叉角厉蝽丝氨酸蛋白酶抑制剂基因EfSPI8、EfSPI31的克隆与表达分析OA

Serine protease inhibitors(SPIs)are involved in regulating key physiological processes in insects,such as coagulation,immune melanization,and growth and development,playing an important role in in-sect immunity and host-pathogen interactions.This study aimed to clone the venom serine protease inhibitor genes EfSPI8 and EfSPI31 from the predatory stink bug Eocan-thecona furcellata and analyze the characteristics and ex-pression patterns of their encoded proteins.The open reading frame(ORF)sequences of EfSPI8 and EfSPI31 were success-fully cloned by PCR,with lengths of 1 083 bp and 2 793 bp,encoding 361 and 931 amino acids,respectively.Bioinformat-ic analysis revealed that the theoretical relative molecular masses of EfSPI8 and EfSPI31 were 41 100 and 104 800,respec-tively.Both proteins contained signal peptide sequences at their N-termini,with lengths of 30 and 26 amino acids,indica-ting that they were secretory proteins.EfSPI8 possessed a typical serine protease inhibitor superfamily(Serpin)domain and a reactive center loop(RCL),belonging to the Serpin superfamily.EfSPI31 contained three pairs of disulfide bonds that stabilized its protein structure,and belonged to the Kazal-type serine protease inhibitor family.Sequence alignment revealed that EfSPI8 shared 39.27%amino acid sequence identity with a Serpin from Halyomorpha halys,while EfSPI31 showed 88.28%identity with a Kazal-type protein from Nezara viridula.Real-time quantitative PCR(RT-qPCR)results showed that EfSPI8 was highly expressed in fifth-instar nymphs,male adults,the anterior lobe of the main salivary gland,and testes,suggesting its potential involvement in prey-related immune suppression and reproductive regulation.EfSPI31 was highly expressed in fourth-to fifth-instar nymphs,testes,and fat body,indicating possible roles in immunity,metabolic reserve,and reproductive system development.These findings provide a molecular basis for understanding the multifunctional roles of these two protease inhibitors in predatory natural enemy insects.