Tc1-like transposase Thm3 of silver carp (Hypophthalmichthys molitrix) can mediate 3 gene transposition in the genome of blunt snout bream (Megalobrama amblycephala)

24 Tc1-like transposons consist of an inverted repeat sequence flanking a transposase gene 25 that exhibits similarity to the mobile DNA element, Tc1, of the nematode, Caenorhabditis 26 elegans. They are widely distributed within vertebrate genomes including teleost fish; 27 however, few active Tc1-like transposases have been discovered. In this study, 17 Tc1-like 28 transposon sequences were isolated from 10 freshwater fish species belongs to the families, 29 Cyprinidae, Adrianichthyidae, Cichlidae and Salmonidae. We conducted phylogenetic 30 analyses of these sequences using previously isolated Tc1-like transposases and report that 16 31 of these elements comprise a new subfamily of Tc1-like transposons. In particular, we show 32 that one transposon, Thm3 from silver carp (Hypophthalmichthys molitrix; Cyprinidae), can 33 encode a 335 a.a. transposase with apparently intact domains, containing 3-5 copies in its 34 genome. We then co-injected donor plasmids harboring 367 bp of the left end and 230 bp of 35 the right end of the non-autonomous silver carp Thm1 cis-element along with capped Thm3 36 transposase RNA into the embryos of blunt snout bream (Megalobrama amblycephala; 1-2 37 cell embryos). This experiment revealed that the average integration rate could reach 50.6% in 38 adult fish. Within the blunt snout bream genome, the TA dinucleotide direct repeat, which is 39 the signature of Tc1-like family of transposons, was created adjacent to both ends of Thm1 at 40 the integration sites. Our results indicate that the silver carp Thm3 transposase can mediate 41 gene insertion by transposition within the genome of blunt snout bream genome, and that this 42 occurs with a TA position preference. 43 44