苹果黄蚜细胞色素P450基因AcCYP6CY14的表达及其在抵抗吡虫啉中的作用
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摘要
【目的】研究苹果黄蚜Aphis citricola细胞色素P450基因AcCYP6CY14的表达及其在抵抗吡虫啉中的作用。【方法】搜索苹果黄蚜转录组数据库,获得细胞色素P450基因AcCYP6CY14的cDNA部分序列,采用RT-PCR技术克隆目的基因cDNA序列全长。利用实时定量PCR(Real-time quantitative PCR,RT-qPCR)技术测定目的基因在苹果黄蚜不同发育阶段的表达水平。采用浸虫法,用浓度为LC30的吡虫啉对苹果黄蚜3龄若蚜进行诱导,RT-qPCR检测吡虫啉诱导不同时间后P450基因的表达情况。利用RNA干扰(RNAi)技术沉默苹果黄蚜3龄若蚜的P450基因,并研究该基因沉默后,苹果黄蚜对吡虫啉的敏感性。【结果】获得苹果黄蚜细胞色素P450基因的cDNA全长序列,为1 542 bp,编码513个氨基酸,命名为AcCYP6CY14(GenBank登录号:MH717248)。研究发现该基因在苹果黄蚜各个发育阶段均有表达,在3龄若蚜中表达量最高。经吡虫啉诱导12 h后,该基因的相对表达量显著高于对照。dsRNA饲喂结果显示,苹果黄蚜取食ds AcCYP6CY14 24 h后,将其转接到新鲜的苹果嫩稍上,24、48、72 h后,该基因的表达量显著下调。沉默该P450基因后,苹果黄蚜对吡虫啉的敏感性极显著升高(P<0.01),死亡率提高了65.2%。【结论】克隆得到了苹果黄蚜P450基因AcCYP6CY14的cDNA全长序列,该基因在苹果黄蚜整个生育期均有表达,且在3龄若蚜中表达量最高,推测该基因可能参与了苹果黄蚜对吡虫啉的解毒代谢。
[Objectives] To study the developmental expression of the cytochrome P450 gene AcCYP6CY14 and its role in imidacloprid resistance in Aphis citricola. [Methods] Partial cDNA sequences of AcCYP6CY14 were obtained by searching the transcriptome database of A. citricola. Full-length cDNA of the target gene was cloned using RT-PCR, and its relative expression in different developmental stages measured using real-time quantitative PCR(RT-qPCR). The 3rd instar of A.citricola was chosen for an induction experiment using the insect-soaking method and the relative expression of AcCYP6CY14 was detected by RT-qPCR after exposing insects to imidacloprid for different periods of time. The target gene was silenced using RNA interference(RNAi) technology by feeding nymphs dsRNA, and the susceptibility of the nymphs to imidacloprid was assessed. [Results] The length of the full-length cDNA sequence of AcCYP6CY14 was 1 542 bp, encoding 513 amino acids(GenBank accession No.: MH717248). AcCYP6CY14 was expressed in all developmental stages of A. citricola but was highest in 3rd instar nymphs. After exposure to an LC30 dose of imidacloprid for 12 h, the relative expression of the AcCYP6CY14 was significantly higher than that of the control group. Feeding nymphs ds AcCYP6CY14 for 24 h significantly decreased transcripts of AcCYP6CY14 when nymphs were transferred to tender stems for 24, 48 and 72 h. A. citricola became more sensitive to imidacloprid(P < 0.01) when AcCYP6CY14 was silenced after which mortality increased by 65.2%.[Conclusion] The full-length cDNA sequence of AcCYP6CY14 was cloned and was found to be expressed in all developmental stages of A. citricola. Expression was highest in 3rd instar nymphs. This gene may play a role in imidacloprid resistance in A. citricola.
[Objectives] To study the developmental expression of the cytochrome P450 gene AcCYP6CY14 and its role in imidacloprid resistance in Aphis citricola. [Methods] Partial cDNA sequences of AcCYP6CY14 were obtained by searching the transcriptome database of A. citricola. Full-length cDNA of the target gene was cloned using RT-PCR, and its relative expression in different developmental stages measured using real-time quantitative PCR(RT-qPCR). The 3rd instar of A.citricola was chosen for an induction experiment using the insect-soaking method and the relative expression of AcCYP6CY14 was detected by RT-qPCR after exposing insects to imidacloprid for different periods of time. The target gene was silenced using RNA interference(RNAi) technology by feeding nymphs dsRNA, and the susceptibility of the nymphs to imidacloprid was assessed. [Results] The length of the full-length cDNA sequence of AcCYP6CY14 was 1 542 bp, encoding 513 amino acids(GenBank accession No.: MH717248). AcCYP6CY14 was expressed in all developmental stages of A. citricola but was highest in 3rd instar nymphs. After exposure to an LC30 dose of imidacloprid for 12 h, the relative expression of the AcCYP6CY14 was significantly higher than that of the control group. Feeding nymphs ds AcCYP6CY14 for 24 h significantly decreased transcripts of AcCYP6CY14 when nymphs were transferred to tender stems for 24, 48 and 72 h. A. citricola became more sensitive to imidacloprid(P < 0.01) when AcCYP6CY14 was silenced after which mortality increased by 65.2%.[Conclusion] The full-length cDNA sequence of AcCYP6CY14 was cloned and was found to be expressed in all developmental stages of A. citricola. Expression was highest in 3rd instar nymphs. This gene may play a role in imidacloprid resistance in A. citricola.
引文
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Elzaki ME,Zhang W,Han Z,2015.Cytochrome P450 CYP4DE1and CYP6CW3v2 contribute to ethiprole resistance in Laodelphax striatellus(Fallen).Insect Mol.Biol.,24(3):368-376.
Feyereisen R,2006.Evolution of insect P450.Biochem.Soc.Trans.,34(Pt 6):1252-1255.
Feng YT,Yu Q,Wang Z,Liu ZF,Xie YP,Fan RJ,2012.Susceptibility of Aphis citricola von der Goot to seven insecticides in Yuncheng Shanxi.Journal of Shanxi Agriciltural Sciences,40(11):1214-1216.[封云涛,庾琴,王振,刘中芳,谢映平,范仁俊,2012.山西运城地区苹果黄蚜对7种杀虫剂的敏感性.山西农业科学,40(11):1214-1216.]
Feng YT,Wang Z,Zhang RX,Yu Q,Liu ZF,Gao Y,Fan RJ,2015.Research on discrimination dose of acetamiprid and bifenthrin in Aphis citricola van der Goot.Chinese Journal of Applied Entomology,52(5):1149-1153.[封云涛,王振,张润祥,庾琴,刘中芳,高越,范仁俊,2015.联苯菊酯和啶虫脒对苹果黄蚜的区分剂量研究.应用昆虫学报,52(5):1149-1153.]
Lv CJ,2005.Susceptibility dynamics of Aphis citricola von der Goot to insecticides and resistance management strategy.Taigu:Shanxi Agricultural University.[吕朝军,2005.苹果黄蚜对杀虫剂的敏感性动态及抗性治理对策研究.硕士学位论文.太谷:山西农业大学.]
Lv CJ,Han JC,Liu HP,Ren YX,Ma L,2007.Influence of host plants to detoxification enzymes and susceptibilities to insecticides on Aphis citricola von der Goot.Journal of Plant Protection,34(5):534-538.[吕朝军,韩巨才,刘慧平,任一新,马林,2007.寄主植物对苹果黄蚜药剂敏感性及解毒酶活性的影响.植物保护学报,34(5):534-538.]
Liu CY,Zeng FR,2014.Cloning of Chrysopa septempunctata P450cDNA fragment and the expression induced by inidacloprid.Chinese Journal of Biological Control,30(3):427-433.[刘昌燕,曾凡荣,2014.大草蛉P450基因cDNA片段的克隆及吡虫啉诱导表达.中国生物防治学报,30(3):427-433.]
Liu HP,Han JC,Xu Q,Lv CJ,Liu HQ,2007.Selective toxicity of insecticides between Aphis citricola and Coccinella septempunctala.Chinese Journal of Eco-Agriculture,15(2):126-129.[刘慧平,韩巨才,徐琴,吕朝军,刘慧芹,2007.杀虫剂对苹果黄蚜与七星瓢虫的毒力及选择性研究.中国生态农业学报,15(2):126-129.
Li XF,Fan J,Sun YW,Wang GP,Chen HM,Yan T,Du WM,Xia LQ,2014.Cloning and RNA interference analysis of the Sitobion avenae salivary protein C002 gene.Chinese Journal of Applied Entomology,51(6):1479-1487.[李雪峰,范佳,孙永伟,王根平,陈红梅,闫婷,杜文明,夏兰琴,2014.麦长管蚜唾液蛋白C002的基因克隆与RNA干扰研究.应用昆虫学报,51(6):1479-1487.]
Li X,Schuler MA,Berenbaum MR,2007.Molecular mechanisms of metabolic resistance to synthetic and natural xenobiotics.Annu.Rev.Entomol.,52:231-253.
Li XY,2015.Mloecular cloning and expression analysis of P450CYP6CY3 genes from Rhopalosiphum padi.Master degree dissertation.Yangling:Northwest A&F University.[李晓叶,2015.禾谷缢管蚜P450 CYP6CY3基因的分子克隆和表达分析.硕士学位论文.杨凌:西北农林科技大学.]
Mutti NS,Park Y,Reese JC,Reeck GR,2006.RNAi knockdown of a salivary transcript leading to lethality in the pea aphid,Acyrthosiphon pisum.J.Insect Sci.,6(38):1-7.
Nelson DR,2013.A world of cytochrome P450s.Philos.Trans.RSoc.Lond B Biol.Sci.,368(1612):20120430.
Peng B,Si SD,Luan BH,Wang KW,Wang YZ,2010.Resistance monitoring of Aphis citricola in major apple producing areas in Shandong province.China Fruits,(5):48-51.[彭波,司树鼎,栾炳辉,王可伟,王英姿,2010.山东省主要苹果产区苹果黄蚜抗药性水平监测.中国果树,(5):48-51.]
Pitino M,Coleman AD,Maffei ME,Ridout CJ,Hogenhout SA,2011.Silencing of aphid genes by dsRNA feeding from plants.PLoS ONE,6(10):e25709.
Pan YW,Liu N,Liu XN,2007.Advances in studies of cytochrome P450 in the insect and its mediated insecticide resistance.Xinjiang Agricultural Sciences,44(4):470-475.[潘贻武,刘宁,刘小宁,2007.昆虫细胞色素P450的研究进展及其介导的抗药性.新疆农业科学,44(4):470-475.]
Qiu XH,2014.Molecular mechanisms of insecticide resistance mediated by cytochrome P450s in insects.Acta Entomologica Sinica,57(4):477-482.[邱星辉,2014.细胞色素P450介导的昆虫抗药性的分子机制.昆虫学报,57(4):477-482.]
Schuler MA,2011.P450s in plant-insect interactions.Biochim.Biophys.Acta,1814(1):36-45.
Tang T,Zhao QC,Feng XY,Liu XY,Qiu LH,2013.Effects of RNAi-mediated silencing of several components of cytochrome P450s on beta-cypermethrin toxicity against Helicoverpa armigera.Journal of Plant Protection,40(4):355-362.[唐涛,赵春青,冯晓云,刘雪源,邱立红,2013.RNAi介导的棉铃虫细胞色素P450酶系几种组分基因沉默对高效氯氰菊酯毒力的影响.植物保护学报,40(4):355-362.]
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