| Bacillus thuringiensis(Bt)is the most widely used and environmentally friendly microbial insecticide in the world.Vegetative Insecticidal Protein(VIP)is a novel and representative insecticidal protein found after Insecticidal Crystal Proteins(ICPs).Vip3Aa protein is the most commonly studied protein in vegetative insecticidal proteins.These proteins have high amino acid sequence homology and high insecticidal activity against lepidopteran insects.However,the insecticidal spectrum and insecticidal activities of Vip3Aa proteins from different Bt strains are different.Vip3Aa11 and Vip3Aa39 were isolated from Bacillus thuringiensis strains C9 and T03B001,respectively.These two proteins present 39 amino acid differential sites and they shared 95.06%amino acid sequence similarity.However,they exhibit different insecticidal activities against some Lepidopteran insects.For example,the insecticidal activities of these two proteins against the Lepidoptera insects Agrotis ipsilon are different.The LC50 of these two proteins against the Agrotis ipsilon are 73.62 ?g/mL(3.20?115.03)and 5.43 ?g/mL(2.34?11.19),respectively.In order to investigate the difference in the insecticidal activity of these two proteins against the same insect,we performed high-throughput RNA-sequencing on cDNA generated from the midguts of Agrotis ipsilon larvae.We generated about 98.87 Gb bases in total on BGISEQ-500 sequencing platform.After assembling all sanples together and filtering the abundance,we got 51,887 unigenes,the total length,average length,N50 and GC content of unigenes are 64,523,651 bp,1,243 bp,2,330 bp and 41.81%respectively.We perform Unigenes functional annotation with 7 functional databases with 25,411(NR:48.97%),14,673(NT:28.28%),18,251(SwissProt:35.17%),18,076(KOG:34.84%),19,743(KEGG:38.05%),2,758(GO:5.32%),and 18,787(Interrro:36.21%)unigenes performed functional annotations.We identity the candidate coding region among the Unigenes and get 23,905 CDS.It also detected 4,168 SSRs,predicted 3,477 Unigenes encoding transcription factors and we detected the SNP.Compared with the Nr database function annotation result,the species distribution of the Agrotis ipsilon is closer to the Amyelois transitella and the Bombyx mori.We annotate the Unigenes with KOG database and calculate the Unigene distribution based on the 25 functional groups.Annotate the Unigenes with KEGG database and calculate the Unigene distribution of KEGG Level 1 and KEGG Level 2 respectively.The unigenes which aligned to NR database has been annotated to GO database with Blast2GO,we calculate the distribution in 3 categories:biological process,cellular component and molecular functions.In this study,differential expressed genes(DEGs)were detected based on the results of gene expression level in each sample.A total of 652 differentially expressed genes were obtained by screening.There are 558 differentially expressed genes were produced by the transcriptome of the Agrotis ipsilon in the midgut by feeding the Vip3Aa11 toxin protein,and 65 differentially expressed genes were produced by feeding the Vip3Aa39 toxin protein.There are 29 differentially expressed genes between these two treatments Vip3Aa11(Vip3Aa11-M-A)and Vip3Aa39(Vip3Aa39-M-A).For differentially expressed genes,we performed cluster analysis,GO function analysis and Pathway function analysis.Statistical analysis of differentially expressed genes revealed that 22 genes had the same up or down-regulation trend in Vip3Aa11-VS-CK and Vip3Aa39-VS-CK.Among these differentially expressed genes,we detected 10 immune-related genes,14 metabolic-related genes,13 Bt toxin potential receptor genes(including 1 Cadherin,1 ALP,1 APN,2 G-protein and 8 ABC transporters)and 11 serine protease genes that may be involved in Vip protein activation or degradation.To investigate whether there is a difference in the sensitivity of the Vip3Aa11 and Vip3Aa39 proteins to the midgut protease(mainly serine proteases).In this study,based on the midgut transcriptome data of the Agrotis ipsilon,we selected trypsin,the main component of serine protease,for the Vip3Aa proteins digestion treatment.In vitro digestion showed that 88 kDa of Vip3Aa11 and Vip3Aa39 proteins were able to be treated at different concentrations of trypsin at the same time(1h)and a core protein of 66 kDa size was produced,and the 66 kDa core fragment is resistant to high concentrations of trypsin(25 mg/mL).Under the same concentration of trypsin(1 mg/mL)and different digestion time,both proteins could be completely digested into 66 kDa core protein fragments in 10 minutes,and the core fragments remained stable after 2 h of digestion.This result of trypsin digestion suggested that these two proteins display same levels of stability and processing rates in the insect midgut,and it is preliminarily indicated that the cause of the difference in insecticidal activity between the two proteins against the larvae may not be due to the activation of the midgut protease.Vip3Aa protein binds to BBMV in insect midgut is mediated by binding sites or specific receptors and shown insecticidal activity.In this study,binding experiments in vitro were performed to investigate whether Vip3Aa11 and Vip3Aa39 share the same receptor in the midsut of the s Agrotis ipsilon.The results of the binding experiments showed that the biotin-labeled proteins Bio-Vip3Aa11 and Bio-Vip3Aa39 could bind to the midgut BBMV(Brush Border Membrane Vesicles)of the Agrotis ipsilon,and the binding ability of Vip3Aa39 protein was slightly higher than that of Vip3Aa11 protein.It indicated that Vip3Aa11 and Vip3Aa39 were present receptor proteins in the midgut of the test insect.Competitive binding experiments showed that one protein(Vip3Aa11)could compete with another protein(Vip3Aa39).These results revealed that these two proteins have some of the same receptor proteins in the midgut of the Agrotis ipsilon.The ligand-blotting assay showed that both biotinylated Vip3Aa toxins primarily showed binding to four proteins of 30,50,70 and 85 kDa,suggesting that these two toxins have the same numbers and size of receptors in the midgut of the Agrotis ipsilon.In summary,we conclude that the causes for the different insecticidal activity between Vip3Aa11 and Vip3Aa39 proteins against Agrotis ipsilon may be due to the difference in binding ability of these two proteins on BBMV.In conclusion,the midgut transcriptome of this study provides a comprehensive sequence of resources for studying the receptors and virulence mechanisms of the Agrotis ipsilon after feeding the Vip3Aa toxin and expanding the transcriptomics resources of the pest.In addition,this transcriptomics data enhances our understanding the response of the Agrotis ipsilon to the Vip3Aa proteins. |
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