Identification Of Protein Components And Functional Analysis Of SgSPH72 From The Venom Of Scleroderma Guani

Venom is a product of the co-evolution of parasitoids and hosts.It has the ability to regulate the immunity,development,nutrition and metabolism of the host,and plays an important role in the manipulation of the host by the parasitoid.To data,although the venom components of some parasitoids have been revealed,they are mainly limited to a few groups,such as Ichneumonidae,Braconidae,Pteromalidae.Moreover,the functions and act mechanisms of few venom proteins from parasitic wasps have been clarified.In this study,the venom protein components,and the function of one of the venom proteins as a serine protease homology gene（SgSPH72）of Scleroderma guani（Hymenoptera:Bethylidae）was characterized.The main results are summarized as follows:1.Identification and expression profile analysis of venom genes of S.guaniBy combined proteomics and transcriptomics approaches,102 venom proteins of S.guani were identified.Among them,there are 26 identidied by both transcriptomics and proteomics,74 by transcriptomics and 2 by proteomics.Except for 16 novel venom proteins with unknown functions,other venom proteins can be divided into 11 functional categories.The category associated with the largest number of proteins is enzymes proteins,of which the serine protease（serineproteases,SPs）and its homologys（serineprotease homologys,SPHs）have the largest number of ten.According to digital gene expression profiles,it was found that all venom genes were expressed at different developmental stages and in different tissues.Among them,55 of venom genes were specifically or highly expressed in venom apparatus and female adults.2.Identification and gene expression analysis of SPs/SPHs of S.guaniBased on the method of Blast homologous alignment,78 SPs/SPHs genes were identified from the genome of S.guani.The SPs/SPHs can be divided into three types including Single domain,Clip domain and Multiple domain.The molecular phylogenetic tree and gene Scaffold mapping results showed that there was gene duplication event occurred in part of the single domain SPs/SPHs and venom SPs/SPHs of the S.guani.According to the gene digital expression profiles,it was found that part of the single domain SP/SPH genes were highly expressed in the digestive tracts and at larval stage,indicating that they are mostly related to digestive function.In addition,some SPs/SPHs genes were highly expressed at the female adult stage,and specifically or highly expressed in venom apparatus.Through the comparative analysis of the gene expression profiles of SPs/SPHs between non-venom and venom tissues,ten SP/SPH genes were identified,They are highly expressed in venom apparatus and at female adults,of which eight dispalys specifically or highly expressed.But non-venom SPs/SPHs genes was not highly expressed in venom apparatus.Quantitative fluorescence real-time PCR（q PCR）verification showed that ten venom SPs/SPHs genes were significantly highly expressed in venom apparatus.Based on the existing SPs/SPHs from venoms of parasitoids and the findings of this study,it was found that venom SgSPH72 is the high molecular weight venom SPH of parasitoids,which has not been functionally characterized.3.Regulation of trehalose metabolism pathway genes of the host by venom SgSPH72 of S.guaniThe gene expression of the host,Tenebrio molitor,pupae in response to the injection of venom SgSPH72 24 h was analyzed by transcriptomic approach.A total of593 genes were differentially expressed.Of them,303 and 290 hosts genes were up-and down-regulated,respectively.Trehalase（TRE）gene is one of the most up-regulated genes of host.Also,q PCR analysis showed that venom SgSPH72 can influence the expression of trehalose-6-phosphate synthase（TPS）,glycogen synthase（GS）and glycogen phosphorylase（GP）genes of the trehalose metabolism pathway.It suggests that venom SgSPH72 can regulate the expression of trehalose metabolism pathway genes in T.molitor,and then regulate its carbohydrate metabolism.4.Effect of venom SgSPH72 of S.guani on host carbohydrate metabolism by T.molitorThe changes of carbohydrate contents in hemolymph and fat body of T.molitor pupae after injection of venom SgSPH72 6 h and 24 h were determined by anthrone method and 3,5-dinitrosalicylic acid colorimetric method,respectively.After injection of venom SgSPH72 6 h,the contents of total carbohydrate,glycogen,reduce sugar and trehalose in hemolymph and fat body were significantly higher than that of control.After injection of venom SgSPH72 24 h,the contents of glycogen and reduce sugar increased in hemolymph and fat body.There is no gignificant change of total carbohydrate and trehalose in hemolymph,and total carbohydrate and trehalose in fat body.In this study,the venom protein components of S.guani were deciphered.The transcriptional patterns of the identified venom proteins at different developmental stages and in different tissues were revealed.SgSPH72 abundant in venom was found to with the ability to regulate carbohydrate metabolism of the host by influnencing the gene expression of the trehalose metabolism pathway.This study laid a foundation for the further investigation of the function of venom proteins of S.guani and the evolution of parasitoid venom genes.Many novel venom genes of parasitoids were reported.The mechanism of venom proteins regulating host carbohydrate metabolism was firstly revealed,which provides insights into the understanding of the interaction between parasitoids and hosts.