沉默豆蚜细胞色素P450基因CYP6CY53和CYP302A1增强对氟啶虫酰胺的敏感性

doi:10.3864/j.issn.0578-1752.2025.18.007

摘要/Abstract

摘要：

【目的】明确豆蚜（Aphis craccivora）关键细胞色素P450（CYP450）基因对氟啶虫酰胺毒力的影响并验证其功能，为害虫抗药性管理以及制定基于基因靶向干扰结合药剂处理的蚜虫绿色防控策略提供理论依据。【方法】氟啶虫酰胺处理豆蚜后测定P450酶活性，以评估整体代谢响应；通过数据库挖掘结合系统发育分析鉴定豆蚜P450基因，并利用qRT-PCR分析筛选响应药剂处理的P450基因；采用AlphaFold 3对上调响应的P450酶进行蛋白质结构预测，随后利用AutoDock对P450酶与氟啶虫酰胺进行分子对接，预测二者结合模式和亲和力；通过RNA干扰（RNAi）技术沉默目标P450基因，评估氟啶虫酰胺对豆蚜的毒力变化。【结果】氟啶虫酰胺处理后，P450酶活性显著上升，在24 h达到最高值，随后逐渐下降。从数据库中筛选到59个豆蚜P450基因，其中CYP3簇的CYP6CY53和Mito簇的CYP302A1在氟啶虫酰胺处理后表达显著上调，揭示其在药剂代谢中可能发挥关键作用。使用AlphaFold 3预测CYP6CY53和CYP302A1结构，置信度分数分别为0.75和0.86。利用AutoDock分别对两种蛋白质与氟啶虫酰胺进行分子对接预测，结果显示氟啶虫酰胺主要通过氢键与这两种P450酶结合，其结合能分别为-15.32和-18.17 kJ·mol^-1。沉默豆蚜CYP6CY53和CYP302A1后，氟啶虫酰胺的致死中浓度（LC₅₀）分别降至2.06和6.08 mg·L^-1，敏感性倍数提高至对照的9.26和3.13倍。【结论】豆蚜通过上调表达CYP6CY53和CYP302A1以响应氟啶虫酰胺胁迫，这两个基因对豆蚜抑制氟啶虫酰胺毒力具有重要作用，将其沉默后可显著增强豆蚜对氟啶虫酰胺的敏感性。

关键词: 豆蚜, 氟啶虫酰胺, P450基因, RNA干扰, 毒力测定

Abstract:

【Objective】The objective of this study is to clarify the effect of key cytochrome P450 (CYP450) genes in Aphis craccivora on the toxicity of flonicamid and validate their functions, thus providing a theoretical basis for managing insect resistance and formulating green control strategies that combine gene-targeted interference with pesticide treatment. 【Method】P450 enzyme activity was measured after flonicamid treatment to evaluate the overall metabolic response. P450 genes of A. craccivora were identified through database mining combined with phylogenetic analysis, and the qRT-PCR was used to screen the P450 genes responsive to insecticide treatment. The protein structures of up-regulated P450 enzymes were predicted using AlphaFold 3, and molecular docking of P450 enzymes with flonicamid was conducted via AutoDock to characterize binding modes and affinities. RNA interference (RNAi) was applied to silence the target P450 genes, and then the alteration of flonicamid toxicity to A. craccivora was evaluated. 【Result】After treatment with flonicamid, the activity of P450 enzymes increased significantly, peaking at 24 h, and then gradually decreased. A total of 59 P450 genes of A. craccivora were screened out, among which the expressions of CYP6CY53 in the CYP3 cluster and CYP302A1 in the Mito cluster were significantly up-regulated after flonicamid treatment, suggesting that they play a crucial role in insecticide metabolism. AlphaFold 3 predicted the structures of CYP6CY53 and CYP302A1 with confidence scores of 0.75 and 0.86, respectively. Molecular docking between the two enzymes and flonicamid was performed using AutoDock, and the results indicated that flonicamid binds to the two enzymes primarily through hydrogen bonds, with binding energy values of -15.32 and -18.17 kJ·mol^-1, respectively. After silencing CYP6CY53 and CYP302A1 in A. craccivora, the median lethal concentration (LC₅₀) of flonicamid decreased to 2.06 and 6.08 mg·L^-1, respectively, and the sensitivity ratios increased to 9.26 and 3.13 times that of the control group. 【Conclusion】The expression of CYP6CY53 and CYP302A1 was up-regulated in A. craccivora in response to flonicamid stress. These two genes play crucial roles in mitigating the toxicity of flonicamid, and the silencing of them can significantly enhance the sensitivity of A. craccivora to flonicamid.

Key words: Aphis craccivora, flonicamid, P450 gene, RNA interference (RNAi), toxicity assay

肖焯丹, 乔家正, 高渝岚, 尚掌印, 刘怀, 王佳. 沉默豆蚜细胞色素P450基因CYP6CY53和CYP302A1增强对氟啶虫酰胺的敏感性[J]. 中国农业科学, 2025, 58(18): 3664-3675.

XIAO ZhuoDan, QIAO JiaZheng, GAO YuLan, SHANG ZhangYin, LIU Huai, WANG Jia. Silencing of Cytochrome P450 Genes CYP6CY53 and CYP302A1 in Aphis craccivora Enhances the Sensitivity to Flonicamid[J]. Scientia Agricultura Sinica, 2025, 58(18): 3664-3675.

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图/表 7

表1

本研究所用引物序列"

登录号/名称Accession number/name	上游引物序列Forward primer sequence	下游引物序列Reverse primer sequence
用于qRT-PCR For qRT-PCR
KAF0713336.1	CGTTCTTGATCGTGTCCAGC	ACGTCCGGAAAGCTAAAGGA
KAF0729120.1	TGGCTGGACACAAGTATGGA	GCGTTTCTGACAGTGGTTCA
KAF0730706.1	CTATGCAACCTCTCGGACCA	TCCGATGATCTCACTCTGCC
KAF0741275.1	GGCAGCTTATCATCACCATGT	GTTTGTGCCACATCGTTCCT
KAF0745846.1	ACCAAAAGCACACGTTCCAA	TCCTGGATTCACGTCGGTTA
KAF0749705.1	TAATTGGGTATGACGGCGGT	ACTTCTAAACATTCAGCGGGC
KAF0750338.1	TAATTGGGTATGACGGCGGT	AACATTCAGCGGGTGGGATA
KAF0751009.1	CGTTGTATCGTTTACTGGCCA	ACAAGAAGAAGCAGTCCAGC
KAF0752653.1	CCTCTCGAACCACCACTGAA	ACGCTGCCAAAAGGAACAAT
KAF0752909.1	TTGTCCTGGTGTCGTCTGAT	GCAAAATGGCCGCATAAACC
KAF0754418.1	TGGACAGATGGCGAAAACAC	ATCCCAGAGGTCAAACGGTT
KAF0756480.1	CAACAACATCGGTGGCGTAT	ATGCGACAAAATCTCAGCCC
KAF0758588.1	CGCACATCAGACGGTACATG	GTTCGAATAGCACGTGGTCC
KAF0759798.1	AAAACTGCAGGATCCCTCGG	ACCGACCAAGAAACTGAAGAA
KAF0760910.1	AGAAAAGCGTTGAGTCCAGC	ATTTGCCCAACATGTCTCGC
KAF0761535.1	GTCGGTGAGCCCAATTGATC	ATCGGGCTCTTTTCGCTAGA
KAF0763819.1	AGTAGGTGCAATGTAGTCCCA	GGCGTAAACATCGTCGTCTT
KAF0766099.1	GAAAGTCCATCGCGTAACCC	ATTCCGAGTGTGAGGTGCAA
KAF0767093.1	GAGTCCTCTTTGTTGGCCAT	TCAATTTTGGCTGCTGGTGG
KAF0767926.1	GTAAGCACGATCGAAGTCAAAA	TCGTATTTGTTCGTGGTTCATTT
KAF0770710.1	AAAGGTCGAAACGCAGAAGT	TTACAGTGCCGAGTCGTGT
KAF0771958.1	GTTTCAGCCGTCGTTCAGAT	ACTGCATGAGATCGTCCCTC
KAF0769200.1	GACACCTGCGTTCACATCTG	GCACAGGTTCCGATGACATC
QQL12303.1	CCGGGCCAAGAAATTGCATA	GCTCGGCAAGTAATCAGCTC
QQL12304.1	ACGCTGCCAAAAGGAACAAT	TAAGCAACCTCTCGGACCAG
QQL12306.1	CAACCTTGTCTACGTCGTGC	GCTATGGTGCACGAGAACAG
QQL12307.1	CATCGGGTCATCTGCTTTCG	AAAATTTAGCCGCGTCCTCC
QQL12308.1	ACGTCCGGAAAGCTAAAGGA	CGTTCTTGATCGTGTCCAGC
QQL12309.1	ACCCGCTAACATTTCAACCG	GTTATGCCAAACTCCCCGTC
QQL12310.1	CACCAGCATTTACTTCAGGCA	TCTACTCCAAACGCACAAGT
QQL12311.1	TGGCGCTCAGATCATACCTT	TTCTCCGACCAACTCCGAAA
QQL12312.1	ATTCCAGCCACACCGTTTTC	TGTGCCGGGATTAGAACTGT
QQL12313.1	GGGAAATCTTGTTGGTCGACC	CTCCGACAACACCTGGTAGT
QQL12314.1	CGTTCACAGACACTATGCGG	AAACCAGCGGCAAACAGAAT
QQL12315.1	ACGACTCTTTACGCCTGACA	AAATATGGGTCTCGCTGGGT
QQL12317.1	TTGAAGATACGGCAACAGCG	GTTCATCTTCAAGGCACCCG
QQL12318.1	GTGCGTATCGTGTTGGTTGT	ACGGCACATTGAGATTTTCCC
QQL12319.1	AGGCCATGACACAACTTCCA	AGCGATTCTTTGATTACGGCT
QQL12320.1	CTATTTTGCCGGGGACATCG	TCGGTCCTGCACTAAATGGT
QQL12321.1	TGCTATGACCAATGCGTGTG	ACACAGGTAAAGGAGGGAACA
QQL12323.1	GAATGGGATGTGCAGTGCTT	ATAAAGTCATTCCGGCGCAC
QQL12324.1	CAGGTACCCGGAAAGACGTA	ATACCCCTTGACGCACTGAA
QQL12326.1	CATAGGGACGTGTGCTTTCG	CGGCGGGGTTGAACTTATTT
QQL12327.1	ATGCATCATGGAGACGTTGC	ACTTCGGGATTGGGGAAAGT
QQL12328.1	TCCCAGCGCTAACTACACAA	TCCCACAAACGGTATCCACT
QQL12329.1	GGTCAGAATTCTTGAGGCACC	CGACCGACCTTCCGAAATTC
QQL12330.1	TGGGCAGGAGATTCGGATTT	CGTCTTCGCTCGCTACAAAT
QQL12331.1	TCAACGATCCGGACTCAGAG	AACGCGTCATGGAAGAATCG
QQL12332.1	CTCGTATTTCACCGACCATGG	CAACTGATCGCTGCACTCTC
QQL12333.1	CGACCGGGTGATGAATAGGA	GACGTGAGCAGCAACAGTTT
QQL12334.1	TGGTGAACTTGCGAGGAGAT	CGCTGCTTCACTCACATCAA
QQL12335.1	GACCCAAGGAACATTACCGC	ATATGCACCAATGGCCCATG
QQL12336.1	CGTCTTGGATGAAAACCCGG	GCTCGATCTTGAACATCCTGG
QQL12337.1	CACCTGGTCTCACATCGGTA	GGCGTCCAATTTGAGTCCAA
QQL12338.1	ATCCGCGATCCAGAACTGAT	AGTTTTCCCGGTGTGAATGC
QQL12339.1	AAGGATTTACTGTGCGGTCG	ACCATCTGCAACGTATGTGC
QQL12340.1	AAAACTGACCCACGACCAGA	TTCAAACCGAAAGCACACGT
用于dsRNA合成For dsRNA synthesis
dsCYP302A1	T7AACGAAATCCCAGGACCAAT	T7AGTCCTCCAGTGTTGTATACGT
dsCYP6CY53	T7CTCGTTCCTCTTCTCCCCAA	T7TTAAAGTACGGCCAGCACTC

表1

图1

图2

图3

图4

图5

表2

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处理 Treatment	回归方程 Regression equation (y=)	致死中浓度 LC₅₀ (mg·L^-1)	95%置信限 95% Confidence limit (mg·L^-1)	卡方值 χ²(df=3)	敏感性倍数 Sensitivity ratio
dsGFP	0.9506+3.7833x	19.05	18.683-19.416	0.2023	—
dsCYP6CY53	1.2093+4.6213x	2.06	0.995-4.254	1.3222	9.26
dsCYP302A1	1.4155+4.6723x	6.08	1.414-1.994	1.2750	3.13