Identification of mth-like Genes in D. helophoroides

The mth-like genes were searched using the D. melanogaster mth sequence as a query in the sequences from the adult D. helophoroides transcriptome database (previously published under the accession GBCX00000000) that had already been identified and annotated by Blastx [28 ] against the non-redundant (NR) protein database at the National Center for Biotechnology Information (NCBI) with a cut-off E-value of 1.0 × 10⁻⁵ [27 (link),29 ]. Based on the cDNA fragments obtained by searching the transcriptome annotation files, gene-specific primers were designed using Primer Premier 5.0 (Premier Biosoft International, Palo Alto, CA, USA) to amplify the full length mth-likes of D. helophoroides. The primers used for PCR-based cloning are shown in Table 1. The rapid amplification of cDNA ends PCR (RACE-PCR) technique was applied to obtain the full-length mth-like cDNAs with a SMARTer™ RACE cDNA Amplification Kit (Clontech, Beijing, China) according to the manufacturer’s instructions. Total RNA isolated from adult insects using a UNIQ-10 Trizol RNA extraction kit (Sangon, Shanghai, China) was used to obtain cDNA templates needed in RACE-PCR. The PCR products were cloned into the pMD-19-T Vector (TaKaRa, Kyoto, Japan) and sequenced by Sangon Biotech (Shanghai, China).