PiggyBac Transposable Elements In Beet Armyworm, Spodoptera Exigua Hübner And Cotton Bollworm, Helicoverpa Armigera (Hübner)

PiggyBac,a classâ…¡transposable element,was originally isolated from a Trichoplusia ni cell line.The piggyBac transposon has been used most widely as an effective gene-transfer vector,because of its mobility in the genomes of a wide range of insect species.Currently,it is considered,homologous endogenous element would certainly affect the stability and function of transgenic insects mediated by transposable elements.So it is necessary to determine whether piggyBac or related sequences exist in potential hosts and a range of other species.In our study,two important lepidopteron pests,beet armyworm and cotton bollworm were surveyed for their endogenous piggyBac.As a result,endogenous piggyBac elements were found in these two insects,and one of the piggyBac elements isolated from the genome of cotton bollworm was intact and thought to be potentially active.1 Clonging and sequence analysis of piggyBac from beet armywormUsing PCR technique,with degenerate primers,a DNA fragment of piggyBac-like element was cloned from the genome of beet armyworm,spodoptera exigua hubner.The DNA fragment was 456bp in length,and the deduced amino acid sequence shares 50%- 78% similarity with other piggyBac elements from insects.But one stop code was found in this DNA fragment.According to the information of this fragment,subsequent inverse PCR yielded two copies of piggyBac,which were designated as SePLE1 and SePLE2.Sequences analysis revealed that SePLE1 and SePLE2 not only lost their ITRs at both ends,but also there were many mutations(stop codes) in their ORF.Thus,we conclude that there are endogenous piggyBac-like elements in the genome of beet armyworm,but the cloned SePLE1 and SePLE2 are both inactive.2 Clonging and sequence analysis of piggyBac from cotton bollwormUsing degenerate PCR,inverse PCR and vectorret PCR techniques,two full-length piggyBac-like elements were isolated from the genome of cotton bollworm,helicoverpa armigera(hubner),and were designated as HaPLE1,HaPLE2.The HaPLE1(GenBank accession number:EF593176 ) was 2500 bp in length with an ORF of 1794 bp encoding a transposase,perfect 16 bp ITRs,and the specific tetranucleotide target-site duplication, TTAA.Amino acid sequences alignment showed that the conceptual translation of HaPLE1 transposase shares 78%amino acid identity and similarity with T.niIFP2,67%with Bactrocera dorsalis-white eye piggyBac,48%with Heliothis virescens HvPLE1.1,and 46% with Bombyx mori yabusame-1,respectively.Candidates of the DDD domain in piggyBac families D268,D346,D447 and D450 were all observed in the HaPLE1 transposase sequence.So,this element was thought to be an active autonomous element.Another copy of full-length HaPLE,HaPLE2,was 982bp with the same ITRs as HaPLE1 just linked to the TTAA tetranucleotide at both ends,but most of its ORF encoding transposase has been deleted.3 Clonging of flanking sequences of piggyBac in cotton bollwormTE display showed that there were likely a small numbers of copies exist in the genome of cotton bollworm with large variation in insertion site.Using vectorret PCR,we obtained a total of twelve 5'- and eight 3'- flanking sequences of HaPLEs(HaPLE1, HaPLE2) from 12 individual CBW.Sequence analysis revealed that eleven 5'- and seven 3'-flanking sequences obtained belong to HaPLE2.A BLAST search of the flanking sequences of the HaPLEs found no significant match to known genes in the database, indicating that the insertions are likely located in non-coding regions.These insertions may not affect the function and expression of the host genes.Thus,we considered it was not very long after the piggyBac invaded the cotton bollworm.HaPLE1 was intact and thought to potentially active.HaPLE2 was thought to occur through internal deletions of intact piggyBac element.Insertion to the function regions,might be lethal to the host insects. 4 Distribution of HaPLE1,HaPLE2 in different population of cotton bollwormA pair of primers designed on the common region of HaPLE1 and HaPLE2 was used to investigate the distribution of the HaPLEs in different populations of H.armigera. Genomic DNA of seventeen individuals from each strain,NJ,GY,Oxford,was used as the PCR templates.As a result,HaPLE2 was found in all the individuals of the three CBW strains,but HaPLE1 was only observed in 35%individuals of NJ strain and GY strain,and 24%individuals of Oxford strain.The conclusion from the results is as follows.Firstly,we consider that the HaPLEs obtained in our experiments have existed in the cotton bollworm genome for more than 20 years.Because the Oxford strain has been kept in laboratories for more than 20 years.Secondly,the intact copy HaPLE1 has potential mobility.In our study, HaPLE2 was observed in all individuals within the three populations,but HaPLE1 was not. Generally,inactive copies of a transposable element will be fixed or lost in the genome of individuals in a population over time if they are neutral.Therefore,the inactive copies should exist in almost all individuals within the population after several generations. However,HaPLE1 was observed in 24-35%individuals in the three populations in our study.5 Phylogenetic analysesA phylogenetic tree was generated using 19 amino acid sequences of PLEs from eleven species including the sequence of HaPLE1.The phylogenetic tree shows that,all the transposase sequences are distributed within three major clades(â… ,â…¡,â…¢) with some bootstrap support.In the first clade,there are HaPLEland other insect PLEs.The second and third clades comprise a mixture of insect and mammal PLEs.Obviously,the evolutionary pattern within the piggyBac family deviates from the phylogeny of their host species,which indicates that horizontal transfer were probably involved in the evolution of PLEs.The phylogenetic tree also showed that piggyBac-like elements from the same species were separated into different clades,indicating that different piggyBac elements may invade the same population.6 Mobility assay of piggyBac from cotton bollworm in embryos of Drosophila melanogasterTwo piggyBac elements,HaPLE1and HaPLE2 isolated from the genome of cotton bollworm were constructed to plasmid p3E1.2-CBW-1 and p3E1.2-CBW-2,respectively. Mobility of the two plasmids was tested in introducing into embryos of Drosophila melanogaster by electroporation.The results revealed that no excision was observed in p3E1.2-CBW-2,but two excisions were observed in p3E1.2-CBW-2,though the frequency was very low(0.00025%).Sequences analysis showed that the excision within p3E1.2-CBW-1 were not in the TTAA sites.So,the piggyBac-like element HaPLE1 might be mobile,but further confirmation is still needed.As described above,two important lepidopteron pests,beet armyworm and cotton bollworm were surveyed and confirmed for existence of endogenous piggyBac elements. An intact piggyBac-like element was also obtained from the genome of cotton bollworm, and was thought to be potentially active.All of these results could found the bases for study on germline transformation of noctuidae,and for further study on construction of highly effective gene-transfer vectors.