Discovery The Autonomous Pogo Transposons In Fish And Its Evolution And Transposition Validation

The Tc1 superfamily Transposons is widely distributed in bacteria,fungi,insects and other organisms,some of them have been developed be used as important genetic tools,such as SB,has been applied in the research of gene therapy and transgenesis.The pogo transposon,as an importatn member of Tc1 superfamily,few reports are avaliable for their distribution,evolution,and transposition.Here we annotated the mobilomes of nine teleost,and investigate the distribution,evolution,and transposition of this transposon in vertebrates,aim to discovery active pogo transposons,and develop genetic tools with intellectual property rights for furtehr use in transgenesis and gene thrapy.The results including:(1)the mobilomes of 9 teleost species were annotated by bioinformatics methods.The results showed that both of the mobilome size and constitute display a significant difference in 9 species of teleost fishes,the species of mobilome content ranking from high to low were zebrafish,medaka,Coelacanth,Platyfish,cod,Stickleback,Tetradon,tilapia and fugu,mobilome content and genome size was positively correlated.The DNA transposons display higher diversity and larger variation in teleost(0.50%-38.37%),is a major determinant of differences in teleost mobilomes,and hAT and Tc/Mariner superfamily are the major DNA transposons in teleost.RNA transposons also exhibit high diversity in teleost,LINE transposons account for 0.53%-5.66%teleost genomic sequences,and 12 superfamilies(I,CR1,L1,L2,R1,R2,Jockey,RTE and Rex)were detected,L1,L2,RTE and Rex retrotransposons obtain significant amplification.While LTR displays low amplication in most teleost with less than 2%of genome coverages,except in zebrafish and stickleback,where LTR reachs 5.58%and 2.51%of genome coverages respectively.And 6 LTR superfamilies(Copia,DIRS,ERV,Gypsy,Ngaro and Pao)were detected in the teleost,and Gypsy exhibits obvious amplication among them.While the SINE represents the weakest ampificationypes in teleost,only within zebrafish and Coelacanth,it represents 3.28%and 5.64%of genome coverages,in the other 7 teleost,it occupies less than 1%of genomes,and tRNA,5S and MIR families of SINE have a certain degree of amplification in some teleosts.This study shows that the teleost display high diversity and large variation of mobilome,there is a strong correlation with the size variations of genomes and mobilome contents in teleost,mobilome is an important factor in determining the teleost genome size.(2)The distribution of pogo transposons in fish was investigated by using the TBlastN program with zebrafish pogo transposase ORF sequence.These searches revealed that the pogo transposons mainly distribute in the fish lineages,totally,we identified 28 pogo transposons within the 28 species of fishes.This transposon also found in 2 species of amphibians and 4 species of reptiles.Whereas in the bird and mammals,we couldn't detect any typical pogo transposons.POGK and POGZ are pogo transposon derived genes,and POGZ distribute extenstively in vertebrates,while POGK main distributes in mammals,both of POGK and POGZ harbor highly conserved DBD and DDE domains of transposase,which indicated they may maitain the transposition activity.(3)To verify pogo transposition activity in a mammal cell(Hela),we constructed the pogo transposon backbone harboring the neomycin resistance gene expression cassette(pPG-PGK-NEO),the expression vector of pogo transposase,POGK,and POGZ genes(pT2-CMV-Pgase,pT2-CMV-POGK,pT2-CMV-POGZ).Then transposon plasmid pPG-PGK-NEO was mixed with transposase,POGK,POGZ expression plasmids(pT2-CMV-Pgase,pT2-CMV-POGK,pT2-CMV-POGZ)respectively,co-transfected Hela cells and obtain the NEO gene position cells.Based on the comparasion of the number of NEO gene position cells we found that the numbers of NEO gene position cells of pT2-CMV-POGK,pT2-CMV-POGZ are significantly higher than that of the control,and that of pT2-CMV-Pgase is significantly higher that of pT2-CMV-POGK,pT2-CMV-POGZ,and the control.And these indicate that the pogo transposase,and POGK?POGZ can are active in transposition.(4)The expression of pogo transposase in zebrafish embryos was also investigate by in situ hybridization detection with snese and antise probes,the result revealed that the pogo transposase expressed in all stages of embryos detected,but with a variaed abundance.Both of the sense and antisense of expression of pogo transposase were observed,and but the antisese expression is sobvious higher than that of sense.Here this study revealed that the pogo transposon mainly distribute in the fish,amphibian,reptile,and the pogo transposase has high transposition activity,and both of POGK and POGZ also display transposition activity.