Cloning, expression and predation function analysis of serine protease EfSP2 gene from Eocanthecona furcellata（Wolff） venom

【Objective】The purpose of the study was to analyze the function of EfSP2 protein by cloning, sequence analysis, expression profile analysis and RNA interference(RNAi) of serine protease(SP) gene EfSP2 from the venom of Eocanthecona furcellata(Wolff), and to provide a scientific basis for the molecular characterization and functional annotation of the EfSP2 gene. 【Method】The EfSP2 gene sequence was obtained from the salivary gland transcriptome of E.furcellata. The complete open reading frame(ORF) sequence of the EfSP2 gene was amplified by PCR technology and analyzed by bioinformatics. Real-time fluorescence quantitative PCR was used to analyze the expression of EfSP2 gene in different developmental stages(eggs, 1st-5th instar nymphs, male and female adults)salivary gland and different tissues(malpighian tube, salivary gland, fat body, ovary, gut, head) of female adults. At the same time, RNAi technology was used to detect its effects on the survival and predation of male and female E. furcellata adults. 【Result】The ORF sequence of EfSP2 gene obtained from cloning was 861 bp, encoding 286 amino acids. The amino acid sequence of EfSP2 contained a complete Tryp-SPc domain and a conserved initial amino acid sequence(IVGG) at the N-terminus of trypsin. It contained a conserved SP triplet catalytic activity center and belonged to the tryptase subfamily of the serine protease family. It had the highest homology with Nezara viridula SP, with an amino acid sequence identity of 44.69%. The EfSP2gene was highly expressed in male and female adults and salivary glands of E. furcellata. The injection of dsEfSP2 significantly inhibited the expression of target genes(P<0.05, the same below), and significantly reduced the survival rate and predation of male and female E. furcellata adults. 【Conclusion】The E. furcellata venom serine protease EfSP2 gene is successfully cloned. The spatiotemporal expression profile and RNAi results show that EfSP2 is likely to be one of the important proteins in the processes of growth, development, predation and digestion of E. furcellata.