Gene cloning and expression of the serine protease and serine protease inhibitor in Eocanthecona furcellata(Wolff)
In order to clarify the gene sequence characteristics and spatio-temporal transcription characteristics of serine protease gene EfSP1 and inhibitor gene EfSPI20 in Eocanthecona furcellata(Wolff),and lay a foundation for its physiological function research,the complete open reading frame(ORF)sequences of EfSPI20 and EfSP1 in the salivary gland of E.furcellata were obtained by PCR cloning,and sequence analysis and phylogenetic analysis were carried out by bioinformatics software.The expression characteristics of EfSPI20 and EfSP1 in different developmental stages and tissues were analyzed by real-time quantitative PCR(RT-qPCR).The results showed that the CDS of EfSPI20 and EfSP1 were 378 bp and 921 bp,encoding 125 amino acids and 306 amino acids.They were both hydrophilic proteins,the theoretical molecular weights were 13.48 kDa and 33.82 kDa.The isoelectric points were 6.68 and 5.80.There were signal peptide sequences of 30 and 23 amino acid residues,EfSPI20 had a transmembrane domain,EfSP1 had no transmembrane domain.Sequence alignment showed that EfSPI20 had the highest homology with Halyomorpha halys PPI,and the amino acid sequence identity was 58％.EfSP1 had the highest homology with Nezara viridula SP,and the amino acid sequence identity was 66％.The phylogenetic tree showed that E.furcellata was closely related to the species of H.halys and N.viridula in the Pentatomidae.The EfSPI20 gene was highly expressed in male and female adults and salivary glands,it was speculated that EfSPI20 may have the function of inhibiting trypsin activity and be related to the predation and digestion of E.furcellata.The EfSP1 gene was highly expressed in the egg stage,ovary and intestine,suggesting that EfSP1 may be related to the reproductive function and protein digestion of E.furcellata.
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