谷胱甘肽S-转移酶介导的朱砂叶螨对炔螨特抗药性研究
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摘要
朱砂叶螨Tetranychus cinnabarinus (Boisduval)属蛛形纲Arachnida蜱螨目Acarina叶螨科Tetranychidae叶螨属Tetranychus,是一种寄主广泛的世界性重要害螨,危害包括棉花、花卉、果树和蔬菜在内的多种植物,朱砂叶螨也是桑园重要的植食性害螨,近年来,发生危害日益严重。目前化学防治仍是害螨的主要防控手段,但由于螨类繁殖力强、世代周期短等自身特点,以及不合理使用杀虫杀螨剂,导致其抗药性问题日趋严重,已经发展成为世界上抗药性最严重的害螨之一。
作为一种传统的杀螨剂,炔螨特具有高效、低毒、广谱的特点,自1964年推出以来便被广泛应用于各种农林害螨的防治。虽然长期使用该药剂,但抗药性监测表明橘全爪螨、朱砂叶螨等螨类对炔螨特并未产生抗性。未来几年内,炔螨特仍是螨类防控的主打药剂之一。目前,对该药剂的研究主要集中在农药残留和环境毒理学等方面,而关于害虫对炔螨特的抗药性却没有系统研究报道。因此,深入了解炔螨特的作用机理,评估螨类对该药剂的抗性风险,对害虫害螨抗药性产生机制和治理对策的深入研究具有重要的基础作用。
本学位论文以重要害螨朱砂叶螨为研究对象,针对螨类防治中可能出现的抗药性这一农业生产实际问题,开展对云南桑园朱砂叶螨不同种群的抗药性测定,首次明确了云南桑园朱砂叶螨抗药性水平；在室内选育了朱砂叶螨炔螨特抗性品系,并对其抗性现实遗传力和抗性风险进行估测,揭示了朱砂叶螨对炔螨特的抗性发展规律；在基于对抗性品系生理生化特性分析的基础上,明确了以朱砂叶螨体内重要解毒代谢酶系GSTs作为炔螨特代谢抗性主导因子,并进一步开展了其分子生物学特性分析,克隆了朱砂叶螨GSTS基因,比较分析抗性和敏感品系mRNA水平表达差异,进行了异源表达研究。从种群生物学到分子生物学综合分析朱砂叶螨对炔螨特抗性机制,明确了以GSTs介导的朱砂叶螨对炔螨特代谢抗性机制,对解决农业生产问题和丰富农业害虫害螨抗药性研究理论等都具有实际意义。通过三年多的研究工作,主要取得以下研究结果：
1.云南桑园朱砂叶螨不同种群抗药性测定
根据国际杀虫剂抗性委员会(Insecti cide Resistance Action Committee, IRAC)对杀虫杀螨剂作用机制分类,选择5类7个常用药剂,采用FAO(1980)推荐的玻片浸渍法(Slide-dip method)对云南7个蚕桑主产区桑园朱砂叶螨种群进行抗药性测定,并检测了主要代谢解毒酶羧酸酯酶(CarE)、谷胱甘肽S-转移酶(GSTs)和细胞色素P450单加氧酶(P450)的活性及动力学参数。结果表明,云南桑园朱砂叶螨种群总体上对7种药剂仍处于低到中抗水平。蒙自和祥云朱砂叶螨田间种群对辛硫磷的相对抗性倍数分别为31.45和26.22,已达到中等抗性水平；陆良、蒙自、鹤庆和巧家田间朱砂叶螨种群对溴虫腈的相对抗性倍数分别为22.52、19.05、16.28和14.98,达到低抗水平。朱砂叶螨各地方种群对阿维菌素比较敏感；各田间种群对长期使用的敌敌畏、灭多威、哒螨灵和炔螨特的抗性倍数在10倍以下,说明上仍可作用于桑树上。将朱砂叶螨田间种群对常用杀虫杀螨剂的相对抗性比进行Spearman's rank相关性分析发现,阿维菌素和灭多威呈显著负相关(rs=-0.857),这对田间药剂轮用具有重要参考价值。分别比较朱砂叶螨各田间种群CarE、GSTs和P450比活力与敏感品系相对比值发现,GSTs比值范围在6.44-117.96之间,CarE为1.53-29.87倍,P450为1.12-2.67倍,各田间种群体内GSTs和CarE的活性相对较高。
2.朱砂叶螨炔螨特抗性品系的选育及抗性特点
2.1朱砂叶螨炔螨特抗性品系的选育
以2000年采自重庆北碚区田间,对在室内未接触任何药剂饲养10多年的朱砂叶螨敏感品系进行抗药性筛选,选育朱砂叶螨炔螨特抗性品系。经炔螨特不连续(F1-F23)和连续(F24-F34)筛选共34代后,得到炔螨特相对抗性品系,其抗性倍数达30.669。在炔螨特选择压力下,朱砂叶螨产生抗性的速率较慢,截止汰选至第34代,其毒力回归方程的斜率b值逐渐减小。分析炔螨特选育过程中不同选育方式(连续用药与不连续用药)对炔螨特抗性进化的影响发现,不连续用药筛选(F1-F23)23代现实遗传力(h2)为0.047,连续筛选(F24-F34)10代的现实遗传力(h2)为0.055,连续药剂选择压力下的朱砂叶螨对炔螨特的现实遗传力(h2)大于不连续用药的抗性遗传力。连续药剂选择压力下的朱砂叶螨对炔螨特的抗性进化速率高于不连续用药选择。根据现实遗传力预测抗性提高10倍出现的时间,若药剂防治平均杀死率为50%-90%时,在不连续药剂情况下,朱砂叶螨抗性提高10倍需要53-21代,连续用药则需要46-18代。
2.2朱砂叶螨炔螨特抗性品系的相对适合度
采用叶碟饲养法组建朱砂叶螨敏感品系(F0)和抗性品系(F34)的生命生殖力表,进行种群参数和适合度分析。朱砂叶螨经炔螨特筛选34代后,其抗性倍数达30.669倍,与敏感品系相比,炔螨特抗性品系卵期、若螨期和产卵前期延长,幼螨期缩短,在整个世代历期上,完成世代大概需要11.117d,敏感品系为10.581d,无显著差异。抗性品系每雌产卵量少于敏感品系,卵孵化率低于敏感品系,但差异并不显著。种群参数分析表明,抗性品系的内禀增长率(rm)为0.243,略低于敏感品系(0.257),抗性品系的净生殖率(Rn)为28.990,略高于敏感品系(25.342)。以Rn比值比较朱砂叶螨抗性品系的适合度,抗性品系适合度为1.144,与敏感品系无明显差异,表明朱砂叶螨敏感品系经过炔螨特筛选后形成的抗性品系(F34)并未出现适合度缺陷。
2.3朱砂叶螨对炔螨特抗性生化机制
使用玻片浸渍法(Slide-dip method)分别对炔螨特抗性选育过程中的朱砂叶螨F26(RR=10.80)和F34(RR=30.669)进行交互抗性测定,结果显示在抗性筛选过程中,朱砂叶螨炔螨特抗性品系对辛硫磷、哒螨灵和溴虫腈表现出不同程度的交互抗性,对丁醚脲、敌敌畏和阿维菌素未产生明显的交互抗性。
在酶特异性抑制实验中,运用玻片浸渍法(Slide-dip method)分别测定了敏感品系和抗性品系(F34)雌成螨中,羧酸酯酶(CarE)、谷胱甘肽S-转移酶(GSTs)和细胞色素P450(P450)的特异性酶抑制剂—磷酸三苯酯(TPP)、顺丁烯二酸二乙酯(DEM)和增效醚(PBO)对炔螨特的增效作用。三种特异性酶抑制剂对敏感品系朱砂叶螨雌成螨没有增效作用,在抗性品系(F34)中,TPP和PBO对炔螨特的增效比分别为1.059和1.471,增效作用不明显,而DEM的增效作用相对明显,增效比达2.583。在此基础上,进一步运用喷雾法(Potter spray method)测定两个品系内各个发育阶段螨态中,DEM对炔螨特的增效作用,结果显示DEM对敏感品系各螨态朱砂叶螨没有增效作用,在抗性品系(F34)的卵、幼螨、若螨中,DEM对炔螨特的增效比分别为1.269、1.164、1.092,增效作用不明显,而在成螨中的增效作用相对明显,增效比达1.9916。根据酶特异性抑制剂实验结果,比较两个品系内GSTs酶活性差异,当底物分别为GSH和CDNB时,朱砂叶螨抗性品系(F34)的GSTs比活力分别是敏感品系的1.982倍和2.719倍。综上,酶特异性抑制剂和酶活性实验表明,GSTs在朱砂叶螨对炔螨特的解毒代谢中发挥了重要作用,可能与其抗性密切相关,且在朱砂叶螨不同品系和不同发育阶段中的作用存在差异,这将在后一部分研究中在分子生物学水平探讨。
3.朱砂叶螨GSTs基因的克隆及其mRNA的表达模式
3.1朱砂叶螨GSTs基因cDNA克隆及序列分析
参考朱砂叶螨的近缘种二斑叶螨全基因组信息,利用RT-PCR技术,从朱砂叶螨体内克隆获得了12个GSTs基因cDNA序列,并比对确定了这12个GSTs基因的开放阅读框,对其编码的氨基酸序列进行了推导。根据与其他物种的序列相似性和聚类分析结果,将其中的2个基因归类于Delta家族,5个基因归类于Mu家族,2个基因归类于Omega家族,以及Kappa和Zeta家族基因各1个,余下的1个基因则不能划分在已知的类型中,暂将其归为未分类家族Unclassified。2个Delta家族基因间的氨基酸同源性为88.58%,5个Mu家族基因间的氨基酸的同源性范围介于41.26-98.67%,2个Omega家族基因的氨基酸同源性为45.38%,而在任意的非同一家族之间,氨基酸序列同源性在5.07-18.52%之间。根据命名法则命名这些GSTs基因并在GenBank上登录,其名称和登录号分别为：TcGSTd1(KF421142)、TcGSTd2(KF421143)、TcGSTm1(KF421144)、TcGSTm2(KF421149)、TcGSTm3(KF421146)、 TcGSTm4(KF421147)、TcGSTm5(KF421148)、TcGSTol (KF421150)、TcGSTo2(KF421151)、TcGSTz1(KF421153)、TcGSTkl (KF421152)、TcGSTu2(KF421145).克隆所获得的12个GSTs基因编码区长度范围是651-735bp,编码216-244个氨基酸,相对分子量为24.19-28.95kDa,理论等电点的范围是5.97-8.99,TMHMM2.0跨膜预测显示12个基因编码的氨基酸序列不存在跨膜结构域,易于分离纯化。ScanProsite推测氨基酸保守功能域表明,这些基因N-端结构域高度保守,具有多个GSH的结合位点。将克隆获得的朱砂叶螨各家族GSTs基因同黑腹果蝇、橘全爪螨、二斑叶螨等物种氨基酸序列进行多重比较,结果显示朱砂叶螨Delta家族的GSTs氨基酸序列相对保守,具有与其他物种相同的催化活性中心丝氨酸S、天冬酰胺N以及决定蛋白折叠的氨基酸脯氨酸P、亮氨酸L、甘氨酸G、天冬氨酸D。朱砂叶螨Mu家族GSTs基因同样相对保守,具有催化活性中心酪氨酸Y,以及决定蛋白折叠的苏氨酸T、苯丙氨酸F以及AILRYLARKH特征基序。朱砂叶螨Omega家族GSTs的关键残基半胱氨酸C代表GSH的催化位点,N-端结构域非常保守,特征基序是ESLIIAEYL。
3.2朱砂叶螨RT-qPCR内参基因筛选
运用geNorm和NormFinder软件,对6个看家基因a-TUB、RPS18、EF1α、5.8SrRNA、SDHA、GAPDH在朱砂叶螨不同发育阶段(卵、幼螨、若螨和雌成螨)和不同品系(敏感品系F0和炔螨特抗性品系F34)中的稳定性进行评价。结果分析表明,在朱砂叶螨不同发育阶段中,稳定性较好的是RPS18和GADPH,在不同品系中,经筛选表达最稳定的是a-TUB和RPS18,综合考虑评价结果,选用RPS18作为内参基因用于后续实验。
3.3朱砂叶螨不同发育阶段GSTs基因的表达模式
运用定量PCR技术(quantitative real-time PCR, qPCR),以朱砂叶螨RPS18作为内参基因,对所克隆的8个GSTs基因在不同发育阶段(卵、幼螨、若螨、雌成螨)mRNA表达水平进行了相对定量分析。
在朱砂叶螨敏感品系中,Delta家族的2个基因TcGSTd1和TcGSTd2在幼螨和若螨阶段的相对卵期的表达量上调,在若螨期明显高于卵期,随后在成螨阶段下降。而在朱砂叶螨抗性品系中,这2个基因在幼螨、若螨和成螨阶段随着发育历期的增加其相对表达量明显上调,在成螨达到最高。朱砂叶螨敏感品系Mu家族5个GSTs基因中,TcGSTm1、TcGSTm2、TcGSTm3、TcGSTm4在幼螨的相对表达量显著高于其余三个发育阶段,随后降低,最终在成螨期的表达量与卵期相当；TcGSTm5基因的相对表达量随着发育阶段的增加而逐渐升高,若螨阶段表达量达到最高；TcGSTz1基因在4个螨态中的相对表达量没有较大波动。在抗性品系中,TcGSTm1、TcGSTm2、TcGSTm4随着发育历期的增加其表达量上调,在若螨期达到高峰,然后在成螨阶段的相对表达量降低,但还是高于卵期的表达量；TcGSTm3在幼螨阶段的表达量显著增高,随后在若螨阶段突然下调后在成螨其恢复到与卵期相当的水平。TcGSTm5基因的相对表达量随着发育阶段的增加而逐渐上调；TcGSTz1基因在朱砂叶螨卵期、幼螨和成螨期的相对表达量略有上调,其显著表达高峰出现在若螨期。由上述基因在朱砂叶螨不同发育阶段的表达模式推测,TcGSTd1和TcGSTd2不仅参与了朱砂叶螨正常的生长发育活动,而且在成螨阶段在该螨对炔螨特的抗性中发挥了重要作用,Mu家族5个GSTs基因也不同程度地参与了朱砂叶螨对炔螨特的抗性。
3.4朱砂叶螨不同品系GSTs基因的表达模式
运用实时定量PCR技术,以朱砂叶螨RPS18作为内参基因,对所克隆的8个GSTs基因在敏感品系和炔螨特抗性品系(F34)mRNA表达水平进行相对定量分析。结果表明,相对于敏感品系,8个GSTs基因在朱砂叶螨抗性品系中总体趋势表现为卵期和幼螨期表达下调,而若螨和成螨期显著上调。在卵期,朱砂叶螨抗炔螨特品系的TcGSTd2基因的相对表达显著下降,推测该基因参与了螨类在这一阶段的生理活动。在幼螨期,朱砂叶螨抗炔螨特品系Delta家族的2个GSTs基因TcGSTd1和Mu家族的4个GSTs基因TcGSTm1、TcGSTm2、TcGSTm4基因相对表达量显著下调,推测这几个基因在这一螨态发挥了重要生理功能。在若螨和成螨期,8个GSTs基因的相对表达量都上调,其中TcGSTd1、TcGSTd2、TcGSTm、 TcGSTm2、TcGSTm4和TcGSTm5显著增加,表明这些基因参与了该螨的生理功能和抗药性的形成。
3.5朱砂叶螨GSTs基因的原核表达载体构建
利用限制性内切酶Nde I和Xho I的双酶切以及DNA重组技术成功构建了朱砂叶螨TcGSTm2基因基于pET30a(+)的原核表达载体,为进一步深入研究GSTm蛋白质特性和功能研究奠定了基础。
综上所述,本研究开展了对云南桑园朱砂叶螨不同田间种群的抗药性测定,在室内继代选育朱砂叶螨炔螨特抗性种群,获得抗性倍数达30.669的相对抗性品系,以该品系和敏感品系为基础,研究了朱砂叶螨对炔螨特的抗性发展趋势,对其抗性现实遗传力和抗性风险进行评估,在对抗性品系酶特异性抑制实验和生理生化特性分析基础上,确定了朱砂叶螨体内重要解毒代谢酶系GSTs是作为炔螨特抗性机制的主导因子,以此为基础,在分子水平上进一步解析GSTs基因在朱砂叶螨对炔螨特的抗性分子机制中的作用。运用分子生物学技术,克隆了12个朱砂叶螨GSTs基因,分析了这些基因的分子生物学特性。并采用geNorm和NormFinder软件确定了朱砂叶螨不同发育阶段和不同品系的最适内参基因。采用qPCR技术,解析了本研究所克隆的8个GSTs基因在朱砂叶螨敏感品系和炔螨特抗性品系mRNA水平表达模式和差异,揭示了这些基因在朱砂叶螨抗性过程中发挥的作用。成功构建了朱砂叶螨TcGSTm2基因基于pET30a(+)的原核表达载体,为GSTs基因的异源表达和蛋白质特性研究奠定基础。本研究应用生物学、生理生化和分子生物学知识和技术,全面解析了GSTs介导的朱砂叶螨对炔螨特的抗性机制,为朱砂叶螨的抗药性治理和炔螨特的可持续应用提供理论依据。
Carmine spider mite Tetranychus cinnabarinus (Boisduval) belongs to Arachnida, Tetranychidae, Tetranychus. It is a phytophagous mite distributed worldwide, and feeds on a large number of host plants including cotton, flowers, fruits and vegetables, as well as the mulberry. In recent years, T. cinnabarinus has caused severe damage in the field. The management of this mite still mainly depends on acaricides, while the resistance of T. cinnabarinus could develop quickly for its high reproductive potentials, short life cycle, and the unreasonable use of acaricides. Nowadays, the resistance problem of T. cinnabarinus has been the severest in the mites.
As a traditional acaricide, propargite has been wide used in control of pest mites in the fields and forests since1964for its high efficiency, low toxicity, and broad-spectrum. Although propargite has been used for a long history, the resistance monitoring showed that the citrus red mite and the carmine spider mite were still sensitive to this acaricide. In future, propargite will still be frequently used in the management of mites. Currently, researches about propargite were mainly focused on its residues and environmental toxicology, whereas basic systematic research on pest resistance was still insufficient. As a result, the mechanism study of propargite, and its resistance risk evaluation are basic works to understand its resistance development and conduct effective resistance management..
This study focused on the important pest mite, T. cinnabarinus, paid attention to its resistance problem, worked on the resistance monitoring of different populations of T. cinnabarinus in mulberry fields in Yunnan province, and reported the resistance level for the first time. A propargite-resistant strain was selected in the laboratory, and its realized heritability and resistance risk were evaluated to reveal the rules of resistance development. Based on the biochemistry study of resistance strain, GSTs were identified as an important factor for the resistance development of propargite. The molecular characterization of GSTs were further analyzed. Genes encoding GSTs of T.cinnabarinus were cloned and their specific expressions were compared between susceptible and resistance strains. Heterogenous expression was also conducted. The resistance mechanism of propargite were synthetically analyzed by population biology and molecular biology methods, and the contribution of GSTs in the resistance mechanism were clarified. The results are important reference to solve production problems in agriculture, and can enrich the resistance theory of pest mites. Through the research work of three years, the findings are presented as follows:
1Acaricide susceptibility of seven T. cinnabarinus strains from mulberry fields in Yunnan
To gain primary data that reflects the resistance levels of CMS in mulberry plantations, we evaluated and compared the susceptibility of one susceptible strain and7field-collected populations to7acaricides of5chemistry classes using FAO (1980) recommended slide dipping method. The activities and kinetics of major detoxification enzymes (cytochrome P450, esterases and glutathione s-transferases) of each population were also determined to understand their potential role in acaricide resistance. The results showed that most of T. cinnabarinus populations in mulberry trees exhibited low and moderate levels of resistance. Mengzi and Xiangyun population had moderate resistance to phoxim (RR=31.45and26.22, respectively). The Qiaojia, Luliang, Mengzi and Heqing population showed moderate resistance to chlorfenapyr (RR=14.98-22.52). T. cinnabarinus was still sensitive to propargite (the LC50was87.34-243.65mg/L and the resistance ratio (RR) was3.62-10.08). The esistant of field populations to insecticides correlation analysis showed that abamectin and methomyl was significantly and negatively correlated (rs=-0.857,p<0.05). Comparing the enzymes activity between the field isolates and the susceptible strain of CMS. All the tesed enzymes were more active in the field populations than that in the susceptible strain, GSTs was6.44-117.96folds, CarE was1.53-29.87folds, and the P450was1.12-2.67folds, which indicating that GSTs played an important role in the detoxification of mulberry T. cinnabarinus. The results provide useful reference data for acaricides application and spider mite management in mulberry plantations.
2Resistance selectivity and characteristics of T. cinnabarinus against Propargite
2.1Resistant strain selectivity of Propargite against carmine spider mite
The susceptible strain of T. cinnabarinus was original collected from Beibei District, Chongqing, China in2000and maintanct in laboratory without exposure to aracricides for10years. The susceptible strain was screened continuously with propargite to obtain the resistant strain and the basic characteristics were investigated in this study. After discontinuous (F1-F23) and continuous (F24-F34) selection over34generations, the resistance ratio increased30.669folds. T. cinnabarinus had a slow rate in resistance development to propargite. The breeding procedure of the T. cinnabarinus resistant strain showed the slope of F34strain was smaller than the forgoing strain (F1) according to log concentration-probit curve. The realized heritability (h2) in the continuous (F24-F34) propargite-selection was greater than the discontinuous one (F1-F23), and the values were0.055and0.047, respectively. According to the realized heritability (h2), theoretically, to obtain a10-fold increase in resistance requires53-21generations under discontinuous selective pressure of propargite, and46-18generations continuous selection under selective pressure of propargite of50%-90%for each selective generation.
2.2Relative fitness of propargite-resistant strains of T. cinnabarinus
Using the leaf disc feeding method, The life table of T. cinnabarinus susceptible strain (F0) and resistant strain (F34) at25℃were constructed, the ecological aspects of two strains were investigated. The age-specific fecundity and survival curves showed no significant difference between two strains, so were the developmental duration, eggs per female and hatchability, etc. The fitness of propargite-resistance strain was also not significant changed compared to SS.
2.3Biochemical mechanism of T. cinnabarinus against propargite
The F26and F34generations of propargite-resistant strain showed increased resistance against propargite11folds and31folds respectively using slide-dip method. Toxicity of other pesticides/acaricides to the propargite-resistance strain (F26and F34) were assayed using same method. The LC50values and resistance ratios to these pesticides/acaricides indicated that the F26and F34still sensentive to avermectin, but exhibit cross-resistance to phoxim, pyridaben and dichlorvos, chlorfenapyr and diafenthiuron. PBO, TPP and DEM were the specific inhibitors of cytochrome P450monooxygenases (P450), carboxylesterase (CarE) and glutathione S-transferase (GSTs), respectively. The synergistic ratios of PBO, TPP and DEM to propargite were1.06,1.47and2.58fold in resistant strain (F34) of female adult T. cinnabarinus, respectively. None of those three synergists showed significant synergistic effect in susceptible strain of T. cinnabarinus. Based on this result, the mite of four developmental stages (eggs, larvae, nymph, adults) were exposed to the synergist DEM in combination with propargite using potter spray method. There were no significant synergistic effect on susceptible among different life-stages in carmin spider mite. The synergistic resistance ratios of the propargite-resistant strain (F34) with DEM were calculated to be1.27,1.16,1.09, and1.99folds respectively in eggs, larvae, nymph and adults. Compared to susceptible strain, the activities of GST in F34increased by1.98and2.72folds to different substrate GSH and CDNB, respectively. In conclusion, the results indicated GSTs might play an important role in carmine spider mite resistance to propargite.
3cDNA cloning and mRNA expression profiles of GSTs genes in T. cinnabarinus
3.1cDNA cloning and characterization
Based on the genome data of Tetranychus urticae,12novel GST genes were cloned from T. cinnabarinus using the reverse transcriptase-PCR (RT-PCR) techniques. Putative proteins have been predicted by Protparam and Scanprosite softwares. Based on sequence similarity and phylogenetic analysis, two genes were classified to the Delta gene family, five genes were classified to the Mu family, two genes were classified to the Omega family, two genes were classified as Kappa and Zeta family genes, and the remaining one gene did not belong to any of known family, temporarily be named as an unclassified GST. The nomenclature of these T. cinnabarinus GSTs cDNA and their deduced amino acid sequences have been deposited in Genbank with the following accession numbers:TcGSTd1(KF421142), TcGSTd2(KF421143), TcGSTm1(KF421144), TcGSTm2(KF421149), TcGSTm3(KF421146), TcGSTm4(KF421147), TcGSTm5(KF421148), TcGSTo1(KF421150), TcGSTo2(KF421151), TcGSTz1(KF421153), TcGSTk1(KF421152), TcGSTu2(KF421145). The length of cDNA of these12GSTs gene contained were ranged from651to735bp, encoding proteins with216-244amino acids. Predicted molecular weight was24.19-28.95kDa and the isoelectric point (pI) was between5.97and8.99. It was predicted that there was no transmembrane domain present in these twelve proteins using TMHMM2.0bio-prediction software suggesting that they could be easily purified.
ScanProsite showed that two distinct functional domains were found in10proteins except TcGSTkl and TcGSTul. N-terminal domains were highly conserved, which was binding sites of GSH, were found both in TcGSTd1,TcGSTd2(residues1-82) and TcGSTml, TcGSTm2, TcGSTm3, TcGSTm4and TcGSTm5(1-88,1-90,1-88,1-88, 1-82). Other TcGSTo1, TcGSTo2and TcGSTz1were:22-101,23-101and3-90. Also, C-terminal region were located at position86-219,88-207,90-211,92-220,90-214,90-209,90-211,105-240and105-234in TcGSTdl, TcGSTd2, TcGSTm1, TcGSTm2, TcGSTm3, TcGSTm4, TcGSTm5, TcGSTol, TcGSTo2and TcGSTzl. Delta family GSTs in T. cinnabarinus had the same catalytic domain as other species containing Serine, and Asparagine in the center and to determine the same amino acids regarding protein folding including Proline,Leucine,Glycine,Aspartic acid. Mu family GSTs in T. cinnabarinus had the center Tyrosine as the catalytic site and amino acids determing protein folding, like Threonine, Phenylalanine as well as the "AILRYLARKH" motif. Omega GSTs contained the key residue, Cysteine as the catalytic site and a very conserved N-terminal, in which characteristic motif is "ESLIIAEYL"
3.2Stability evaluation of housekeeping genes
The stability of reference genes used for the quantification of mRNA can be affected by the experimental condition. Therefore, the evaluation of reference genes is critical for gene expression profiling. The stabilities of six candidate reference genes (a-TUB, RPS18, EFlα,5.8SrRNA, SDHA, GAPDH) of T.cinnabarinus were analyzed at different developmental stages and strains using geNorm and NormFinder software, respectively. RPS18and GADPH were identified as the most stable genes in defferent developmental stages, while a-TUB and RPS18were found to be the most stable in different strains. Thus, RPS18genes was used as reference gene in RT-qPCR in this study.
3.3Developmental stages expression profiles of the eight GSTs genes
The expression of eight GSTs genes in T. cinnabarinus were analyzed by real-time quantitative PCR (RT-qPCR) using RPS18as an reference gene. The assayed developmental stages included eggs, larvae, nymph and female adults. The results showed that2Delta family genes,5Mu family genes and TcGSTzl were differently expressed at different stages of mites. The relative expression level of TcGSTdl and TcGSTd2were increased in the larvae and nymph stage compared with egg stage, peaked in nymph stage and declined in adult stage in susceptable strain. But in the resistant strain, the results indicated that the trend of relative expression rose along with growth and development, and reached maximum point in the adult stage. In suscepitable strains, the highest expression levels of four Mu family GST genes(TcGSTm1, TcGSTm2, TcGSTm3and TcGSTm4) were observed at larvae stage. Another Mu family GST genes TcGSTm5showed higher expression level in the nymph stage. In resistant strains, The expression level of three Mu GST genes(TcGSTml,TcGSTm2and TcGSTm4) increased at larvea, showed highest expression in nymph, but reduced in adult stage. Another TcGSTm3showed higher expression levels in the larvae stages, followed by a sudden reduction in the nymph stage, and a recovery in adult. The expression level of TcGSTzl were not significantly different during the development in susceptible strain but showed higher expression in nymph stage in resistant strain. The result indicated that two Delta family GST genes(TcGSTdl and TcGSTd2) and five Mu family GST genes(TcGSTm1,TcGSTm2,TcGSTm3and TcGSTm4) might not only involved in T. cinnabarinus growth and development, but also may played an important role in propargite resistant development in T.cinnabarinus.
3.4Expression profiles of the eight GSTs genes between two different strains
In order to verify whether GSTs are over-expressed in propargite-resistant strain, qPCR with RPS18as the reference gene was employed to determine the relative expression quantity of two Delta GST genes(TcGSTd1,TcGSTd2) and five Mu GST genes(TcGSTml,TcGSTm2,TcGSTm3and TcGSTm4in four life-stage (eggs, larvae, nymph and adults) of T. cinnabarinus in both susceptible and propargite-resistant strain. Expression for eight GST genes in propargite-resistant strain of T. cinnabarinus showed down regulation in eggs and larvae stage, but up regulation in nymph and adult stage in comparison with susceptible strain. In eggs, the relative expression of TcGSTd2was significantly lower than susceptible strain. In larvae, the expression of TcGSTd1,TcGSTd2,TcGSTm1,TcGSTm2,TcGSTm3and TcGSTm4were significantly lower than susceptible strain. In nymph and adults, all of eight GST genes were up regulated than susceptible strain, which implied that these GST genes were possibly involved in propargite resistance.
3.5Prokaryotic expression vector of TcGSTm2
The cDNA of TcGSTm2was cloned into expression vector pET30a (+) by the restriction sites of Nde I and Xho I, which provided the basis for GSTs expression in E. coli.
In summary, this study determined the resistant levels of T. cinnabarinus in Yunnan mulberry plantations. A resistance strain ofpropargite was selected in the laboratory.. After34generations, resistance ratio has increased to30.89folds. The reality of resistant riskment, physiological and biochemical synergist characteristic were analyzed.Biochemical and toxicological characterization of GSTs of T. cinnabarinus were studied; based on genome of T. urticae,12GST genes were cloned from T. cinnabarinus using RT-PCR technique; GSTs genes expression profiles were assayed using qPCR technique from different strains T. cinnabarinus at developmental stages; the expression vector for TcGSTm2was constructed. The results provide insights into exploring the functions of GSTs system in physiology, and it showed evidence for clarifying the resistance mechanisms of carmine spider mite to propargite. Meanwhile, the results also enlighten the resistance mechanism of other insect/mite pests.
作为一种传统的杀螨剂,炔螨特具有高效、低毒、广谱的特点,自1964年推出以来便被广泛应用于各种农林害螨的防治。虽然长期使用该药剂,但抗药性监测表明橘全爪螨、朱砂叶螨等螨类对炔螨特并未产生抗性。未来几年内,炔螨特仍是螨类防控的主打药剂之一。目前,对该药剂的研究主要集中在农药残留和环境毒理学等方面,而关于害虫对炔螨特的抗药性却没有系统研究报道。因此,深入了解炔螨特的作用机理,评估螨类对该药剂的抗性风险,对害虫害螨抗药性产生机制和治理对策的深入研究具有重要的基础作用。
本学位论文以重要害螨朱砂叶螨为研究对象,针对螨类防治中可能出现的抗药性这一农业生产实际问题,开展对云南桑园朱砂叶螨不同种群的抗药性测定,首次明确了云南桑园朱砂叶螨抗药性水平；在室内选育了朱砂叶螨炔螨特抗性品系,并对其抗性现实遗传力和抗性风险进行估测,揭示了朱砂叶螨对炔螨特的抗性发展规律；在基于对抗性品系生理生化特性分析的基础上,明确了以朱砂叶螨体内重要解毒代谢酶系GSTs作为炔螨特代谢抗性主导因子,并进一步开展了其分子生物学特性分析,克隆了朱砂叶螨GSTS基因,比较分析抗性和敏感品系mRNA水平表达差异,进行了异源表达研究。从种群生物学到分子生物学综合分析朱砂叶螨对炔螨特抗性机制,明确了以GSTs介导的朱砂叶螨对炔螨特代谢抗性机制,对解决农业生产问题和丰富农业害虫害螨抗药性研究理论等都具有实际意义。通过三年多的研究工作,主要取得以下研究结果：
1.云南桑园朱砂叶螨不同种群抗药性测定
根据国际杀虫剂抗性委员会(Insecti cide Resistance Action Committee, IRAC)对杀虫杀螨剂作用机制分类,选择5类7个常用药剂,采用FAO(1980)推荐的玻片浸渍法(Slide-dip method)对云南7个蚕桑主产区桑园朱砂叶螨种群进行抗药性测定,并检测了主要代谢解毒酶羧酸酯酶(CarE)、谷胱甘肽S-转移酶(GSTs)和细胞色素P450单加氧酶(P450)的活性及动力学参数。结果表明,云南桑园朱砂叶螨种群总体上对7种药剂仍处于低到中抗水平。蒙自和祥云朱砂叶螨田间种群对辛硫磷的相对抗性倍数分别为31.45和26.22,已达到中等抗性水平；陆良、蒙自、鹤庆和巧家田间朱砂叶螨种群对溴虫腈的相对抗性倍数分别为22.52、19.05、16.28和14.98,达到低抗水平。朱砂叶螨各地方种群对阿维菌素比较敏感；各田间种群对长期使用的敌敌畏、灭多威、哒螨灵和炔螨特的抗性倍数在10倍以下,说明上仍可作用于桑树上。将朱砂叶螨田间种群对常用杀虫杀螨剂的相对抗性比进行Spearman's rank相关性分析发现,阿维菌素和灭多威呈显著负相关(rs=-0.857),这对田间药剂轮用具有重要参考价值。分别比较朱砂叶螨各田间种群CarE、GSTs和P450比活力与敏感品系相对比值发现,GSTs比值范围在6.44-117.96之间,CarE为1.53-29.87倍,P450为1.12-2.67倍,各田间种群体内GSTs和CarE的活性相对较高。
2.朱砂叶螨炔螨特抗性品系的选育及抗性特点
2.1朱砂叶螨炔螨特抗性品系的选育
以2000年采自重庆北碚区田间,对在室内未接触任何药剂饲养10多年的朱砂叶螨敏感品系进行抗药性筛选,选育朱砂叶螨炔螨特抗性品系。经炔螨特不连续(F1-F23)和连续(F24-F34)筛选共34代后,得到炔螨特相对抗性品系,其抗性倍数达30.669。在炔螨特选择压力下,朱砂叶螨产生抗性的速率较慢,截止汰选至第34代,其毒力回归方程的斜率b值逐渐减小。分析炔螨特选育过程中不同选育方式(连续用药与不连续用药)对炔螨特抗性进化的影响发现,不连续用药筛选(F1-F23)23代现实遗传力(h2)为0.047,连续筛选(F24-F34)10代的现实遗传力(h2)为0.055,连续药剂选择压力下的朱砂叶螨对炔螨特的现实遗传力(h2)大于不连续用药的抗性遗传力。连续药剂选择压力下的朱砂叶螨对炔螨特的抗性进化速率高于不连续用药选择。根据现实遗传力预测抗性提高10倍出现的时间,若药剂防治平均杀死率为50%-90%时,在不连续药剂情况下,朱砂叶螨抗性提高10倍需要53-21代,连续用药则需要46-18代。
2.2朱砂叶螨炔螨特抗性品系的相对适合度
采用叶碟饲养法组建朱砂叶螨敏感品系(F0)和抗性品系(F34)的生命生殖力表,进行种群参数和适合度分析。朱砂叶螨经炔螨特筛选34代后,其抗性倍数达30.669倍,与敏感品系相比,炔螨特抗性品系卵期、若螨期和产卵前期延长,幼螨期缩短,在整个世代历期上,完成世代大概需要11.117d,敏感品系为10.581d,无显著差异。抗性品系每雌产卵量少于敏感品系,卵孵化率低于敏感品系,但差异并不显著。种群参数分析表明,抗性品系的内禀增长率(rm)为0.243,略低于敏感品系(0.257),抗性品系的净生殖率(Rn)为28.990,略高于敏感品系(25.342)。以Rn比值比较朱砂叶螨抗性品系的适合度,抗性品系适合度为1.144,与敏感品系无明显差异,表明朱砂叶螨敏感品系经过炔螨特筛选后形成的抗性品系(F34)并未出现适合度缺陷。
2.3朱砂叶螨对炔螨特抗性生化机制
使用玻片浸渍法(Slide-dip method)分别对炔螨特抗性选育过程中的朱砂叶螨F26(RR=10.80)和F34(RR=30.669)进行交互抗性测定,结果显示在抗性筛选过程中,朱砂叶螨炔螨特抗性品系对辛硫磷、哒螨灵和溴虫腈表现出不同程度的交互抗性,对丁醚脲、敌敌畏和阿维菌素未产生明显的交互抗性。
在酶特异性抑制实验中,运用玻片浸渍法(Slide-dip method)分别测定了敏感品系和抗性品系(F34)雌成螨中,羧酸酯酶(CarE)、谷胱甘肽S-转移酶(GSTs)和细胞色素P450(P450)的特异性酶抑制剂—磷酸三苯酯(TPP)、顺丁烯二酸二乙酯(DEM)和增效醚(PBO)对炔螨特的增效作用。三种特异性酶抑制剂对敏感品系朱砂叶螨雌成螨没有增效作用,在抗性品系(F34)中,TPP和PBO对炔螨特的增效比分别为1.059和1.471,增效作用不明显,而DEM的增效作用相对明显,增效比达2.583。在此基础上,进一步运用喷雾法(Potter spray method)测定两个品系内各个发育阶段螨态中,DEM对炔螨特的增效作用,结果显示DEM对敏感品系各螨态朱砂叶螨没有增效作用,在抗性品系(F34)的卵、幼螨、若螨中,DEM对炔螨特的增效比分别为1.269、1.164、1.092,增效作用不明显,而在成螨中的增效作用相对明显,增效比达1.9916。根据酶特异性抑制剂实验结果,比较两个品系内GSTs酶活性差异,当底物分别为GSH和CDNB时,朱砂叶螨抗性品系(F34)的GSTs比活力分别是敏感品系的1.982倍和2.719倍。综上,酶特异性抑制剂和酶活性实验表明,GSTs在朱砂叶螨对炔螨特的解毒代谢中发挥了重要作用,可能与其抗性密切相关,且在朱砂叶螨不同品系和不同发育阶段中的作用存在差异,这将在后一部分研究中在分子生物学水平探讨。
3.朱砂叶螨GSTs基因的克隆及其mRNA的表达模式
3.1朱砂叶螨GSTs基因cDNA克隆及序列分析
参考朱砂叶螨的近缘种二斑叶螨全基因组信息,利用RT-PCR技术,从朱砂叶螨体内克隆获得了12个GSTs基因cDNA序列,并比对确定了这12个GSTs基因的开放阅读框,对其编码的氨基酸序列进行了推导。根据与其他物种的序列相似性和聚类分析结果,将其中的2个基因归类于Delta家族,5个基因归类于Mu家族,2个基因归类于Omega家族,以及Kappa和Zeta家族基因各1个,余下的1个基因则不能划分在已知的类型中,暂将其归为未分类家族Unclassified。2个Delta家族基因间的氨基酸同源性为88.58%,5个Mu家族基因间的氨基酸的同源性范围介于41.26-98.67%,2个Omega家族基因的氨基酸同源性为45.38%,而在任意的非同一家族之间,氨基酸序列同源性在5.07-18.52%之间。根据命名法则命名这些GSTs基因并在GenBank上登录,其名称和登录号分别为：TcGSTd1(KF421142)、TcGSTd2(KF421143)、TcGSTm1(KF421144)、TcGSTm2(KF421149)、TcGSTm3(KF421146)、 TcGSTm4(KF421147)、TcGSTm5(KF421148)、TcGSTol (KF421150)、TcGSTo2(KF421151)、TcGSTz1(KF421153)、TcGSTkl (KF421152)、TcGSTu2(KF421145).克隆所获得的12个GSTs基因编码区长度范围是651-735bp,编码216-244个氨基酸,相对分子量为24.19-28.95kDa,理论等电点的范围是5.97-8.99,TMHMM2.0跨膜预测显示12个基因编码的氨基酸序列不存在跨膜结构域,易于分离纯化。ScanProsite推测氨基酸保守功能域表明,这些基因N-端结构域高度保守,具有多个GSH的结合位点。将克隆获得的朱砂叶螨各家族GSTs基因同黑腹果蝇、橘全爪螨、二斑叶螨等物种氨基酸序列进行多重比较,结果显示朱砂叶螨Delta家族的GSTs氨基酸序列相对保守,具有与其他物种相同的催化活性中心丝氨酸S、天冬酰胺N以及决定蛋白折叠的氨基酸脯氨酸P、亮氨酸L、甘氨酸G、天冬氨酸D。朱砂叶螨Mu家族GSTs基因同样相对保守,具有催化活性中心酪氨酸Y,以及决定蛋白折叠的苏氨酸T、苯丙氨酸F以及AILRYLARKH特征基序。朱砂叶螨Omega家族GSTs的关键残基半胱氨酸C代表GSH的催化位点,N-端结构域非常保守,特征基序是ESLIIAEYL。
3.2朱砂叶螨RT-qPCR内参基因筛选
运用geNorm和NormFinder软件,对6个看家基因a-TUB、RPS18、EF1α、5.8SrRNA、SDHA、GAPDH在朱砂叶螨不同发育阶段(卵、幼螨、若螨和雌成螨)和不同品系(敏感品系F0和炔螨特抗性品系F34)中的稳定性进行评价。结果分析表明,在朱砂叶螨不同发育阶段中,稳定性较好的是RPS18和GADPH,在不同品系中,经筛选表达最稳定的是a-TUB和RPS18,综合考虑评价结果,选用RPS18作为内参基因用于后续实验。
3.3朱砂叶螨不同发育阶段GSTs基因的表达模式
运用定量PCR技术(quantitative real-time PCR, qPCR),以朱砂叶螨RPS18作为内参基因,对所克隆的8个GSTs基因在不同发育阶段(卵、幼螨、若螨、雌成螨)mRNA表达水平进行了相对定量分析。
在朱砂叶螨敏感品系中,Delta家族的2个基因TcGSTd1和TcGSTd2在幼螨和若螨阶段的相对卵期的表达量上调,在若螨期明显高于卵期,随后在成螨阶段下降。而在朱砂叶螨抗性品系中,这2个基因在幼螨、若螨和成螨阶段随着发育历期的增加其相对表达量明显上调,在成螨达到最高。朱砂叶螨敏感品系Mu家族5个GSTs基因中,TcGSTm1、TcGSTm2、TcGSTm3、TcGSTm4在幼螨的相对表达量显著高于其余三个发育阶段,随后降低,最终在成螨期的表达量与卵期相当；TcGSTm5基因的相对表达量随着发育阶段的增加而逐渐升高,若螨阶段表达量达到最高；TcGSTz1基因在4个螨态中的相对表达量没有较大波动。在抗性品系中,TcGSTm1、TcGSTm2、TcGSTm4随着发育历期的增加其表达量上调,在若螨期达到高峰,然后在成螨阶段的相对表达量降低,但还是高于卵期的表达量；TcGSTm3在幼螨阶段的表达量显著增高,随后在若螨阶段突然下调后在成螨其恢复到与卵期相当的水平。TcGSTm5基因的相对表达量随着发育阶段的增加而逐渐上调；TcGSTz1基因在朱砂叶螨卵期、幼螨和成螨期的相对表达量略有上调,其显著表达高峰出现在若螨期。由上述基因在朱砂叶螨不同发育阶段的表达模式推测,TcGSTd1和TcGSTd2不仅参与了朱砂叶螨正常的生长发育活动,而且在成螨阶段在该螨对炔螨特的抗性中发挥了重要作用,Mu家族5个GSTs基因也不同程度地参与了朱砂叶螨对炔螨特的抗性。
3.4朱砂叶螨不同品系GSTs基因的表达模式
运用实时定量PCR技术,以朱砂叶螨RPS18作为内参基因,对所克隆的8个GSTs基因在敏感品系和炔螨特抗性品系(F34)mRNA表达水平进行相对定量分析。结果表明,相对于敏感品系,8个GSTs基因在朱砂叶螨抗性品系中总体趋势表现为卵期和幼螨期表达下调,而若螨和成螨期显著上调。在卵期,朱砂叶螨抗炔螨特品系的TcGSTd2基因的相对表达显著下降,推测该基因参与了螨类在这一阶段的生理活动。在幼螨期,朱砂叶螨抗炔螨特品系Delta家族的2个GSTs基因TcGSTd1和Mu家族的4个GSTs基因TcGSTm1、TcGSTm2、TcGSTm4基因相对表达量显著下调,推测这几个基因在这一螨态发挥了重要生理功能。在若螨和成螨期,8个GSTs基因的相对表达量都上调,其中TcGSTd1、TcGSTd2、TcGSTm、 TcGSTm2、TcGSTm4和TcGSTm5显著增加,表明这些基因参与了该螨的生理功能和抗药性的形成。
3.5朱砂叶螨GSTs基因的原核表达载体构建
利用限制性内切酶Nde I和Xho I的双酶切以及DNA重组技术成功构建了朱砂叶螨TcGSTm2基因基于pET30a(+)的原核表达载体,为进一步深入研究GSTm蛋白质特性和功能研究奠定了基础。
综上所述,本研究开展了对云南桑园朱砂叶螨不同田间种群的抗药性测定,在室内继代选育朱砂叶螨炔螨特抗性种群,获得抗性倍数达30.669的相对抗性品系,以该品系和敏感品系为基础,研究了朱砂叶螨对炔螨特的抗性发展趋势,对其抗性现实遗传力和抗性风险进行评估,在对抗性品系酶特异性抑制实验和生理生化特性分析基础上,确定了朱砂叶螨体内重要解毒代谢酶系GSTs是作为炔螨特抗性机制的主导因子,以此为基础,在分子水平上进一步解析GSTs基因在朱砂叶螨对炔螨特的抗性分子机制中的作用。运用分子生物学技术,克隆了12个朱砂叶螨GSTs基因,分析了这些基因的分子生物学特性。并采用geNorm和NormFinder软件确定了朱砂叶螨不同发育阶段和不同品系的最适内参基因。采用qPCR技术,解析了本研究所克隆的8个GSTs基因在朱砂叶螨敏感品系和炔螨特抗性品系mRNA水平表达模式和差异,揭示了这些基因在朱砂叶螨抗性过程中发挥的作用。成功构建了朱砂叶螨TcGSTm2基因基于pET30a(+)的原核表达载体,为GSTs基因的异源表达和蛋白质特性研究奠定基础。本研究应用生物学、生理生化和分子生物学知识和技术,全面解析了GSTs介导的朱砂叶螨对炔螨特的抗性机制,为朱砂叶螨的抗药性治理和炔螨特的可持续应用提供理论依据。
Carmine spider mite Tetranychus cinnabarinus (Boisduval) belongs to Arachnida, Tetranychidae, Tetranychus. It is a phytophagous mite distributed worldwide, and feeds on a large number of host plants including cotton, flowers, fruits and vegetables, as well as the mulberry. In recent years, T. cinnabarinus has caused severe damage in the field. The management of this mite still mainly depends on acaricides, while the resistance of T. cinnabarinus could develop quickly for its high reproductive potentials, short life cycle, and the unreasonable use of acaricides. Nowadays, the resistance problem of T. cinnabarinus has been the severest in the mites.
As a traditional acaricide, propargite has been wide used in control of pest mites in the fields and forests since1964for its high efficiency, low toxicity, and broad-spectrum. Although propargite has been used for a long history, the resistance monitoring showed that the citrus red mite and the carmine spider mite were still sensitive to this acaricide. In future, propargite will still be frequently used in the management of mites. Currently, researches about propargite were mainly focused on its residues and environmental toxicology, whereas basic systematic research on pest resistance was still insufficient. As a result, the mechanism study of propargite, and its resistance risk evaluation are basic works to understand its resistance development and conduct effective resistance management..
This study focused on the important pest mite, T. cinnabarinus, paid attention to its resistance problem, worked on the resistance monitoring of different populations of T. cinnabarinus in mulberry fields in Yunnan province, and reported the resistance level for the first time. A propargite-resistant strain was selected in the laboratory, and its realized heritability and resistance risk were evaluated to reveal the rules of resistance development. Based on the biochemistry study of resistance strain, GSTs were identified as an important factor for the resistance development of propargite. The molecular characterization of GSTs were further analyzed. Genes encoding GSTs of T.cinnabarinus were cloned and their specific expressions were compared between susceptible and resistance strains. Heterogenous expression was also conducted. The resistance mechanism of propargite were synthetically analyzed by population biology and molecular biology methods, and the contribution of GSTs in the resistance mechanism were clarified. The results are important reference to solve production problems in agriculture, and can enrich the resistance theory of pest mites. Through the research work of three years, the findings are presented as follows:
1Acaricide susceptibility of seven T. cinnabarinus strains from mulberry fields in Yunnan
To gain primary data that reflects the resistance levels of CMS in mulberry plantations, we evaluated and compared the susceptibility of one susceptible strain and7field-collected populations to7acaricides of5chemistry classes using FAO (1980) recommended slide dipping method. The activities and kinetics of major detoxification enzymes (cytochrome P450, esterases and glutathione s-transferases) of each population were also determined to understand their potential role in acaricide resistance. The results showed that most of T. cinnabarinus populations in mulberry trees exhibited low and moderate levels of resistance. Mengzi and Xiangyun population had moderate resistance to phoxim (RR=31.45and26.22, respectively). The Qiaojia, Luliang, Mengzi and Heqing population showed moderate resistance to chlorfenapyr (RR=14.98-22.52). T. cinnabarinus was still sensitive to propargite (the LC50was87.34-243.65mg/L and the resistance ratio (RR) was3.62-10.08). The esistant of field populations to insecticides correlation analysis showed that abamectin and methomyl was significantly and negatively correlated (rs=-0.857,p<0.05). Comparing the enzymes activity between the field isolates and the susceptible strain of CMS. All the tesed enzymes were more active in the field populations than that in the susceptible strain, GSTs was6.44-117.96folds, CarE was1.53-29.87folds, and the P450was1.12-2.67folds, which indicating that GSTs played an important role in the detoxification of mulberry T. cinnabarinus. The results provide useful reference data for acaricides application and spider mite management in mulberry plantations.
2Resistance selectivity and characteristics of T. cinnabarinus against Propargite
2.1Resistant strain selectivity of Propargite against carmine spider mite
The susceptible strain of T. cinnabarinus was original collected from Beibei District, Chongqing, China in2000and maintanct in laboratory without exposure to aracricides for10years. The susceptible strain was screened continuously with propargite to obtain the resistant strain and the basic characteristics were investigated in this study. After discontinuous (F1-F23) and continuous (F24-F34) selection over34generations, the resistance ratio increased30.669folds. T. cinnabarinus had a slow rate in resistance development to propargite. The breeding procedure of the T. cinnabarinus resistant strain showed the slope of F34strain was smaller than the forgoing strain (F1) according to log concentration-probit curve. The realized heritability (h2) in the continuous (F24-F34) propargite-selection was greater than the discontinuous one (F1-F23), and the values were0.055and0.047, respectively. According to the realized heritability (h2), theoretically, to obtain a10-fold increase in resistance requires53-21generations under discontinuous selective pressure of propargite, and46-18generations continuous selection under selective pressure of propargite of50%-90%for each selective generation.
2.2Relative fitness of propargite-resistant strains of T. cinnabarinus
Using the leaf disc feeding method, The life table of T. cinnabarinus susceptible strain (F0) and resistant strain (F34) at25℃were constructed, the ecological aspects of two strains were investigated. The age-specific fecundity and survival curves showed no significant difference between two strains, so were the developmental duration, eggs per female and hatchability, etc. The fitness of propargite-resistance strain was also not significant changed compared to SS.
2.3Biochemical mechanism of T. cinnabarinus against propargite
The F26and F34generations of propargite-resistant strain showed increased resistance against propargite11folds and31folds respectively using slide-dip method. Toxicity of other pesticides/acaricides to the propargite-resistance strain (F26and F34) were assayed using same method. The LC50values and resistance ratios to these pesticides/acaricides indicated that the F26and F34still sensentive to avermectin, but exhibit cross-resistance to phoxim, pyridaben and dichlorvos, chlorfenapyr and diafenthiuron. PBO, TPP and DEM were the specific inhibitors of cytochrome P450monooxygenases (P450), carboxylesterase (CarE) and glutathione S-transferase (GSTs), respectively. The synergistic ratios of PBO, TPP and DEM to propargite were1.06,1.47and2.58fold in resistant strain (F34) of female adult T. cinnabarinus, respectively. None of those three synergists showed significant synergistic effect in susceptible strain of T. cinnabarinus. Based on this result, the mite of four developmental stages (eggs, larvae, nymph, adults) were exposed to the synergist DEM in combination with propargite using potter spray method. There were no significant synergistic effect on susceptible among different life-stages in carmin spider mite. The synergistic resistance ratios of the propargite-resistant strain (F34) with DEM were calculated to be1.27,1.16,1.09, and1.99folds respectively in eggs, larvae, nymph and adults. Compared to susceptible strain, the activities of GST in F34increased by1.98and2.72folds to different substrate GSH and CDNB, respectively. In conclusion, the results indicated GSTs might play an important role in carmine spider mite resistance to propargite.
3cDNA cloning and mRNA expression profiles of GSTs genes in T. cinnabarinus
3.1cDNA cloning and characterization
Based on the genome data of Tetranychus urticae,12novel GST genes were cloned from T. cinnabarinus using the reverse transcriptase-PCR (RT-PCR) techniques. Putative proteins have been predicted by Protparam and Scanprosite softwares. Based on sequence similarity and phylogenetic analysis, two genes were classified to the Delta gene family, five genes were classified to the Mu family, two genes were classified to the Omega family, two genes were classified as Kappa and Zeta family genes, and the remaining one gene did not belong to any of known family, temporarily be named as an unclassified GST. The nomenclature of these T. cinnabarinus GSTs cDNA and their deduced amino acid sequences have been deposited in Genbank with the following accession numbers:TcGSTd1(KF421142), TcGSTd2(KF421143), TcGSTm1(KF421144), TcGSTm2(KF421149), TcGSTm3(KF421146), TcGSTm4(KF421147), TcGSTm5(KF421148), TcGSTo1(KF421150), TcGSTo2(KF421151), TcGSTz1(KF421153), TcGSTk1(KF421152), TcGSTu2(KF421145). The length of cDNA of these12GSTs gene contained were ranged from651to735bp, encoding proteins with216-244amino acids. Predicted molecular weight was24.19-28.95kDa and the isoelectric point (pI) was between5.97and8.99. It was predicted that there was no transmembrane domain present in these twelve proteins using TMHMM2.0bio-prediction software suggesting that they could be easily purified.
ScanProsite showed that two distinct functional domains were found in10proteins except TcGSTkl and TcGSTul. N-terminal domains were highly conserved, which was binding sites of GSH, were found both in TcGSTd1,TcGSTd2(residues1-82) and TcGSTml, TcGSTm2, TcGSTm3, TcGSTm4and TcGSTm5(1-88,1-90,1-88,1-88, 1-82). Other TcGSTo1, TcGSTo2and TcGSTz1were:22-101,23-101and3-90. Also, C-terminal region were located at position86-219,88-207,90-211,92-220,90-214,90-209,90-211,105-240and105-234in TcGSTdl, TcGSTd2, TcGSTm1, TcGSTm2, TcGSTm3, TcGSTm4, TcGSTm5, TcGSTol, TcGSTo2and TcGSTzl. Delta family GSTs in T. cinnabarinus had the same catalytic domain as other species containing Serine, and Asparagine in the center and to determine the same amino acids regarding protein folding including Proline,Leucine,Glycine,Aspartic acid. Mu family GSTs in T. cinnabarinus had the center Tyrosine as the catalytic site and amino acids determing protein folding, like Threonine, Phenylalanine as well as the "AILRYLARKH" motif. Omega GSTs contained the key residue, Cysteine as the catalytic site and a very conserved N-terminal, in which characteristic motif is "ESLIIAEYL"
3.2Stability evaluation of housekeeping genes
The stability of reference genes used for the quantification of mRNA can be affected by the experimental condition. Therefore, the evaluation of reference genes is critical for gene expression profiling. The stabilities of six candidate reference genes (a-TUB, RPS18, EFlα,5.8SrRNA, SDHA, GAPDH) of T.cinnabarinus were analyzed at different developmental stages and strains using geNorm and NormFinder software, respectively. RPS18and GADPH were identified as the most stable genes in defferent developmental stages, while a-TUB and RPS18were found to be the most stable in different strains. Thus, RPS18genes was used as reference gene in RT-qPCR in this study.
3.3Developmental stages expression profiles of the eight GSTs genes
The expression of eight GSTs genes in T. cinnabarinus were analyzed by real-time quantitative PCR (RT-qPCR) using RPS18as an reference gene. The assayed developmental stages included eggs, larvae, nymph and female adults. The results showed that2Delta family genes,5Mu family genes and TcGSTzl were differently expressed at different stages of mites. The relative expression level of TcGSTdl and TcGSTd2were increased in the larvae and nymph stage compared with egg stage, peaked in nymph stage and declined in adult stage in susceptable strain. But in the resistant strain, the results indicated that the trend of relative expression rose along with growth and development, and reached maximum point in the adult stage. In suscepitable strains, the highest expression levels of four Mu family GST genes(TcGSTm1, TcGSTm2, TcGSTm3and TcGSTm4) were observed at larvae stage. Another Mu family GST genes TcGSTm5showed higher expression level in the nymph stage. In resistant strains, The expression level of three Mu GST genes(TcGSTml,TcGSTm2and TcGSTm4) increased at larvea, showed highest expression in nymph, but reduced in adult stage. Another TcGSTm3showed higher expression levels in the larvae stages, followed by a sudden reduction in the nymph stage, and a recovery in adult. The expression level of TcGSTzl were not significantly different during the development in susceptible strain but showed higher expression in nymph stage in resistant strain. The result indicated that two Delta family GST genes(TcGSTdl and TcGSTd2) and five Mu family GST genes(TcGSTm1,TcGSTm2,TcGSTm3and TcGSTm4) might not only involved in T. cinnabarinus growth and development, but also may played an important role in propargite resistant development in T.cinnabarinus.
3.4Expression profiles of the eight GSTs genes between two different strains
In order to verify whether GSTs are over-expressed in propargite-resistant strain, qPCR with RPS18as the reference gene was employed to determine the relative expression quantity of two Delta GST genes(TcGSTd1,TcGSTd2) and five Mu GST genes(TcGSTml,TcGSTm2,TcGSTm3and TcGSTm4in four life-stage (eggs, larvae, nymph and adults) of T. cinnabarinus in both susceptible and propargite-resistant strain. Expression for eight GST genes in propargite-resistant strain of T. cinnabarinus showed down regulation in eggs and larvae stage, but up regulation in nymph and adult stage in comparison with susceptible strain. In eggs, the relative expression of TcGSTd2was significantly lower than susceptible strain. In larvae, the expression of TcGSTd1,TcGSTd2,TcGSTm1,TcGSTm2,TcGSTm3and TcGSTm4were significantly lower than susceptible strain. In nymph and adults, all of eight GST genes were up regulated than susceptible strain, which implied that these GST genes were possibly involved in propargite resistance.
3.5Prokaryotic expression vector of TcGSTm2
The cDNA of TcGSTm2was cloned into expression vector pET30a (+) by the restriction sites of Nde I and Xho I, which provided the basis for GSTs expression in E. coli.
In summary, this study determined the resistant levels of T. cinnabarinus in Yunnan mulberry plantations. A resistance strain ofpropargite was selected in the laboratory.. After34generations, resistance ratio has increased to30.89folds. The reality of resistant riskment, physiological and biochemical synergist characteristic were analyzed.Biochemical and toxicological characterization of GSTs of T. cinnabarinus were studied; based on genome of T. urticae,12GST genes were cloned from T. cinnabarinus using RT-PCR technique; GSTs genes expression profiles were assayed using qPCR technique from different strains T. cinnabarinus at developmental stages; the expression vector for TcGSTm2was constructed. The results provide insights into exploring the functions of GSTs system in physiology, and it showed evidence for clarifying the resistance mechanisms of carmine spider mite to propargite. Meanwhile, the results also enlighten the resistance mechanism of other insect/mite pests.
引文
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