- suppressalis may be related to changes in the transcription levels of enzymes involved in hydrolysis, digestive, catalytic and. - The assembled and annotated transcriptome data provide valuable genomic resources for further study of the molecular mechanisms of C. - The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. - If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. - Based on the pore forma- tion model, protoxin is solubilized in the alkaline gut, digested by gut proteinase, and converted to the acti- vated toxin, which then binds to the cadherin receptor located in the cell membrane of the insect midgut. - In contrast, the signal transduction model considers that the binding of the activated Bt toxin to specific receptors stimulates the G-protein coupled signalling pathway, leading to the activation of protein kinases [14]. - Regardless of the model, conversion of the protoxin to the activated toxin by insect midgut proteases and the binding of the activated toxins to re- ceptors on the surface of midgut cells are recognized as essential steps for toxicity [15]. - Thus, decreased conver- sion of the protoxin to activated toxin and reduced toxin binding of Cry toxin to receptors are considered the most common mechanisms resulting in insect resistance to Cry toxins due to downregulation or mutation of the midgut proteinase and receptors [15, 16]. - For example, decreased expression of the trypsin gene leading to in- complete activation of protoxin results in Cry1A resist- ance in Helicoverpa armigera and Ostrinia nubilalis [17–21]. - Additionally, reduced binding ability of Cry toxin to midgut receptors via a decrease in the activity and tran- scription of ALP or APN as well as mutations of APN, cadherin, and ABCC lead to Cry1A re- sistance in H. - Moreover, Cry1C and Cry1A toxins specifically bind to distinct isoforms of APN in the brush border membrane of Manduca sexta [34]. - In our previous study, we ob- served that Cry1A and Cry1C were able to recognize dif- ferent binding proteins in the midgut of C. - Therefore, clarifi- cation of the molecular mechanism of Cry1C resistance in C. - Among these, 86,912 unigenes were greater than 500 bp, accounting for 62.43% of the assembled unigenes.. - Annotation of the 139,206 unigene sequences from the midgut transcriptome of the susceptible (FZS) and re- sistant (FZ1C) strains of C. - suppressalis was performed by Blast searches with a cut-off E-value of 1e-5 in the following databases: NR (NCBI non-redundant protein sequences), NT (NCBI non-redundant nucleotide se- quences), PFAM (Protein family), KOG/COG (Clusters of Orthologous Groups of proteins), Swiss-Prot (a manually annotated and reviewed protein sequence data- base), KO (KEGG Orthology) and GO (Gene Ontology).. - According to the results, 40.18% of the unigenes were annotated in NR, 27.72% in NT, 31.41% in PFAM, 15.17% in KOG, 25.9% in Swiss-Prot, 14% in KO and 31.45% in GO (Additional file 1: Figure S1. - In Blastx homology searches with a cut-off E-value of 1e and 27.5% of the unigenes matched with B. - To further test the integrity and effectiveness of the annotation process, the unigenes annotated in the NR database were classified in KOG. - Before the analysis of differentially expressed unigenes be- tween the resistant and susceptible strains, the hierarchical clustering analysis on the midgut samples collected for Table 1 Summary of reads of the midgut transcriptome in Cry1C-resistant (FZ1C) and -susceptible (FZS) strains of C. - Table 2 Summary of the length of assembled unigenes of the midgut transcriptome in Cry1C-resistant (FZ1C) and -susceptible (FZS) strains of C. - A total of 21,127 unigenes were assigned to 25 categories in the KOG classification. - The right legend shows a description of the 25 functional categories. - (DEGs) were analysed by comparing the midgut transcrip- tome of the FZ1C and FZS strains. - The results revealed 5328 unigenes to be significantly differentially expressed in the midgut transcriptomes of the FZ1C and FZS strains (padj<. - The DEGs encoding potential Cry toxin receptors or those involved in the Cry toxin action pathway were fur- ther investigated (Table 3), including aminopeptidase-N, the ABC transporter family, alkaline phosphatase-like,. - Among them, nine uni- genes encode APNs, but they were all upregulated in the FZ1C strain. - Two unigenes annotated as an aminopepti- dase P-like protein (APP-like) were both downregulated in the FZ1C strain. - moreover, one of them was downreg- ulated 10.741-fold compared with that in the FZS strain.. - Seventeen unigenes were associated with the ABC trans- porter family, 11 of which were upregulated and 6 downregulated in the FZ1C strain compared with the FZS strain. - Ten unigenes encode ALPs: seven of them were downregulated and 3 of them upregulated in the FZ1C strain. - The heatmap of DEGs encoding potential Cry toxin receptors or those involved in pathways of the mode of Cry toxin action are displayed (Additional file 4: Figure S4).. - Seven of them were upregu- lated and 10 downregulated in the resistant strain com- pared with the susceptible strain. - Twenty-two unigenes encode serpin protease inhibitors: two of them were up- regulated and the other 20 downregulated in the Cry1C- resistant strain compared to the susceptible strain (Table 3). - To further analyse the biological functions of the sig- nificant DEGs between the resistant (FZ1C) and suscep- tible (FZS) strains of C. - upregulated unigenes were annotated to 1727, 378 and 761 GO terms in the biological process, cellular compo- nent and molecular function categories, respectively.. - 4 Changes in the distribution of differentially expressed unigenes between Cry1C-resistant (FZ1C) and Cry1C-susceptible (FZS) strains of C. - AFQ01141.1 serine protease inhibitor 005 [H. - ACD44927.1 serine protease [B. - E-18 EHJ63159.1 serine protease inhibitor 28 [D. - serine protease inhibitor 28 [B. - EHJ63159.1 serine protease inhibitor 28 [D. - AEW46890.2 serine protease inhibitor 004, partial [C. - AFQ01141.1 serine protease inhibitor 005 [C. - AEW46891.2 serine protease inhibitor 012 [C. - AEW46893.2 serine protease inhibitor 002 [C. - Validation of differentially expressed genes by qRT-PCR To validate the expression patterns of the unigenes, 12 differentially expressed unigenes (including Cluster and were selected for qRT-PCR according to their fold change values in the expression profiles from the dataset. - Overall, relative expression of the top 8 unigenes was significantly upregulated in the resistant FZ1C strain of C. - suppressalis, whereas that of the last 4 unigenes was remarkably reduced in the resist- ant strain (Fig. - The mode of Cry toxin action is a very complex process involving the toxin structure, many enzymes and recep- tors, among others, and a change in any step of the toxi- cology process inevitably leads to insect resistance. - A total of 139,206 unigenes were de novo assembled from 373 million clean reads in the C. - suppressalis lar- vae to Cry1C and Cry1Ab/Cry1Ac fusion proteins expressed by transgenic Bt rice, suggesting that APNs are involved in the mode of Cry1Ab/Cry1Ac and Cry1C ac- tion. - Nine unigenes en- coding APNs in the current study were all upregulated in the resistant strain (FZ1C) compared with the susceptible strain (FZS) of C. - In addition, we observed signifi- cant downregulation of two unigenes annotated as amino- peptidase P-like proteins in the resistant strain (FZ1C) of C. - in particular, expression of one unigene was decreased 10.74-fold in the FZ1C strain compared with the FZS strain. - These results indicate that an aminopeptidase P-like protein is likely involved in the mode of Cry1C toxin action and accounts for C. - Another important group of receptors involved in the mode of Cry toxin action is ABC transporters. - xylostella showed that eight unigenes annotated as ABCC2 were detected in the Cry1Ac-resistant strain, with the majority being downregulated [41]. - Furthermore, six downregulated unigenes anno- tated as ABC transporters (Cluster Cluster Cluster Cluster Cluster and Cluster were identified in the C. - Nevertheless, further study should be conducted to reveal the exact function of ABC transporters in the resistant strain of C. - Three unigenes were annotated as cadherin (Cluster Cluster and Cluster but they were all upregulated in the resistant strain. - which found that cadherin did not play a major role in the mode of Cry 1C action in C. - The conversion of the Cry protoxin to the cytotoxic form by proteases is an inevitable process for Cry tox- icity. - In this study, 12 unigenes annotated as trypsin or trypsin/chymotryp- sinogen-like proteinase were overexpressed in the Cry1C-resistant strain compared with the susceptible strain. - Over-transcription of these proteinases in the resistant strain of C. - In the midgut transcriptome of O. - Thus, the exact role of P450 in the mode of Cry toxin ac- tion is uncertain, as they are generally thought to be in- volved in the degradation of xenobiotics [58]. - In the present study, a large proportion of unigenes (12/21) an- notated as detoxification enzymes were upregulated in the Cry1C-resistant strain. - GO and KEGG analyses provided important clues to re- veal the potential mechanisms involved in the develop- ment of resistance. - sup- pressalis resistance to Cry1C toxin may be associated with increased digestive activity, catalytic activity, detoxification activity and hydrolase activity in the larval midgut. - Insect rearing and selection of the resistant strain. - The Cry1C-selected strain (FZ1C) was initially exposed to a sublethal dose of the Cry1C diet throughout larval development. - The toxin concentration was steadily increased in succeeding generations to achieve 40–60% mortality of the exposed larvae. - In parallel, the susceptible strain (FZS) reared in the absence of Cry1C toxin was used as the negative control.. - Dissection of the midgut and extraction of RNA. - After assessment of the quantity and purity of the total RNA, the RNA samples were delivered to Beijing Novogene Technology Com- pany for RNA sequencing.. - RNA-Seq library preparation and Illumina sequencing The RNA-Seq libraries were generated from a total amount of 1.5 μg RNA per biological replicate using NEB- Next® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) according to the manufacturer’s instructions, and index codes were added to identify the sequences of each. - The remaining overhangs of the cDNA was converting into blunt ends by exonuclease/polymerase activities of DNA polymerase I, the 3′ ends of the DNA fragments were adenylated and NEBNext adaptors with hairpin loop structures were ligated. - At the same time, the Q20, Q30, GC content and sequence duplication level of the clean reads were calculated in this step. - All of the downstream ana- lyses were conducted with clean reads of high quality.. - Twelve genes (Additional file 7) identi- fied as differentially expressed were selected based on their fold changes in the expression profile. - Each of the three biological replicates was measured with four technical replicates, and the expres- sion levels of candidate genes were normalized with the EF-1 gene.. - (F) Serine protease inhibitors. - This research was funded by the National Genetically Modified Organisms Key Breeding Projects of China (2016ZX ZX0801101B and 2016ZX the National Natural Science Foundation of China and the Scientific and Technological Innovation Project of the Chinese Academy of Agricultural Sciences.. - LZH and FJC conceived of the study, participated in its design and coordination and helped to revise the manuscript. - The raw reads of the two transcriptomes in this study have been deposited in the NCBI SRA database under accession numbers SRR12170219 (midgut transcriptome of susceptible strain of Chilo suppressalis) and SRR12170218 (midgut transcriptome of Cry1C resistant strain of Chilo suppressalis).. - Chilo suppressalis is a common agricultural pest, which is not included in the “ List of Endangered and Protected Animals in China. - All animal procedure in this study was performed according to guidelines developed by the ethics committee of the State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences and the Experimental Animal Welfare Ethics Committee of Nanjing Agricultural University. - Characterization of cysteine protease- like genes in the striped rice stem borer, Chilo suppressalis. - Effects of insect-resistant transgenic Bt rice with a fused cry1Ab+cry1Ac gene on population dynamic of the stem borers, Chilo suppressalis and Sesamia inferens, occurring in paddy field. - Resistance of Helicoverpa armigera to Cry1Ac toxin from Bacillus thuringiensis is due to improper processing of the protoxin. - Mis-splicing of the ABCC2 gene linked with Bt toxin resistance in Helicoverpa armigera.. - Single amino acid mutation in an ATP-binding cassette transporter gene causes resistance to Bt toxin Cry1Ab in the silkworm, Bombyx mori. - RNAi in the striped stem borer, Chilo suppressalis, establishes a functional role for aminopeptidase N in Cry1Ab intoxication. - Midgut transcriptome response to a cry toxin in the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae). - Differential role of Manduca sexta aminopeptidase-N and alkaline phosphatase in the mode of action of Cry1Aa, Cry1Ab, and Cry1Ac toxins from Bacillus thuringiensis. - Down-regulation of a novel ABC transporter gene (Pxwhite) is associated with Cry1Ac resistance in the diamondback moth, Plutella xylostella (L. - Binding characteristic to mosquito-larval midgut proteins of the cloned domain II-III fragment from the Bacillus thuringiensis Cry4Ba toxin. - Cytochromes P450 of insects: the tip of the iceberg. - Involvement of nonbinding site proteinases in the development of resistance of Helicoverpa armigera (Lepidoptera: Noctuidae) to Cry1Ac
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