Transcriptome profiling analysis of the intoxication response in midgut tissue of Agrotis ipsilon larvae to Bacillus thuringiensis Vip3Aa protoxin

Abstract

Vip insecticidal proteins are produced by Bacillus thuringiensis (Bt) during its vegetative growth phase. In the present study, Vip3Aa11 and Vip3Aa39 proteins were investigated. These two proteins present 39 amino acid differential sites and they shared 95.06% amino acid sequence similarity. They are effective against some Lepidoptera insect larvae. In a previous study, using artificial diet bioassays, we estimated the LC50 of Vip3Aa11 and Vip3Aa39 strains against Agrotis ipsilon larvae were 73.41 μg/mL (with 95% confidence interval of 2.34–11.19) and 5.43 μg/mL (with 95% confidence interval of 43.20–115.03), respectively. To investigate the response of Agrotis ipsilon transcriptome in defending against Vip3Aa11 and Vip3Aa39 toxins, we performed high-throughput RNA-sequencing on cDNA generated from the midguts of Agrotis ipsilon larvae that consumed a control diet (CK-M-A), Vip3Aa11 (Vip3Aa11-M-A) and Vip3Aa39 (Vip3Aa39-M-A) proteins. We generated about 98.87 Gb bases in total on BGISEQ-500 sequencing platform. After assembling all samples 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, 1243 bp, 2330 bp and 41.81% respectively. We revealed 558 midgut genes differential expressed in Vip3Aa11-M-A and 65 midgut genes differentially expressed in Vip3Aa39-M-A. The differentially expressed genes were enriched for serine proteases and potential Bt Vip toxin midgut receptor genes. Eleven serine proteases related genes and 13 Bt toxin potential receptor genes with differential expression were found. Based on transcriptome profiling, we focused on validation the sensitivity of these two Vip3Aa proteins to trypsin and their binding properties to Agrotis ipsilon midgut BBMV (Brush Border Membrane Vesicles). The results show that the sensitivity of the two proteins to trypsin is similar. Binding experiments revealed that both proteins can bind to Agrotis ipsilon midgut BBMV, and there is a competitive binding between them. This transcriptome dataset provided a comprehensive sequence resource of Agrotis ipsilon and provides a foundation for comparative studies with other species of insects.