昆虫致死基因siRNA在靶标和非标靶昆虫中的脱靶效应

doi:10.1016/S2095-3119(20)63394-9

Journal of Integrative Agriculture ›› 2022, Vol. 21 ›› Issue (1): 170-177.DOI: 10.1016/S2095-3119(20)63394-9

昆虫致死基因siRNA在靶标和非标靶昆虫中的脱靶效应

收稿日期:2020-06-12 接受日期:2020-08-08 出版日期:2022-01-01 发布日期:2022-01-01

Using transcriptome Shannon entropy to evaluate the off-target effects and safety of insecticidal siRNAs

MA Wei-hua¹, WU Tong^{1, 2}, ZHANG Zan³, LI Hang³, SITU Gong-ming³, YIN Chuan-lin², YE Xin-hai², CHEN Meng-yao², ZHAO Xian-xin², HE Kang², LI Fei²

¹ College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, P.R.China
²State Key Laboratory of Rice Biology/Ministry of Agricultural and Rural Affairs Key Laboratory of Molecular Biology of Crop Pathogens and Insect Pests/Institute of Insect Sciences, Zhejiang University, Hangzhou 310058, P.R.China
³College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, P.R.China

Received:2020-06-12 Accepted:2020-08-08 Online:2022-01-01 Published:2022-01-01
About author:MA Wei-hua, E-mail: weihuama@mail.hzau.edu.cn; Correspondence LI Fei, E-mail: lifei18@zju.edu.cn
Supported by:
This work was funded by grants from the National Science and Technology Major Project of China (2016ZX08011002). We also thank the DBMediting Company for professional English language editing services.

摘要/Abstract

摘要：

RNAi介导的有害生物防控策略是农业生物技术领域的最新突破之一。但是，目前对RNAi防控策略可能产生的脱靶效应仍未完全了解。本文中，我们研究了两种昆虫致死基因siRNA在靶标和非标靶昆虫中的脱靶效应。结果表明，致死基因siRNA的脱靶效应广泛存在于靶标昆虫和非靶标昆虫中。我们根据基因的同源性、相关KEGG途径以及与siRNA序列的连续匹配度，对所有表达量受影响的基因进行了分类。出人意料的是，非靶标基因表达量受影响的程度与序列的连续匹配度并不一直。而少部分同源基因和KEGG相关基因的表达量正如预期的一样发生了显著变化。通过计算转录组熵值，结果表明尽管在siRNA处理后数百个基因的表达受到影响，但转录组的熵值保持不变，这表明转录组的表达模式在整体上是平衡的。本文的结果表明，siRNA与非靶标生物中的单个基因发生交叉反应，但在基因组水平上对靶标和非靶标生物中转录组完整性并没有显着影响。同时，本文提出了一种评估昆虫致死基因siRNA脱靶效应的体系，将有助于评估RNAi害虫防控策略的安全性。

Abstract: A recent breakthrough in agricultural biotechnology is the introduction of RNAi-mediated strategies in pest control. However, the off-target effects of RNAi pest control are still not fully understood. Here, we studied the off-target effects of two insecticidal siRNAs in both target and non-target insects. The results revealed that off-target effects of insecticidal siRNAs occur widely in both target and non-target insects. We classified the expression-changed genes according to their homology to the siRNA-targeted gene, related KEGG pathways with the siRNA-targeted gene and continuous matches with siRNAs. Surprisingly, the unintended significant changes in gene expression levels did not strictly match with the number of contiguous nucleotides in the siRNAs. As expected, the expression of small portions of the homologous and KEGG-related genes were significantly changed. We calculated the Shannon entropy of the transcriptome profile of the insects after injecting them with insecticidal siRNAs. Though hundreds of genes were affected in their expression levels post siRNA-treatment, the Shannon entropy of the transcriptome remained unchanged, suggesting that the transcriptome expression was balanced. Our results provide evidence that siRNAs cross-reacted with individual genes in non-target species, but did not have significant effects on the integrity of the transcriptome profiles in either target or non-target species on a genomic scale. The metric we proposed can be used to estimate the off-target effects of insecticidal siRNAs, which might be useful for evaluating the safety of RNAi in pest control.

Key words: RNAi , off-target effect , transcriptome entropy , non-target organisms , RNAi-mediated pest management

. 昆虫致死基因siRNA在靶标和非标靶昆虫中的脱靶效应 [J]. Journal of Integrative Agriculture, 2022, 21(1): 170-177.

MA Wei-hua, WU Tong, ZHANG Zan, LI Hang, SITU Gong-ming, YIN Chuan-lin, YE Xin-hai, CHEN Meng-yao, ZHAO Xian-xin, HE Kang, LI Fei . Using transcriptome Shannon entropy to evaluate the off-target effects and safety of insecticidal siRNAs[J]. Journal of Integrative Agriculture, 2022, 21(1): 170-177.

参考文献

Bally J, Mcintyre G J, Doran R L, Lee K, Perez A, Jung H, Naim F, Larrinua I M, Narva K E, Waterhouse P M. 2016. In-plant protection against Helicoverpa armigera by production of long hpRNA in chloroplasts. Frontiers in Plant Science, 7, e47534.
Baum J A, Bogaert T, Clinton W, Heck G R, Feldmann P, Ilagan 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.
Bolger A M, Lohse M, Usadel B. 2014. Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics, 30, 2114–2120.
Conesa A, Madrigal P, Tarazona S, Gomez-Cabrero D, Cervera A, McPherson A, Szcześniak M W, Gaffney D J, Elo L L, Zhang X, Mortazavi A. 2016. A survey of best practices for RNA-seq data analysis. Genome Biology, 17, 181.
Davidson B L, Mccray P B. 2011. Current prospects for RNA interference-based therapies. Nature Reviews Genetics, 12, 329–340.
Duan J, Li R, Cheng D, Fan W, Zha X, Cheng T, Wu Y, Wang J, Mita K, Xiang Z, Xia Q. 2010. SilkDB v2.0: A platform for silkworm (Bombyx mori) genome biology. Nucleic Acids Research, 38, 453–456.
Fedorov Y, Anderson E M, Birmingham A, Reynolds A, Karpilow J, Robinson K, Leake D, Marshall W S, Khvorova A. 2006. Off-target effects by siRNA can induce toxic phenotype. RNA, 12, 1188–1196.
Fire A, Xu S, Montgomery M K, Kostas S A, Driver S E, Mello C C. 1998. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, 391, 806.
Gatehouse J A. 2008. Biotechnological prospects for engineering insect-resistant plants. Plant Physiology, 146, 881–887.
Gordon K H J, Waterhouse P M. 2007. RNAi for insect-proof plants. Nature Biotechnology, 25, 1231–1232.
Grabherr M G, Haas B J, Yassour M, Levin J Z, Thompson D A, Amit I, Adiconis X, Fan L, Raychowdhury R, Zeng Q, Chen Z, Mauceli E, Hacohen N, Gnirke A, Rhind N, di Palma F, Birren B W, Nusbaum C, Lindblad-Toh K, Friedman N, Regev A. 2011. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nature Biotechnology, 29, 644–652.
Guo M, Bao E L, Wagner M, Whitsett J A, Xu Y. 2017. SLICE: Determining cell differentiation and lineage based on single cell entropy. Nucleic Acids Reserch, 45, e54.
He K, Xiao H, Sun Y, Ding S, Situ G, Li F. 2019. Transgenic microRNA-14 rice shows high resistance to rice stem borer. Plant Biotechnology Journal, 17, 461–471.
Jackson A L, Bartz S R, Schelter J, Kobayashi S V, Burchard J, Mao M, Li B, Cavet G, Linsley P S. 2003. Expression profiling reveals off-target gene regulation by RNAi. Nature Biotechnology, 21, 635–637.
Jin S, Singh N D, Li L, Zhang X, Daniell H. 2015. Engineered chloroplast dsRNA silences cytochrome P450 monooxygenase, V-ATPase and chitin synthase genes in the insect gut and disrupts Helicoverpa armigera larval development and pupation. Plant Biotechnology Journal, 13, 435–446.
Klümper W, Qaim M. 2014. A meta-analysis of the impacts of genetically modified crops. PLoS ONE, 9, e111629.
Kumar P, Pandit S S, Steppuhn A, Baldwin I T. 2014. Natural history-driven, plant-mediated RNAi-based study reveals CYP6B46’s role in a nicotine-mediated antipredator herbivore defense. Proceedings of the National Academy of Sciences of the United States of America, 111, 1245–1252.
Langmead B, Salzberg S L. 2012. Fast gapped-read alignment with Bowtie 2. Nature Methods, 9, 357–359.
Li B, Dewey C N. 2011. RSEM: Accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics, 12, 323.
Li H, Jiang W, Zhang Z, Xing Y, Li F. 2013. Transcriptome analysis and screening for potential target genes for RNAi-mediated pest control of the beet armyworm, Spodoptera exigua. PLoS ONE, 8, e65931.
Li R, Zhu H, Ruan J, Qian W, Fang X, Shi Z, Li Y, Li S, Shan G, Kristiansen K, Li S, Yang H, Wang J, Wang J. 2010. De novo
assembly of human genomes with massively parallel short read sequencing. Genome Research, 20, 265–272.
Love M I, Huber W, Anders S. 2014. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biology, 15, 550.
Lundgren J G, Duan J J. 2013. RNAi-based insecticidal crops: Potential effects on nontarget species. Bioscience, 63, 657–665.
Luo J, Liang S, Li J, Xu Z, Li L, Zhu B, Li Z, Lei C, Lindsey K, Chen L, Jin S, Zhang X. 2017. A transgenic strategy for controlling plant bugs (Adelphocoris suturalis) through expression of double-stranded RNA homologous to fatty acyl-coenzyme A reductase in cotton. New Phytologist, 215, 1173–1185.
Mao Y B, Cai W J, Wang J W, Hong G J, Tao X Y, Wang L J, Huang Y P, Chen X Y. 2007. Silencing a cotton bollworm P450 monooxygenase gene by plant-mediated RNAi impairs larval tolerance of gossypol. Nature Biotechnology, 25, 1307.
Martinez J, Patkaniowska A, Urlaub H, Lührmann R, Tuschl T. 2002. Single-stranded antisense siRNAs guide target RNA cleavage in RNAi. Cell, 110, 563–574.
Martínez O, Reyes-Valdés M H. 2008. Defining diversity, specialization, and gene specificity in transcriptomes through information theory. Proceedings of the National Academy of Sciences of the United States of America, 105, 9709–9714.
Mogren C L, Lundgren J G. 2017. In silico identification of off-target pesticidal dsRNA binding in honey bees (Apis mellifera). PeerJ, 5, e4131.
Ni M, Ma W, Wang X, Gao M, Dai Y, Wei X, Zhang L, Peng Y, Chen S, Ding L, Tian Y, Li J, Wang H, Wang X, Xu G, Guo W, Yang Y, Wu Y, Heuberger S, Tabashnik B E, et al. 2017. Next-generation transgenic cotton: Pyramiding RNAi and Bt counters insect resistance. Plant Biotechnology Journal, 15, 1204–1213.
Price D R G, Gatehouse J A. 2008. RNAi-mediated crop protection against insects. Trends in Biotechnology, 26, 393–400.
Qiu S, Adema C M, Lane T. 2005. A computational study of off-target effects of RNA interference. Nucleic Acids Research, 33, 1834–1847.
Schiemann J, Dietz-Pfeilstetter A, Hartung F, Kohl C, Romeis J, Sprink T. 2019. Risk assessment and regulation of plants modified by modern biotechniques: Current status and future challenges. Annual Review of Plant Biology, 70, 699–726.
Sun Y, Wang P, Abouzaid M, Zhou H, Liu H, Yang P, Lin Y, Hull J J, Ma W. 2020. Nanomaterial-wrapped dsCYP15C1, a potential RNAi-based strategy for pest control against Chilo suppressalis. Pest Management Science, 76, 2483–2489.
Teschendorff A E, Enver T. 2017. Single-cell entropy for accurate estimation of differentiation potency from a cell’s transcriptome. Nature Communications, 8, 15599.
Vermeulen A, Behlen L, Reynolds A, Wolfson A, Marshall W S, Karpilow J, Khvorova A. 2005. The contributions of dsRNA structure to Dicer specificity and efficiency. RNA, 11, 674–682.
Worrall E A, Bravo-Cazar A, Nilon A T, Fletcher S J, Robinson K E, Carr J P, Mitter N. 2019. Exogenous application of RNAi-inducing double-stranded RNA inhibits aphid-mediated transmission of a plant virus. Frontiers in Plant Science, 10, 265–265.
Yan S, Qian J, Cai C, Ma Z, Li J, Yin M, Ren B, Shen J. 2020a. Spray method application of transdermal dsRNA delivery system for efficient gene silencing and pest control on soybean aphid Aphis glycines. Journal of Pest Science, 93, 449–459.
Yan S, Ren B, Shen J. 2020b. Nanoparticle-mediated double-stranded RNA delivery system: A promising approach for sustainable pest management. Insect Science, doi: 10.1111/1744–7917.12822.
Yan S, Ren B, Zeng B, Shen J. 2020c. Improving RNAi efficiency for pest control in crop species. BioTechniques, 68, 283–290.
Zambelli F, Mastropasqua F, Picardi E, D’Erchia A M, Pesole G, Pavesi G. 2018. RNentropy: An entropy-based tool for the detection of significant variation of gene expression across multiple RNA-Seq experiments. Nucleic Acids Research, 46, e46.
Zhang J, Khan S A, Hasse C, Ruf S, Heckel D G, Bock R. 2015. Full crop protection from an insect pest by expression of long double-stranded RNAs in plastids. Science, 347, 991–994.
Zhang J, Khan S A, Heckel D G, Bock R. 2017. Next-generation insect-resistant plants: RNAi-mediated crop protection. Trends in Biotechnology, 8, 871–882.
Zheng Y, Hu Y, Yan S, Zhou H, Song D, Yin M, Shen J. 2019. A polymer/detergent formulation improves dsRNA penetration through the body wall and RNAi-induced mortality in the soybean aphid Aphis glycines. Pest Management Science, 75, 1993–1999.
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.

昆虫致死基因siRNA在靶标和非标靶昆虫中的脱靶效应

Using transcriptome Shannon entropy to evaluate the off-target effects and safety of insecticidal siRNAs

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