Negligible transcriptome and metabolome alterations in RNAi insecticidal maize against Monolepta hieroglyphica

Abstract

RNAi-based genetically modified maize resistant to Monolepta hieroglyphica (Motschulsky) was demonstrated with negligible transcriptome and metabolome alterations compared to its unmodified equivalent. As one of the most prevalent insect pests afflicting various crops, Monolepta hieroglyphica (Motschulsky) causes severe loss of agricultural and economic productivity for many years in China. In an effort to reduce damages, in this study, an RNA interference (RNAi)-based genetically modified (GM) maize was developed. It was engineered to produce MhSnf7 double-stranded RNAs (dsRNAs), which can suppress the Snf7 gene expression and then lead M. hieroglyphica to death. Field trail analysis confirmed the robustly insecticidal ability of the MhSnf7 GM maize to resist damages by M. hieroglyphica. RNA sequencing analysis identified that only one gene was differentially expressed in the MhSnf7 GM maize compared to non-GM maize, indicating that the transcriptome in MhSnf7 GM maize is principally unaffected by the introduction of the MhSnf7 dsRNA expression vector. Likewise, metabolomics analysis identified that only 8 out of 5787 metabolites were significantly changed. Hence, the integration of transcriptomics and metabolomics demonstrates that there are negligible differences between MhSnf7 GM maize and its unmodified equivalent. This study not only presents a comprehensive assessment of cellular alteration in terms of gene transcription and metabolite abundance in RNAi-based GM maize, but also could be used as a reference for evaluating the unintended effect of GM crops.

Keywords: Metabolome; Monolepta hieroglyphica (motschulsky); RNAi insecticidal maize; Transcriptome; Unintended effect.

Fig.1

Schematic map of the MhSnf7 dsRNA expression vector DBN11048. The vector contains eCaMV 35S promoter, which promotes the MhSnf7 dsRNA with a high expression level. The selection marker is bar gene

Fig. 2

Relative expression analysis of MhSnf7 dsRNA detected by qRT-PCR in the tissues of GM maize and non-GM maize. Mean ± SE was calculated for each tissue type. The values shown in this figure are the averages of three independent experiments. Error bars represent the SD (n = 3) of relative expression levels of MhSnf7 dsRNA

Fig. 3

Insecticidal field trail analysis of MhSnf7 GM maize. M. hieroglyphica larvae fed on GM maize and non-GM maize at time points as the x-axis. The mortalities as the y-axis are shown in red line and black line, respectively. Error bars represent the mortality SD (n = 10) of M. hieroglyphica larvae

Fig. 4

Differentially expressed genes analysis in GM and non-GM maize. a PCA analysis indicates the similarity of RNA-Seq datasets between the two groups. b Volcano plot with -log₁₀ (FDR-value) from the t test as the y-axis and log₂ (fold change) as the x-axis. The blue dot represents down-regulated gene with |log₂ fold change|> 1 and FDR < 0.05, and the gray dots represent non-differentially expressed genes

Fig. 5

Differential metabolites analysis in GM and non-GM maize. a Score plot from PCA model. b Score plot from OPLS-DA model. c Corresponding validation plot from OPLS-DA model. Correlation coefficient as the x-axis represents the replacement reservation degree of replacement test, and the y-axis represents the value of R²Y (green dots) and Q² (blue square dots). The two dashes represent the regression lines of R²Y and Q², respectively

Fig. 6

Volcano plot analysis for differential metabolite expression. log₂ Fold change as the x-axis represents the multiple change of each metabolite and − log₁₀ (FDR-value) from the t test as the y-axis. The significantly up-regulated metabolites are shown in red, while the significantly down-regulated metabolites are shown in blue, and the non-significantly different metabolites are shown in gray. The dots size represents the VIP value of the OPLS-DA model, and the larger the dot, the greater the VIP value