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A survey on the off-target effects of insecticidal double-stranded RNA targeting the Hvβ´COPI gene in the crop pest Henosepilachna vigintioctopunctata through RNA-seq
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| LÜ Jing1, Satyabrata NANDA2, CHEN Shi-min1, MEI Yang3, HE Kang3, QIU Bao-li1, ZHANG You-jun4, LI Fei3, PAN Hui-peng1 |
1 Key Laboratory of Bio-pesticide Innovation and Application of Guangdong Province, South China Agricultural University, Guangzhou 510642, P.R.China
2 MS Swaminathan School of Agriculture, Centurion University of Technology and Management, Paralakhemundi 761211, India
3 Institute of Insect Sciences, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, P.R.China
4 Department of Plant Protection, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R.China
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摘要
本研究比较了分别摄取了dsHvβ′COPI（外壳蛋白复合物I，β′亚基）和dsGFP的茄二十八星瓢虫（Henosepilachna viginitoctopunctata）转录组，以期了解外源dsRNA在茄二十八星瓢虫体内引起的脱靶效应。RNA-Seq结果显示，与dsGFP对照组相比，dsHvβ′COPI处理组中分别有63个上调和44个下调的差异表达基因（DEG）。通过荧光定量PCR（RT-qPCR）分析验证选取的DEG的差异表达，证实了转录组分析结果是可靠的。进一步的分析表明，在茄二十八星瓢虫转录组中没有与Hvβ'COPI同源的基因。此外，在转录组中未发现与dsHvβ'COPI连续匹配>11 bp的基因。我们选择了6个可能参与茄二十八星瓢虫中Hvβ'COPI所参与的代谢路径基因（Hvcitron、Hvhelicase、Hvtransportsase、Hvserine、Hvdynein和HvE3 ubiquitin）来检测dsHvβ'COPI可能引起的脱靶效应。利用RNAi评估这6个脱靶基因的沉默效率。然而，RNAi结果表明这6个基因的表达量虽下调，但对茄二十八星瓢虫无明显的致死作用。此外，本研究的结果将有助于将来RNAi介导的害虫防治的风险分析。
Abstract
The specificity of the double-stranded RNA (dsRNA) used in the RNA interference (RNAi) technique is crucial for the success of sequence-specific gene silencing. Currently, RNAi-mediated insect control is a trending research topic. However, the off-target effects of the dsRNA in RNAi are a major concern. In this study, the dsHvβ´COPI (coat protein complex I, β´ subunit)-treated and untreated transcriptomes of the 28-spotted potato lady beetle (Henosepilachna vigintioctopunctata) were compared to understand its off-target gene silencing effects. The RNA-seq results revealed that 63 and 44 differentially expressed genes (DEGs) were upregulated and downregulated, respectively, in the dsHvβ´COPI treated group as compared with the control. Validation of the differential expressions of some selected DEGs via reverse transcription-quantitative PCR (RT-qPCR) analysis confirmed the reliability of the transcriptome analysis results. Further downstream analysis revealed that there were no genes homologous with Hvβ´COPI in H. vigintioctopunctata. Additionally, no genes with a >11 bp continuous match with dsHvβ´COPI were found in the H. vigintioctopunctata transcriptome. Six genes (Hvcitron, Hvhelicase, Hvtransposase, Hvserine, Hvdynein, and HvE3 ubiquitin) were selected to examine the off-target activity of dsHvβ´COPI based on their potential involvement in various H. vigintioctopunctata metabolic pathways. The severity of silencing these six off-target genes was evaluated by employing RNAi. The RNAi results confirmed the downregulation of the expression of all six genes, although there was no significant lethality. The findings of this study will be helpful in the risk analysis of future RNAi-mediated pest control experiments.
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Received: 28 June 2021
Accepted: 30 August 2021
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Fund: This research was supported by the National Natural Science Foundation of China (31972269 and 32172500) and the National Key R&D Program of China (2017YFD0200900).
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| About author: Correspondence PAN Hui-peng, E-mail: panhuipeng@scau.edu.cn; LI Fei, E-mail: lifei15@zju.edu.cn |
Cite this article:
LÜ Jing, Satyabrata NANDA, CHEN Shi-min, MEI Yang, HE Kang, QIU Bao-li, ZHANG You-jun, LI Fei, PAN Hui-peng.
2022.
A survey on the off-target effects of insecticidal double-stranded RNA targeting the Hvβ´COPI gene in the crop pest Henosepilachna vigintioctopunctata through RNA-seq
. Journal of Integrative Agriculture, 21(9): 2665-2674.
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