Isolation Of The Flanking Sequences Of Insecticide Resistance-associated Cytochrome P450Genes In Helicoverpa Armigera And Identification Of Transposable Element Insertions

The cotton bollworm, Helicoverpa armigera, is one of the most important cotton pests in the world. Cytochrome P450s (CYP) are one of the major phase I-type classe of detoxification enzymes found in all organisms, and play an important role in the development of insecticide resistance. Recent studies have shown that the insertion of transposable elements might lead to the over-expression of the cytochrome P450s. In this study, the5’-and3’-flanking sequences of insecticide resistance-associated cytochrome P450genes in the cotton bollworm were obtained by genome walking, and three novel families of short interspersed nuclear elements (SINEs) were identified using bioinformatic methods and molecular techniques, two of them, HaSE1and HaSE2, are tRNA-derived SINEs, and HaSE3is5S rRNA-derived SINE. These results might assist in understanding the evolutionary adaptation of insect cytochrome P450genes as well as the roles of retroelements in H. armigera genome evolution.To gain information about possible regulatory elements involved in P450expression in the cotton bollworm, flanking sequences upstream and downstream the open reading frame (ORF) region of CYP9A12, CYP9A14and CYP6AE12as well as orthologous CYP321A1gene of Helicoverpa Zea were obtained by genome walking based on the full length cDNA sequence of these P450genes. Several potential cis-acting element binding sites were predicted in upstream flanking sequences, including GATA-1, Oct-1, C/EBP, BR-C Z, CdxA and HSF, suggesting that these cis-acting elements may play a role in the specific regulation of the expression of these resistance-associated P450s. Additionally, one non-autonomous Lep1Helitron element and one SINE element HaSEl.1were found to insert into the upstream and downstream flanking sequences of CYP6AE12gene, respectively.One cytochrome P450gene was found in the5’-end of upstream flanking region of CYP6AE12gene, and its full-length cDNA sequence of1730bp was obtained by RT-PCR. Sequence analysis showed that this novel P450gene belongs to CYP6family, encodes521amino acid residues with the predicted protein molecular weight being14.35kD and the isoelectric point being4.98. Sequence alignment showed that this novel P450gene has74%amino acid identity with CYP6AE12.The3’-flanking sequence of the P450gene CYP6AE12was compared with non-redundant databases using the NCBI server with blastn, a total of21full length sequences with high homology to HaSE1.1were identified from non-redundant database, and named as HaSE1.2-HaSE1.22. Sequence annnalysis showed that these sequences present the typical structural features of the tRNA-derived SINE elements:all HaSE1copies are flanked by5to18bp short direct repeats (DRs) or target site duplications (TSDs), presumably generated during retroposition; the A and B boxes of the RNA polymerase III promoter as well as a tRNA-like region were found at the5’-end; various numbers of perfect or imperfect TGA trinucleotide repeats were found at the3’-end. The consensus sequence of the HaSE1family members is386bp long. It includes a short A-tag followed by a74-bp tRNA-related region at the5’-end (58%identity to72-bp tRNA of Drosophila melanogaster). Computer assisted similarity search showed that the3’-terminal43bp fragment of the HaSEl consensus sequence was very similar (82%identity) to the3’-end of HaRTE1.1, a member of the novel LINE family HaRTE1identified in a BAC clone of H. armigera. The HaRTE1.1element was1645bp long, flanked by15bp TSDs, encoded an RT domain with42%amino acid sequence identity to that of the RTE-3-BM in Bombyx mori, and was terminated by a region of TGA trinucleotide repeats in the short3’UTR.Using74-bp tRNA-related region of HaSE1as a query, a total of7sequences (HaSE2.1-HaSE2.7) of the second tRNA-derived SINE family, HaSE2, were identified. The5’-structure of the286bp consensus sequence of HaSE2is highly similar to that of HaSEl (94%identity over a132-nt region), including a70-bp tRNA-related region immediately followed by a62bp conserved central domain. Note that the154bp3’-region showed high identity (98%) with one mariner-like element, Hamarl.3, in H. armigera. It is the first report of a SINE family with its3’-region derived from DNA transposon. It is quite likely that HaSE2elements resulted from integration of reverse transcriptase transcripts into the genomic copies of mariner-like elements or recombination of tRNA and transcripts of mariner-like elements during the template switching common for reverse transcriptases.Using blastn and PCR, a total of16different repeat sequences of the5S rRNA-derived SINE family were obtained, and named as HaSE3.1-HaSE3.16. Comparative analysis showed that the3’-region of HaSE3is very similar to that of HaSE1(94%identity between the two consensus sequences) suggested that the novel HaSE3family might be generated by template switching from the tRNA-derived HaSE1RNA to5S rRNA during the process of cDNA synthesis in retroposition. Thus, this study may represent the first description of a chimera of a5S rRNA and a tRNA-derived SINE in insect species.Searches against various GenBank databases revealed the presence of HaSE1in Helicoverpa zea and Heliothis subflexa, two closely-related species of Helicoverpa armigera. Likewise, HaSE2were found in Heliothis subflexa, Heliothis virescens, Spodoptera frugiperd, Spodoptera littoralis, Spodoptera litura and Danaus plexippus, and HaSE3were found in Heliothi subflexa, Heliothi virescens, Trichoplusia ni and Mamestra brassicae. Interestly, while all the three SINE families were not identified in the fully-sequenced Bombyx mori, the finding of one HaSE2element from the Aphis gossypii, a non-lepidopteran species, suggests the occurrence of horizontal transfer of HaSE2.