Transcriptome-based cry Gene Cloning

Approximately 100 male heads and 100 female heads were pooled together from which total RNA was extracted using TRIzol reagent (Invitrogen, USA) according to the manufacturer's protocol. The RNA was treated with DNase (Takara, Japan) and then reverse transcribed to cDNA using PrimeScript 1st strand cDNA synthesis kit (Takara, Japan).
Previously, one transcriptome dataset for L. striatellus was built by Zhang et al. (2010 (link)) and another one had been built in our laboratory (unpublished data). Based on the sequence data obtained from transcriptome searching using the basic local alignment search tool (BLAST), gene-specific primers were designed and used for cloning cry cDNA (Table S1). PCR were conducted using KOD FX DNA polymerase (Toyobo, Japan). The PCR conditions were determined empirically for the amplification of each gene. PCR products were verified by 1% agarose gel electrophoresis. Target bands were excised and purified using DNA gel extraction kit (Axygen, USA). The DNA fragments were TA ligated into pMD18-T vector (Takara, Japan) and transformed into Escherichia coli DH5α (TransGen, China). Positive clones were sequenced by SunnyBio (China).