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解毒酶和转运蛋白介导的害虫抗药性分子机制研究进展

徐莉 王建华 梅宇 李冬植

引用本文:
Citation:

解毒酶和转运蛋白介导的害虫抗药性分子机制研究进展

    作者简介: 徐莉,女,博士,讲师,研究方向为害虫抗药性机制,E-mail:xuli-apple-love@163.com.
    通讯作者: 李冬植, lidongzhi1989@163.com
  • 中图分类号: Q965.9

Research progress on the molecular mechanisms of insecticides resistance mediated by detoxification enzymes and transporters

    Corresponding author: Dongzhi LI, lidongzhi1989@163.com
  • CLC number: Q965.9

  • 摘要: 害虫抗药性是导致杀虫剂防效降低的一个重要因素,而抗性机制的阐明是害虫抗药性综合治理的基础。研究表明,代谢能力增强是害虫抗药性产生的重要原因,害虫对杀虫剂等外源物质的代谢需要细胞色素P450酶系 (P450s)、羧酸酯酶 (CarEs)、谷胱甘肽S-转移酶 (GSTs)、UDP-葡萄糖醛酸转移酶 (UGTs) 和ATP结合盒转运蛋白 (简称ABC转运蛋白) 等解毒酶和转运蛋白的参与。结合近年来对害虫抗药性分子机制的研究进展,本文综述了上述解毒酶和转运蛋白参与杀虫剂抗药性的分子机制,并对害虫抗药性治理的新方法进行了展望。
  • 图 1  解毒酶和转运蛋白参与外源化合物代谢的示意图

    Figure 1.  Schematic drawing of the metabolism of xenobiotics by detoxification enzymes and transporters

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出版历程
  • 收稿日期:  2019-06-05
  • 网络出版日期:  2019-11-16
  • 刊出日期:  2020-02-01

解毒酶和转运蛋白介导的害虫抗药性分子机制研究进展

    通讯作者: 李冬植, lidongzhi1989@163.com
    作者简介: 徐莉,女,博士,讲师,研究方向为害虫抗药性机制,E-mail:xuli-apple-love@163.com
  • 1. 河南科技学院 资源与环境学院,河南 新乡 453003
  • 2. 新疆农业科学院 生物质能源研究所,乌鲁木齐 830091

DOI: 10.16801/j.issn.1008-7303.2020.0016

摘要: 害虫抗药性是导致杀虫剂防效降低的一个重要因素,而抗性机制的阐明是害虫抗药性综合治理的基础。研究表明,代谢能力增强是害虫抗药性产生的重要原因,害虫对杀虫剂等外源物质的代谢需要细胞色素P450酶系 (P450s)、羧酸酯酶 (CarEs)、谷胱甘肽S-转移酶 (GSTs)、UDP-葡萄糖醛酸转移酶 (UGTs) 和ATP结合盒转运蛋白 (简称ABC转运蛋白) 等解毒酶和转运蛋白的参与。结合近年来对害虫抗药性分子机制的研究进展,本文综述了上述解毒酶和转运蛋白参与杀虫剂抗药性的分子机制,并对害虫抗药性治理的新方法进行了展望。

English Abstract

  • 杀虫剂的施用是防治农业及卫生害虫的重要手段,但长期大量不合理使用杀虫剂已引发严重的害虫抗药性问题,由此造成杀虫剂防效降低、使用次数和剂量增加、作物产量降低、环境污染以及虫媒人畜疾病的流行等问题,给人类造成巨大损失。如何有效治理害虫抗性是全球亟待解决的难题,而抗性机制的阐明是抗性治理的基础。

    一般而言,害虫对杀虫剂产生抗性的原因包括:靶标敏感性降低、解毒酶系代谢能力增强和表皮穿透能力下降等,其中由解毒酶介导的代谢能力增强是抗性产生的一个重要原因。外源物质在昆虫体内的代谢一般可分为3个阶段[1]:第1阶段,在细胞色素P450酶系 (简称P450s) 和羧酸酯酶 (carboxylesterases,CarEs) 等解毒酶的作用下,催化亲核官能团 (羟基、羧基或氨基) 加入到外源化合物中,使其极性增加,易溶于水;第2阶段,在UDP-葡萄糖醛酸转移酶 (UDP-glycosyltransferases,UGTs) 和谷胱甘肽S-转移酶 (glutathione S-transferases,GSTs) 等转移酶作用下,将第1阶段的产物与内源性分子 (如糖分子和谷胱甘肽) 轭合,使之更易溶于水;第3阶段,在ATP结合盒转运蛋白(ATP-binding cassette),简称ABC转运蛋白的作用下,将轭合物运出细胞膜,最终排出体外(图1)。由P450s、CarEs、UGTs、GSTs和ABC等解毒酶和转运蛋白介导的害虫抗药性机理是代谢抗性研究的重要内容,而有关P450s、CarEs和GSTs介导的害虫抗药性已有多篇综述[2-6]。在此基础上,本文对近年来一些新的研究进展,以及UGTs和ABC介导的害虫抗药性的国内外研究报道进行综述,以期为制定害虫综合治理策略提供依据。

    图 1  解毒酶和转运蛋白参与外源化合物代谢的示意图

    Figure 1.  Schematic drawing of the metabolism of xenobiotics by detoxification enzymes and transporters

    • P450是一种广泛存在于动物、植物、微生物中的解毒酶,是P450酶系 (P450s) 的重要组成部分,具有种类和功能多样、底物广泛等特点,由P450介导的杀虫剂代谢解毒作用的增强是害虫产生抗药性常见且重要的机制之一。随着对害虫抗药性分子机制研究的深入,研究对象逐渐从单基因或多基因转变到基因组,邱星辉[7]、郭亭亭等[8]已经就单个或多个P450基因过表达导致的害虫抗药性及其调控机制进行过综述,本节就近年来P450基因参与害虫抗药性的研究进展以及借助转录组技术对P450的研究成果进行论述。

    • 细胞色素P450是昆虫体内各种外源和内源性化合物的主要代谢酶,其含量的增加和 (或) 活性的提高是大多数重要害虫形成抗药性的主要机制。在棉铃虫Helicoverpa armigera中,CYP6B7基因在氰戊菊酯汰选品系和田间种群中均显著上调表达,对其进行RNAi处理,可以显著增加汰选品系对氰戊菊酯的敏感性[9-11]。Li等[12]测定了多个P450基因在氯虫苯甲酰胺抗性小菜蛾Plutella xylostella中的相对表达水平,发现CYP6BG1显著过量表达,用RNAi技术将其沉默,能够增加小菜蛾对氯虫苯甲酰胺的敏感性,将该基因转入到果蝇Drosophila melanogaster中,可以降低氯虫苯甲酰胺对果蝇的毒力。已有研究表明,致倦库蚊Culex quinquefasciatus对菊酯类杀虫剂的抗性与CYP9M10的过量表达有关[13-14]。Itokawa等[15]通过CRISPR/Cas9系统打乱抗性品系中CYP9M10的基因序列,使该基因的所有拷贝沉默。生物测定结果表明,沉默品系对氯菊酯的敏感性比抗性品系增加了100多倍,有力证明了CYP9M10的扩增和过量表达是致倦库蚊对菊酯类杀虫剂产生抗性的重要原因。Zhang等[16]分析了抗吡虫啉褐飞虱Nilaparvata lugens中54个P450基因的相对表达水平,其中有6个基因在抗性品系中显著高表达;RNAi结果表明,抑制CYP6AY1CYP6ER1CYP4CE1CYP6CW1的表达可显著增加抗性褐飞虱对吡虫啉的敏感性。转录组和qRT-PCR结果表明,CYP6AA1基因在抗溴氰菊酯和氯菊酯的不吉按蚊Anopheles funestus中的表达量显著高于其敏感种群,将该基因转到果蝇体内,导致试虫对溴氰菊酯和氯菊酯抗性水平提高[17]。用转录组技术分析用吡虫啉处理前后韭菜迟眼蕈蚊Bradysia odoriphaga基因的表达水平,发现4个P450基因在处理后显著上调表达,对以上基因进行沉默,可以显著增加该虫对吡虫啉的敏感性[18]。对氯虫苯甲酰胺汰选前后的甜菜夜蛾Spodoptera exigua品系进行转录组分析,发现汰选后品系中上调表达的4个P450基因 (CYP9A21v1CYP9A21v2CYP9A21v3CYP9A21v4) 在另外2个抗氯虫苯甲酰胺的田间种群中也显著上调表达,用RNAi技术对CYP9A21v3进行沉默,可以显著增加其对药剂的敏感性[19]。以上研究均表明P450基因的过量表达参与了害虫对杀虫剂的抗性。

    • P450基因的异源表达产物能够代谢杀虫剂是其参与该杀虫剂抗性的最直接证据。将褐飞虱中过量表达的CYP6AY1CYP6ER1CYP4CE1CYP6CW1在大肠杆菌中异源表达,其重组蛋白对吡虫啉有明显的代谢作用[16]。Ibrahim等[17]将不吉按蚊中过量表达的CYP6AA1在大肠杆菌中异源表达,提取的重组蛋白可以代谢氯菊酯、溴氰菊酯和恶虫威。此外,Joußen等[20]CYP337B1为探针,从采自澳大利亚昆士兰的TWB种群棉铃虫BAC文库中筛选出CYP337B2CYP337B3基因,研究发现CYP337B3是由CYP337B1CYP337B2嵌合而来,但只有CYP337B3与该种群棉铃虫对氰戊菊酯的抗药性密切相关,其在Ha2302细胞中的表达产物能将氰戊菊酯代谢为无毒的4′-羟基氰戊菊酯。随后Rasool等[21]发现,CYP337B3在Ha2302细胞中的重组蛋白也可以将氯氰菊酯代谢为毒性较小的4´-羟基氯氰菊酯。近来,Tian等[22]CYP6B6CPR成功地在大肠杆菌中表达,将其重组蛋白与顺式氰戊菊酯孵育,发现重组蛋白可以将顺式氰戊菊酯代谢为4′-羟基氰戊菊酯,表明CYP6B6可以催化顺式氰戊菊酯羟基化,在氰戊菊酯代谢中发挥重要作用。上述研究有力地证明了P450不仅可通过过量表达参与害虫抗性,还可通过基因重组产生有利变异而参与害虫抗性。

    • 与害虫抗药性相关的P450基因的过量表达受顺式作用元件、反式作用因子等的控制。Amenya等[23]研究发现,不吉按蚊对拟除虫菊酯类杀虫剂的抗性是由于CYP6P9基因过量表达导致的,且其表达受顺式元件调控。但与抗性相关的家蝇Musca domestica CYP6A1[24]和果蝇CYP6A2CYP6A8[25-26]的过量表达则是受反式作用因子的调控。Maitra等[25-26]通过基因杂交和染色体置换研究了位于果蝇Ⅱ号染色体上的CYP6A2CYP6A8的过量表达机制,发现在敏感种群中这2个基因的表达均受到位于Ⅲ号染色体上的反式作用因子的抑制,而抗性种群中的反式作用因子因发生突变而失去抑制功能,从而导致过量表达。而与拟除虫菊酯类杀虫剂抗性相关的家蝇CYP6D1基因的过量表达则受到位于Ⅰ号染色体的顺式作用元件和位于Ⅱ号染色体上的反式作用因子的共同调控[27-28]

      顺式作用元件存在于基因的5′非翻译区,一些调控元件可以直接影响基因的表达量,进一步影响昆虫抗药性的产生。Daborn等[29]通过微阵列分析 (microarray analysis) 证明5′端Accord转座子的插入引起了Cyp6g1基因在果蝇中的过量表达,从而产生了对DDT的抗性。在拟果蝇Drosophila simulans中,Cyp6g1基因5′端Doc转座子的插入也和DDT抗性相关[30-31]。致倦库蚊中,在核心5′非翻译区域一个碱基的突变导致了拟除虫菊酯类杀虫剂抗性相关P450基因CYP9M10的过量表达[32]。Pu等[33]发现在灰飞虱中Laodelphax striatellus,多个顺式作用元件参与了溴氰菊酯抗药性相关基因CYP6FU1的上调表达。

      转录因子通过与顺式作用元件的特异性结合进而调控靶标基因的转录。关于昆虫细胞色素P450基因的转录因子目前已有一些报道。Kalsi等[34]研究发现,在赤拟谷盗Tribolium castaneum中,cap n collar isoform C(CncC) 和muscle aponeurosis fibromatosis(Maf) 转录因子调节溴氰菊酯抗性相关基因CYP6BQ9的过量表达,将CncC和Maf分别敲除后再用溴氰菊酯处理均能显著增加赤拟谷盗的死亡率,说明二者在CYP6BQ9的转录调控中起重要作用;该作者随后还发现,马铃薯甲虫Leptinotarsa decemlineata (Say) 参与吡虫啉和植物次生物质代谢的多个P450基因也是由CncC和Maf调控[35]

      对抗性相关P450基因调控机制的研究,有助于了解害虫抗性产生和发展的内在分子机制,为研究制定害虫抗性预防和治理策略奠定基础。

    • 羧酸酯酶 (CarEs) 的基因扩增、上调、编码序列突变或这些机制的组合都参与了昆虫对有机磷、氨基甲酸酯和拟除虫菊酯类杀虫剂的抗药性。

    • 羧酸酯酶基因编码序列的G137D和W251L突变主要发生在双翅目昆虫中。已有研究表明:铜绿蝇Lucilia cuprina、丝光绿蝇Lucilia sericata、螺旋锥蝇Cochliomyia hominivorax和家蝇羧酸酯酶这2个位点的突变导致了其对有机磷类杀虫剂的抗性[36-40];在一个马拉硫磷抗性寄生蜂Anisopteromalus calandrae中也发现了W251G突变[41]。Cui等[42]通过克隆棉蚜Aphis gossypii、褐飞虱、赤拟谷盗、斜纹夜蛾Spodoptera litura、异色瓢虫Harmonia axyridis、家蚕Bombyx mori和蜜蜂Apis mellifera的羧酸酯酶基因,并对其进行定点突变,异源表达后测定了羧酸酯酶突变体对β-乙酸萘酯和对氧磷、毒虫畏的降解活性。结果表明,G/A151D和W271L突变能够显著降低羧酸酯酶对β-乙酸萘酯的活性,增加对对氧磷和毒虫畏的水解活性,且W271L突变对上述结果影响更大,证明了昆虫通过羧酸酯酶点突变获得抗药性的进化途径具有普遍性。

      在对氧化乐果表现出120倍抗性的棉蚜品系中,通过RNAi技术发现1个CarE基因和有机磷抗性密切相关,且在该基因上存在4个突变位点,分别是H104R、A128V、T333P和K484R,该CarE酶对底物α-乙酸萘酯的活性降低[43-44];使用定点突变的方法对敏感品系该基因的4个位点分别进行突变,在Sf9细胞中进行异源表达和酶活检测,发现4个位点同时突变和H104R、A128V、T333P的单独突变都增加了其对有机磷类杀虫剂的水解活性,而H104R与A128V或T333P同时突变对水解活性的促进作用更为明显;K484R突变则降低了其对有机磷类杀虫剂的水解活性;分子模拟对接结果与代谢结果基本一致[45]。以上结果表明基因的结构变化是导致蚜虫产生抗药性的分子基础。

    • 羧酸酯酶基因扩增导致的抗药性发生在包括以桃蚜Myzus persicae、麦二叉蚜Schizapis graminum和褐飞虱为代表的半翅目及以埃及伊蚊Aedes aegypti和白纹伊蚊Aedes albopictus为代表的双翅目等昆虫中。在桃蚜中,基因扩增导致羧酸酯酶E4或FE4蛋白的过量产生,该蛋白能够在杀虫剂到达靶标之前起到水解和隔离作用,与包括有机磷、氨基甲酸酯和拟除虫菊酯在内的多种类型杀虫剂的代谢增强相关[46]。在抗双硫磷的白纹伊蚊种群中,qRT-PCR发现酯酶基因CCEae3aCCEae6a在抗性品系中存在近10倍的扩增[47],后续通过对全球多地区白纹伊蚊研究发现,还存在CCEae3a的单独扩增[48]。在埃及伊蚊中,对双硫磷的抗性也和CCEae3a基因的扩增相关[49]。表明羧酸酯酶基因扩增是介导半翅目和双翅目昆虫抗药性的重要分子机制之一。

    • 除了基因扩增,转录水平的上调表达也导致了酯酶的过量产生。在抗甲氰菊酯 (FeR) 和抗氟啶虫酰胺 (CyR) 的朱砂叶螨Tetranychus cinnabarinus品系中,分别有8个和4个酯酶基因在转录水平过量表达,在DNA水平没有扩增。在FeR品系中,有4个基因可以被甲氰菊酯诱导;在CyR品系中,有2个基因可以被氟啶虫酰胺诱导;敏感种群中这些酯酶基因均未被诱导[50]。对FeR品系中可被诱导的4个酯酶基因进行RNAi处理,发现沉默其中任何一个均能降低朱砂叶螨对甲氰菊酯的抗性,说明这4个酯酶基因和朱砂叶螨对甲氰菊酯的抗性密切相关[51]。在抗马拉硫磷的东方果实蝇Bactrocera dorsalis中,酯酶基因BdCarE4BdCarE6在转录水平显著过量表达,在DNA水平无扩增,编码序列也无基因突变。将这2个基因进行异源表达,发现重组蛋白对马拉硫磷有一定的降解效果,对2个基因进行RNAi处理后能够增加抗性品系对马拉硫磷的敏感程度[52]。说明酯酶基因上调表达导致其活性升高参与了害虫抗药性的发生和发展。

      昆虫抗药性机制复杂多样,通常是多种机制共同起作用。在抗马拉硫磷的家蝇品系中,发现在酯酶基因MdαE7上存在Ser250-Thr、Trp251-Ser、Met303-Ile、Leu354-Phe、Ser357-Leu、Trp378-Arg和Ser383-Thr等多个点突变,且该基因在抗性品系中过量表达[53]。褐飞虱对有机磷和氨基甲酸酯类杀虫剂的抗药性和羧酸酯酶基因Nl-EST1的扩增有关,在抗性品系中,发现该基因在转录水平也有一定程度的过量表达,共同导致酯酶活性的提高[54]

    • GSTs可以通过直接作用或者代谢第1阶段所得产物而参与解毒,催化谷胱甘肽 (GSH) 和杀虫剂的轭合,增加其水溶性,降低产物毒性。GSTs还具有过氧化物酶活性,可以缓解杀虫剂导致的氧化应激和减少脂质过氧化产物的产生。

    • GSTs通过基因上调表达和突变参与昆虫对有机氯类杀虫剂的抗药性,通过脱氯化氢作用将DDT代谢为DDE。冈比亚按蚊Anopheles gambiae GSTs活性的升高被报道和DDT抗性相关[55],对代谢DDT的GSTs同工酶进行表征,发现31个GSTs中,AgGSTD5AgGSTD6AgGSTE2在大肠杆菌中异源表达后可代谢DDT[56-58]GSTe2及其同源基因在抗DDT的不吉按蚊和埃及伊蚊中均呈现上调表达[59-60],将不吉按蚊中的GSTe2基因转到果蝇中,能够提高果蝇对DDT的抗性水平,同时对拟除虫菊酯类杀虫剂产生交互抗性,且在抗DDT品系的GSTe2基因编码区存在一个与DDT和拟除虫菊酯类杀虫剂抗性相关的点突变L119F,该突变增大了GSTe2与DDT的结合腔,使DDT更易进入和代谢[59]。在抗DDT的埃及伊蚊GG品系中,AaGSTD 1在抗性品系GG中的过量表达主要是由一个未识别的反式作用抑制子的功能缺失突变所致,它抑制了AaGSTD 1的转录和/或降低了AaGSTD 1在敏感品系中的稳定性[61]。表明GSTs可通过多种方式参与有机氯类杀虫剂的代谢。

    • GSTs在有机磷抗药性中发挥重要作用。Qin等[62]将飞蝗Locusta migratoria中的LmGSTs5LmGSTu1沉默可使幼虫对马拉硫磷和毒死蜱的死亡率分别增加28%和16%。将棉铃虫的HaGST-8在毕赤酵母中异源表达,其重组蛋白对毒死蜱和敌敌畏有较好的代谢作用[63]。Yamamoto等[64]从家蚕中克隆得到能够代谢二嗪磷的bmGSTu2。在抗马拉硫磷的东方果实蝇中,BdGSTe2BdGSTe4BdGSTe9显著过量表达,将这3个基因和BdGSTe3在Sf9细胞中表达,发现对马拉硫磷有代谢作用,采用RNAi沉默BdGSTe2BdGSTe3BdGSTe4,能够增加对马拉硫磷的敏感性,表明这4个GST基因的过量表达在东方果实蝇对马拉硫磷的抗药性中发挥重要作用[65]

    • GSTs活性的提高与几种昆虫对拟除虫菊酯的抗性有关,但GSTs不直接代谢拟除虫菊酯类药剂。在冈比亚按蚊和阿拉伯按蚊Anopheles arabiensis中,GSTE4在抗性种群中过量表达,异源表达后的重组蛋白对拟除虫菊酯类药剂没有直接代谢作用,而是对药剂起隔离贮存作用[66]。在用拟除虫菊酯类药剂汰选得到的抗性褐飞虱中,GSTs酶活性升高,部分纯化的GSTs蛋白对拟除虫菊酯类药剂或其初级代谢产物却没有代谢作用,进一步研究发现,拟除虫菊酯暴露可引起虫体的脂质过氧化和还原型谷胱甘肽的损耗,抗性品系GSTs的升高可缓解拟除虫菊酯引起的脂质过氧化,降低死亡率,推测GSTs升高是通过保护组织免受氧化损伤而赋予褐飞虱抗药性[67]。随后从抗性品系中分离到nlGST1-1,Northern blot结果表明,抗性品系的nlGST1-1转录水平高于敏感品系,将该基因异源表达,测定其重组蛋白的过氧化物酶活性,结果表明,nlGST1-1可以通过清除拟除虫菊酯诱导的脂质过氧化产物而产生抗药性;Southern分析发现,抗性品系中该基因条带较敏感品系更亮,表明nlGST1-1通过基因扩增提高抗性品系中GSTs的活性而产生了对拟除虫菊酯的抗性[68]

      GSTs还被报道参与了对噻虫嗪和阿维菌素的抗性。Yang等[69]比较了对噻虫嗪抗性和敏感品系烟粉虱Bemisia tabaci的解毒酶活性差异,发现抗性品系中GSTs的酶活性显著高于敏感品系,还有7个GST基因在抗性品系中高表达;沉默GST14可显著增加抗性烟粉虱在噻虫嗪处理后的死亡率。PcGSTm5基因在抗阿维菌素柑橘全爪螨Panonychus citri品系中显著过量表达,沉默后可以增加抗性品系对阿维菌素敏感性,但异源表达的重组蛋白不能直接代谢阿维菌素[70]

      GSTs通过基因突变、扩增和上调表达参与了昆虫对有机氯、有机磷、拟除虫菊酯、新烟碱和阿维菌素等杀虫剂的抗药性。其中,有关GSTs突变和扩增的报道相对较少,上调表达是GSTs参与抗性的主要途径。

    • UGTs是一种广泛存在于动物、植物和微生物中的重要生物转化酶,可以催化活化糖供体中的糖基加到受体分子的糖苷配基上,产生可被有效排泄的水溶性物质,从而使毒物得到解毒和消除。

      目前对哺乳动物中的UGTs解毒外源物质研究较多,在昆虫解毒代谢中的作用近年来逐渐增多。在棉贪夜蛾Spodoptera littoralis的触角上,UGT46A6基因的表达量可被亚致死剂量的溴氰菊酯诱导表达2.2倍[71]。Li等[72]发现,在23个 (包括2个突变体)UGT基因中,只有UGT2B17在抗氯虫苯甲酰胺的小菜蛾品系中过量表达,RNA干扰结果表明,将该基因沉默能够显著增加小菜蛾对氯虫苯甲酰胺的死亡率。随后Li等[73]发现,9个UGT基因在多抗小菜蛾品系中过量表达,且其中的UGT40V1UGT45B1UGT33AA4(即UGT2B17) 可以被5~7种杀虫剂诱导表达。在抗吡虫啉的马铃薯甲虫中,转录组测序筛选出7个过量表达的代谢相关酶,RNAi结果表明,CYP4Q3UGT2的沉默显著增加了对吡虫啉的抗药性[74]。在甜菜夜蛾中,Hu等[75]测定了多种杀虫剂对UGT的诱导表达情况,发现高效氯氟氰菊酯、氯虫苯甲酰胺、氰氟虫腙和茚虫威都能够诱导脂肪体细胞中4个UGT基因 (UGT42B5UGT40D5UGT33J3UGT33T3) 的表达,而阿维菌素则抑制了大部分UGT基因的表达。以上研究表明,UGT基因可能参与拟除虫菊酯类、双酰胺、烟碱和氨基甲酸酯等杀虫剂的抗性。

    • ABC转运蛋白广泛存在于真核生物中,在昆虫中可分为A~H 8个亚家族,其中ABCH亚家族目前只在部分昆虫中发现。ABCB、ABCC和ABCG亚家族的基因多被报道参与对杀虫剂的抗性。

      P-糖蛋白 (P-glycoproteins, P-gps) 由ABCB亚家族编码,包含多个药物抗性蛋白[76],也被报道参与昆虫对多种杀虫剂的抗性,其中P-gpABCB1编码。Lanning等[77]证明,P-gp在抗氯氰菊酯和硫双威的烟芽夜蛾Heliothis virescens幼虫中表现出2~6倍的高表达。P-gp在抗拟除虫菊酯和有机磷杀虫剂的棉铃虫中的表达明显升高[78-79]。用0.000 7 mg/L的双硫磷处理埃及伊蚊幼虫48 h后,P-gp mRNA的表达量增加了8倍,在P-gp基因沉默的幼虫中,双硫磷的毒力提高了57%[80]。室内汰选得到的对阿维菌素产生6.9倍抗性的果蝇品系中,P-gp的表达量和P-gp ATP酶的活性比敏感品系提高了2~3倍[81]。Zuo等[82]借助CRISPR/Cas9系统敲除了甜菜夜蛾的P-gp基因,得到该基因开放阅读框上删除4 bp的甜菜夜蛾品系,生物测定结果表明,P-gp基因敲除品系对阿维菌素和甲维盐的敏感性显著提高。在抗伊维菌素的雌微小牛蜱Rhipicephalus microplus成虫中,RmABCB 10的表达显著高于敏感品系,且伊维菌素处理可使抗性品系微小牛蜱RmABCB 10的表达上调,而敏感品系中没有显著变化[83]。表明P-gps的上调表达是多种害虫产生抗性的重要原因。

      ABC转运蛋白作为药物受体,其基因突变也可导致害虫的抗药性。在烟芽夜蛾、小菜蛾和粉纹夜蛾Trichoplusia ni中,虽然利用遗传作图将Cry1Ac抗性和ABCC2基因突变联系了起来,但是缺乏直接的证据[84-85]。Atsumi等[86]在家蚕中克隆得到抗Cry1Ab毒素的ABC候选基因007792–93,在抗性品系中该基因在氨基酸234位点存在酪氨酸 (Tyr) 插入。将敏感品系的该基因转入抗性品系,导致其对Cry1Ab毒素的敏感性显著增加,有力证明了ABC转运蛋白参与对Bt毒素的抗性。将敏感 (BmABCC2_S) 和抗性 (BmABCC2_R) 家蚕ABCC2基因分别在Sf9细胞中异源表达,观察到表达BmABCC2_R的Sf9细胞对Cry1Ab和Cry1Ac具有抗性;构建引入酪氨酸的敏感突变体 (BmABCC2_S + Tyr234) 和缺少酪氨酸的抗性突变体 (BmABCC2_R–Tyr234),分别转入Sf9细胞,发现敏感突变体对Cry1Ab和Cry1Ac产生了抗性,而抗性突变体对Cry1Ab和Cry1Ac失去了抗性;体外结合试验表明,BmABCC2_S + Tyr234不能与Cry1A毒素结合。以上结果证明ABCC2是Cry1A毒素的受体,酪氨酸插入导致Cry1A毒素不能与之结合,从而产生抗药性[87]。在棉铃虫LF60品系中,ABCC2基因的突变导致143个氨基酸的丢失和功能缺失,该突变与棉铃虫对Cry1Ac的高水平抗性密切相关[88]。在草地贪夜蛾Spodoptera frugiperda中,ABCC2基因开放阅读框中2个碱基的插入,导致该基因功能突变,从而产生了对Cry1Fa和Cry1A.105的抗性[89]

      ABCG亚家族被报道参与对拟除虫菊酯、DDT等杀虫剂的抗性。Epis等[90]发现,用ABC抑制剂戊脉安处理能够增加氯菊酯对史氏按蚊Anopheles stephensi的毒力,进一步从其转录组数据中筛选出5个ABC基因,其中ABCG4可以被氯菊酯显著诱导表达。ABCG基因在DDT抗性果蝇和按蚊品系中也明显过量表达[91-92]。基因表达微阵列分析表明,ABCG转运蛋白基因在抗噻虫嗪烟粉虱成虫中上调表达[93-94]。此外,ABCA2基因也被报道参与了对Bt毒素的抗性。Tay等[95]报道,棉铃虫对Cry2Ab的抗性与ABCA2基因突变密切相关;Wang等[96]利用CRISPR/Cas9系统对SCD品系棉铃虫的ABCA2基因进行编辑,得到了敲除ABCA2基因的2个纯合品系。生物测定结果表明,与SCD品系相比,这2个品系对Cry2Aa和Cry2Ab均产生了100倍以上的抗性,说明ABCA2基因在棉铃虫对Cry2Aa和Cry2Ab的抗性中具有重要作用。ABC转运蛋白作为运输载体参与外源物质第3阶段的代谢,但其在昆虫中的代谢途径有待进一步研究。

    • 综上所述,害虫对杀虫剂的代谢抗性源于多种解毒酶和转运蛋白基因的突变、上调表达或者扩增,这些基因的变化通常可以遗传给后代,导致抗性在种群中传播。通过对害虫抗药性机制的深入研究,采用RNAi和CRISPR/Cas9等分子生物学技术进行害虫防治已成为可能。RNAi技术不仅可以用于基因功能的研究,也为害虫防治提供了新的思路,如Mao等[97]在实验室条件下用表达了CYP6AE14 dsRNA的烟草和拟南芥饲喂棉铃虫幼虫,可以降低其体内CYP6AE14的表达量,阻碍幼虫生长发育;利用CRISPR/Cas9技术可以在基因组中对靶标位点进行定点突变,从而使靶基因功能改变或者丧失并稳定遗传给后代,是近年来发展前景最为广阔的技术之一。但由于两项技术存在不稳定、脱靶效应等问题,对其在大田中的应用仍在研究中。此外,害虫抗药性的产生往往伴随着适合度代价,即抗性个体表现出发育速率较慢、存活率和生殖力较低等现象[98],对抗性适合度代价的研究有利于寻找抗性发展规律及其综合治理策略,如在转Bt玉米田附近设置非转基因玉米种植带,利用抗性个体的适合度代价使相对敏感的害虫个体扩大繁殖,并通过自然杂交使抗性个体基因型由纯合子变成杂合子,进而被转基因玉米产生的毒素蛋白杀死,减少抗性个体的比例,达到延缓抗性发展的目的[99-100]

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