Functional Research Of Immune Response About PxGNBP3 In Plutella Xylostella(L.)

The diamondback moth(DBM),Plutella xylostella(L.),is a worldwide and destructive pest that damage cruciferous crops.Due to its wide feeding habits and short generation cycles,it has brought great losses to crop production.For a long time,the abuse of chemical drugs has caused serious resistance.Drug resistance,while increasing the difficulty of prevention and control.In order to effectively control the DBM and provide new ideas for the prevention and control of other pests,we have determined the expression profile data of the transcriptome and the pest-infecting larvae of DBM at different times after infection by the laboratory.Based on this data,the differentially expressed immune-associated genes infested by the entomogenous fungi of DBM were screened by heat map analysis.The immune-related genes were analyzed by bioinformatics including pattern recognition,signal modulation,conduction pathways and terminationeffect factor and so on.In this study,the Gram-negative bacterial binding protein PxGNBP3 is screened from the pattern recognition family,and be useand as a research object to study the kinetics and functions of the spatio-temporal expression pattern of microorganisms in detail.The result is as follows:1.Based on the unigene fragments obtained from DBM,the PxGNBP3 gene which form DBM is obtained by RT-PCR combined with RACE technology and named as PxGNBP3(Gene ID: MH319939).The full-length cDNA sequence of PxGNBP3 is 1510 bp with an open reading frame of 1299 bp and encoded 433 amino acid residues.The protein molecular weight is 48.3 kDa and the isoelectric point is 5.79.The PxGNBP3 gene sequence contains beta-1,3-glucan recognition site,an N-terminal saccharide binding domain,a glycosyl hydrolase domain,and a laminin G protein domain.Phylogenetic tree analysis and sequence alignment showed that the PxGNBP3 gene of DBM had high homology with GNBPs of other species.2.In order to further clarify the temporal and spatial expression pattern of PxGNBP3 gene and its sensitiity to microorganisms,RT-qPCR is used to detect the PxGNBP3 gene of the DBM during the entire developmental period(eggs,larvae,pre-pupa,pupa and adults)and larval stage.The analysis of expression showed that PxGNBP3 gene is expressed at different developmental stages,and with the highest expression in the pupal stage.The expression level of PxGNBP3 in the pupal stage was 11.6 times than that in the egg stage.The results of differential tissue expression showed that the PxGNBP3 gene was expressed in different tissues,with the highest expression in the midgut and the second in the epidermis.The 4th instar larvae of P.xylostella were treated with Gram-positive bacteria,Gram-negative bacteria and fungi.The results of RT-qPCR showed that PxGNBP3 gene could be induced by three kinds of microorganisms,among which PxGNBP3 gene is strongly induced by entomopathogenic fungi,and Beauveria bassiana induced had a better expression.PxGNBP3 had a high expression at 6h which induced by Bacillus thuringiensis,and 48 h which induced by Staphylococcus aureus,Serratia marcescens and Escherichia coli both induced large expression of PxGNBP3 gene at 18 h.3.In order to study the function of the PxGNBP3 gene,in this paper,two RNAi methods were used to deal with the DBM:(1)dsRNA was injected into the body in vitro.The expression level of PxGNBP3 was detected by RT-qPCR.After 36 h of RNA interference,PxGNBP3 gene was significantly inhibited.(2)Construction of PxGNBP3 interference vectors pSilent-dsGNBP3,a recombinant B.bassiana,was infested by the epidermis of DBM.The three tissues include midgut,epidermis and fat body of DBM were dissected at different time points after inoculation.The dynamic expression of PxGNBP3 in these three tissues was detected by RT-qPCR.The results showed that the expression of PxGNBP3 gene in the midgut after infection with B.bassiana was down-regulated by 24% to 90%.And PxGNBP3 gene was down-regulated in the fat body from 43% to 94%.The PxGNBP3 gene was found in the epidermis.The highest reduction of 85%,the lowest reduction of 56%.The key ligand,Sp?tzle,in Toll signaling pathway was further tested.RT-qPCR results showed that Sp?tzle gene was always inhibited in fat body and epidermis,and Sp?tzle gene was also inhibited in the midgut,but the expression level of Sp?tzle gene at 36 h was higher than that of the control.RT-qPCR was performed on the end effector antimicrobial peptide genes(Cecropin1,Cecropin2,Cecropin3,Lysozyme1,Lysozyme2,and Moricin).The results showed that the six antibacterial peptide genes were inhibited in body wall and fat bodies at 12-24 h,while some antibacterial peptide genes were highly expressed at 36 h or 48 h,and the expression patterns of all antibacterial peptide genes were basically the same;for the midgut There is no regularity in expression.4.In order to obtain recombinant proteins and their polyclonal antibodies,the prokaryotic expression vector pGEX-4T-PxGNBP3 was constructed and expressed in host strain BL21.The SDS-PAGE electrophoresis detection of PxGNBP3 protein induced by IPTG induced a large number of expression,molecular weight of 55 kDa.The fusion protein GST-PxGNBP3 was purified using the purified tag GST and the protein rPxGNBP3 was purified.The titer of the immunized New Zealand white rabbit was 1:512000.The polyclonal antibody anti-PxGNBP3 could specifically recognize the target protein.5.To further study the function of PxGNBP3 protein,the purified recombinant protein rPxGNBP3 was incubated with microorganisms.Electron microscopy revealed that the recombinant protein rPxGNBP3 had agglutination effect on Gram-negative bacteria,Gram-positive bacteria and fungi.When the calcium ion was present,the agglutination effect was observed.The different cell wall components(PGN,LTA,LPS)and parasite-infected DBM plasma are incubated to determine their PO activity.The results showed that these cell wall components can strongly increase PO activity,and LTA has the greatest effect on PO activity.When the polyclonal antibody anti-PxGNBP3 was added to the plasma,the PO activity decreased,indicating that the polyclonal antibody anti-PxGNBP3 can inhibit the protein in the hemolymph,thereby further reducing the recognition of the fungal cell wall by PxGNBP3.6.The cyst of blood cells of DBM showed that Sephadex A-25 sepharose beads were added to the blood cells and examined under an optical microscope.The results showed that the blood cells could quickly aggregate on the surface of the gel beads to form one or more layers.The sheath material encapsulates the gel beads.After incubation with recombinantprotein rPxGNBP3,the effect of encapsulation was more obvious,indicating that protein rPxGNBP3 can promote the cyst of blood cells.In this dissertation,the pattern recognition receptor PxGNBP3 is screened out from the DGE detected by an entomogenous fungus infecting DBM,and its function was studied in detail from the aspects of mRNA and protein.The results provided a new idea for the novel fungicides and methods.