Functional Analysis of RNA Interference-Related Soybean Pod Borer (Lepidoptera) Genes Based on Transcriptome Sequences

Abstract

RNA interference (RNAi) is useful for controlling pests of agriculturally important crops. The soybean pod borer (SPB) is the most important soybean pest in Northeastern Asia. In an earlier study, we confirmed that the SPB could be controlled via transgenic plant-mediated RNAi. Here, the SPB transcriptome was sequenced to identify RNAi-related genes, and also to establish an RNAi-of-RNAi assay system for evaluating genes involved in the SPB systemic RNAi response. The core RNAi genes, as well as genes potentially involved in double-stranded RNA (dsRNA) uptake were identified based on SPB transcriptome sequences. A phylogenetic analysis and the characterization of these core components as well as dsRNA uptake related genes revealed that they contain conserved domains essential for the RNAi pathway. The results of the RNAi-of-RNAi assay involving Laccase 2 (a critical cuticle pigmentation gene) as a marker showed that genes encoding the sid-like (Sil1), scavenger receptor class C (Src), and scavenger receptor class B (Srb3 and Srb4) proteins of the endocytic pathway were required for SPB cellular uptake of dsRNA. The SPB response was inferred to contain three functional small RNA pathways (i.e., miRNA, siRNA, and piRNA pathways). Additionally, the SPB systemic RNA response may rely on systemic RNA interference deficient transmembrane channel-mediated and receptor-mediated endocytic pathways. The results presented herein may be useful for developing RNAi-mediated methods to control SPB infestations in soybean.

RNA interference-mediated silencing of genes involved in the immune responses of the soybean pod borer Leguminivora glycinivorella (Lepidoptera: Olethreutidae)

“…The cDNA library was sequenced on the Illumina HiSeq 2000 system (Illumina, San Diego, CA, USA). The unigenes from six samples were combined to create the SPB unigene database ( Chen et al, 2014 ; Meng et al, 2017c ). All raw transcriptome data have been deposited in the NIH Short Read Archive (accession numbers SRR5985984 , SRR5985985 , SRR5985986 , SRR5985987 , SRR5985988 , SRR5985989 ).…”

“…The efficiencies of the qRT-PCR primer pairs were greater than 90% ( Table S2 ). The qRT-PCR analysis contained three biological replicates, each having three technical replicates ( Meng et al, 2017c ). Each target gene’s qRT-PCR products were sequenced to confirm their identities.…”

Peer Review #1 of "RNA interference-mediated silencing of genes involved in the immune responses of the soybean pod borer Leguminivora glycinivorella (Lepidoptera: Olethreutidae) (v0.1)"

“…The cDNA library was sequenced on Illumina HiSeq 2000 system. The unigenes from six samples were combined to create the SPB unigene database (Chen et al, 2014;Meng et al, 2017c). All raw transcriptome data have been deposited in the NIH Short Read Archive (accession numbers SRR5985984-SRR5985989).…”

Untitled

“…cDNA fragments of a suitable length (300-500 bp) were obtained by agarose gel electrophoresis and amplified byPCR to construct the final cDNA libraries for paired-end sequencing using the Illumina HiSeq 2000 system. The unigenes from six samples were combined to create the SPB unigene database (Chen et al, 2014;Meng et al, 2017c). All raw transcriptome data have been deposited in the NIH Short Read Archive (accession numbers SRR5985984-SRR5985989).…”