| Venom is one of the virulent factors associated with parasitoids that was used to manipulate the hosts.The vestigation of its physiological functions were focused on the parasitoids belonging to Ichneumonidae and Braconidae.Up to date,only few venom proteins of parasitoids have been characterazied.In this study,the physiological funcion of Scleroderma guani belong to Bethylidae has been chararcterzied.Venom gene encodeing metalloproteinases and serine protease were cloned from this species.In additon,the physiological funcions of these two venom genes were deciphered.The results were as follows:Venom of S.guani can paralyze Tenebrio molitor pupae,inhibit its development and reduce its eclosion.Additionally,it was with the ability to enhance the number of total haemocytes,granular haemocytes(GRs)and plasmatocytes(PLs),inhibit the spreading of GRs and PLs,induce the death of haemocytes,and inhibit the activity of phenoloxidase in the hemolymph of T.molitor pupae.Using RT-PCR strategy,cDNAs of venoms metalloproteinase and serine protease genes of S.guani were cloned.Their open reading frames were 1872 bp and 798 bp,enconding 623 and 265 amino acid residues.Their predicted molecular weights were 71.84 kDa and 30.53 kDa.Phylogenetic tree analysis showed that metalloproteinase in venom of S.guani belongs to M13 family of gluzincin superfamily,which differs from other metalloproteinases reported in venom of other parasitoid wasps,belonging to M12 family of metzincin superfamily.The fluorescent quantitative real time PCR analysis revealed that the transcripts of these two venom genes were significantly higher in the venom apparatus than in the head,thorax and abdomen without venom apparatus.And their transcripts were only detected adult,which was not observed in other developmental stages including egg,larva and pupa.The cloned venoms metalloproteinase and serine protease genes were successfully expressed in the vectors of pCzn1 and pSUMO-Mut,respectively.Their expression products were purified.By the assays of injection these two venom proteins in vitro,it was found that they were able to inhibit development,enhance the haemocyte count,inhibit haemocyte spreading,and induce haemocyte death of the hosts.In this study,the physiological functions of S.guani venom were defined.The venom genes of metalloproteinases and serine protease in this parasitoid were cloned,and their physiological functions were unraveled.To some extent,these results revealed the physiological mechanism of how S.guani venom regulate its hosts.Additionally,they are helpful for guiding the development and utilization of venom proteins of this species in the future. |
