黏虫CYP9A134基因的克隆及其解毒功能

doi:10.16409/j.cnki.2095-039x.2022.11.015

摘要/Abstract

摘要： 昆虫体内的细胞色素P450酶系统在昆虫解毒过程中发挥着重要作用。本次试验通过转录组测序获得一条新的黏虫P450基因，经国际委员会命名为CYP9A134（登录号为MT990973）。该序列全长为1801 bp，开放序列长度为1596 bp，编码531个氨基酸，分子质量为61.42 kDa，等电点为4.90。在黏虫幼虫阶段用2.5%高效氯氟氰菊酯和20%氯虫苯甲酰胺诱导时该基因表达量会有不同程度上升，最高的可分别达对照组的2.6倍和6.5倍。RNA干扰后，该基因表达量最低下降了70%，以上2种杀虫剂LD₃₀杀虫效果分别提高了13%和25%；在黏虫成虫阶段用20%氯虫苯甲酰胺LD₃₀诱导时，该基因表达量最高可达7.8倍。RNA干扰后，该基因表达量最低下降了61%，LD₃₀剂量的氯虫苯甲酰胺杀虫效果提高26%。结果表明，该基因可能在杀虫剂诱导的解毒代谢过程中发挥重要作用。

关键词: 黏虫, 细胞色素P450基因, 诱导, RNA干扰, 死亡率

Abstract: The cytochrome P450 monooxygenase system in insects plays an important role in detoxification. A novel P450 gene obtained from Mythimna separata by transcriptome sequencing was nominated CYP9A134 by international P450 Nomenclature Committee (Genbank accession number MT990973). The full-length CYP9A134 cDNA sequence was 1801 bp with an open reading frame of 1596 bp, which encoded a 531-amino acid polypeptide. The predicted molecular weight of the deduced amino acids was 61.42 kDa and its isoelectronic point was 4.90. The expression level of CYP9A134 was increased in the M. separata larvae upon induction with 2.5% beta-cyfluthrinand and 20% chlorantraniliprole, with the highest values reaching 2.6 times and 6.5 times that of the control group, respectively. After RNA interference, CYP9A134 expression level was decreased by 70%, and the mortalities of M. separata was increased by 13% and 25% after treatment with LD₃₀ofbeta-cyfluthrinand and 20% chlorantraniliprole, respectively. CYP9A134 expression was upregulated 7.8 times when the adults were induced with LD₃₀of 20% chlorantraniliprole . After RNA interference, CYP9A134 expression level was decreased by 61% and the mortality of M. separata adults was increased by 26% after treatment with chlorantraniliprole. The results suggest that CYP9A134 may play an important role in insecticide-induced detoxification metabolism.

Key words: Mythimna separata, cytochrome P450, induction, RNA interference, mortality

中图分类号:

张良, 杨洪佳, 李泽, 张春雨, 胡育硕, 樊东. 黏虫CYP9A134基因的克隆及其解毒功能[J]. 中国生物防治学报, 2023, 39(1): 130-140.

ZHANG Liang, YANG Hongjia, LI Ze, ZHANG Chunyu, HU Yushuo, FAN Dong. Cloning of CYP9A134 Gene and Its Detoxification Function in Mythimna separata[J]. Chinese Journal of Biological Control, 2023, 39(1): 130-140.

参考文献

[1] Guo Y, Zhang X, Wu H, et al. Identification and functional analysis of a cytochrome P450 gene CYP9AQ2 involved in deltamethrin detoxification from Locusta migratoria[J]. Pesticide Biochemistry and Physiology, 2015, 122:1-7.
[2] 宋展, 高鑫, 吴冕, 等. 细胞色素P450酶的结构、功能与应用研究进展[J]. 微生物学通报, 2020, 47(7):2245-2254.
[3] 于月, 殷硕, 刘高峰. 中药注射剂对细胞色素P450的调控与药物相互作用预测[J]. 中国药师, 2014, 17(1):99-103.
[4] 冯纪年, 田宏刚, 郭付振, 等. 昆虫细胞色素P450研究进展[A]. 当代昆虫学研究——中国昆虫学会成立60周年纪念大会暨学术讨论会论文集[C]. 中国昆虫学会, 2004, 7.
[5] 蔡双虎, 程立生. 昆虫细胞色素P-450基因的研究进展[J]. 热带农业科学, 2002, 6:76-82.
[6] 艾均文, 孟繁利, 贾孟周, 等. 昆虫细胞色素P450的研究进展[A]. 中国蚕学会第六届青年学术研讨会论文集(3)[C]. 中国蚕学会, 2009, 10.
[7] Li X, Schuler M A, Berenbaum M R. Molecular mechanisms of metabolic resistance to synthetic and natural xenobiotics[J]. Annual Review of Entomology, 2007, 52(1):231-253.
[8] 艾均文, 龚昕, 薛宏, 等. 不同鳞翅目昆虫细胞色素P450基因(CYPs)的比较基因组学分析[J]. 农业生物技术学报, 2015, 23(2):244-252.
[9] Liu N. Insecticide resistance in mosquitoes:impact, mechanisms, and research directions[J]. Annual Review of Entomology, 2015, 60(1):537-559.
[10] 第五进学, 梁锴, 杨俭, 等. 拟除虫菊酯类杀虫剂急性中毒防治[J]. 医学动物防治, 2004, 20(8):1.
[11] Zhu Q, Yang Y, Zhong Y, et al. Synthesis, insecticidal activity, resistance, photo degradation and toxicity of pyrethroids (A review)[J]. Chemosphere, 2020, 254(1):126779.
[12] 张一宾. 鱼尼汀受体及以其为靶标的杀虫剂的作用机理[J]. 世界农药, 2007, 29(4):1-6.
[13] 刘宴弟, 孙丽娜, 张怀江, 等. 二酰胺类杀虫剂作用机制和抗性机理研究综述[J]. 中国植保导刊, 2020, 40(11):65-68, 79.
[14] Fire A, Xu S, Montgomery M, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans (see comments)[J]. Nature, 1998, 391(6669):806-811.
[15] 陈颖, 朱明华. RNA干扰[J]. 中国生物工程杂志, 2003, 23(3):39-43.
[16] 施秀珍, 吴青君, 王少丽, 等. RNA干扰在昆虫中的作用机理及应用进展[J]. 生物安全学报, 2012, 21(3):229-235.
[17] 王伟伟, 刘妮, 陆沁, 等. RNAi技术的最新研究进展[J]. 生物技术通报, 2017, 33(11):35-40.
[18] 李光博. 我国粘虫研究概况及主要进展[J]. 植物保护, 1993, 4:2-4.
[19] 祁欣. 细胞色素氧化酶P450介导的昆虫抗药性研究进展[J]. 北京农业, 2015(20):79-80.
[20] 潘贻武, 刘宁, 刘小宁. 昆虫细胞色素P450的研究进展及其介导的抗药性[J]. 新疆农业科学, 2007, 4:470-475.
[21] 朱江, 邱星辉. 昆虫抗药性相关细胞色素P450基因的表达调控机制[J]. 昆虫学报, 2021, 64(1):109-120.
[22] 董杰, 岳瑾, 乔岩, 等. 5种杀虫剂对北京地区黏虫的室内毒力测定[J]. 中国植保导刊, 2014, 34(10):71-73.
[23] Vandesompele J, Preter K D, Pattyn F. Accurate normalization ofreal-time quantitative RT-PCR data by geometric averaging of multiple internal control genes[J]. Genome Biology, 2002, 3(7):467-470.
[24] Livak, K J, Schmittgen, T D. Analysis of relative gene expressiondata using real-time quantitative PCR and the 2−△△Ct method[J]. Methods, 2001, 25(4):402-408.
[25] Scott J G. Cytochromes P450 and insecticide resistance[J]. Insect Biochemistry and Molecular Biology, 1999, 29:757-777.
[26] Sutherland D T, Unnithan G C, Andersen J F, et al. A cytochrome P450 terpenoid hydroxylase linked to the suppression of insect juvenile hormone[J]. Proceedings of the National Academy of Sciences of the United States of America, 1998, 95(22):12884-12889.
[27] Winter J, Bilbe G, Richener H, et al. Cloning of a cDNA encoding a novel cytochrome P450 from the insect Locusta migratoria:CYP6H1, a putative ecdysone 20-Hydroxylase[J]. Biochemical and Biophysical Research Communications, 1999, 259(2):305-310.
[28] Omura T, Gotoh O. Evolutionary origin of mitochondrial cytochrome P450[J]. Journal of Biochemistry, 2017, 161(5):399-407.
[29] Dillen S, Chen Z, Broeck J V. Nutrient-dependent control of short neuropeptide F transcript levels via components of the insulin/IGF signaling pathway in the desert locust, Schistocerca gregaria[J]. Insect Biochemistry and Molecular Biology, 2016, 68:64-70.
[30] 李恒, 周知恩, 陈勇, 等. 西花蓟马不同RNA干扰技术比较研究[J]. 福建农业学报, 2019, 34(10):1179-1184.
[31] 张爽. 棉铃虫P450基因CYP9A12和CYP9酯代谢[D]. 南京:南京农业大学, 2008.
[32] 黄水金, 秦文婧, 陈琼. 斜纹夜蛾P450基因CYP4M14和CYP4S9的克隆与mRNA表达水平研究[J]. 中国农业科学, 2010, 43(15):3115-3124.
[33] 李芬. 棉蚜细胞色素P450基因应答植物次生物质的转录调控[D]. 北京:中国农业大学, 2019.
[34] 白琪. CsCYP6A2、CsCYP306A1基因在香根草对二化螟致死过程的作用研究[D]. 杭州:浙江师范大学, 2018.
[35] 赵斯斯. 家蚕P450基因CYP9A19和CYP9A22的启动子分析[D]. 苏州:苏州大学, 2012.
[36] 梁晓, 伍春玲, 陈青, 等. 拟除虫菊酯A17的功能表达与拟除虫菊类杀虫剂对赤拟谷盗CYP4基因的诱导表达特性[J]. 热带作物学报, 2018, 39(7):1402-1409.
[37] 陈羿渠. 氯虫苯甲酰胺对甜菜夜蛾细胞色素P450基因的诱导表达及RNA干扰研究[D]. 雅安:四川农业大学, 2017.
[38] 赵鹏. 斜纹夜蛾四条P450基因克隆和功能研究[D]. 武汉:华中农业大学, 2020.