Cloning and Sequencing of MAPK Genes in C. suppressalis

Total RNA was isolated from actively feeding 4^th instar C. suppressalis larvae using RNAiso reagent (TaKaRa, Dalian, China). Contaminating genomic DNA was eliminated with RNase-free DNase, and the RNA preparation was then subjected to reverse transcription using a PrimeScript^TM RT reagent Kit (TaKaRa, China) according to the manufacturer’s instructions. Partial p38, JNK, ERK1 and ERK2 cDNA sequences had been obtained from the C. Suppressalis transcriptome determined in previous studies35 (link). A 5′ and 3′ RACE kit (TaKaRa, Dalian, China) was used to amplify full-length MAPK genes from C. Suppressalis larvae, and pairs of gene-specific and degenerate primers were designed based on the partial sequences using Primer 5.0 software (Supplementary Table S1). PCR products were subcloned into a PMD (18)-T vector (Takara, Dalian, China) and sequenced by the Nanjing Genscript Company, China.