中国农业科学 ›› 2021, Vol. 54 ›› Issue (14): 3004-3016.doi: 10.3864/j.issn.0578-1752.2021.14.007

• 植物保护 • 上一篇    下一篇

新疆马铃薯甲虫对噻虫嗪的抗性监测及其细胞色素P450基因表达分析

石鑫1(),李莎1,王志敏1,付开赟2,付文君3,姜卫华1()   

  1. 1南京农业大学植物保护学院,南京 210095
    2新疆农业科学院植物保护研究所/农业农村部西北荒漠绿洲作物有害生物综合治理重点实验室,乌鲁木齐 830091
    3新疆伊犁州农业技术推广总站,新疆伊宁 835000
  • 收稿日期:2020-10-30 接受日期:2020-11-21 出版日期:2021-07-16 发布日期:2021-07-26
  • 通讯作者: 姜卫华
  • 作者简介:石鑫,E-mail: 907094441@qq.com
  • 基金资助:
    国家重点研发计划(2018YFD0200802)

Resistance Monitoring to Thiamethoxam and Expression Analysis of Cytochrome P450 Genes in Leptinotarsa decemlineata from Xinjiang

SHI Xin1(),LI Sha1,WANG ZhiMin1,FU KaiYun2,FU WenJun3,JIANG WeiHua1()   

  1. 1College of Plant Protection, Nanjing Agricultural University, Nanjing 210095
    2Research Institute of Plant Protection, Xinjiang Academy of Agricultural Sciences/Key Laboratory of Intergraded Management of Harmful Crop Vermin of China North-Western Oasis, Ministry of Agriculture and Rural Affairs, Urumqi 830091
    3Agricultural Technology Extension Master Station of Yili Prefecture, Yining 835000, Xinjiang
  • Received:2020-10-30 Accepted:2020-11-21 Online:2021-07-16 Published:2021-07-26
  • Contact: WeiHua JIANG

摘要:

【目的】以世界性害虫马铃薯甲虫(Leptinotarsa decemlineata)为研究对象,筛选其参与噻虫嗪解毒的细胞色素P450主效基因。【方法】于2018、2019年利用点滴法监测新疆察布查尔县、伊宁县、塔城市、乌鲁木齐市和吉木萨尔县11个马铃薯甲虫田间种群对噻虫嗪的抗性水平,并测定分析噻虫嗪LD50处理72 h后成虫中3种主要解毒酶细胞色素P450酶(P450)、谷胱甘肽S-转移酶(GST)和酯酶(EST)的活性变化。利用Illumina HiSeqTM 2500 高通量测序技术进行转录组测序,获得抗性和敏感种群之间的差异表达基因(DEG),运用实时荧光定量PCR(qRT-PCR)技术验证3个P450基因并分析6个P450基因(CYP4C1、CYP9e2CYP305a1CYP9Z25、CYP9YaCYP9Yc)在不同种群及噻虫嗪处理后的表达情况。【结果】抗性监测发现,2018年的YN1、URMQ1、TC和QPQL1以及2019年的YN2和JMS种群成虫对噻虫嗪抗性倍数(RR)分别达到5.99、8.81、10.86和20.33倍和11.82、14.05倍,为低至中水平抗性。解毒酶活性测定结果表明,2个敏感种群URMQ2、URMQ3以及2个抗性种群YN2、JMS成虫分别经噻虫嗪LD50处理72 h后其P450活性均显著增加,分别为对照的1.76、2.75、1.91和1.66倍。另外,URMQ3种群的GST(CDNB和DCNB为底物)及URMQ2、YN2种群的EST活性显著升高,分别为对照的1.19、2.10倍和1.35、1.91倍。转录组分析表明,通过合并组装分别获得噻虫嗪敏感和抗性种群样品56 872 051和62 249 136个原始序列数据以及55 903 706和61 082 076个过滤后的序列数据,过滤后的序列长度分别为8.39和9.16 G,碱基错误率均为0.03%。抗性种群中差异表达的P450基因13个,其中2个基因显著上调。3个上调的P450基因CYP4C1、CYP9e2CYP305a1的qRT-PCR检测结果与转录组测序结果基本一致,qRT-PCR分析还发现CYP9Ya、CYP9Yc、CYP4C1、CYP305a1CYP9Z25在抗性种群成虫中表达量显著增加,噻虫嗪LD50处理均可引起URMQ2种群4龄幼虫和成虫CYP9Ya、CYP9Yc表达量显著上调。相关性分析发现成虫对噻虫嗪的抗性水平与CYP9Ya表达量呈显著正相关。【结论】CYP9Ya可能在马铃薯甲虫中对噻虫嗪具有重要的解毒作用,其他基因的作用也不能排除。

关键词: 马铃薯甲虫, 噻虫嗪, 抗药性, 细胞色素P450, 基因表达

Abstract:

【Objective】Colorado potato beetle (CPB), Leptinotarsa decemlineata, is a significant agricultural pest in potato worldwide. The objective of the study is to identify the main cytochrome P450 genes involved in metabolic regulation of neonicotinoid insecticide thiamethoxam in L. decemlineata.【Method】In this study, the resistance level to thiamethoxam was assayed using topical applications in 11 field populations of L. decemlineata from Qapqal (QPQL), Yining (YN), Tacheng (TC), Urumqi (URMQ), Jimusar (JMS) of Xinjiang in 2018 and 2019. The activities of three detoxifying enzymes including cytochrome P450 monooxygenase (P450), glutathioneS-transferase (GST) and esterase (EST), exposed to LD50of thiamethoxam for 72 h were analyzed by in vitro enzyme activity assay. The differentially expressed genes (DEGs) of L. decemlineata adults susceptible and resistant to thiamethoxam were detected by Illumina HiSeqTM 2500 sequencing platform. The expression verification of three P450 genes and the expression change of six P450 genes in different populations and the beetles exposed to LD50 of thiamethoxam for 72 h were performed by real-time quantitative PCR (qRT-PCR).【Result】Resistance monitoring results showed that the adults of YN1, URMQ1, TC and QPQL1 populations in 2018 and YN2 and JMS in 2019 developed low to moderate level resistance to thiamethoxam with resistance ratio (RR) ranging from 5.99 to 20.33 times. The P450 activities of adults from two sensitive populations URMQ2 and URMQ3 and two resistant populations YN2 and JMS after treatment with thiamethoxam LD50for 72 h were significantly increased to 1.76, 2.75, 1.91 and 1.66 times as high as the control, respectively. In addition, the GST (CDNB and DCNB as substrates) of URMQ3 population and the EST activities of URMQ2 and YN2 populations were obviously increased to 1.19, 2.10 and 1.35, 1.91 times of the control, respectively. Based on Illumina RNA sequencing, the raw reads (56 872 051 and 62 249 136), clean reads (55 903 706 and 61 082 076), clean bases (8.39 and 9.16 G) and error of basic group (0.03%) were obtained for the thiamethoxam-susceptible and -resistant samples of adult populations, respectively. Thirteen differentially expressed P450 genes were identified in the thiamethoxam-resistant sample, of which two genes were significantly up-regulated. The expression levels of three P450 genes, namely CYP4C1,CYP9e2and CYP305a1by RNA sequencing were validated by qRT-PCR analysis. Furthermore, CYP9Ya, CYP9Yc, CYP4C1, CYP305a1 and CYP9Z25 were up-regulated in the thiamethoxam-resistant adults, and the expression of CYP9Ya, CYP9Yc increased significantly in the 4th instar larvae and adults under thiamethoxam treatment. It was found by correlation analysis that there was a significant positive correlation between resistance level to thiamethoxam and CYP9Ya expression of adults. 【Conclusion】CYP9Ya may be involved in the production of thiamethoxam resistance in L. decemlineata, and the role of other genes cannot be ruled out.

Key words: Leptinotarsa decemlineata, thiamethoxam, resistance, cytochrome P450, gene expression