| Spodoptera litura(Fab.)(Noctuidae:Lepidoptera)is notorious as one of the most destructive pests of agricultural crops.The species is responsible for huge yield losses annually in many crops due to its vigorous defoliation.It has shown resistance to numerous synthetic insecticides,which has led to frequent control failure in crops.As a consequence,considerable attention has been paid to biological insecticides as alternatives for controlling this pest.Vegetative insecticidal proteins(Vips)synthesized during the vegetative stage of growth by several strains of Bacillus thuringiensis,were proved to share no sequence homology with Cry toxins,which contribute to amplify range of activity spectra and delay insect resistance of this bacterium against insects.In this study,a Vip3Aa protein was obtained by cloning and expression from B.thuringiensis WB5 which was isolated and stored in our laboratory.The insecticidal activities of Vip3Aa protein against three lepidopteran pests(Helicoverpa armigera,Spodoptera exigua and Spodoptera litura)were tested.Based on transcriptional profiling analysis,the complex response of S.litura larvae to Vip3Aa toxin was investigated and relevant analysis of the action mechanism of the toxin was carried out.Using genomic DNA as template and Vip3A-F/Vip3A-R as primers,the entire coding region of the vip3Aa gene of B.thuringiensis WB5 was amplified by PCR with High Fidelity Phusion DNA polymerase.The PCR product was purified,cloned into pMD18-T vector and sequenced.Nucleotide sequence of the vip3Aa gene had been registered in GenBank(accession no.AAM22456).It consists of 2370 bases which encodes a protein of 789 amino acid residues with a calculated molecular mass of 88 kDa.The deduced amino acid sequence of the Vip3Aa from B.thuringiensis WB5 showed more than 99%identity with those of the similar protein reported on NCBI.This vip3Aa gene was inserted into multiple cloning sites of pCznl vector to generate the recombinant expression construct pCznl-vip3Aa,which was then transformed into E.coli BL21.The transformants were induced with 0.2 mmol/L IPTG for the Vip3Aa expression.SDS-PAGE analysis indicated that molecular mass of the expressed product was 88 kDa and appeared in precipitation and supernatant of the cultures.The expressed product was purified by affinity chromatography with Ni-column.The insecticidal activities of Vip3Aa toxin were tested by artificial diet feeding.Bioassay results indicated that S.litura larvae were most sensitive to Vip3Aa toxin,followed by S.exigua,but H.armigera larvea were not sensitive to this toxin.Vip3Aa toxin showed high toxicity to neonatal and first to third instar S.litura larvae with LC50 of 2.609 ng/cm2,28.778 ng/cm2,70.460 ng/cm2 and 200.627 ng/cm2 artificial diet respectively.The histopathological changes of S.litura larvae fed with Vip3Aa mainly included vacuolization of the cytoplasm,cells swelling and brush border membrane destruction,the karyotheca of cell nucleus crinkled and the chromatins unevenly distributed,mitochondria swelling and deformation with disintegration and lysis of cristae.The complete transcriptome information of S.litura was obtained.A total of 50.09 Gb Clean Data was generated.After assembly using the Trinity method,56,498 unigenes with an N50 value of 1,853 bp and a mean length of 827.6 bp were obtained,and 21,701 unigenes were successfully annotated by Nr?SwissProt?Pfam?KOG?KEGG?GO and COG database.Nr database queries revealed that a high percentage of S.litura sequences closely matched sequences of silkworm and monarch butterfly.GO distributions of the unigenes were classified into three categories--cellular components,molecular functions and biological processes.The potential pathways of unigene could participated in were annotated by KEGG database.A genome-wide high-throughput transcriptome sequencing was used for comparison of Vip3Aa-treated and non-treated of S.litura whole larvae and midgut.Results of transcriptome abundance showed that Vip3Aa toxin provoked a wide transcriptional response of the S.litura midgut.To investigate the complex response of S.litura larvae to Vip3Aa toxin,the differentially expressed genes between Vip3Aa toxin treatment and untreatmen S.litura whole larvae were enriched for immunity-related,metabolic-related genes,and DEGs between Vip3Aa toxin treatment and untreatmen S.litura larvae midgut were enriched for Bt-related genes.In total,37 differentially expressed immunity-related genes were identified,including 12 genes for recognition,8 genes for melanization processes,7 genes for signal transduction,7 genes for antimicrobial effector and 3 genes for other immune-related proteins.Most of immunity-related genes were shown up-regulated,which indicates that the S.litura larvae can activate immune response.Meanwhile,127 metabolic genes were differentially expressed between control and Vip3Aa-treated S.litura whole larvae.Five types of mainly basal metabolic systems were identified,including amino acid metabolism,metabolism of cofactors and vitamins,lipid metabolism,xenobiotics biodegradation and metabolism,and carbohydrate metabolism.Thirty-six differentially expressed detoxification metabolism genes were identified,including glutathione S-transferase genes and cytochrome P450 genes.And the majority of these genes were up-regulated,which indicates that the S.litura larvae can activate detoxification metabolism response.Forty-seven serine proteases in S.litura larvae midgut were found to be differentially expressed when treatment with Vip3Aa toxin,which was mainly annotated as trypsin,chymotrypsin and serine proteases.Four kinds of general characterized receptors genes of B.thuringiensis Cry toxin were found differentially expressed in S.litura larvae midgut,involved in 4 ALP,4 G-protein,2 cadherin and 1 APN transcripts.Most of the serine proteases genes in midgut,which were up-regulated relative to whole larvae,were found differentially expressed when S.litura larvae fed with Vip3Aa toxin.It provided a preliminary proof that serine proteases maybe involved in the solubilization of Vip3Aa toxin.Due to the complexity of serine protease family,we select two of serine protease,trypsin and chymotrypsin,for the investigation of in vitro proteolytic processing of Vip3Aa toxin.Using casein as a general substrate,zymogram analysis of the soluble proteases and membrane-bound proteases of S.litura larvae showed the presence of many active proteases including trypsin and chymotrypsin.The results of in vitro proteolytic processing of Vip3Aa toxin by the soluble proteases,membrane-bound proteases,trypsin and chymotrypsin revealed that all of them can digest the toxin.Bioassay results indicated that the digested products of trypsin and chymotrypsin were toxic to first instar S.litura larvae,with LCso of 77.052 ng/cm2 and 118.26 ng/cm2 artificial diet respectively.Moreover,the observations of midgut sections showed wide damage to S.litura larvae midgut epithelium induced by the above two digested products.These results demonstrated that trypsin and chymotrypsin are involved in the activation of Vip3Aa toxin.The ability of Vip3Aa toxin bound to S.litura gut BBMV was investigated by in vitro pull-down assay.Obvious bands at about 62 kDa,55 kDa,43 kDa,30 kDa were detected by SDS-PAGE.A 62 kDa bioactive core component of Vip3Aa toxin was found by Western Blot.Then some putative receptors of Vip3Aa toxin(129 kDa,87 kDa,52 kDa,47 kDa)were detected by Ligand Blot with Biotin-Vip3Aa and DyLight-Avidin as antibody.Proteins obtained by pull-down assay were identified by Shotgun mass spectrometry and blasted with Uniprot database and transcriptome data.Eleven and 168 sequence were matched to the Uniprot database and the transcriptome data respectively.Combining differential expression analysis of general characterized receptors genes of B.thuringiensis toxin in S.litura midgut when treatment with Vip3Aa toxin,we can speculate that APN(cl8942;c18980;c18685;c5990),G protein(cl9499)and ABCC2(c19529)will probably be the putative receptors of the Vip3Aa toxin. |
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