Molecular Characteristics And Functions Of Two Salivary Proteins Of Larve Of Pteromalts Puparum

Parasitic wasps are important natural enemy insect resources for biological control of agricultural pests,and they are abundant in nature.At present,the research in our laboratory focuses on some key parasitic active factors,such as venom and polydnavirus(PDVs),which are carried by female parasitic wasps and can regulate a series of important physiological processes such as the host’s natural immune response.However,studies on the active substances secreted by parasitic wasp larvae such as saliva,functions and their interaction with the host’s natural immune response are almost blank.The research on insect salivary proteins mainly focuses on herbivorous insects and blood-sucking insects.Their salivary proteins have a defense against plant defenses,act as plant-insect effectors,and help spread viruses.Preliminary studies on the saliva of the larvae of Pteromalus puparum in our laboratory indicate that it has the function of inhibiting hemolymph melanosis of the host,which means,it may regulate the natural immunity of the host pest.Therefore,in this paper,the salivary protein group was prepared and the salivary components and enrichment pathways were identified.Combined with the transcriptome data of the salivary glands of the larvae,two salivary protein encoding genes with signal peptide and specific high expression were selected,and then gene cloning,analysis of expression patterns and preliminary exploration of its functional research system were carried out.1.Analysis of the salivary proteome of the larvae of P.puparumA total of 38 salivary proteins were identified by analysis of the salivary proteome of the larvae of P.puparum.According to sequence and domain information,they are divided into protease,protease inhibitor,recognition and binding protein,carbohydrate metabolism,esterase,other proteins and unknown proteins.They were subjected to GO(Gene Ontology)and KEGG(Kyoto Encyclopedia of Genes and Genomes)analysis.Using the transcriptome data of the salivary glands of the larvae of P.puparum,the 508 genes highly expressed in the salivary glands of the larvae were screened.Combined with the results of the proteome analysis,two salivary protein encoding genes of the larvae of P.puparum with specific high expression and signal peptides were selected for subsequent experiments.2.Cloning and expression pattern analysis of two salivary protein encoding genesThe full length of CDS of PP05924 and PP16911 was obtained from the genome of P.puparum by sequence alignment,and sequence analysis and phylogenetic tree analysis were carried out.The results showed that the open reading frame of PP05924 was 1884 bp,encoding 628 amino acids,and the predicted molecular weight was 75KDa,with signal peptide.Evolutionary analysis showed that PP05924 was closely related to arylphorin-like(XP 014234947.1)of Trichogramma pretiosum.The open reading frame of PP16911 is 459 bp,encoding 153 amino acids,and the predicted molecular weight was 18.5KDa,with signal peptide.Evolutionary analysis showed that PP16911 was closely related to two unknown functional proteins.Then successfully cloned the coding regions of these two genes after de-signaling peptides,and they were prokaryotic expression using the Pcoldl expression system,thereafter,purification,dialysis and preparation of polyclonal antibodies.The results of real-time PCR and Western blot analysis showed that the PP05924 gene was highly expressed on fifth day at larval stage(abbreviated as L5).The results of real-time PCR and Western blot analysis of tissue distribution at L5 stage showed that PP05924 is highly expressed in salivary glands,saliva and intestines,both at the mRNA level and at the protein level.Quantitative analysis of the tissue distribution on first day at adult stage(abbreviated as A1)showed that PP05924 was highly expressed in the salivary glands.The above analysis of the PP16911 gene also showed that the PP16911 gene was also highly expressed at L5 stage.The results of real-time PCR and Western blot analysis of tissue distribution at L5 stage showed that PP16911 was highly expressed in salivary glands and saliva,both at the mRNA level and at the protein level.Quantitative analysis of the tissue distribution at Al stage showed that PP16911 was highly expressed in the salivary glands.3.Preliminary study on the functional research system of larval salivary proteinSince these two salivary protein encoding genes of P.puparum are highly expressed in the L5 phase,the expression level in the adult stage is low.Therefore,by injecting dsRNA into the host pupa to interfere with the salivary protein gene,the purpose of establishing a larval high expression gene function research system is achieved.The results showed that on the second day(L2)of larvae of P.puparum,9000 ng/μl and 7μl of dsRNA were injected into the host pupa,and qPCR was detected 48 h later.The transcription level of PP16911 was significantly decreased,indicating that the research system can be used to interfere the gene highly expressed in the larvae of P.puparum,and lay a foundation for the subsequent functional study of the gene.