Characterization And Gene Expression Pattern Analysis Of Two Protease Inhibitors From Pteromalus Puparum

Parasitoid wasps play an important role in the biological control. During the oviposition process, parasitoid wasps inject their eggs into the host hemocoel, accompanied with many maternal factors, such as, venom, polydnaviruses, virus-like particles, teratocyte and other parasitic factors. For sucessful development of their offspring in host hemocoel, these parasitic factors can interefere host immune response and regulate host development. Pteromalus puparum is pupal advantage gregarious endoparasitoid of Pieris rapae, which is a cruciferous vegetable pest. In the previous study,60venom proteins, which including proteases, protease inhibitors, recognition or binding protein and functions unknown proteins, have been identified from venom gland of Pteromalus puparum. In this studies, the characterization and gene expression pattern analysis of two serpins, PpSPN4and PpSPN6were studied.1. Characterization and gene expression pattern analysis of PpSPN4from Pteromalus puparumThe full length cDNA of PpSPN4was cloned by using RACE technology. The full length of the cDNA is2623bp, including a35bp5’untranslated region （UTR）,665bp3’UTR and1923bp open reading frame （ORF）. The ORF encodes641amino acids including23-aa signal peptide. The protein molecular predicted weight was72.3kDa and the theoretical isoelectric point was7.34. Phylogenetic analysis of PpSPN4compared with serpins in other species showed that the nearest homolog is Nasonia vitripennis serpin4（XM₀01603896.2）. SDS-PAGE result showed that pET28a-PpSPN4can be producted by auto-induction in Escherichia coli. Real-time quantitative PCR and Western blot results showed that PpSPN4is highly expressed in the venom, but low expression in head, thorax, carcass and ovary. In time expression pattern, western blot result showed that PpSPN4expressed in all seven days post emergence, and with two peak at the day4and day6post emeregence. And, the quantitative PCR result also showed two peak at day1and day5.2. Characterization and gene expression pattern analysis of PpSPN6in the venom of Pteromalus puparumThe full length cDNA of PpSPN6was cloned by RACE technology. The full length of the cDNA is1806bp, including a220bp5’UTR,389bp3’UTR and1197bp ORF. The ORF encodes399amino acids including19-aa signal peptide. The protein molecular predicted weight was44.1kD and the theoretical isoelectric point was4.8. Phylogenetic analysis of PpSPN6compared with serpins in other species showed that the nearest homolog is Nasonia vitripennis serpin3/4（XP₀08201843.1） and alaserpin （XP₀08201838.1）. SDS-PAGE result showed that pET28a-PpSPN6can be producted by auto-induction in Escherichia coli. Semi-quantitative PCR and Western blot results showed that PpSPN6is highly expressed in the venom, but low expression in head, thorax, carcass and ovary. In time expression pattern, western blot result showed that PpSPN6expressed in all seven days post emergence, and semi-quantitative PCR reasult showed four peak at the day2、day3、day5and day6post emergence.