are as follows:(1) Through bioinformatics analysis, we found five Tc1/Mariner family DNA transposons with structural integrity in the zebrafish genome. Genetic divergence comparison showed that one transposon was most active and youngest transposon, named ZB. ZB transposon is 1.4kb full-length, and contains a 1026 bp-coding transposase gene flanked by 203 bp-TIRs. There are at least 21 copies in zebrafish genome, of which 16 copies are full length copies of the homology of DNA sequence and the ORF amino acid sequence are 99.80% and 99.47%, respectively. And the identity of TIR is up to 100%. Its ORF contains typical conserved domains of Tel transposon (such as HTH, NLS, DD34E, etc.).(2) The results of qPCR and in situ hybridization detection showed that, ZB transposase was expressed in 11 stages detected of zebrafish embryonic development, and the expression strength over time increased. The expression in Adult zebrafish tissues (brain, heart, muscle, testis and ovary) also were expressed, and the highest expression was in the brain, the lower expression was in heart, testis and ovary.(3) To verify ZB activity in a mammal cell (Hela), we cloned ZB upstream and downstream TIR transposable element and the ORF of transposase, resulting in the frame vector (pZB-Msc) and transposase helper plasmid (pCMV-ZB), respectively. Then the neomycin resistance gene expression cassette (PGK-NEO) was cloned into the frame vector (pZB-Msc), thus constructed the transposon donor plasmid (pZB-PGK-NEO). Then transposon plasmid pZB-PGK-NEO was mixed with transposase expression plasmid pCMV-ZB co-transfected Hela cells at different mass ratio, with PB, SB and Tol2 transposon as comparision. Experimental results showed that the number of cells expressing the neomycin resistance gene of four transposons in the experimental group were higher than the one of the negative control group, and ZB transposon at a mass ratio of 1:1 containing the largest number of resistant cells that turn seat for maximum efficiency. When ZB was at a mass ratio of 10:1,2:1,1:1,1:2, the number of resistant cells was higher than the SB transposon, and was significant difference at a mass ratio of 1:1 (P<0.01). ZB transposition efficiency was similar with the PB, and higher than SB and TOL2(P<0.05).(4) To verify ZB transposon activity in zebrafish embryoic level, we cloned GFP gene expression cassette (FAG-GFP) containing the carp beta-actin promoter and green fluorescent protein gene into the frame vector pZB-Msc, resulting in a transposon donor plasmid. Then transposon plasmid (containing FAG-GFP expression cassette) and transposase mRNA were co-injected into zebrafish one-cell stage embryos, detection of GFP was done by fluorescence microscope after injection to compare transposition efficiency of four transposons, the results showed that GFP-positive rate of ZB transposon was higher than the other three transposons at 24hpf and 5dpf after injection, and ZB was significant higher than the SB transposon (P<0.05).(5) To verify ZB transposon activity in mouse embryoic level, we cloned GFP gene expression cassette (CAG-GFP) containing the chicken beta-actin promoter and green fluorescent protein into the frame vector pZB-Msc, resulting in a transposon donor plasmid. Then transposon plasmid (containing CAG-GFP expression cassette) and t transposase mRNA were co-injected into mice fertilized eggs, and detection of GFP expression at embryo development to the blastocyst stage, each group with three times repeated, the results showed that all three groups embryos could expressthe green fluorescent protien, and the positive rate of embryos in different groups which co-injected with ZB-mRNA, ZB DNA and no trasposase were 31.76% (n=110),33.43%(n=147) and 4.34%(n=162), respectively. And the efficiency of transposase group was significant higher than the no transposase group (P<0.05), the difference between embryonic positive rate of transposase groups was not significant (P> 0.05).This study showed that the newly discovered ZB transposon was a hyperactive transposon, the transposon can efficiently mediate exogenous gene transfer in mammalian cells, mice and zebrafish embryos. It has a good prospect in the production of transgenic animals and gene therapy. |
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