Population Insertion Polymorphisms Of The Piggybac-like Element PgPLE1

The pink bollworm (PBW), Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae), is one of the most destructive pests in cotton-growing areas of the world. Transgenic PBW strains have been successfully produced using the Trichoplusia ni piggyBac transposable element, and the EGFP transgenic PBW has been registered for the field release test as the only genetically modified agricultural pest. piggyBac is a TTAA-insertion site specific DNA transposon, which was originally discovered in Trichoplusia ni.. The piggyBac element is capable of precise excision and shows high frequency of transformation in insect genome, and its activity is less restricted by host factors. These characteristics make piggyBac the most widely gene vector in insect transgenesis. In this paper, the insertion site preference, transcription and transposition activity and population insertion polymorphism of piggyBac-like element, PgPLE1, in pink bollworm were studied, and population genetic differentiation of pink bollworm was also investigated based on PgPLE1 insertion polymorphism and mitochondrial DNA CO I sequences.Flanking sequences of piggyBac-like element, PgPLE1, in the pink bollworm, have been cloned and sequenced using vectorette PCR, and the insertion site preference was analyzed. The results showed that most copy of PgPLE1 specifically insert into the TTAA target site,flanking sequences of six PgPLE1 copy showed no significantly homologous sequences in the GenBank nucleotide collection (nr/nt), while the insertion regions of PgPLE1.F3 and PgPLE1.F8 had 30% amino acid identity to gag protein of Tirant retrotransposon from Drosophila melanogaster and 53% to reverse transcriptase of non-LTR retrotransposon from Bombyx mori, respectively.The transcription and transposition activity of piggyBac-like element, PgPLE1, in pink bollworm, were analyzed by RT-PCR and vectorette PCR. Three kinds of transcripts of PgPLE1 were detected by RT-PCR, and the cloning and sequencing results showed that one of the transcripts encodes a putative intact transposase. Analysis of 3′vectorette PCR amplicons in parents and F1 offsprings by agarose electrophoresis showed that the insertion sites of PgPLE1 in two F1 offsprings have changed, suggested that PgPLE1 is still active in transposition.Insertion polymorphisms of the piggyBac-like element PgPLE1 were detected by PCR in five pink bollworm populations, including Jurong of Jiangsu Province, Xiantao of Hubei Province, Anxiang and Yueyang of Hunan Province and Arizona State of USA, and genetic relationship of pink bollworm populations was analyzed. The genotype distribution of five insertion locus of PgPLE1 is in good agreement with Hardy-Weinberg equilibrium in most testing populations, the average gene frequency of each locus varied from 0.0050 to 0.2400. Based on allele frequency, genetic distance and phylogenetic relationship among pink bollworm populations were studied. The result showed that the genetic relationship was consistent with geographic distribution. Our data indicated that transposon insertion polymorphism could be used in the evolution research of insect populations.Partial mitochondrial DNA (mtDNA) Cytochrome Oxidase I (COⅠ)sequences were analyzed in 40 individuals of pink bollworm, which were sampled from Jurong of Jiangsu Province, Honghu and Xiantao of Hubei Province, Anxiang and Yueyang of Hunan Province and Arizona State of USA, respectively. In total of 5 polymorphic sites were identified, and 6 haplotypes were defined, four of which were unique for population, suggesting the genetic differentiation among these populations. The AMOVA analysis revealed that the higher level of genetic differentiation existed among Arizona and Jurong population and other populations. The genetic distance analysis showed that Hubei and Hunan populations had the closest relationship.