JIA-2021-1127 利用RNA-Seq研究具有杀虫功能的Hvβ’COPI基因dsRNA在茄二十八星瓢虫体内的脱靶效应

doi:10.1016/j.jia.2022.07.015

Journal of Integrative Agriculture ›› 2022, Vol. 21 ›› Issue (9): 2665-2674.DOI: 10.1016/j.jia.2022.07.015

JIA-2021-1127 利用RNA-Seq研究具有杀虫功能的Hvβ’COPI基因dsRNA在茄二十八星瓢虫体内的脱靶效应

收稿日期:2021-06-28 接受日期:2021-08-30 出版日期:2022-09-01 发布日期:2021-08-30

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

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

Received:2021-06-28 Accepted:2021-08-30 Online:2022-09-01 Published:2021-08-30
About author:Correspondence PAN Hui-peng, E-mail: panhuipeng@scau.edu.cn; LI Fei, E-mail: lifei15@zju.edu.cn
Supported by:
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).

摘要/Abstract

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.

Key words: RNAi , dsRNA , off-target effects , RNA-Seq , transcriptome analysis

. JIA-2021-1127 利用RNA-Seq研究具有杀虫功能的Hvβ’COPI基因dsRNA在茄二十八星瓢虫体内的脱靶效应[J]. Journal of Integrative Agriculture, 2022, 21(9): 2665-2674.

LÜ Jing, Satyabrata NANDA, CHEN Shi-min, MEI Yang, HE Kang, QIU Bao-li, ZHANG You-jun, LI Fei, PAN Hui-peng.

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 [J]. Journal of Integrative Agriculture, 2022, 21(9): 2665-2674.

参考文献

Baum J A, Bogaert T, Clinton W, Heck G R, Feldmann P, llagan O, Johnson S, Plaetinck G, Munyikwa T, Pleau M, Vaughn T, Roberts J. 2007. Control of coleopteran insect pests through RNA interference. Nature Biotechnology, 25, 1322–1326.
Birmingham A, Anderson E M, Reynolds A, Ilsley-Tyree D, Leake D, Fedorov Y, Baskerville S, Maksimova E, Robinson K, Karpilow J, Marshall W S, Khvorova A. 2006. 3´UTR seed matches, but not overall identity, are associated with RNAi off-targets. Nature Methods, 3, 199–204.
Chen J, Peng Y, Zhang H, Wang K, Zhao C, Zhu G, Palli S R, Han Z J. 2021. Off-target effects of RNAi correlate with the mismatch rate between dsRNA and non-target mRNA. RNA Biology, 4, 1–13.
Christiaens O, Whyard S, Vélez A M, Smagghe G. 2020. Double-stranded RNA technology to control insect pests: Current status and challenges. Frontiers in Plant Science, 11, 451.
Cooper A M, Silver K, Zhang J, Park Y, Zhu K Y. 2019. Molecular mechanisms influencing efficiency of RNA interference in insects. Pest Managment Science, 75, 18–28.
Gong L, Chen Y, Hu Z, Hu M. 2013. Testing insecticidal activity of novel chemically synthesized siRNA against Plutella xylostella under laboratory and field conditions. PLoS One, 8, e62990.
Guo M J, Nanda S, Chen S M, Lü J, Yang C X, Liu Z Q, Guo W, Qiu B L, Zhang Y J, Zhou X G, Pan H P. 2021a. Oral RNAi toxicity assay suggests clathrin heavy chain as a promising molecular target for controlling the 28-spotted potato ladybird, Henosepilachna vigintioctopunctata. Pest Management Science, 9, doi: 10.1002/ps.6594.
Guo W, Guo M J, Yang C X, Liu Z Q, Chen S M, Lü J, Qiu B L, Zhang Y J, Zhou X G, Pan H P. 2021b. RNA interference-mediated silencing of vATPase subunits A and E affect survival and development of the 28-spotted ladybeetle, Henosepilachna vigintioctopunctata. Insect Science, 28, 1–13.
Haley B, Zamore P D. 2004. Kinetic analysis of the RNAi enzyme complex. Nature Structural & Molecular Biology, 11, 599–606.
Head G P, Carroll M W, Evans S P, Rule D M, Willse A R, Clark T L, Storer N P, Flannagan R D, Samuel L W, Meinke L J. 2017. Evaluation of SmartStax and SmartStax PRO maize against western corn rootworm and northern corn rootworm: Efficacy and resistance management. Pest Management Science, 73, 1883–1899.
Isoe J, Collins J, Badgandi H, Day W A, Miesfeld R L. 2011. Defects in coatomer protein I (COPI) transport cause blood feeding-induced mortality in Yellow Fever mosquitoes. Proceedings of the National Academy of Sciences of the United States of America, 108, E211–E217.
Liu B, Fang L, Liu F, Wang X, Chen J, Chou K C. 2015. Identification of real microRNA precursors with a pseudo structure status composition approach. PLoS ONE, 10, e0121501.
Livak K J, Schmittgen T D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2–∆∆CT method. Methods, 25, 402–408.
Lundgren J G, Duan J J. 2013. RNAi-based insecticidal crops: Potential effects on nontarget species. Bioscience, 63, 657–665.
Lü J, Chen S M, Guo M J, Ye C Y, Qiu B L, Wu J H, Yang C X, Pan H P. 2018. Selection and validation of reference genes for RT-qPCR analysis of the ladybird beetle Henosepilachna vigintioctomaculata. Frontiers in Physiology, 9, 1614.
Lü J, Guo W, Chen S M, Guo M J, Qiu B L, Yang C X, Zhang Y J, Pan H P. 2020a. Double-stranded RNAs targeting HvRPS18 and HvRPL13 reveal potential targets for pest management of the 28-spotted ladybeetle, Henosepilachna vigintioctopunctata. Pest Management Science, 76, 2663–2673.
Lü J, Liu Z Q, Guo W, Guo M J, Chen S M, Yang C X, Zhang Y J, Pan H P. 2020b. Oral delivery of dsHvlwr is a feasible method for management of the pest Henosepilachna vigintioctopunctata (Coleoptera: Coccinellidae). Insect Science, 28, 509–520.
Lü J, Yang C X, Liu Z Q, Vélez A M, Guo M J, Chen S M, Qiu B L, Zhang Y J, Zhou X G, Pan H P. 2021. Dietary RNAi toxicity assay suggests α and γ subunits of HvCOPI as novel molecular targets for Henosepilachna vigintioctopunctata, an emerging coccinellid pest. Journal of Pest Science, 94, 1473–1486.
Ma F, Jiang S, Zhang C. 2020. SiRNA-directed self-assembled quantum dot biosensor for simultaneous detection of multiple microRNAs at the single-particle level. Biosensors and Bioelectronics, 157, 112177.
Pan H P, Yang X W, Romeis J, Siegfried B D, Zhou X G. 2020. Dietary RNAi toxicity assay exhibits differential responses to ingested dsRNAs among lady beetles. Pest Management Science, 76, 3606–3614.
Poreddy S, Li J, Baldwin I T. 2017. Plant-mediated RNAi silences midgut-expressed genes in congeneric lepidopteran insects in nature. BMC Plant Biology, 17, 1–10.
Tu X Y, Wang G H. 2010. Research progress of bio-control of Henosepilachna vigintioctopunctata. Chinese Plant Protection, 30, 13–16. (in Chinese)
Vélez A M, Fishilevich E. 2018. The mysteries of insect RNAi: A focus on dsRNA uptake and transport. Pesticide Biochemistry and Physiology, 151, 25–31.
Vélez A M, Fishilevich E, Rangasamy M, Khajuria C, McCaskill D G, Pereira A E, Gandra P, Frey M L F, Worden S E, Whitlock S L, Lo W, Schnelle K D, Lutz J R, Narva K E, Siegfried B D. 2020. Control of western corn rootworm via RNAi traits in maize: Lethal and sublethal effects of Sec23 dsRNA. Pest Management Science, 76, 1500–1512.
Wang Q, Shen B, Zheng P, Feng H, Chen L, Zhang J, Zhang C, Zhang G, Teng J, Chen J. 2010. Silkworm coatomers and their role in tube expansion of posterior silk gland. PLoS ONE, 5, e13252.
Willow J, Sulg S, Taning C N T, Silva A I, Christiaens O, Kaasik R, Prentice K, Lovei G L, Smagghe G, Veromann E. 2021. Targeting a coatomer protein complex-I gene via RNA interference results in effective lethality in the pollen beetle Brassicogethes aeneus. Journal of Pest Science, 94, 703–712.
Yan S, Ren B Y, Shen J. 2021. Nanoparticle-mediated double-stranded RNA delivery system: A promising approach for sustainable pest management. Insect Science, 28, 21–34.
Zhou G, Isoe J, Day W A, Miesfeld R L. 2011. Alpha-COPI coatomer protein is required for rough endoplasmic reticulum whorl formation in mosquito midgut epithelial cells. PLoS ONE, 6, e18150.
Zotti M, Dos Santos E A, Cagliari D, Christiaens O, Taning C N T, Smagghe G. 2018. RNA interference technology in crop protection against arthropod pests, pathogens and nematodes. Pest Management Science, 74, 1239–1250.

JIA-2021-1127 利用RNA-Seq研究具有杀虫功能的Hvβ’COPI基因dsRNA在茄二十八星瓢虫体内的脱靶效应

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

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 0

编辑推荐

Metrics