【作者】 丁天波；

【导师】 王进军；

【作者基本信息】 西南大学 ， 农业昆虫与害虫防治， 2014， 博士

【摘要】 柑橘全爪螨Panonychus citri (McGregor)是我国柑橘产区的第一大害螨,其大量发生严重影响柑橘的产量和品质,且以化学防治为主的控制措施加之该螨自身的特点(繁殖速度快、体小、活动范围小等),导致其抗性问题日益凸显。目前,对于相橘全爪螨解毒代谢及抗药性机理的报道相对较少,且多集中在生化水平及有限基因的鉴定方面。细胞色素P450酶系作为昆虫(螨)体内一类关键解毒酶系,可代谢多种内源和外源化合物,在昆虫(螨)对杀虫(螨)剂解毒代谢过程中起着十分重要的作用。诸多研究表明,蜱螨P450与拟除虫菊酯类、有机磷类、METIs、季酮酸类、生物源类等多种杀螨剂抗性有关。国内外有关细胞色素P450酶系的研究多针对P450的特性展开,而对于酶系发挥作用的关键因子NADPH-细胞色素P450还原酶(CRP)和细胞色素b5(CYB5)的作用地位涉及相对较少。因此,对细胞色素P450酶系进行整体研究,将有利于全面理解细胞色素P450酶及其相关基因在解毒代谢过程中的作用方式和具体定位,从而为全面解析其解毒代谢及介导的代谢抗性机制奠定理论基础。本学位论文以柑橘全爪螨为研究对象,围绕细胞色素P450对药剂胁迫的响应机制这一科学问题,开展了系统地研究工作。具体内容包括：从柑橘全爪螨田间种群敏感性监测出发,在掌握柑橘全爪螨抗性水平的基础上,在生化水平上比较分析了三种主要代谢酶的酶学特性,锁定细胞色素P450作为研究重点；建立了柑橘全爪螨全螨态转录组数据库,筛选鉴定出包括细胞色素P450在内的诸多解毒代谢酶基因信息；进一步克隆P450、CPR及CYB5基因的cDNA全长序列,解析这些基因在不同螨态、不同药剂胁迫及诱导条件下的表达模式,初步建立柑橘全爪螨细胞色素P450基因的真核表达体系。研究结果对于全面揭示柑橘全爪螨P450酶系的解毒代谢机制及其介导的抗药性机制具有重要的理论意义,在实践上对于制订柑橘全爪螨抗药性的治理策略亦具有重要的指导意义。本学位论文的主要研究结果总结如下：1柑橘全爪螨抗药性监测及主要解毒酶活性比较1.1柑橘全爪螨4个田间种群的抗药性测定通过改进后的叶碟浸渍法,对采自重庆4个地区(北碚、璧山、忠县、武隆)的柑橘全爪螨对阿维菌素、三唑锡、哒螨灵和螺螨酯的抗性水平进行了测定。结果表明,四种不用类型杀螨剂相比较,阿维菌素的杀螨活性最强,4个种群对其敏感性水平最高,螺螨酯和哒螨灵次之,三唑锡的杀螨活性最差,4个种群对其敏感性水平最低。不同种群间相比较发现,柑橘全爪螨璧山种群对阿维菌素最为敏感,忠县和武隆种群对其敏感性最低,LC5o分别是璧山种群的11倍和12倍；忠县种群对三唑锡的抗性水平最低,璧山种群抗性水平最高,但总体差异不大；对哒螨灵而言,璧山种群对其敏感性最高,武隆种群、忠县种群与璧山种群的LCso十分接近,而北碚种群对哒螨灵的抗性水平最高,与璧山种群相比其相对抗性倍数达4倍；重庆4个柑橘全爪螨种群对螺螨酯的抗性水平差异比较明显,抗性水平由高到底依次为：璧山种群、忠县种群、武隆种群和北碚种群。1.2柑橘全爪螨三种主要解毒酶活性比较1.2.1不同发育阶段主要解毒酶活性比较通过微量酶标板法对柑橘全爪螨不同螨态P450s、GSTs和CarEs活性进行了测定。比较分析发现,幼螨期P450s比活力最高,分别是成螨、若螨和卵P450s活力的2.66、2.70和16.02倍,且差异显著；柑橘全爪螨若螨和成螨期GSTs活力均显著高于其它发育阶段,卵期GSTs活力最低；柑橘全爪螨幼螨期CarEs比活力最高,其次是成螨和若螨,卵期最低。以上结果也暗示P450s和CarEs可能在幼螨期主要参与对外界化合物的代谢过程,而GSTs则可能主要在成螨和幼螨期行使解毒代谢功能。1.2.2不同种群主要解毒酶活性比较研究表明,P450s、GSTs和CarEs的活力分别在北碚种群、忠县种群、忠县种群最高,但总体来看代谢酶活性变化与抗性水平之间相关程度不高,分析可能与4个地区施药背景不同、杀螨剂类型及作用靶标不同、以及不同种群问的遗传背景存在差异有关。1.2.3不同药剂胁迫下主要解毒酶活性比较在生物测定的基础上,选择5种杀螨剂(阿维菌素、三唑锡、甲氰菊酯、哒螨灵和螺螨酯)及柑橘次生挥发物(柠檬醛),以LC30浓度对柑橘全爪螨处理,24h后测定三种代谢酶的活性。结果表明,经不同药剂处理后P450活性均有不同程度的提高,其中经螺螨酯处理后P450的活力最高；对于GSTs而言,除阿维菌素外,其余5种药剂均能显著诱导GSTs活力升高,其中螺螨酯处理后GSTs的活力最高；然而,经螺螨酯处理后,CarEs的活力显著降低,其余5种药剂处理后,其活力无显著变化。综上分析发现,P450s和GSTs对以上6种药剂的胁迫反应比较灵敏,而CarEs则较为迟钝,也暗示P450s和GSTs在一定程度上参与柑橘全爪螨对上述药剂的代谢过程。1.2.4巴比妥酸钠对主要解毒酶的诱导效应柑橘全爪螨经P450酶特异性诱导剂巴比妥酸钠(19.96mg/L)诱导24h后,P450s和GSTs活性显著上升,而CarEs活性则被显著抑制。2柑橘全爪螨转录组分析及解毒酶基因鉴定2.1全螨态转录组数据库建立及数据分析为尽可能全面获得柑橘全爪螨的基因转录本信息,提取获得柑橘全爪螨4个螨态的高质量RNA,并基于Illumina HiseqTM2000平台进行测序,将数据提交至NCBI的SRA数据库,登录号为SRA045743.1。序列分析发现共获得51,214,018条clean reads,经过一系列组装拼接后,得到65,149条Unigene。将所有Unigene提交至nr、Swiss-Prol、KEGG、COG和GO数据库进行BLASTX比对,以E=10-5为阈值,分别有32,535、25,813、22,725、15,412和10,421条Unigene成功得到注释。对nr数据库成功注释的Unigene进行同源性分析,发现柑橘全爪螨Unigene与昆虫纲相关基因的亲缘关系最近,其中9%的Unigene与赤拟谷盗Tribolium castaneum相关基因的同源性最高,其次为印度跳蚁Harpegnathos saltator (6%)和佛罗里达弓背蚁(5%),与蛛形纲的肩突硬蜱Ixodes scapularis同源性亦达1.7%。对柑橘全爪螨Unigene进行GO分类发现,共有10,421个Unigene在GO数据库得到注释,且被划分到生物过程、细胞组分和分子功能3大主要类别的50个功能组中。其中注释基因最多的功能组是细胞组分中的cell,有7,035个Unigene,注释基因最少的是分子功能中的channel regulator activities和细胞组分中的virion,两个功能组均只有2个Unigene。将所有Unigene在COG中比对,有15,412个Unigene被成功注释,并且被划分到25个不同的功能家族,其中注释Unigene最多的功能家族是general function prediction(3,642个),注释Unigene最少的功能家族是nuclear structure(11个)。2.2解毒酶基因筛选及代谢通路分析根据nr数据库比对结果,共筛选鉴定出柑橘全爪螨与解毒代谢相关的P450Unigene79个,且分别有12、9、26和7个Unigene与CYP2、CYP3、CYP4和CYP6家族基因亲缘关系最近,其余25个Unigene与其它家族(CYP9、CYP18、CYP314和线粒体P450)基因的同源性较高。有58个P450Unigene在KEGG数据库得到注释到,共参与16条代谢通路；筛选鉴定出柑橘全爪螨24个GSTs Unigene,分属于7个家族(delta、kappa、mu、omega、sigma、theta和zeta),其中有19个Unigene通过在KEGG数据库注释到10条代谢通路中；筛选鉴定出柑橘全爪螨13个CarE Unigene,根据同源性分析,有4个Unigene归于Clade A,分别有3个和1个Unigene归于Clade B和D；代谢通路分析发现,仅有4个Unigene能够成功注释,且均属于同1条代谢通路。总体来看上述三种解毒酶所涉及通路大多与外源物质或药剂的代谢有关,从另一角度佐证了解毒酶的主要功能。3柑橘全爪螨P450及其相关基因的克隆与序列分析基于转录组数据库中鉴定出的P450Unigene信息,利用RACE结合RT-PCR技术,从柑橘全爪螨体内分离克隆出了11个P450基因的cDNA全长序列,提交至细胞色素P450命名委员会命名后,登录入至GenBank,其名称和登陆号分别为：CYP4CL2(JQ690089)、CYP384A2(KC201279)、CYP385C7(KJ173877)、CYP385C8(KJ173878)、CYP389D1(KC201278)、CYP392A22(KC201275)、CYP392A23(KC201276)、CYP392A24(KC201277)、CYP392A25(KJ173880)、CYP392B6(KJ173879)和CYP392F1(KC201274)。这11个P450基因的开放阅读框长度范围为1,494-1,758bp,编码氨基酸序列在497-585aa之间。此外,利用ProParaam、 TMHMM等生物信息学软件对P450蛋白组成、理化性质、跨膜区段及信号肽位点进行分析和预测,结果表明这11个P450基因均为微粒体型,也暗示可能与外源化合物的解毒代谢相关。同时,柑橘全爪螨11个P450氨基酸序列中均含有经典血红素结合位点、C螺旋、Ⅰ螺旋、K螺旋和meander等保守区域的特征基序,且CYP4CL2序列中含有CYP4家族特征序列EVDTFMFA/GHDTT。通过对本研究克隆获得的11个及之前本课题组获得的1个柑橘全爪螨P450基因全长序列进行同源性比对,结果发现这12个P450基因核苷酸同源性在36.47%-96.39%之间,氨基酸同源性在16.29%-96.39%之间,且相同家族基因序列的同源性较高。以从二斑叶螨基因组数据库中选取所有的75个P450基因序列作为参照,利用MEGA5.05的最大似然法构建系统发育树,结果发现柑橘全爪螨12个P450基因分属于3个集团(CYP2Clan、CYP3Clan和CYP4Clan),且相同家族基因聚在一起。根据转录组数据库信息,克隆获得了柑橘全爪螨CPR及CYB5基因cDNA全长序列,GenBank登陆号分别为KJ173881和KJ173882。PcCPR和PcCYB5基因完整开放阅读框分别包含2,061和324个碱基,编码686和107个氨基酸。序列多重比对发现,柑橘全爪螨PcCPR与二斑叶螨CPR同源性最高,且在氨基酸序列中发现P450还原酶的保守功能区包括FMN结合区、FAD结合区和NADPH结合区。此外,与其它20个物种进行系统发育分析,发现按照蛛形纲、昆虫纲、哺乳动物纲和双子叶植物纲聚为4大类,PcCPR与二斑叶螨亲缘关系最近,节点自展值达100%。对CYB5进行序列分析后发现,靠近N段的血红素结合区十分保守,且PcCYB5与二斑叶螨相关基因的同源性最高。4柑橘全爪螨P450及其相关基因的表达模式解析4.1细胞色素P450基因表达模式解析4.1.1柑橘全爪螨不同发育阶段P450基因的表达模式运用qPCR技术完成了对柑橘全爪螨12个具有全长序列的P450基因和14个P450Unigene片段在不同发育阶段的表达模式解析。结果发现,26个P450基因的表达伴随柑橘全爪螨的整个生命历程,CYP4CF1、CYP4CL2、CYP392A25和CYP392B6在幼螨阶段表达量均最高,CYP385C7和CYP389D1基因则在若螨特异高表达,此外有10个Unigene也在幼、若螨阶段表达水平较高,说明以上16个基因可能过多参与到幼螨和若螨阶段对内外源物质的代谢过程中；CYP384A2和两个Unigene表达水平随龄期增长而逐渐升高,暗示基因随螨体的生长发育,解毒代谢能力也逐渐增强；此外还发现CYP392A22、CYP392A24及Unigene1146、Unigene27622在卵期高表达,推测可能与卵期抵御外界次生物质胁迫有关；而CYP385C8和CYP392F1则在螨体内呈组成型表达,在整个生长发育过程中均可能行使重要作用。4.1.2柑橘全爪螨P450基因对药剂胁迫的响应表达模式在前期生物测定基础上,完成了阿维菌素、甲氰菊酯、哒螨灵和柠檬醛对柑橘全爪螨不同时间和不同浓度的胁迫处理,进而对26个P450基因进行了表达模式解析。qPCR结果发现,柑橘全爪螨P450对阿维菌素响应幅度最大,CYP385家族基因(CYP385C7和CYP385C8)上调倍数均在25倍以上,而CYP392家族四个基因(CYP392A22、CYP392A23、CYP392A25和CYP392B6)的相对表达量也分别达到了对照的41.46、68.66、25.56和11.34倍。在时间效应方面,本研究中大多数P450基因均在药剂胁迫24h后表达量达到最高,仅两个Unigene在胁迫前期(12h)均发生响应,CYP4CL2和CYP392F1表现出的诱导效应较为滞后；P450基因在特定浓度的高表达呈家族分布,CYP385家族两个基因(CYP385C7和CYP385C8) mRNA水平在低浓度LC10胁迫下达到最高,CYP392家族5个基因(CYP392A22、CYP392A23、CYP392A24、CYP392A25和CYP392F1)均能被亚致死浓度LC30最大程度诱导,致死中浓度胁迫下,CYP4家族基因(CYP4CF1和CYP4CL2)相对表达量达最高峰值。甲氰菊酯胁迫下,大多数P450基因表达均能被诱导上调,CYP4CL2表达量上升倍数最大(4.48倍)。CYP392家族4条基因(CYP392A24、CYP392A25、CYP392B6和CYP392F1)及CYP384A2、CYP389D1均在12h后表达水平最高,CYP4家族基因的最大诱导值出现在24h；经LC30胁迫24h后,CYP4CF1、CYP4CL2、 CYP392A22、CYP392B6和CYP392F1基因表达量均达到最大值,且显著高于对照,CYP385C8能够被甲氰菊酯LCso浓度显著诱导,暗示了以上基因在很大程度上参与了柑橘全爪螨对甲氰菊酯的代谢过程。同上述两种杀螨剂不同,柑橘全爪螨P450基因对哒螨灵的响应数目和幅度都有所降低,CYP385C7、CYP385C8和CYP392A22在哒螨灵胁迫12h后,表达量即达到最大值,说明这三个基因在哒螨灵胁迫初期发挥主要作用,CYP4CL2和CYP392家族4个基因(CYP392A23、CYP392A24、CYP392A25和CYP392B6)表达最高峰则分别推至24h和36h,暗示其分别在中后期参与对螨体内哒螨灵的解毒过程；低浓度胁迫下,仅CYP4CL2表达量最高,而CYP392家族3个基因和CYP385C8基因均可能参与到柑橘全爪螨对较高剂量哒螨灵的代谢过程中。柠檬醛特定条件胁迫处理下,柑橘全爪螨12个具有全长序列的P450基因表达量均显著上调。绝大多数P450基因表达水平在柠檬醛胁迫24h和36h后达到最高峰值,说明柑橘全爪螨P450对柠檬醛响应模式相对比较滞后；此外定量结果发现高浓度柠檬醛对P450的诱导效果较为明显,LCso浓度处理下,10个P450全长基因和8个Unigene的相对表达量均达到最高峰。总体来看CYP385C7、 CYP385C8、CYP392A23、CYP392B6和CYP384A2上调幅度较大,均超过8倍,相对于其它P450基因而言,可推测上述5个基因在介导柑橘全爪螨对柠檬醛解毒代谢及适应寄主环境过程中发挥主要作用。4.1.3柑橘全爪螨P450基因对巴比妥酸钠诱导响应的表达模式巴比妥酸钠对多数P450基因均表现出显著的诱导效应,且诱导最大值多出现在24h和36h后,此外发现CYP392A23、CYP392A25、CYP392B6、CYP384A2和Unigene9596的表达量同诱导浓度之间呈正相关。4.2PcCPR和PcCYB5基因表达模式解析4.2.1柑橘全爪螨不同发育阶段PcCPR和PcCYB5基因的表达模式PcCPR基因在幼螨和若螨中表达水平均显著高于其它两个发育阶段,在幼螨阶段表达量最高,卵期最低,PcCPR表达模式同P450不同发育阶段酶活性变化-致,也说明了其在幼螨和若螨阶段主要参与了P450的解毒代谢过程,也反映出CPR同P450间的协同作用。PcCYB5基因则在若螨期表达量最高,其次为卵和幼螨、成螨阶段表达水平最低,暗示PcCYB5基因在若螨阶段保持较为活跃的电子传递活动。4.2.2柑橘全爪螨PcCPR和PcCYB5基因对药剂胁迫响应的表达模式在阿维菌素胁迫下,PcCPR基因反应较为迅速,LC10浓度处理12h后表达量均达最大值；低浓度和高浓度的甲氰菊酯均能抑制CPR基因的表达,仅亚致死浓度能显著诱导PcCPR的表达上调；哒螨酮对PcCPR的诱导效应最为强烈,且柠檬醛不同浓度和不同时间胁迫均能使PcPCR基因表达水平升高。上述结果也说明PcCPR在很大程度上参与到P450对外源物质的代谢过程中。对于PcCYB5而言,阿维菌素、甲氰菊酯、哒螨灵和柠檬醛均能够显著诱导其表达水平升高,并且其表达模式同PcCPR基因表达量变化在时间和浓度梯度上呈现一定的互补效应,暗不PcCYB5和PcCPR协同作用保证电子传递的有序进行,共同参与到P450酶系抵御外界环境压力的过程中。4.2.3柑橘全爪螨PcCPR和PcCYB5基因巴比妥酸钠诱导响应的表达模式qPCR结果发现,巴比妥酸钠均能显著诱导PcCPR和PcCYB5基因的表达,且表现出良好的诱导时间效应和浓度效应,二者表达量均随时间和浓度的增加而升高。5柑橘全爪螨P450基因的真核表达运用Bac-to-Bac昆虫杆状病毒表达系统,选择柑橘全爪螨2个细胞色素P450基因CYP392A24和CYP392F1,采用双酶切及DNA重组技术,构建了Bacmid-CYP392A24和Bacmid-CYP392F1病毒表达载体,实现对草地贪夜蛾Spodoptera frugiperda Sf9细胞的侵染和蛋白表达,后续对表达产物进行P450含量测定时,发现两个蛋白均以失活态存在,说明仍需进一步完善表达及蛋白纯化体系。而真核表达体系的构建将为今后柑橘全爪螨P450基因功能的验证和目的基因的解毒代谢能力研究奠定基础。综上所述,本学位论文研究运用叶碟浸渍法对重庆4个地区柑橘全爪螨田间种群的抗性水平进行了监测,同时完成了对代谢抗性相关的三大解毒酶活性的比较研究；基于高通量测序平台,构建了柑橘全爪螨全螨态转录组数据库,针对性地筛选鉴定出了包括P450在内的主要解毒酶基因,并进行了代谢通路分析；克隆获得了柑橘全爪螨11个P450基因cDNA全长序列,以及P450酶系重要功能基因CPR和CYB5的cDNA全长序列；运用qPCR技术,系统解析了柑橘全爪螨26个P450基因(12个含有全长序列的P450基因、14个P450Unigene片段)和CPR、CYB5基因在不同发育阶段、不同药剂胁迫及诱导条件下的表达模式,鉴定出一系列与杀螨剂和植物次生物质代谢相关的P450基因；基于Bac-to-Bac系统,建立了柑橘全爪螨P450真核表达体系。研究结果丰富了柑橘全爪螨P450酶系的基础生物学信息,为筛选与柑橘全爪螨抗性相关P450基因提供了基础数据支撑,并对从P450氧化还原作用系统上全面阐述其介导的代谢抗性机制具有重要的理论和实践意义。更多还原

【Abstract】 The citrus red mite, Panonychus citri (McGregor)(Acari:Tetranychidae), is re-garded as the major citrus mite in China. The outbreak of this pest and injury to the plants caused by mite feeding can affect citrus harvest quantity and quality. Control of this mite depends largely on spraying of acaricides. However, frequent acaricides exposure combined with several biology characteristics of this mite (such as high fecundity, short life cycle), facilitate the development of strong resistance in this species to commonly used acaricides. To date, few studies have been attributed to the resistance mechanisms in P. citri, which mostly focus on the biochemical analysis and the identification of resistance genes. As a class of key detoxification enzymes in insects/mites, cytochrome P450monooxygenases (P450s) can metabolize a wide range of endogenous and exogenous chemical substances. Many studies have suggested that P450s in ticks and mites are involved in the resistance to many acaricides/insecticides, including pyrethroids, organophosphates (OPs), Mitochondrial Electron Transport Inhibitors (METI’s), tetronic acids, as well as biological pesticides. Researches around world have been focused particularly on cytochrome P450, while the important P450redox partners NADPH cytochrome P450reductase (CPR) and cytochrome b5are often ignored. Thus, carrying out study on P450and its redox system will be conducive to obtaining a comprehensive understanding of the action modes and roles of P450, which can help to reveal the metabolism resistance mechanism mediated by P450s.Therefore, our study was aimed at the response pattern of P. citri P450s under the stress of xenobiotics. At the beginning, we conducted the monitoring of resistance to four acaricides in P. citri collected from different citrus orchards in Chongqing and compared the activities of three major detoxification enzymes. As a result, cytochrome P450was chosen as the focus of our research. Secondly, we generated a cDNA library containing four different developmental stages of P. citri and identified a number of detoxification enzyme genes, which would provide abundant information for the study on the molecular resistance mechanisms of P. citri. Based on the transcriptome data, we amplified several full-lengths P450s, as well as CPR and CYB5genes. In addition, the expression reaction of P450, CPR and CYB5genes under different chemical stress was further examined. Finally, the eukaryotic expression system of P450genes in P. citri was established preliminarily, which would contribute to the functional verification of cytochrome P450system. The main results are as follows: 1Monitoring of resistance to four acaricides in different field populations of Chongqing and comparison of the activities of major detoxification enzymes1.1Investigation of susceptibility to four acaricides of different field populations of P. citriA modified leaf-dip bioassay method was used to measure the toxicity of four acaricides (abamectin, azocyclotin, pyridaben, spirodiclofen) on four P. citri popula-tions (Beibei, Bishan, Wulong, Zhongxian) sampled from Chongqing. The result showed that abamectin exhibited the highest acaricidal activity among these four acaricides, while the four field populations showed a less susceptibility to spirodiclofen and pyridaben. However, the citrus red mites in Chongqing showed a highest level of resistance to azocyclotin. We learned from the monitoring data for abamectin that, the Bishan population was more susceptible to this acaricide than other three populations. Zhongxian and Wulong populations showed greater tolerance to abamectin and their LC50values were11-and12-folds higher than that of Bishan population. The resistance levels of four populations to azocyclotin were closed and ranged from209.9to379.9mg/L. Samples collected from Zhongxian showed the lowest resistance level to azocyclotin and Bishan population was more tolerant than other populations. Beibei population showed the highest resistance to pyridaben with a LC50value of25.5mg/L, which was4-fold higher than that of the most sensitive population (Bishan). The resistance levels of four populations to spirodiclofen showed great variability and the LC50values were arranged in descending order as follows:Bishan population, Zhongxian population, Wulong population and Beibei population.1.2Comparison of the activities of three major detoxification enzymes1.2.1Comparison of the major detoxification enzymes activities in different developmental stagesWe assayed and compared the P450s, GSTs and CarEs activities in eggs, larvae, nymphs, and adults though a96-well microplate reader. The results suggested that the highest specific activity of P450s was observed in larvae, which was2.66-,2.70-and16.02-folds significantly higher than that in adults, nymphs and eggs. GSTs activity in adults and nymphs were much higher than that in other developmental stages, and the specific activity of GSTs was lowest in eggs. The CarEs specific activity in four stages ranged from0.46to13.60nmol/min/μg, and the value was largest in larva stage and least in egg stage, respectively. In summary, the results indicated that P450s and CarEs might play important roles in metabolism of xneobiotic during the larva stage, while GSTs was involved in protection against environmental stress during the nymph and adult stages.1.2.2Comparison of the major detoxification enzymes activities of different field populationsThe specific activities of P450s was highest in Beibei population, while GSTs and CarEs activities were both highest in Zhongxian population. However, there was no significant correlation between the activities of detoxification enzymes and the re-sistance levels to different acaricides, which might be caused by different background of pesticide application, targets of various acaricides and genetic structures of four populations.1.2.3Comparison of the activities of major detoxification enzymes after xenobiotics treatmentsBased on the bioassay, the mites were treated with LC30of five acaricides (abamectin, azocyclotin, fenpropathrin, pyridaben and spirodiclofen) and one kind of volatile secondary metabolites from citrus (citral) for24h. P450s activities increased after exposure to different chemicals, and reached the maximum value when treated by spirodiclofen. Except abamectin, other five different xenobiotics could significantly induce the GSTs activities. Similarly, GSTs activity was also the highest after treatment with spirodiclofen. However, when stimulated by spirodiclofen, CarEs activity was significantly lower than that of control, while the activities stayed still after exposure to other acaricides and citral. Compared to CarEs, P450s and GSTs could respond to the stress of xenobiotics more quickly and sensitively, which indicated that these two enzymes might take part in the metabolism process of the above xenobiotics.1.2.4Comparison of the major detoxification enzymes activities induced by sodium barbiturateAfter induction with19.96mg/L sodium barbiturate for24h, the activities of both P450s and GSTs increased, and CarEs activity was significantly inhibited by this chemical. 2Transcriptome analysis of P. citri and identification of detoxification enzymes genes2.1Generation and analysis of the transcriptome database of P. citriIn order to obtain a P. citri comprehensive transcriptome, the cDNA library was constructed using the mixture of RNA extracted from four developmental stages and generated by Illumina Hi seqTM2000. As a result, a total of51,214,018clean reads were generated and further assembled into65,149Unigenes. Using BLASTX with a cut-off E-value of10-5, there were32,535Unigenes successfully annotated in nr,25,813in Swiss-Prot,22,725in KEGG,15,412in COG and10,421in GO databases, respectively. The homology analysis of the Unigenes annotated in nr suggested that, P. citri Unigenes had higher similarity to Insecta than other classes;9%of the mite Unigenes showed the highest homology to sequences from Tribolium castaneum, followed by Harpegnathos saltator (6%) and Camponotus floridanus (5%). Additionally,1.7%of the annotated Unigenes were best matched to genes from Ixodes scapularis, which also belonged to Arachnida.The result of GO classification showed that10,421Unigenes which were annotated in nr were divided into three categories and50functional groups. The most abundant group was "cell"(7,035Unigenes) and the smallest group was "channel regulator activities"(2Unigenes). A total of15,412Unigenes annotated in COG were classified into25families. The largest family "general function prediction" contained3,642Unigenes, while the smallest group was "nuclear structure"(11Unigenes).2.2Identification of genes encoding detoxification enzymes and analysis of metabolic pathwaysThere were79P450Unigenes identified from the nr annotation of the P. citri transcriptome. Among these P450-related unique transcripts,12,9,26and7Unigenes were divided to CYP2, CYP3, CYP4and CYP6families, respectively. The remaining25Unigenes belonged to other families, including CYP9, CYP18, CYP314and Mitochondrial CYP family. All the P450Unigenes were screened against KEGG database and58were found to be involved in16pathways. We also identified24GSTs Unigenes from nr annotation of the P. citri transcriptome, which were assigned to the delta, kappa, mu, omega, sigma, theta and zeta families. In the KEGG annotation database,19GSTs Unigenes involved in10pathways were detected. A total of13 sequences encoding CarE were identified from the nr annotation results of P. citri transcriptome, among which there were four, three and one Unigenes were divided into Clade A, Clade B and Clade D, respectively. Only four CarE Unigenes could be annotated in KEGG database and involved in one pathway.3Full-length molecular cloning and sequences analysis of P450and its relative genes from P. citriBased on the transcriptome data,11novel P450genes were cloned from P. citri using the RT-PCR and RACE techniques. All the full-length cDNA sequences were deposited in GenBank and submitted to the P450nomeclature committee. The P450names and the corresponding GenBank accession numbers were as follows:CYP4CL2(JQ690089), CYP384A2(KC201279), CYP385C7(KJ173877), CYP385C8(KJ173878), CYP389D1(KC201278), CYP392A22(KC201275), CYP392A23(KC201276), CYP392A24(KC201277), CYP392A25(KJ173880), CYP392B6(KJ173879), CYP392F1(KC201274). The P450cDNAs contained opening reading frames (ORFs) of1,494-1,758bp that encoding497-585amino acid residues. Furthermore, the bioinformatics software such as ProParam, TMHMM and SignalP3.0were used to analyze the components of protein, physicochemical characteristics, transmembrane structure and signal peptide. As a result, the11P450proteins were considered to be microsomal P450s, which would play important roles in the detoxification of xenobiotics. Protein sequence alignments revealed the typical conserved P450motifs, including the heme binding domain, Helix C, Helix I, Helix K and the meander region. Moreover, the CYP4signature motif EVDTFMFA/GHDTT was present at the amino acid sequence of CYP4CL2.We compared the pairwise percentage identities of both nucleotide and predicted amino acid sequences among12P. citri P450genes (11P450s obtained in this research and one P450gene CYP4CF1cloned in our previous study). The percentages of nucleotide sequence identities were36.47-96.39%and the pairwise percentages of their encoded proteins were16.29-96.39%. To analyze the phylogenetic relationships,75P450s of Tetranychus urticae were downloaded from BIOINFORMATICS&SYSTEM BIOLOGY and aligned to the12P. citri P450genes. A phylogenetic tree was constructed with MEGA5.05applying the Maximum Likelihood method and revealed that the12P. citri P450s belonged to3Clans (CYP2Clan, CYP3Clan and CYP4Clan). Additionally, CYPs within the same family were clustered in the same branch. The CPR and CYB5genes were also isolated from P. citri with the GenBank accession numbers of KJ173881and KJ173882. The PcCPR cDNA sequence contained an ORF of2,061nucleotides encoding686amino acids, while the PcCYB5cDNA sequence contained an ORF of324nucleotides encoding107amino acids. A multiple alignment of the amino acid sequences was conducted among PcCPR and other three arthropod CPRs. The result showed that PcCPR shared the greatest identity with T. urticae CPR and consisted of three conserved domains such as FMN, FAD and NADPH binding regions. Phylogenetic analysis suggested that CPRs from the same class were grouped together. PcCPR originated from a same evolutionary root with the CPR in T. urticae with the bootstrap value of100, which was consistent with the result of homology analysis. In addition, we generated the protein sequence alignment among CYB5s and found the typical conserved motif (the heme binding region) at the N terminus. Moreover, PcCYB5also had the highest homology with T. urticae CYB5.4Transcription profiles of P450and its relative genes from P. citri4.1Transcription profiles of P450genes4.1.1Developmental expression profiles of P450genesDevelopmental expression profiles of12P450genes containing entire coding region and14Unigenes were analyzed by RT-qPCR. The results showed that the transcripts of26P450genes were detected at all the life stages of P. citri. CYP4CF1, CYP4CL2, CYP392A24and CYP392B6were highly expressed at larval stage, while higher mRNA levels of CYP385C7and CYP389D1were observed at nymphal stage. Additionally,10P450Unigenes had the highest relative expression quantities in the larvae or nymphs. It was inferred that the16P450genes above might be involved in the detoxification process during larval and nymphal stages. The expression levels of CYP384A2and two Unigenes were increased during the development of P. citri, which indicated that the metabolic capacity also increased in ontogeny of the citrus red mite. We also found the expression levels of CYP392A22, CYP392A24, Unigene1146and Unigene27622were significant higher in the eggs, which suggested that these four P450genes might be associated with xenobiotic metabolism in the egg stage. The expression quantities of CYP385C8and CYP392F1were relatively stable, which suggested that the two genes might play important roles during the whole life of P. citri.4.1.2Response profiles of P450genes under xenobiotic stress To determine the response of P450genes, three acaricides (abamectin, fenpropathrin and pyridaben) and citral were chosen in this study. Based on the bioassay, the mites were treated in two different manners:time treatment (12,24and36h) and concentration treatment (LC10, LC30and LC50). The RT-qPCR analyses revealed that the transcription response of P450genes was very apparent after treatment with abamectin. Two CYP385members (CYP385C7and CYP385C8) was up-regulated more than25-folds and four CYP392genes (CYP392A22, CYP392A23, CYP392A25and CYP392B6) transcripts increased41.46,68.66,25.56and11.34-folds, respectively. For time course effect of abamectin on the expression patterns of P450s, the majority of the26genes had a highest mRNA level after24h and only two Unigenes transcripts reached the peak12h later. Compared to the P450genes above, CYP4CL2and CYP392F1exhibited a slower response. Two CYP385genes(CYP385C7and CYP385C8) expression levels were highest when treated with LQ10abamectin, the relative expression quantities of five CYP392members (CYP392A22, CYP392A23, CYP392A24, CYP392A25and CYP392F1) increased up to the maximum value after exposure to sub-lethal concentration (LC30) abamectin. Under the tress of abamectin at a median lethal concentration (LC50), the CYP4s(CYP4CF1and CYP4CL2) transcripts reached the highest level.Most of the detected P450genes could be induced by fenpropathrin, and the relative quantity of CYP4CL2was higher than other genes (4.48-folds). Following exposure to fenpropathrin for12h, four CYP392genes (CYP392A24, CYP392A25, CYP392B6and CYP392F1) as well as CYP384A2and CYP389D1reached the maximum expression. The highest transcription levels of two CYP4s were both observed24h later. After treatment with LC30fenpropathrin, the five genes CYP4CF1, CYP4CL2, CYP392A22, CYP392B6and CYP392F1were significantly up-regulated and the expression levels of them reached the peak. The results also showed that CYP385C8could be significant induced by the LC50of fenpropathrin. It could be indicated that these genes might participated in the detoxification process for fenpropathrin of P. citri.Unlike treatments with abamectin and fenpropathrin, the number of P450genes which were up-regulated reduced and the changes of relative quantities were less obvious after exposure to pyridaben. CYP385C7, CYP385C8and CYP392A22transcripts reached the peak after pyridaben treatment for12h, which suggested that the three CYPs might be involved in the detoxification of this acaricide during the early stage. The expression levels of CYP4CL2and four CYP392members (CYP392A23, CYP392A24, CYP392A25and CYP392B6) reached a maximum after24h or36h treatment and these genes might play roles in resisting the pyridaben stress during the middle and late stages. Only the mRNA level of CYP4CL2could be induced to the peak by LCio pyridaben concentration, while three CYP392genes and CYP385C8might be associated with the metabolism of high concentration pyridaben.The expression of all the12P450genes containing full-length cDNA increased significantly when treated by citral. The highest mRNA levels of most P450genes were detected after24h or36h treatment, which indicated that the response to citral of P450s in P. citri had a lag-effect. Moreover, qPCR analyses revealed that the high concentration of citral could lead to an obvious induction effect on P450s. There were ten P450genes containing entire ORF and eight Unigenes reached the highest expression level after exposure to citral at the concentration of LC50. The results also showed that the expression changes of CYP385C7, CYP385C8, CYP392A23, CYP392B6and CYP384A2were higher than other P450s (>8-folds increase), which could be inferred that the five CYPs might have important roles in the detoxification of citral and adaptation to the host environment.4.1.3Response profiles of P450genes induced by sodium barbiturateThe sodium barbiturate exhibited significant induction effects on the majority of P450genes in P. citri and the highest induction levels were detected after treatment for24h or36h in most cases. It was also found that a positive correlation existed between the relative expression quantities of five CYPs (CYP392A23, CYP392A25, CYP392B6, CYP384A2and Unigene9596) and the induction concentrations.4.2Transcription profiles of PcCPR and PcCYB5genes4.2.1Developmental expression profiles of PcCPR and PcCYB5genesThe expression levels of PcCPR at larval and nymphal stages were significantly higher than that in other two stages. The highest expression quantity was observed in larvae, while the mRNA level was the lowest in eggs. The developmental expression profile of PcCPR was consistent with the P450s activities during the life stages, indicating that PcCPR was mainly involved in the detoxification process mediated by P450s during larval and nymphal stages. Moreover, this phenomenon also suggested that a cooperative action existing between CPR and P450s in P. citri. The highest mRNA level of PcCYB5was detected in nymphs, which indicated that PcCYB5was likely to play an important role in delivering the electrons to P450catalysis.4.2.2Response profiles of PcCPR and PcCYB5genes under xenobiotic stressThe qPCR results showed that PcCPR exhibited a rapid response under the abamectin stress. The PcCPR transcripts reached a maximum after exposure to LC10abamectin for12h. The expression of PcCPR was inhibited by fenpropathrin at the concentrations LC10and LC50, while the sub-lethal concentration (LC30) could significantly induced PcCPR transcripts. Pyridaben showed the strongest induction effect on PcCPR and the citral treatments with different concentrations and times resulted in the up-regulation of PcCPR expression. Thus we concluded that PcCPR could be involved in the metabolic process for xenobiotics of P450. The expression of PcCYB5was significantly up-regulated after treatment with three acaricides and citral. It was also found that a complementary effect existed between the expression profiles of PcCYBS and PcCPR, which indicated that the cooperation of the two genes would ensure the high-speed transfer of electrons.4.2.3Response profiles of PcCPR and PcCYBS genes induced by sodium barbiturateThe qPCR analyses showed that both PcCPR and PcCYBS expression levels increased significantly after sodium barbiturate induction. These two genes transcripts rose with the increase of treatment time and concentration.5Eukaryotic expression of P450genes from P. citriIn order to study the function of CYP392A24and CYP392F1, we conducted the eukaryotic expression experiment with Bac-to-Bac baculovirus expression system. Two recombinant virus expression vectors Bacmid-CYP392A24and Bacmid-CYP392F1were successfully constructed in the use of double digestion of XhoⅠ/HindⅢ and BamHI/PstI restriction enzymes as well as the DNA recombination technology. Transfecting Sf9cells with the recombinant vectors, we finally obtained the expression products. The P450contents were determined by reduced CO-difference spectrum, but the CYP392A24and CYP392F1proteins were proved to be inactive. Therefore, the expression and protein purification system is still need to be improved in the future. However, the generation of eukaryotic expression system for P. citri P450s will provide the technical foundation for the functional verification of P450s and research on the detoxification capacity of target genes.In summary, we conducted the resistance monitoring in different populations of P. citri and compared the major detoxification enzymes activities. The transcriptome database of P. citri was generated via high throughput sequencing technology. Based on the transcriptome data, we identified the genes related to detoxification and analyzed their putative metabolic pathways. Using RT-PCR and RACE techniques,11P450genes as well as PcCPR and PcCYB5were isolated from P. citri. Transcription profiles of26P450genes (12CYPs containing entire coding region and14Unigenes), PcCPR and PcCYB5were determined through qPCR technique from P. citri including different developmental stages, acaricides and citral stress and sodium barbiturate induction. As a result, a series of P450genes involving in the detoxification of xenobiotics were identified. Furthermore, we generated the eukaryotic expression system for P. citri P450s and constructed two recombinant virus expression vectors. The results enriched the P450genes information of P. citri and will provide basic data for exploring the resistance related P450genes. Additionally, our study will promote the research on the metabolism resistance mechanism mediated by P450s.更多还原