Study On Structure And Function Of Insect PiggyBac Transposon

PiggyBac is a kind of DNA transposon via a "cut-and-paste" mechanism, inserting exclusively at TTAA tetranucleotide target-site duplications. The transformation system mediated by piggyBac transposon has been successfully used in transformation of a variety of animal species, and become the most widely adaptable transformation system. Thus, piggyBac transposon become now one of the hotspots of transgenic research. PiggyBac-like transposons has been proved to distribute widely in the genome of various organisms, but most of them were found inactive and few with intact structure. Recently, though some new active piggyBac transposons were discovered, little about their structure and function was studied. In this work, the transposition frequency, inverted terminal repeat sequences (ITR) interaction, sub-terminal inverted repeat sequences (sub-IR) interaction of several different insect piggyBac transposons were studied and the effect of the integrity of ITR sequences on the activity of transposons was also tested in order to revealed the relations between structure and function of the transposon.The results were summarized as follows:1. The binary vector/helper transformation system was employed to determine the transposition efficiency of AyPLE, HaPLE and AaPLE. The results showed that AyPLE displayed significant transposition activity in Drosophila S2 cells, and the transposition efficiency was approximately 65% that of IFP2 element. However, no obvious transposition activity was detected with HaPLE and AaPLE.2. By test of mobility with the binary vector/helper transformation system, the cross-action among the active transposases (from IFP2 and AyPLE) and the transposon ITR sequences from HvPLE1.1,TcPLE1,AgoPLE1.1,Uribo2,BmPBLE04,BmPBLE07 and BmPBLE69 was studied. The results showed that the functional transposase of IFP2 or AyPLE couldn't excise elements with the ITR sequences from other resources but its own. It revealed that ITRs determined the speciality of a functional transposase of piggybac transposons. 3. With the Helper/Donor binary transformation system in the Drosophila S2 cell lines, the cross-action among the active transposases (from IFP2, AyPLE and AgoPLE) and the transposon sub-IR sequences from IFP2, TcPLE1 and PgPLE was also studied. The results showed that the transposase of IFP2, AyPLE or AgoPL could recognize all the elements with the sub-IR sequence from IFP2, TcPLE1 or PgPLE, and make precise cut. It indicated that sub-IR sequences of piggyBac transposons were not specific. However, statistics analysis revealed that the rate of exact cut was affected by alternation of different sub-IR sequences.4. With point mutation and activity test using the Hepler/Donor binary transposition system the significance of the integrity of ITR sequences was studied. The results showed that every base in ITR sequences was important for the activity of transposons. Deletion of any base could result in failture of identification and cut of the transposons. Thus, the integrity of ITR played an important effect in the piggyBac transposition activity.This work revealed that the speciality of functional piggyBac transposons were determined by their ITR sequences, but not the sub-IR sequences. The integrity of ITRs was also the key for the activity of piggyBac transposons. Our results provided new ideas for the study of piggyBac transposon structure and functional relationships, and for development of safe and efficient transgenic systems.