Molecular Characteristic And Functional Analyses Of Two Salivary Proteins In Nasonia Vitripennis

Nasonia vitripennis is a high specificity exoparasite in the pupal stage of flies which is an important health pest.The female adults lay eggs in the pupa shell of the host,and the larvae feed on the host for nutrition to ensure themselves successful development.N.vitripennis is one of the ideal model insects because it has fewer chromosomes,can reproduce indoors all year round and is easy to raise.The study on the function of their saliva during feeding can not only develop new technologies and provide new ideas for the prevention and control of flies,but also provide a good model for the further study on the mechanism of the saliva of natural enemies on the host.This study takes N.vitripennis and Musca domestica.as the research object,the molecular characteristics and functions of two proteins in the saliva of N.vitripennis were studied in this research,the main research contents and results are as follows:1.Functional correlation analysis of alcohol dehydrogenase NvAdh in larval saliva of N.vitripennis.An open reading frame of an alcohol dehydrogenase gene of saliva protein was obtained by cloning based on the design of primers for the genome sequence of N.vitripennis and the gene was named NvAdh.This gene encodes 349 amino acid sequences and contains a signal peptide sequence of 19 amino acid residues at the n-terminal.Through qPCR and Western blot experiments,the expression pattern of NvAdh in N.vitripennis was studied.The results showed that the transcription and expression levels of NvAdh were relatively high in the larvae on the fourth day after the parasitism of the N.vitripennis,and were the highest in the salivary glands.Meanwhile,pCold I DNA expression vector was used for prokaryotic expression and protein purification of NvAdh.The purified protein was used for the study of enzyme kinetics.Results show that NvAdh optimum reaction temperature is 37 ?,the pH value is around 9,Km of isopropyl alcohol value is 1.4185,the maximum reaction rate is 0.5507 nmol/min/mg.At the same time,we exposed the female adults of N.vitripennis to the pupae of M.domestica which were feed on 0%and 4%alcohol respectively,and then examined the biological parameters of offspring.After parasitization for 5 h,this gene was upregulated in the N.vitripennis larvae which were emerged from M.dome.stica pupae with 4%alcohol.Moreover,there was no difference in the biological parameters of the offspring of N.vitripennis.2.Functional correlation analysis of antimicrobial protein NvAMP in larval saliva of N.vitripennis.An open reading frame of an antimicrobial protein gene of saliva protein was obtained by cloning based on the design of primers for the genome sequence of N.vitripennis and the gene was named NvAMP.This gene encodes 135 amino acid sequences and contains a signal peptide sequence of 21 amino acid residues at the n-terminal.The results of evolutionary tree showed that the relationship between NvAMP and Trichomalopsis sarcophagae was the closest,the rest of the results were bacteria.The expression pattern of NvAMP was studied by qPCR,Western blot and immunofluorescence localization.The results showed that the transcriptional and expression levels of NvAMP were the highest in the salivary glands of larvae on the fourth day after the parasitization of N.vitripennis.Meanwhile,pCold I DNA expression vector was used for prokaryotic expression and protein purification of NvAMP.The purified protein was used in the antibacterial experiment.The results showed that NvAMP could promote Micrococcus luteus,restrain Escherichia coli,Salmonella enteritidis and Bacillus subtilis,but there was no effect on Herbaspirillum sp.