基于壳聚糖和星形聚阳离子纳米载体的比较评估：旨在提高Cnaphalocrocis medinalis中CmFibL基因沉默效果的RNAi技术

《Pest Management Science》：Comparative evaluation of chitosan-based and star polycation nanocarriers for enhanced RNAi efficacy targeting CmFibL in Cnaphalocrocis medinalis

　　摘要

背景

稻叶卷叶虫（Cnaphalocrocis medinalis）通过幼虫卷叶的行为导致水稻产量大幅下降。RNA干扰（RNAi）提供了一种可持续的解决方案，但其在大蛾类昆虫中的应用受到双链RNA（dsRNA）降解和细胞吸收不良的限制。本研究开发了一种纳米载体介导的dsRNA递送方法以克服这些限制。

结果

比较了三种纳米载体——壳聚糖（CS）、壳聚糖-三聚磷酸盐（CS-TPP）和星形聚阳离子（SPc）——在提高针对C. medinalis纤维蛋白轻链基因（CmFibL）的RNAi效率方面的效果。CS-TPP和SPc分别实现了61%和55%的沉默效率，比裸露的dsRNA（23%）提高了2.7倍。所有纳米载体都能保护dsRNA免受RNase A（30分钟）和中肠液（6小时）的降解。在温室试验中，CmFibL的敲低导致严重的丝质缺陷，蛹期延长了23%，蛹体重减少了33%，卷叶损害减少了31%。转录组学分析显示氨基酸代谢下调以及内质网（ER）应激和免疫反应的激活。在人类基因组以及任何共享相同生态位的捕食者或寄生蜂中均未检测到脱靶效应。

结论

CS-TPP和SPc纳米载体有效提高了对大蛾类害虫的RNAi效率。靶向CmFibL可以破坏丝质介导的取食庇护所，同时具有最小的生态风险，为基于RNAi的生物农药在田间应用提供了实用框架。? 2026 化学工业学会。利益冲突

作者声明没有利益冲突。

数据可用性声明

支持本研究结果的数据可向通讯作者索取。

文件名 描述
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ps70983-sup-0001-Supinfo.docx Word 2007文档，2 MB
表S1. dsRNA合成、定量实时PCR（qRT-PCR）和质粒构建所需的引物。
表S2. 样本分组信息。
表S3. 用于验证差异表达基因的qRT-PCR引物。
表S4. CmFibL沉默对Cnaphalocrocis medinalis发育时间、化蛹率和成虫羽化的影响。
图S1. dsRNA表达的IPTG诱导条件优化。(a) 不同IPTG浓度对细菌系统中dsRNA产量的影响。(b) 不同IPTG诱导时间对细菌系统中dsRNA产量的影响。(c) 不同IPTG浓度诱导的dsRNA产物的琼脂糖凝胶电泳。M，DL2000 DNA标记物。(d) 不同诱导时间诱导的dsRNA产物的琼脂糖凝胶电泳。M，DL2000 DNA标记物。
图S2. dsRNA的特性。dsCmibL的扫描电子显微镜（SEM）形态。
图S3. 纳米颗粒–dsRNA复合物在RNase A中的稳定性。通过琼脂糖凝胶电泳评估纳米颗粒–dsRNA复合物在RNase A暴露下的稳定性。复合物与RNase A孵育30分钟后收集样品并通过1%琼脂糖凝胶电泳进行分析。
图S4. CmFibL基因被纳米颗粒–dsRNA复合物沉默后48小时，Cnaphalocrocis medinalis丝腺的形态。
图S5. dsCmFibL核苷酸序列在NCBI上的BLAST结果。
图S6. dsCmFibL与(a) Haritalodes derogata和(b) Ostrinia furnacalis的Fib-L基因序列的比对。黑色区域表示相同序列。
图S7. 不同组间差异表达基因（DEGs）的维恩图。
图S8. 通过qRT-PCR验证DEGs。

参考文献

1. Zhang ZJ, Xia L, Du J, Li SW and Zhao F, Cloning, characterization, and RNAi effect of the chitin synthase B gene in Cnaphalocrocis medinalis. J Asia Pac Entomol 24: 486–492 (2021).
2. Zhang F, Zhang Z, Wu Y and Zhang W, Adult oviposition preferences and larval performance of rice leaf folder, Cnaphalocrocis medinalis on rice and non-rice graminaceous plants. Arthropod-Plant Interact 13: 31–40 (2019).
3. Wei X, Pan X, Xin C, Zheng X, Gao J, Xi Q et al., Cross-resistance pattern and basis of resistance in a thiamethoxam-resistant strain of Aphis gossypii glover. Pestic Biochem Physiol 138: 91–96 (2017).
4. Bruce TJA and Pickett JA, Plant volatiles in insect pest management: manipulating host-plant location behaviour. Curr Opin Insect Sci 67: 101–108 (2025).
5. Wang C, Mao J, Zhang J, Li F and Chen X, RNA interference in agriculture: delivery mechanisms, challenges and prospects. Plant Biotechnol J 23: 123–138 (2025).
6. Mahanta DK, Komal J, Bhoi TK, Samal I, Dash S and Jangra S, RNA interference (RNAi) for insect pest management: understanding mechanisms, strategies, challenges and future prospects. Biol Futura 76: 465–477 (2025).
7. Zielińska A, Insect double-stranded RNA-binding proteins: structure, function and RNAi efficiency. J Insect Physiol 167:104910 (2025).
8. Li J, Shi Y, Xue Q, Smagghe G, Kristof DS, De Schutter K et al., Identification and functional analysis of gut dsRNases in the beet armyworm Spodoptera exigua. Insect Biochem Mol Biol 175:104206 (2024).
9. Hu S, Guan R, Li H and Miao X, Application of RNAi in insect pest management: important progress and problems. Acta Entomol Sin 62: 506–515 (2019).
10. Han X, Li H, Xu S, Miao X and Guan R, Bacillus secretes nucleases to degrade dsRNA, thereby reducing host's susceptibility to RNAi. NPJ Biofilms Microbiomes 11: 127 (2025).
11. Liu J, He Q, Lin X and Smagghe G, Recent progress in nanoparticle-mediated RNA interference in insects: unveiling new frontiers in pest control. J Insect Physiol 167:104884 (2025).
12. Kunte N, McGraw E, Bell S, Held D and Avila LA, Prospects, challenges and current status of RNAi through insect feeding. Pest Manag Sci 76: 26–41 (2020).
13. Li M, Zhao L, Chen Y, Zhang Y and Xi Z, RNAi-mediated knockdown of Nanos3 eliminates female germline stem cells in Aedes aegypti. Nat Commun 15: 8213 (2024).
14. Gao Z, Rensing C, Wang J, Shen C, Elzaki MEA, Li X et al., Increased and synergistic RNAi delivery using MOF polydopamine nanoparticles for biopesticide applications. Nat Commun 16: 5136 (2025).
15. Li S, Zhang J, Wang H, Zhao X, Yang L, Chai L et al., Poly-L-lysine functionalized melanin nanocarrier boosts dsRNA delivery and RNAi efficiency in the migratory locust. Chem Eng J 502:170751 (2025).
16. Yan S, Ren BY and Shen J, Nanoparticle-mediated double-stranded RNA delivery system: a promising approach for sustainable pest management. Insect Sci 28: 21–34 (2021).
17. Kumar S, Nehra M, Dilbaghi N, Marrazza G, Hassan AA and Kim KH, Nano-based smart pesticide formulations: emerging opportunities for agriculture. J Control Release 294: 131–153 (2019).
18. Yan S, Qian J, Cai C, Ma Z, Li J, Yin M et al., Spray method application of transdermal dsRNA delivery system for efficient gene silencing and pest control on soybean aphid Aphis glycines. J Pest Sci 93: 449–459 (2020).
19. Zheng Y, Hu Y, Yan S, Zhou H, Song D, Yin M et al., A polymer/detergent formulation improves dsRNA penetration through the body wall and RNAi-induced mortality in the soybean aphid Aphis glycines. Pest Manag Sci 75: 1993–1999 (2019).
20. Chuan D, Jin T, Fan R, Zhou L and Guo G, Chitosan for gene delivery: methods for improvement and applications. Adv Colloid Interface Sci 268: 25–38 (2019).
21. Zhang X, Mysore K, Flannery E, Michel K, Severson DW, Zhu K et al., Chitosan/interfering RNA nanoparticle mediated gene silencing in disease vector mosquito larvae. J Vis Exp 97: 52523 (2015).
22. Dhandapani RK, Gurusamy D, Howell JL and Palli SR, Development of CS-TPP-dsRNA nanoparticles to enhance RNAi efficiency in the yellow fever mosquito, Aedes aegypti. Sci Rep 9: 8775 (2019).
23. Shan Y, Ma Y, Wu C, Ren X, Song X, Wang D et al., Preparation of Cu-CS@IN@MIL-100 (Fe) for controlled indoxacarb release and reduced non-target organism risks. J Environ Chem Eng 11:109967 (2023).
24. Qiao H, Jiang R, Zhong G, Tang S, Zhu-Salzman K, Tan Y et al., Ecdysone receptor B1 isoform/ultraspiracle protein 1-mediated ecdysteroid signaling regulates the growth and development: potential targets for efficient Hyphantria cunea management. Int J Biol Macromol 333:148594 (2025).
25. Lu Y-H, Liu Y, Lin Y-C, Su Y-J, Henry D, Lacarriere V et al., Lipoplex-based RNA delivery system enhances RNAi efficiency for targeted pest control in Spodoptera litura. Int J Biol Macromol 319:145091 (2025).
26. Li M, Ma Z, Peng M, Li L, Yin M, Yan S et al., A gene and drug co-delivery application helps to solve the short life disadvantage of RNA drugs. Nano Today 43:101452 (2022).
27. Chao Z, Ma Z, Zhang Y, Yan S and Shen J, Establishment of star polycation-based RNA interference system in all developmental stages of fall armyworm Spodoptera frugiperda. Entomologia 43: 127–137 (2023).
28. Zhao J, Yan S, Li M, Sun L, Dong M, Yin M et al., NPFR regulates the synthesis and metabolism of lipids and glycogen via AMPK: novel targets for efficient corn borer management. Int J Bio Macromol 247:125816 (2023).
29. Jiang Q, Lin Z, Peng M, Zhou B, Liu Z, Li Z et al., A nano-delivery system enhances the stomach toxicity of Methoxyfenozide against Spodoptera litura by suppressing the synthesis of insect cuticle protein. ACS Appl Nano Mater 6: 13524–13532 (2023).
30. Qiao H, Jiang Q, Zhao J, Xiao L, Zhu-Salzman K, Xu D et al., Nano-delivery platform with strong protection and efficient delivery: preparation of self-assembled RNA pesticide with dual RNAi targets against Apolygus lucorum. J Nanobiotechnol 23: 123 (2025).
31. Tsubota T, Yoshioka T, Jouraku A, Suzuki TK, Yonemura N, Yukuhiro K et al., Transcriptomic analysis of the bagworm moth silk gland reveals a number of silk genes conserved within Lepidoptera. Insect Sci 28: 885–900 (2021).
32. Moreno-Tortolero RO, Luo Y, Parmeggiani F, Skaer N, Walker R, Serpell LC et al., Molecular organization of fibroin heavy chain and mechanism of fibre formation in Bombyx mori. Commun Biol 7: 786 (2024).
33. Cui Y, Zhu Y, Lin Y, Chen L, Feng Q, Wang W et al., New insight into the mechanism underlying the silk gland biological process by knocking out fibroin heavy chain in the silkworm. BMC Genomics 19: 1–10 (2018).
34. Ye X, Tang X, Zhao S, Ruan J, Wu M, Wang X et al., Mechanism of the growth and development of the posterior silk gland and silk secretion revealed by mutation of the fibroin light chain in silkworm. Int J Biol Macromol 188: 375–384 (2021).
35. Chen CX, Cloning and Functional Verification of the Silk Fibroin Light Chain in Sylepta Derogata Fabricius. Dissertation, Yangzhou University, Yangzhou, Jiangsu, China (2018).
36. Li X, Liu R, Li G, Jin D, Guo J, Ochoa R et al., Identification of the fibroin of Stigmaeopsis nanjingensis by a nanocarrier-based transdermal dsRNA delivery system. Exp Appl Acarol 87: 31–47 (2022).
37. Li J, Du J, Li S and Wang X, Identification and characterization of a double-stranded RNA degrading nuclease influencing RNAi efficiency in the rice leaf folder Cnaphalocrocis medinalis. Int J Mol Sci 23: 3961 (2022).
38. Ma Z, Shen J and Yan S, An efficient dsRNA production method based on engineering bacteria for targeted insect genes. Chin J Appl Entomol 56: 342–347 (2019).
39. Kumar DR, Kumar PS, Gandhi MR, Al-Dhabi NA, Paulraj MG and Ignacimuthu S, Delivery of chitosan/dsRNA nanoparticles for silencing of wing development vestigial (vg) gene in Aedes aegypti mosquitoes.《国际生物大分子杂志》86: 89–95 (2016)。
10.1016/j.ijbiomac.2016.01.030
CAS
PubMed
Web of Science?
Google Scholar

40. Xue X, Yu X, Zhang J, Xu J, Qin M, Cao M 等人，《RNAi技术在作物保护中的昆虫控制应用》。《作物杂志》13: 1009–1021 (2025)。
10.1016/j.cj.2025.05.006
Google Scholar

41. He L, Huang Y 和 Tang X，《基于RNAi的害虫控制：双链RNA的生产、应用及其命运》。《前沿生物工程与生物技术》10: 1–14 (2022)。
10.3389/fbioe.2022.1080576
Web of Science?
Google Scholar

42. Jiang Y, Zong S, Wang X, Zhu-Salzman K, Zhao J, Xiao L 等人，《pH响应性纳米颗粒用于口服递送RNAi以持续防治Spodoptera exigua》。《国际生物大分子杂志》306:141763 (2025)。
10.1016/j.ijbiomac.2025.141763
CAS
PubMed
Web of Science?
Google Scholar

43. Ma Z, Zheng Y, Chao Z, Chen H, Zhang Y 和 Yin M，《纳米载体介导的基因传递过程可视化：基因的稳定化、内吞作用及内体逃逸》。《纳米生物技术杂志》20: 124 (2022)。
10.1186/s12951-022-01336-6
CAS
PubMed
Web of Science?
Google Scholar

44. Liu Y, Zhang J, Li S, Chai L, Chang B, Malak M 等人，《壳聚糖纳米颗粒介导的双链RNA递送以提高Locusta migratoria中的RNAi效率》。《害虫管理科学》81: 5260–5269 (2025)。
10.1002/ps.8880
CAS
PubMed
Web of Science?

45. Xie J, Mo Q, Chen L, Zhu Z, Liu X, Smagghe G 等人，《水稻叶卷虫中主要丝素基因成分Fib-L的鉴定与功能研究》。《昆虫分子生物学》34: 228–238 (2025)。
10.1111/imb.12965
CAS
PubMed
Web of Science?

46. Sun L, Wang R, Ma S, Shi R, Zhao P 和 Xia Q，《完全敲除BmFib-L基因揭示其在丝纤维形成中的不可或缺作用》。《国际生物大分子杂志》294:138759 (2025)。
10.1016/j.ijbiomac.2024.138759
CAS
PubMed
Web of Science?

47. Cunha JCS, Rivera Vega LJ, Torres JB, Suh CPC 和 Sword GA，《真菌种子处理对棉铃象虫发育的影响》。《害虫管理科学》80: 1566–1576 (2024)。
10.1002/ps.7890
CAS
PubMed
Web of Science?

48. Li H, Guo Y, Yin J, Chen W, Xu H, Cheng T 等人，《小丝素分子在再生丝素自组装及性质中的关键作用》。《国际生物大分子杂志》284:137926 (2025)。
10.1016/j.ijbiomac.2024.137926
CAS
PubMed
Web of Science?

49. Zhang T, Ma S, Zhang Z, Guo Y, Yang D 和 Lu W，《昆虫丝素重链（FibH）的概述与进化》。《国际分子科学杂志》25: 7179 (2024)。
10.3390/ijms25137179
CAS
PubMed
Web of Science?

50. Castellanos NL, Smagghe G, Taning CNJT, Oliveir EE 和 Christiaens O，《基于RNAi的农药对非目标生物的风险评估：以寄生蜂Telenomus podisi为例研究序列相似性的影响》。《科学总环境》832:154746 (2022)。
10.1016/j.scitotenv.2022.154746
CAS
PubMed
Web of Science?

51. Abrar M, Sun C 和 Shao Y，《鳞翅目模式昆虫家蚕Bombyx mori L.对微生物病原体的体液免疫反应》。《当前昆虫科学研究》6:100097 (2024)。
10.1016/j.cris.2024.100097
PubMed
Google Scholar

52. Hetz C 和 Paschen W，《昆虫分泌组织中的蛋白质 unfolded 反应：从稳态到病理》。《细胞与分子生命科学》81: 45 (2024)。
PubMed
Google Scholar

53. Chen G, Chen J, Wen N, Liu L, Yang X, Li F 等人，《利用转录组多样性评估miRNA介导的转基因水稻的脱靶效应》。《农业与食品化学杂志》73: 16573–16583 (2025)。
10.1021/acs.jafc.4c11433
CAS
PubMed
Web of Science?

54. Wu Q, Gao G, Li Y, Zhang C, Yan H, Xu N 等人，《用于农药的纳米颗粒负载双链RNA递送系统：现状与展望》。《农业化学进展》4: 235–248 (2025)。
10.1016/j.aac.2025.08.003
CAS
Web of Science?

55. Shangguan W, Chen H, Zhao P, Cao C, Yu M, Huang Q 等人，《面向未来的纳米农药：为未来农业设计纳米农药》。《农业化学进展》3: 265–278 (2024)。
10.1016/j.aac.2024.07.002
CAS
Web of Science?