中国农业科学 ›› 2022, Vol. 55 ›› Issue (13): 2562-2571.doi: 10.3864/j.issn.0578-1752.2022.13.007

• 植物保护 • 上一篇    下一篇

P450基因在氯虫苯甲酰胺不同抗性品系小菜蛾中的表达及功能分析

尹飞(),李振宇,SAMINA Shabbir,林庆胜()   

  1. 广东省农业科学院植物保护研究所/广东省植物保护新技术重点实验室,广州 510640
  • 收稿日期:2021-12-01 接受日期:2022-02-03 出版日期:2022-07-01 发布日期:2022-07-08
  • 通讯作者: 林庆胜
  • 作者简介:尹飞,E-mail: feier0808@163.com
  • 基金资助:
    国家自然科学基金(31701819);广东省农业科学院“十四五”学科团队建设项目(202105TD);广东省企业科技特派员(GDKTP2020025700);广东省自然科学基金(2021A1515012385);广东省自然科学基金(2022A1515012395)

Expression and Function Analysis of Cytochrome P450 Genes in Plutella xylostella with Different Chlorantraniliprole Resistance

YIN Fei(),LI ZhenYu,SAMINA Shabbir,LIN QingSheng()   

  1. Plant Protection Research Institute, Guangdong Academy of Agricultural Sciences/Guangdong Provincial Key Laboratory of High Technology for Plant Protection, Guangzhou 510640
  • Received:2021-12-01 Accepted:2022-02-03 Online:2022-07-01 Published:2022-07-08
  • Contact: QingSheng LIN

摘要:

【目的】以十字花科蔬菜害虫小菜蛾(Plutella xylostella)为研究对象,筛选参与小菜蛾代谢氯虫苯甲酰胺的主要细胞色素P450解毒基因,为阐明不同抗性水平小菜蛾对氯虫苯甲酰胺的抗性机理提供依据。【方法】利用叶片药膜法测定不同小菜蛾种群3龄幼虫对氯虫苯甲酰的抗性水平,通过转录组测序、insectbase数据库和小菜蛾基因组数据库筛选得到52个细胞色素P450基因。应用MEGA5.10软件对52个细胞色素P450基因进行进化分析,获得与抗药性密切相关的CYP3和CYP4家族P450基因。利用实时荧光定量PCR方法分析目的基因在小菜蛾室内筛选种群(HZY)和田间抗性种群惠州种群(HZ)、连州种群(LZ)、东升种群(DS)、钟落潭种群(ZLT)中的表达量,选用RNA干扰技术,采用注射法,验证在抗性种群中显著上调表达的CYP6BF1V4在小菜蛾抗氯虫苯甲酰胺中的功能。【结果】抗药性测定结果表明,LZ和HZ小菜蛾种群为中等水平抗性,ZLT、DS和HZY小菜蛾种群为高水平抗性。对52个细胞色素P450基因的系统发育树分析发现,小菜蛾拥有10个CYP4家族基因,28个CYP3家族基因,其中2个CYP4家族基因在HZ和HZY种群中的表达量显著高于敏感种群,4个CYP3家族基因表达量与小菜蛾抗性呈正相关,8个基因在中等水平抗性小菜蛾体内的表达量高于在高水平抗性小菜蛾体内的表达量。对田间种群进一步筛选得到6个与小菜蛾抗氯虫苯甲酰胺密切相关,在不同抗性种群中均上调表达的细胞色素P450基因,其中4个为CYP6家族基因(CYP6BF1V4CYP6BF1V3CYP6f1CYP6B6),2个为CYP9家族基因(CYP9G2.1CYP9G2.2),以CYP6BF1V4的表达量最高,其在抗性种群中的表达量是在敏感种群中表达量的3.5—6.3倍。RNA干扰结果显示,沉默CYP6BF1V4能够显著提高小菜蛾对氯虫苯甲酰胺的敏感性。【结论】CYP6BF1V4CYP6BF1V3CYP6f1CYP6B6CYP9G2.1CYP9G2.2可能在小菜蛾体内协同调控多功能氧化酶的表达,从而加快小菜蛾代谢氯虫苯甲酰胺的速度,提高小菜蛾对氯虫苯甲酰胺的抗性。

关键词: 小菜蛾, 抗药性, 氯虫苯甲酰胺, CYP6, CYP9, 代谢活性, RNA干扰

Abstract:

【Objective】The Plutella xylostella is a global pest of cruciferous plants. The objective of this study is to identify the key cytochrome P450 genes involved in chlorantraniliprole metabolic regulation in P. xylostella, and to provide a basis for understanding the resistance mechanism concerning chlorantraniliprole in different resistance levels of P. xylostella.【Method】The resistance of the 3rd instar larvae of different P. xylostella populations to chlorantraniliprole was determined using the leaf dip method. On the strength of insectbase database and P. xylostella genome database together with transcriptome sequencing data, 52 relevant cytochrome P450 genes were screened. MEGA5.10 software was used to analyze the evolution of 52 cytochrome P450 genes, and CYP3 and CYP4 family P450 genes closely related to insecticide resistance were obtained. The expression levels of genes in CYP3 and CYP4 families were compared in HZ, HZY, LZ, DS and ZLT populations by qRT-PCR. The RNA interference experiments through injection were used to verify the function of CYP6BF1V4, which was significantly up-regulated in the resistant population of P. xylostella to chlorantraniliprole. 【Result】 The leaf dip bioassays showed that the populations of LZ and HZ were at medium level of resistance, while ZLT, DS and HZY populations were at high level of resistance. Phylogenetic tree analysis of 52 cytochrome P450s showed that ten genes from the CYP4 family and 28 genes from the CYP3 family potentially associated with insecticide resistance. The expression levels of two CYP4 family genes in the resistant induced population, HZ and HZY, were significantly higher than those in the susceptible population. Among the genes of CYP3 family, the expression levels of four genes were positively correlated with the resistance of P. xylostella. The expression levels of eight genes in P. xylostella with medium resistance were higher than those with high resistance. Six genes were up-regulated in all resistant field populations, among which four genes were CYP6 family genes (CYP6BF1V4, CYP6BF1V3, CYP6f1 and CYP6B6) and two genes were CYP9 family genes (CYP9G2-1 and CYP9G2-2). The expression of CYP6BF1V4 was the highest, and it was 3.5-6.3 times higher in the resistant populations than that in sensitive populations. The results of RNAi showed that knockdown of CYP6BF1V4 could significantly increase the sensitivity of P. xylostella to chlorantraniliprole.【Conclusion】CYP6BF1V4, CYP6BF1V3, CYP6f1, CYP6B6, CYP9G2-1 and CYP9G2-2 may help regulate the expression of multifunctional oxidases, thus accelerating the metabolize of chlorantraniliprole and enhancing the resistance of P. xylostella to chlorantraniliprole.

Key words: Plutella xylostella, insecticide resistance, chlorantraniliprole, CYP6, CYP9, metabolic activity, RNA interference (RNAi)