PiggyBac Transposable Elements In Lepidopteran Insects

PiggyBac is a kind of classⅡtransposable elements,and its first sequence(IFP2) was originally isolated from a cell line of Trichoplusia ni.This piggyBac transposon has been modified and widely used as an effective gene-transfer vector to transform the germ-line of more than a dozen species of insects spanning five different orders.Recently,it was reported that piggyBac also efficiently transposes in vertebrate embryos and cell lines. Actually,previous studies had revealed that piggyBac-like elements(PLEs) were widespread in a variety of organisms.However,among all of these hundreds of PLEs recovered,few were found active or even with intact structure.Furthermore,knowledge of the biological characteristics and evolutionary history of piggyBac elements is still limited.This work focused on cloning new intact and potential functional PLEs sequences from lepidopteran insects where the active piggyBac was originally found.And a naturally automobile McrPLE element was isolated,which belongs to the highly conserved IFP2 class.Another moderate related IFP2 sequence amplified was AyPLE1.1,which encoding a functional transposase.Cross-mobility assay showed that the sub-inverted terminal repeat sequence was essential for the transposase recognition,while the inverted terminal repeat determines the speciality of a functional transposase.1 Investigation piggyBac-like element in lepidopteran insectsWith nested degenerate PCR,41 lepidopteran insect species spanning thirteen different families have been surveyed for the presence of endogenous PLEs.Only in five noctuid species,Argyrogramma agnate,Agrotis ypsilon,Macdunnoughia confuse,M.crassisigna and Spodoptera litura,the degenerate primers successfully amplified target fragments about 350bp～380bp in length.Sequence analysis revealed these partial fragments sharing 57-97%identities with each other and with IFP2. The southern dot blot analysis using degenerate PCR fragments as probes showed clear hybridization signals,confirming the presence of endogenous piggyBac-like element in the genome in five noctuid species.2 Cloning of full-length PLEsWith inverse PCR and ITR PCR,the full length of piggyBac-like element in M. crassisigna,A.ypsilon and A.agnate were cloned and designated as McrPLE,AyPLE and AaPLE respectively.McrPLE is 2472 bp in length with a single open reading frame(ORF) encoding for a transposase of 595 amino acid residues.It shares 99.5%identity with active IFP2 element,and belongs to highly conserved IFP2 class.AyPLE1.1 and AaPLE1.1 encode transposases of 598 and 600 amino acid residues respectively.Sequence alignment showed that AaPLE1.1 shares 79%similarity with AyPLE1.1.These two elements are moderated related IFP2 sequences,and share the same 16bp inverted terminal repeats.3 Phylogenetic analysisPhylogenetic tree was made with a total 25 transposase sequences from 14 species, including Daphnia pulicaria,Homo sapiens,Mus musculus and insect species from Lepidoptera,Diptera and Coleoptera.The phylogenetic tree reveals three main clades(Ⅰ,ⅡandⅢ),these three clades contain mixture of elements from both insect and vertebrate. Obviously,the evolutionary pattern within the piggyBac family deviates from the phylogeny of their host species,and the existence of nearly identical piggyBac sequence in reproductively isolated species indicate that horizontal transfer were probably involved in the evolution of PLEs.According to the phylogenetic tree,McrPLE formed the common clade with IFP2 sequence,which was strongly supported by 100%bootstrapping value.AaPLE1.1,HaPLE1 and AyPLE1.1 clustered in the other same branch,considering these elements sharing the same ITR sequence,it is likely that they might be members of a new sub-family of piggyBac transposon.4 Mobility assay of McrPLE,AyPLE1.1 and AaPLE1.1Excision and transposition assays were performed in Drosophila S2 cell culture to verify the activity of PLEs.McrPLE was proved to be an active automobile transposable element and transposition in the characteristic cut-and-paste and TTAA target-site specific manner.The transposase of AyPLE1.1 can't recognize the donor pHa[KOα]which had no sub-terminal repeats,while it successfully mediated the excision and transposition of pHB[KOα]donor plasmid into the target pGDV1,which indicated AyPLE1.1 encoding a functional transposase,and sub-terminal repeat sequences were essential for the functional transposon.Although the element AaPLE1.1 shows no active signal in our mobility assay,it is worthy of further study.5 Cross-mobility assay of IFP2,McrPLE and AyPLE1.1Cross-mobility assay showed that functional IFP2 transposase couldn't recognized the donor pHB[KOα],and the active AyPLE1.1 couldn't mobilize the pB[KOα]either.The difference between two donor plasmids were the inverted terminal repeat sequences,the ITR of pB[KOα]was 13bp and the ITR of pHB[KOα]was 16bp.It seems the inverted terminal repeats determine the speciality of a functional transposase.In the negative control of excision assay,the excision of donor pHB[KOα]in the absence of piggyBac transposase in S2 cell culture led us to propose that S2 cells contains related factors that could cross-mobilized with piggyBac vector.This phenomenon is worthy of further study.