转dsAllim和转dsAsfar抗盲蝽棉花对异色瓢虫的脱靶效应评价

doi:10.16409/j.cnki.2095-039x.2025.09.023

摘要/Abstract

摘要： 脱靶效应是RNAi技术的主要弊端，对于利用RNAi策略进行害虫防治的转dsRNA作物来说，dsRNA对非靶标生物的脱靶效应是其安全性评价的重要指标之一。本研究选取异色瓢虫Harmonia axyridis作为代表天敌昆虫类的非靶标生物，采用饲喂法将dsRNA导入非靶标生物体内，通过qRT-PCR探究dsRNA摄取效率与取食天数的关系，并结合转录组测序分析技术和定量分析，综合评价转dsAsfar棉花和dsAllim棉花对天敌昆虫的脱靶效应。研究发现：异色瓢虫幼虫对dsRNA的摄取效率与取食时间长短无直接显著关系，而成虫对dsRNA的摄取效率较高。转dsRNA棉花中表达的dsAsfar或dsAllim未对异色瓢虫far或lim基因的表达量产生显著影响；异色瓢虫幼虫和成虫取食dsAsfar后下调差异基因分别为2个和8个，取食dsAllim后下调差异基因分别为3个和26个。异色瓢虫幼虫取食dsAllim后，KEGG和GO富集分析结果显示，差异基因明显富集到肌肉发育相关的KEGG通路及表皮结构组成的GO条目，且这33个差异基因均为上调表达。异色瓢虫成虫取食dsAllim，以及成虫和幼虫取食dsAsfar后，KEGG和GO富集分析结果显示，差异基因在far基因和lim基因相关的KEGG通路及GO条目无明显富集。本研究对两种转dsRNA棉花的脱靶效应评价可为转dsRNA棉花的安全性提供有效的支撑。

关键词: RNA干扰, 转基因棉花, 脱靶效应, 异色瓢虫, 转录组

Abstract: Off-target effects are a major drawback of RNAi, and for transgenic dsRNA crops using RNAi strategy for pest control, the off-target effects of dsRNA on non-target organisms (NTOs) are an important indicator for their safety evaluation. In this study, Harmonia axyridis was selected as NTOs representing the natural enemy insect group, and dsRNA was introduced into NTOs by feeding. The relationship between the efficiency of dsRNA uptake and feeding days was explored by qRT-PCR. The off-target effects of transgenic dsAsfar cotton and transgenic dsAllim cotton were evaluated by RNA-Seq combined with quantitative analysis for validation. The uptake efficiency of dsRNA by H. axyridis larvae was not directly related to the duration of dsRNA feeding, and the uptake efficiency of dsRNA by adults was significantly higher than that in larvae. In addition, neither dsAsfar nor dsAllim expressed in the two transgenic dsRNA cotton plants had a significant effect on the expression of homologous genes in H. axyridis. The number of down-regulated differentially expressed genes (DEGs) was 2 and 8 in H. axyridis larvae and adults feeding on dsAsfar, and 3 and 26 on dsAllim, respectively. KEGG and GO enrichment analyses of H. axyridis larvae fed on dsAllim revealed that DEGs were significantly enriched in KEGG pathways related to muscle development and GO terms associated with cuticle structural components, with all 33 DEGs showing upregulated expression. The results of the KEGG and GO enrichment analyses for dsAllim in adults, as well as for dsAsfar in both adults and larvae, showed that DEGs were not significantly enriched in the corresponding KEGG pathways and GO entries of far and lim genes. Overall, the results of this study provide valid support for the safety of transgenic dsRNA cotton.

Key words: RNA interference, transgenic cotton, off-target effects, Harmonia axyridis, transcriptome

中图分类号:

S476.2

曹丹阳, 姚海芹, 陈利珍, 马伟华. 转dsAllim和转dsAsfar抗盲蝽棉花对异色瓢虫的脱靶效应评价[J]. 中国生物防治学报, 2026, 42(2): 412-424.

CAO Danyang, YAO Haiqin, CHEN Lizhen, MA Weihua. Evaluation of Off-target Effects of Anti-Miridae Transgenic dsAllim and Transgenic dsAsfar Cottons on Harmonia axyridis[J]. Chinese Journal of Biological Control, 2026, 42(2): 412-424.

参考文献

[1] Koch A, Wassenegger M. Host-induced gene silencing - mechanisms and applications[J]. New Phytologist, 2021, 231: 54-59.
[2] Whyard S. Insecticidal RNA, the long and short of it[J]. Science, 2015, 347: 950-951.
[3] Yen S, Ren BY, Zeng B, et al. Improving RNAi efficiency for pest control in crop species[J]. Biotechniques, 2020, 68: 283-290.
[4] 傅淑, 刘昭霞, 陈金芝, 等. 植物介导的害虫RNA干扰[J]. 昆虫学报, 2019, 62: 1448-1468.
[5] Coleman A D, Wouters R H M, Mugford S T, et al. Persistence and transgenerational effect of plant-mediated RNAi in aphids[J]. Journal of Experimental Botany, 2015, 66: 541-548.
[6] Kanakala S, Kontsedalov S, Lebedev G, et al. Plant-mediated silencing of the whitefly Bemisia tabaci cyclophilin B and heat shock protein 70 impairs insect development and virus transmission[J]. Frontiers In Physiology, 2019, 10: 557.
[7] Liu H, Shen E, Wu H, et al. Transgenic kingdom expression of an insect endogenous microRNA in rice enhances resistance to striped stem borer Chilo suppressalis[J]. Pest Management Science, 2022, 78: 770-777.
[8] Wu K M, Lu Y H, Feng H Q, et al. Suppression of cotton bollworm in multiple crops in China in areas with Bt toxin-containing cotton[J]. Science, 2008, 321: 1676-1678.
[9] Luo J, Liang S, Li J, et al. A transgenic strategy for controlling plant bugs (Adelphocoris suturalis) through expression of double-stranded RNA homologous to fatty acyl-coenzyme A reductase in cotton[J]. New Phytologist, 2017, 215: 1173-1185.
[10] Liang S, Luo J, Alariqi M, et al. Silencing of an LIM gene in cotton exhibits enhanced resistance against Apolygus lucorum[J]. Journal Of Cellular Physiology, 2021, 236: 5921-5936.
[11] Kleter G A, Kuiper H A, Kok E J. Gene-edited crops: Towards a harmonized safety assessment[J]. Trends in Biotechnology, 2019, 37: 443-447.
[12] Casacuberta J M, Devos Y, du Jardin P, et al. Biotechnological uses of RNAi in plants: risk assessment considerations[J]. Trends in Biotechnology, 2015, 33: 145-147.
[13] 王园园, 李云河, 陈秀萍, 等. 抗虫转基因植物对非靶标节肢动物生态影响的研究进展[J]. 生物安全学报, 2011, 20: 100-107.
[14] Obrycki J J, Kring T J. Predaceous Coccinellidae in biological control[J]. Annual Review of Entomology, 1998, 43: 295-321.
[15] Roberts A F, Devos Y, Lemgo G N Y, et al. Biosafety research for non-target organism risk assessment of RNAi-based GE plants[J]. Frontiers in Plant Science, 2015, 6: 958.
[16] 王甦, 张润志, 张帆. 异色瓢虫生物生态学研究进展[J]. 应用生态学报, 2007: 2117-2126.
[17] Islam Y, Shah F M, Güncan A, et al. Temperature-induced effects on development, reproduction, and predation of Harmonia axyridis fed on first instar larvae Spodoptera litura[J]. Bulletin of Entomological Research, 2024, 114: 244-253.
[18] Jackson A L, Bartz S R, Schelter J, et al. Expression profiling reveals off-target gene regulation by RNAi[J]. Nature Biotechnology, 2003, 21: 635-637.
[19] Jalaluddin N S M, Othman R Y, Harikrishna J A. Global trends in research and coμMercialization of exogenous and endogenous RNAi technologies for crops[J]. Critical Reviews in Biotechnology, 2019, 39: 67-78.
[20] Christiaens O, Whyard S, Velez A M, et al. Double-stranded RNA technology to control insect pests: current status and challenges[J]. Frontiers in Plant Science, 2020, 11: 451.
[21] Naito Y, Yamada T, Matsumiya T, et al. dsCheck: highly sensitive off-target search software for double-stranded RNA-mediated RNA interference[J]. Nucleic Acids Research, 2005, 33: W589-W591.
[22] Good R T, Varghese T, Golz J F, et al. OfftargetFinder: a web tool for species-specific RNAi design[J]. Bioinformatics, 2016, 32: 1232-1234.
[23] Allen M L. Comparison of RNAi sequences in insect-resistant plants to expressed sequences of a beneficial lady beetle: a closer look at off-target considerations[J]. Insects, 2017, 8: 27.
[24] Haller S, Widmer F, Siegfried B D, et al. Responses of two ladybird beetle species (Coleoptera: Coccinellidae) to dietary RNAi[J]. Pest Management Science, 2019, 75: 2652-2662.
[25] Pan H, Yang X, Romeis J, et al. Dietary RNAi toxicity assay exhibits differential responses to ingested dsRNAs among lady beetles[J]. Pest Management Science, 2020, 76: 3606-3614.
[26] Chen J, Wang H, Yang X, et al. Consumption of miRNA-mediated insect-resistant transgenic rice pollen does not harm Apis mellifera adults[J]. Journal of Agricultural and Food Chemistry, 2021, 69: 4234-4242.
[27] Lu J, Nanda S, Chen S M, et al. A survey on the off-target effects of insecticidal double-stranded RNA targeting the Hv betáCOPI gene in the crop pest Henosepilachna vigintioctopunctata through RNA-seq[J]. Journal of Integrative Agriculture, 2022, 21: 2665-2674.
[28] Ma W H, Wu T, Zhang Z, et al. Using transcriptome Shannon entropy to evaluate the off-target effects and safety of insecticidal siRNAs[J]. Journal of Integrative Agriculture, 2022, 21: 170-177.
[29] Tschuch C, Schulz A, Pscherer A, et al. Off-target effects of siRNA specific for GFP[J]. BMC Microbiology, 2008, 9: 60.