The Effect Of Sub-Itr Of Piggyback Transposon To Its Activity

Piggybac transposon is a kind of DNA transposon, and characterized by inserting gene into a specific target site and excising them exactly from target site without leaving any trace. Thus, this kind of transposon is widely used as a tool in animal transformation. Nowadays, the researches about piggybac transposon are mianly concentrated on exploring new transposons and new application, little work has been done on the relations between structure and the activity of transposons. Here we try to use different groups of recombinant vector to explore the impaction of piggybac transposon sub-ITRs to translocation activity in different cells and organisms. The results are summarized as follows:1. The two different helper plasmids were first constructed with the transposons from the piggyBac-like-element of Helicoverpa armigera (HaPLE) and Argyrogramma agnate (AaPLE). Then, the binary vector/helper transformation system in Drosophila S2cells was employed to determine the excising activity of these two transposase to the donor plasmids reconstructed with different combinations of Sub-ITRs and ITRs. The results showed that AaPLE transposase had no activity, but HaPLE transposase could recognize the donors with the ITRs of its own and show transposition activity with the donor plasmid constrcted with its own ITRs and the sub-ITRs from Tribolium castaneum. Therefor, HaPLE transposase was active, can identify the donor which is constructed withits own ITRs and appropriate Sub-ITRs. However, it was found that the excising accuracy might be controlled by sub-ITRs.2. With the donors constructed with different sub-ITRs, the activity of the transposons from Agrotis ypsilon (AyPLE) and Aphis gossypii (AgPLE) in Drosophila S2cells was detected. The result showed that both AyPLE and AgPLE transposon have significant excising activity, The donors with different sub-ITRs could change a little of the excising activity of certain transposase, but not significant in statistics. This is possible caused by transposase self-regulation adapted to different sub-ITRs. It was also probably because the sorts of used sub-ITRs were limited, and the effect was not checked out.3. In BHK cells, binary vector/helper transformation system was also employed to detect the excising activity of IFP2, AyPLE and AgPLE transposase to the donors constructed with different sub-ITRs. The results showed that AyPLE could excise the donor constructed with its own ITRs and Pectinophora. gossypiella sub-ITRs (Ay-Pg) in BHK cells. AgPLE also showed well transposition activity with the donors constructed with Aphis gossypii ITRs and the sub-ITRs from Tribolium castaneum (Ag-Tc) or Trichoplusia ni (Ag-d). Analysis together with the results obtained in drosophila S2cells came to the conclusion that the excising accuracy of piggyBac transposases is regulated by both the host cells and the sub-ITRs in the donors.4. The binary vector/helper transformation system and microinjection was employed to determine the in vivo excising activity of IFP2, AyPLE, AgPLE transposase to the donor plasmids reconstructed with different Sub-ITRs in different insect species. The results demonstrated that IFP2could excise precisely in all kinds of insects tested. AyPLE and AgPLE also showed excising activity in different insects, but no precise excision was observed The possible reason might be that no donors were constructed with the sub-ITRs from AyPLE or AgPLE, or proper ones. These results implied that transposons will lose their capability for precise excising in vivo when lost their own sub-ITRs.The binary vector/helper transformation system was employed to compare the activity of several transposases on the donors with different sub-ITRs in various host insects and cells. It was further confirmed that sub-ITRs could impact the activity and precision of trasposase excision. The results should provide a new materials and new ideas for the study of piggyBac transposons for their structure and functional relationships, and for the development of safe and efficient transgenic system.