Study of Insertional Mutagenesis in Grass Carp (Ctenopharyngodon idellus), Crucian Carp (Carassius auratus) and Blunt Snout Bream (Megalobrama amblycephala) Mediated by Tgf2 Transposon
Abstract：Transposon mediated insertional mutagenesis has great value in the discovery and interpretation of functional genes in aquaculture fish. In this paper, a preliminary study of insertion mutagenesis was carried out in 3 Cyprinidae fish mediated by Tgf2 transposon. On the basis of high efficient Tgf2 transposon, 4 donor plasmids pTgf2-β-actin-eGFP, pTgf2-EF1α-eGFP, pTgf2-MyoD-eGFP, pTgf2-Krt8-eGFP and the in vitro expression of mRNA helper plasmid gfTP in pCS2-gfTP were constructed. The donor plasmids and in vitro expressed gfTP transposase mRNA were co-injected into 1~2 cell stage fertilized eggs of grass carp (Ctenopharyngodon idellus), crucian carp (Carassius auratus) and blunt snout bream (Megalobrama amblycephala). By detection and screening of the eGFP gene integration rate, the insertional mutagenesis library mediated by Tgf2 transposon was initially established. In the early stage of embryos, the fluorescence ratio was very high. The fluorescence ratio in both blunt snout bream and crucian carp reached 90%, and even 95% in grass carp. According to the different donor plasmid, there were three types of fluorescence, such as in the muscle, skin and whole body. The integration ratio in larva fish was 50%~53%, and the integration ratio in the one year old fish was 33.0%~36.1%. In total, in grass carp, 162 positive mutants, and 45 individuals were got in them had obvious phenotypes. There were 60 positive individuals in crucian carp, including 14 obvious phenotypes. In blunt snout bream, there were 51 positive individuals, including 10 obvious phenotypes. These results suggest that insertional mutagenesis could be realized in grass carp, blunt snout bream and wild crucian carp by Tgf2 transposon inserted into fish genome. Our studies can accumulate some basis for the study of mining and functional mechanism of target character gene function.
