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害虫遗传不育技术中致死基因的研究与应用

2015-12-17王玉生蔡玉音张桂芬刘桂清万方浩

生物安全学报 2015年2期

王玉生, 蔡玉音, 武 强 ,严 盈,3,4, 张桂芬, 刘桂清,5, 万方浩,6*

1中国农业科学院植物保护研究所,植物病虫害生物学国家重点实验室,北京 100193; 2天津市宝坻区

大白街道办事处农业办公室,天津 301802; 3Department of Entomology, North Carolina State University,

Raleigh, NC, USA, 27606; 4Genetic Engineering and Society Center and W.M. Keck Center for

Behavioral Biology, North Carolina State University, Raleigh, NC, USA, 27606;

5广东省昆虫研究所,广东 广州 510260; 6青岛农业大学

农学与植物保护学院,山东 青岛 266109



害虫遗传不育技术中致死基因的研究与应用

王玉生1+, 蔡玉音1,2+, 武强1,严盈1,3,4, 张桂芬1, 刘桂清1,5, 万方浩1,6*

1中国农业科学院植物保护研究所,植物病虫害生物学国家重点实验室,北京 100193;2天津市宝坻区

大白街道办事处农业办公室,天津 301802;3Department of Entomology, North Carolina State University,

Raleigh, NC, USA, 27606;4Genetic Engineering and Society Center and W.M. Keck Center for

Behavioral Biology, North Carolina State University, Raleigh, NC, USA, 27606;

5广东省昆虫研究所,广东 广州 510260;6青岛农业大学

农学与植物保护学院,山东 青岛 266109

摘要:基于遗传修饰手段的昆虫不育技术(SIT)作为一类物种特异、环境友好、科学高效的新兴策略,在害虫防治中具有广阔的应用前景。释放携带显性致死基因昆虫的技术(RIDL)是改进传统SIT的重要手段之一,主要包括四环素调控系统、特异性启动子、性别特异剪接系统和特异性致死基因等重要元件,其中根据不同昆虫的特点选择合适的特异性致死基因对于构建遗传不育品系至关重要。这些致死基因或受到阻遏调控系统的控制、或特异的在雌虫中表达、亦或直接作用于X染色体,导致后代在特定发育阶段或特定性别中条件致死。本文综述了RHG家族(reapr、hid、grim、michelobx)细胞凋亡基因、转录激活因子tTA及Nipp1Dm、归巢内切酶基因等在害虫遗传不育技术中的研究和应用,讨论了特定致死基因的效应机理和应用特点,并对其可能的发展方向进行了展望。由于不同效应基因的致死作用和调控机理尚未完全明晰,因此深入研究特异致死基因的凋亡机制和在不同物种中的兼容作用,将为害虫遗传防控提供更多的研究思路和手段。

关键词:昆虫不育技术; 致死基因; 细胞凋亡基因; 转录激活因子; 归巢内切酶基因

Used of lethal genes in sterile insect technique

for pest control: a review

Yu-sheng WANG1+, Yu-yin CAI1,2+, Qiang WU1, Ying YAN1,3,4, Gui-fen ZHANG1,

Gui-qing LIU1,5, Fang-hao WAN1,6*

1State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural

近年来,昆虫遗传修饰技术的飞速发展为农林害虫和卫生害虫的防治提供了更为新颖有效的思路,特别是与昆虫不育技术(Sterile insect technique,SIT)相结合,具有环境友好、物种特异和防治高效等优点,弥补了传统SIT技术的缺点,在害虫防治方面取得了一系列进展,形成了以释放携带显性致死基因昆虫(Release of insects carrying a dominant lethal,RIDL)技术为代表的害虫遗传控制新策略。RIDL通过向害虫种群中引入携带致死基因纯合子的昆虫,在与野生型昆虫交配后产生的后代中,这些致死基因或受到阻遏调控系统的控制、或特异地在雌虫中表达、亦或直接作用于X染色体,导致后代在特定条件/发育阶段/性别中致死,从而引起害虫种群瓦解,达到高效控制重大农林害虫的目的。构建一个成功的RIDL品系需要特异的调控元件、特异的致死基因和高效的遗传转化系统等关键因子。

基于四环素系统(tet-off)调控致死基因的表达以实现对靶标昆虫的特异性致死,是目前RIDL品系构建中最常用的昆虫遗传调控系统。tet-off依据的原理是:大肠杆菌E.coli转座子Tn10的四环素阻遏因子(Tetracycline repressor,tetR)与四环素结合,导致其不能阻抑四环素抗性操纵子(Tetracycline-resistance operon,tetO),下游转录随即启动(廖伟,1998)。Gossen & Bujard(1992)将tetR的部分序列与单纯疱疹病毒VP16的转录活化区域组成四环素转录激活因子(Tetracycline transcriptional activator,tTA),tTA与特异性启动子构建为tet-off驱动载体,将tetO与CMV启动子构成四环素响应元件(Tetracycline response element,TRE),TRE与致死基因组合成效应载体,由此便得到了tet-off双元件系统。缺乏四环素时,tTA与tetO结合引发致死基因表达;四环素存在时,tTA与四环素结合而不与tetO结合,进而无法激活下游基因的表达。

特异致死基因的筛选对于构建昆虫种群遗传调控系统至关重要,这些基因通常需要满足以下几个特征:能被阻抑调控、能在特异性启动子作用下在特异性的组织/发育时期/性别中表达、表现为显性致死。目前在害虫遗传调控技术中最常用的致死基因主要包括细胞凋亡基因(如reapr、hid、grim、michelob_x等RHG家族基因)、转录激活因子tTA、Nipp1Dm和归巢内切酶基因等,本文将分别讨论这几类致死基因的特点及其在不同物种中的应用情况(表1)。

1 细胞凋亡相关基因

细胞凋亡(Apoptosis)即I型细胞程序性死亡(Programmed cell death,PCD)是由多层次严格调控的细胞自主有序死亡的过程,在机体的生命活动中具有重要的生物学意义(Bakeevaetal.,2007; Kerretal.,1972; Wyllie,1980),与多种疾病的发生密切相关(张金叶,2011; Vila & Przedborski,2003)。分子生物学的飞速发展使人们对细胞凋亡有了深入理解,细胞凋亡涉及一系列基因的激活、表达和调控等(Liuetal.,1996; Morizaneetal.,2005),迄今其复杂的分子机理尚不完全明晰。

半胱氨酸天冬氨酸特异性蛋白酶(Cysteinyl asparate specific proteinase,Caspase)是细胞凋亡调控网络的核心,分为起始和效应2类。死亡受体和线粒体通路是依赖Caspase细胞凋亡的主要途径(Juoetal.,1998; Lavriketal.,2005; Li & Yuan,2008)。哺乳动物中线粒体释放的细胞色素C(Cytochrome,CytC)与凋亡蛋白酶活化因子(Apoptosis protease activating factor 1,Apaf-1)结合,形成凋亡体,激活起始Caspase-9,进而活化效应Caspase-3和Caspase-7,启动Caspase级联反应(图1)(Cashioetal.,2005; Chipuk & Green,2008; Fritzetal.,2006; Sleeetal.,1999)。凋亡蛋白抑制因子(Inhibitor of apoptosis proteins,IAPs)家族具有抗凋亡作用,能阻断CytC释放,其baculovirus IAP repeat(BIR)结构域与凋亡体小亚基结合从而抑制Caspase-9(LaCasseetal.,1998; Ryooetal.,2004; Yanetal.,2004)。但是当细胞接受凋亡信号时,线粒体释放的Smac等引起IAPs的泛素化,进而阻抑IAPs对Caspase-9的活性抑制(Cashioetal.,2005)。模式生物黑腹果蝇Drosophilamelanogaster(Meigen)在细胞凋亡时,促凋亡基因RHG家族转录激活,引起凋亡蛋白抑制因子DIAP1的自我泛素化,解除其对Caspase-9同源物Dronc酶原的抑制作用,释放的Dronc则与Apaf-1的同源物Dark(Hawkinsetal., 2000; Lee & Baehrecke, 2000)结合活化,进而激活与Caspase-3,7功能相似的Drice和Dcp-1,细胞开始凋亡(Aramaetal.,2006; Salvesen & Duckett,2002; Scheteligetal.,2011b; Steller,2008; Taitetal.,2004)。该过程与哺乳动物Smac等阻抑IAPs对Caspase-9的活性抑制类似,与哺乳动物不同的是,果蝇中Apaf-1的同源物Dark的活化不需要CytC参与(Dorstynetal.,2002; Jiang & Wang,2000; Lietal.,1997; Varkeyetal.,1999)。RHG家族的reaper、hid和grim基因分别控制不同的信号通路(Danial & Korsmeyer,2004),是果蝇细胞凋亡最主要的调控因子,与凋亡抑制蛋白协调作用共同决定细胞凋亡(图1)。

表1 昆虫遗传控制中的致死基因

1.1 hid基因

头部退化缺陷基因(Head involution defective,hid),又称wrinkled(W)基因。果蝇的reaper、hid、grim基因均位于其3号染色体的75CI1, 2基因座中(Whiteetal.,1994)。Gretheretal.(1995)通过克隆得到了果蝇hid基因,并对hid的生物信息学及其在细胞凋亡过程中的功能等进行了详细研究。结果表明,果蝇hid蛋白由410个氨基酸残基组成,其N-末端有一段RHG家族蛋白相对保守的IAP-binding-motif(IBM)结构域,能够与果蝇凋亡蛋白抑制因子DIAP1等中的BIR结构域特异结合,解除其对Caspase的阻抑(Yin & Thummel,2004; Zachariouetal.,2003)。Caspase诱导的细胞开始凋亡,是hid特异性诱导凋亡的必要条件(Scheteligetal.,2011b);同时N-末端存在grim helix 3(GH3)基序,有助于hid在线粒体的定位,使其在细胞凋亡中发挥辅助作用,与IBM协同促进细胞凋亡;IBM和GH3的共同存在使细胞凋亡效果明显提高(Bryantetal.,2009)。研究证实,加勒比按实蝇Anastrephasuspensa(Loew)和果蝇的IBM、GH3缺失,能大幅降低细胞凋亡效果(Scheteligetal.,2011b)。hid蛋白还是丝裂原激活的蛋白激酶信号通路(Mitogen-activated protein kinase,MAPK)的靶分子,果蝇和加勒比按实蝇的hid蛋白有若干个MAPK磷酸化活性位点,MAPK可能通过yan和pnt等转录调节因子抑制hid的转录,阻断下游细胞凋亡(Bergmannetal.,1998、 2002)。将果蝇hid基因的5个磷酸化位点突变后,DmhidAla5比没有突变的hid基因的致死效率高(Horn & Wimmer,2002)。此外,microRNA和转录因子E2F能分别与果蝇hid的3′UTR和5′增强子区域结合,调节其活性(Brenneckeetal.,2003; Tanaka-Matakatsuetal.,2009)。

Gretheretal.(1995)研究发现,hid突变体胚胎中的细胞凋亡明显减少,导致中枢神经系统产生大量额外细胞;用hsp70的启动子表达hid时发现,hid能诱导野生型和hid缺陷的H99果蝇的胚胎细胞凋亡,且在胚胎细胞凋亡处能检测到其mRNA。在眼部特异性启动子的作用下,pGMR-hid在果蝇视网膜处能异位表达并导致眼睛消融,而凋亡抑制蛋白p-35的共表达则抑制hid基因诱导的凋亡。hid基因表达分析表明,在果蝇幼虫成熟过程中hid表达上调,使唾液腺等组织细胞迅速凋亡,形态发生变化(Yin & Thummel,2004);然而,由于细胞凋亡在昆虫生命活动中起重要作用,因此hid基因在成虫期也会有一定水平的稳定表达;而在高日龄的果蝇成虫中hid基因表达量的上升,可能与hid基因在果蝇衰老中发挥作用有关(Zhengetal.,2005)。此外,Bergmannetal.(1998)发现,hid不仅在果蝇注定死亡的细胞中表达,在部分正常存活的细胞中也有表达,而同属RHG家族的reaper和grim则仅在将要死亡的细胞中表达,这可能与RHG家族的不同基因控制不同的信号通路有关(Danial & Korsmeyer,2004)。

图1 哺乳动物和果蝇细胞凋亡途径(Cashio et al.,2005)

除果蝇外,hid基因已在加勒比按实蝇(Scheteligetal.,2011b)和橘小实蝇BactroceradorsalisHendel(蔡玉音等, 2014)等昆虫中克隆得到,并进行了生物信息学与表达分析,推导的氨基酸序列与果蝇hid蛋白有较高的相似性,这表明昆虫hid蛋白存在一定的保守性,且均具有RHG蛋白家族特异的IBM和GH3结构域。Scheteligetal.(2011b)和蔡玉音等(2014)研究发现,加勒比按实蝇和橘小实蝇幼虫化蛹过程中,在蜕皮激素诱导下hid基因的表达迅速上调,这与果蝇相关研究结果类似,即在此阶段hid基因诱导细胞迅速更新,进而完成“变态”发育。此外,Scheteligetal.(2011b)还验证了加勒比按实蝇As-hid基因对加勒比按实蝇胚胎AsE01和果蝇S2细胞系的促凋亡能力,并发现,同物种来源的hid基因在细胞系水平的致死效果优于异种hid基因。

目前,应用hid基因进行RIDL品系的构建已经取得了长足进展。Horn & Wimmer(2002)基于tet-off调控系统和piggyBac转座子基因驱动系统,通过驱动载体中细胞囊胚期特异性表达的serendipityα(sryα)及nullo的启动子/增强子,调控效应载体中磷酸化位点突变的DmhidAla5基因表达,得到了果蝇胚胎条件致死品系,该品系在地中海实蝇Ceratitiscapitata(Wiedenmann)和加勒比按实蝇上(Scheteligetal.,2009a; Schetelig & Handler,2012a)也成功构建。实验室饲养时,添加四环素能阻抑致死基因的表达,释放的不育雄虫与野生雌虫交配后产生的子代由于缺乏四环素,均在胚胎期死亡。在此基础上,若将雌性特异剪接的内含子片段插入到效应载体以驱动hid在雌虫的特异表达,将获得雌性胚胎特异致死品系。目前,已在加勒比按实蝇(Schetelig & Handler,2012b)和地中海实蝇(Ogaugwuetal.,2013)中获得成功,其中地中海实蝇纯合子品系与野生型交配后的雌性后代全部在卵期死亡,获得的单一雄性RIDL品系与SIT结合将有助于实现目标害虫的有效防控。

1.2 reaper基因

果蝇3号染色体的75CI1,2区段为其胚胎凋亡所必需,该区段的缺失将抑制细胞凋亡的产生,reaper基因是在该区段克隆的第一个细胞凋亡调控基因(Whiteetal.,1994)。目前,reaper基因已在黑腹果蝇(Whiteetal.,1994)、家蚕BombyxmoriL.(Bryantetal.,2009)、加勒比按实蝇(Scheteligetal.,2011b)和铜绿蝇Luciliacuprina(Gmelin.)(Chenetal.,2004)等昆虫中克隆得到,并对其进行了功能研究。reaper基因缺失的果蝇胚胎与hid基因缺陷时类似,细胞凋亡明显受抑,中枢神经系统也有大量额外细胞产生,reaper基因表达则恢复细胞凋亡(Whiteetal.,1994)。在野生型胚胎中,reaper基因在注定死亡的细胞中特异表达,与hid基因在部分正常细胞中也有所表达不同(Bergmannetal.,1998)。此外,reaper基因在眼部的异位表达也具有与hid基因类似的结果,能导致眼睛消融。凋亡抑制蛋白p-35蛋白的共表达则抑制reaper基因的活性(Chenetal.,1996; Gretheretal.,1995)。同时,reaper基因还参与外界因素诱导的细胞凋亡,X射线、紫外线和蜕皮激素等都能刺激果蝇中reaper基因的表达(Liuetal.,2011; Zhangetal.,2008)。

reaper蛋白不仅能阻抑果蝇DIAP1等凋亡抑制蛋白对细胞凋亡的抑制能力,还可激活DIAP1的泛素化,降解或抑制DIAP1的翻译(Holleyetal.,2002),使DIAP1不能阻抑Caspase凋亡。据报道,果蝇的Morgue、UbcD1和UbcD2等泛素连接酶与reaper介导的DIAP1等的泛素化有关(Ryooetal.,2002; Wingetal.,2002)。也有研究发现,由于物种间的reaper存在一定保守性,果蝇的reaper能导致哺乳动物细胞凋亡(Abrams,1999),也能与非洲爪蟾属Xenopus的Scythe蛋白作用,而果蝇体内也存在Scythe类似物,表明reaper可能介导Scythe参与的CytC凋亡途径(Thressetal.,1998)。

果蝇reaper基因编码的蛋白质包括65个氨基酸残基(Whiteetal.,1996),与hid蛋白的氨基酸序列相似性较低,但其N-末端也具有RHG家族蛋白相对保守的IBM结构域,该结构域与reaper蛋白对IAP活性的抑制密切相关(Yin & Thummel,2004; Zachariouetal.,2003),果蝇、加勒比按实蝇和铜绿蝇reaper蛋白IBM结构域的缺失能大幅影响reaper的促凋亡活性(Chenetal.,2004; Scheteligetal.,2011b; Zhouetal.,2005)。reaper的GH3结构域为其在线粒体中定位所必需,在DIAP1的泛素化降解中也发挥重要作用(Olsonetal.,2003),保证了IBM缺失时对细胞凋亡的诱导,也能诱导CytC释放并促使细胞凋亡(Freeletal.,2008)。此外,Sanduetal.(2010)和Scheteligetal.(2011b)研究发现,果蝇和加勒比按实蝇的reaper蛋白均具有一个中央螺旋结构域,该结构域在reaper与hid的互作过程中发挥重要作用,hid和reaper的结合使得reaper能更稳固于线粒体上,而hid与grim则不具有类似结构(蔡玉音等,2014; Scheteligetal.,2011b),reaper与hid的另一区别在于reaper的活性不受MAPK途径的抑制。

reaper与hid基因的功能还具有诸多相似之处,在幼虫化蛹时reaper的表达上调,促使组织细胞迅速更新换代,完成“幼虫—蛹”的变化。reaper基因的表达也能诱导正常细胞的凋亡,如Scheteligetal.(2011b)研究发现,果蝇和加勒比按实蝇的reaper基因能诱导加勒比按实蝇胚胎AsE01和果蝇S2细胞系的凋亡,且与hid基因作用结果相似,即同源物种来源的reaper基因的致死效果优于异种reaper基因。进一步研究还发现,reaper和hid共表达促凋亡的作用更明显(Scheteligetal.,2011b; Yooetal.,2002),表明reaper可能与hid控制不同的凋亡途径,二者之间协同作用,共同调控细胞凋亡,进而推测reaper和hid的联合应用可能会取得更好的遗传调控效果。

1.3 grim基因

grim基因同样位于果蝇3号染色体75C1,2这一横跨约300 kb的区段上,grim位于reaper和hid之间。grim基因与RHG家族的hid、reaper基因功能类似,其表达也能恢复H99缺陷型胚胎细胞凋亡,且凋亡处能明显检测到该基因的表达(Chenetal.,1996)。grim在果蝇胚胎中的表达模式与reaper、hid的表达相似,与果蝇胚胎发育过程中细胞凋亡的进程统一,不过在早期胚胎细胞凋亡时能检测到grim表达,但reaper则无;此外,grim与reaper一样仅在将要死亡的细胞中表达(Bergmannetal.,1998; Chenetal.,1996)。grim基因在眼部特异性启动子的诱导下的异位表达也能导致果蝇眼睛的消融,在细胞系水平的研究也证实了grim基因的功能,其对细胞凋亡的诱导不需要reaper和hid基因的参与,且对细胞凋亡的诱导受到共表达的p-35蛋白的抑制(Chenetal.,1996; Clem,2007)。据Chenetal.(1996)推测,grim同hid和reaper基因一样,对凋亡的调控位于p-35上游,grim、reaper和hid可能分别参与多个调控途径,以及同一下游凋亡途径,下游途径受到p-35蛋白的抑制。

果蝇grim编码的蛋白质序列有138个氨基酸残基,具有RHG蛋白家族保守的IBM和GH3结构域,其N-末端IBM结构域发挥保守的抑制凋亡蛋白抑制因子DIAP1等活性的功能。其GH3结构域与reaper蛋白的相似性较高,功能也与reaper的GH3类似:辅助其在线粒体的定位,激活凋亡蛋白抑制因子DIAP1等的降解(Olsonetal.,2003),从而解除DIAP1对Dronc等Caspase的抑制,导致细胞凋亡。

1.4 michelob_x基因

michelob_x基因是凋亡抑制蛋白IAP的拮抗基因,其推导的氨基酸序列与果蝇RHG家族蛋白质整体序列相似度较低,但是已报道的冈比亚按蚊Anophelesgambiae(L.)(Zhouetal.,2005)、埃及伊蚊Aedesaegypti(L.)(Zhouetal.,2005)、致倦库蚊CulexquinquefasciatusSay(Liuetal.,2011)和三带喙库蚊CulextritaeniorhynchusGiles(徐瑶,2012)的michelob_x编码的蛋白同RHG蛋白一样,具有较为保守的N-端IBM结构域并可归入RHG家族,为reaper在蚊虫中的同源基因,不过michelob_x蛋白不具有GH3结构域(徐瑶,2012; Liuetal.,2011; Zhouetal.,2005)。

冈比亚按蚊、埃及伊蚊、致倦库蚊和三带喙库蚊中的michelob_x基因在果蝇的S2细胞或白纹伊蚊Aedesalbopictus(Skuse)的C6/36细胞中的表达都能诱导细胞快速调亡(徐瑶,2012; Liuetal.,2011; Zhouetal.,2005)。埃及伊蚊michelob_x蛋白的促凋亡活性依赖其N-末端的IBM结构域,IBM结构域的功能及促凋亡机制类似于果蝇RHG蛋白家族(Bryantetal.,2008; Liuetal.,2011; Wangetal.,2008),能够移除凋亡抑制蛋白AeIAP1对Caspase-9同源的AeDronc的阻抑,活化的AeDronc与AeArk结合,激活Caspase-16,引发凋亡(图2A; Ocampoetal.,2013)。当IBM缺失时,michelob_x蛋白的促凋亡活性也丧失,这可能与michelob_x蛋白不具有GH3结构域的凋亡辅助作用有关(徐瑶,2012; Liuetal.,2011; Zhouetal.,2005)。此外,michelob_x在蚊虫发育和紫外照射(Zhouetal.,2005)、病毒感染(Liuetal.,2011; Wangetal.,2008)等外因导致的细胞凋亡中起中枢调节作用。将含有埃及伊蚊michelob_x的重组SINVs病毒感染埃及伊蚊C6/36细胞系时,michelob_x能引起细胞大量凋亡,阻抑病毒持续性裂解导致的感染(Wangetal.,2008)。michelob_x在杆状病毒CuniNPV引起的致倦库蚊和埃及伊蚊细胞凋亡中也发挥重要作用,其表达与病毒感染导致的细胞凋亡进程协调(Liuetal.,2011)。michelob_x参与蚊虫病毒感染诱导的免疫反应,与凋亡抑制蛋白p-35或IAP的共表达则抑制michelob_x引起的Caspase反应(Liuetal.,2011; Wangetal.,2008; Zhouetal.,2005)。

细胞凋亡过程涉及一系列机理尚未完全明晰的基因的相互作用,并与其他代谢途径交互作用形成复杂的代谢调控网络,以维持机体组织稳态及正常生命活动。研究发现,细胞凋亡在昆虫防御病毒等病原体感染过程中发挥重要作用(Clarke & Clem,2003; Zhouetal.,2005)。除michelob_x外,昆虫体内的另外一种起始Caspase Dredd也参与此过程(图2B; Ocampoetal.,2013),果蝇和埃及伊蚊的Dredd均具有2个死亡结构域(Death domain,DD),当接受死亡信号后与其配体FADD(Fas associated death domain containing protein)通过DD结构域结合,多聚化形成死亡诱导信号复合体(Death-inducing signaling complexes,DISCs),活化Dredd,进而开启下游细胞的凋亡。虽然果蝇和埃及伊蚊的Dredd均因诱导细胞凋亡作用而被发现,但近年来的研究表明,Dredd主要在机体免疫途径发挥作用,但其机理尚未完全明晰。

图2 埃及伊蚊细胞凋亡途径及其参与免疫反应(Ocampo et al.,2013)

michelob_x基因在登革热的主要媒介昆虫埃及伊蚊的遗传控制研究中已经取得了显著进展。Fuetal.(2010)采用基于tet-off调控系统的昆虫遗传控制技术,利用在雌蚊间接飞行肌特异性表达的肌动蛋白AeAct-4基因的启动子调控促凋亡基因michelob_x的表达,获得了无翅型不育雌蚊,后代雌蚊中65.8%~98.3%的个体为无翅型,雄蚊则无该表型,这一研究为医学媒介蚊虫的遗传控制提供了新思路。

2 转录激活因子tTA的致死效应

研究表明,低水平表达的tTA蛋白对细胞无害,高水平表达的tTA对细胞则有毒害作用,该毒害作用可能是由于高水平的tTA干扰依赖泛素的蛋白质降解所致。Gongetal.(2005)利用高表达的tTA对细胞的毒性作用,将tet-off简化为单元件系统,tTA除发挥转录激活因子功能外,还发挥致死基因功能:存在四环素时,tTA的表达被抑制,低水平的tTA对昆虫无害;不存在四环素时,tTA与tetO结合,进一步促进tTA的表达,高水平累积的tTA导致昆虫死亡。利用该单元件系统,目前已构建了地中海实蝇Ceratitiscapitata(Wiedenmann)(Gongetal.,2005)、埃及伊蚊(Phucetal.,2007)和棉红铃虫Pectinophoragossypiella(Saunders)(Morrisonetal.,2012)等害虫的胚胎条件致死品系。其中Phucetal.(2007)构建的埃及伊蚊RIDL品系OX513A已经实现了田间开放条件的释放研究(Harrisetal.,2011、 2012)。Fuetal.(2007)基于tet-off单元件系统,将地中海实蝇雌性特异内含子片段插入tTA编码区,构建了雌性特异致死品系。不含四环素时,选择性剪切内含子在雌虫体内调控下游tTA蛋白高表达,雄虫则不表达,从而导致雌虫死亡,由此获得的雄虫可以与SIT技术相结合,并进行野外释放。transformer和doublesex等雌性特异剪切基因的深入研究,使得雌性特异致死品系取得了广泛成功,目前,小菜蛾PlutellaxylostellaL.、棉红铃虫(Jinetal.,2013)、橄榄实蝇Bactroceraoleae(Gmelin.)(Antetal.,2012)、家蚕(Tanetal.,2013)和铜绿蝇(Lietal.,2014)的相关品系已被成功构建。此外,Fuetal.(2010)和Labbéetal.(2012)分别用雌蚊飞行肌特异性启动子Act-4驱动单元件系统中tTA的高表达,得到了埃及伊蚊和白纹伊蚊的雌蚊无翅型品系。

3 其他致死基因

3.1 Nipp1Dm基因

Nipp1(Nuclear inhibitor of protein phosphatase type 1)是细胞核内Ⅰ型(丝氨酸/苏氨酸型)蛋白磷酸酶(Protein phosphatase type 1,PP1)的抑制剂(Parkeretal.,2002)。PP1能与许多蛋白质形成复合体辅助其进行亚细胞定位,调控蛋白在特异性部位发挥功能,而且与细胞周期、糖原代谢和RNA合成等功能密切相关(Linetal.,1999)。果蝇Nipp1Dm基因位于果蝇2号染色体53F4-5区段,Nipp1Dm蛋白具有多个功能性结构域,其保守的中心RVXF结构域能专一性地结合PP1,抑制PP1的活性,导致细胞死亡,C-端结构域有RNA结合功能,且Nipp1定位于核小点(Nuclear speckles)(Parkeretal.,2002)。

Parkeretal.(2002)分析了Nipp1Dm在果蝇胚胎、幼虫头部和成虫器官芽的表达模式,发现其在各发育阶段均表达,异位表达将导致有丝分裂异常、细胞凋亡、肌肉不能正常发育、翅发育异常和子代不育等(Bennettetal.,2003; Parkeretal.,2002),上述现象同时也受到了PP1共表达的抑制。

Nipp1Dm基因在蚊媒昆虫的遗传控制中也有一定的应用,Fuetal.(2010)和Marinottietal.(2013)分别在埃及伊蚊和斯氏按蚊Anophelesstephensi(Liston)中用同源的AeAct-4和AsAct-4启动子,以Nipp1Dm为致死基因,获得了雌性后代无翅的RIDL品系。

3.2 homing endonuclease genes

对于XY染色体决定型的双翅目昆虫,归巢内切酶(Homingendonucleasegenes,HEG)也是其遗传控制品系构建的一个有应用前景的致死基因。HEG属于自私基因,能特异地识别染色体上的2段特定序列,并定向插入在这2段序列之间,当同源染色体中的一条具有HEG时,HEG酶将切割另一条染色体,并以前者为模板进行复制(Sinkins & Gould,2006)。由于homing endonuclease Ⅰ-PpoⅠ能高度特异的靶定与X染色体连锁的28S核糖体基因的重复序列(Nolanetal.,2011),如将HEG基因置于雄虫精子发生时特异表达的β2tubulin控制下,在精子发生时切割X染色体,当其转入胚胎时还能切割母系来源的X染色体,导致后代雌虫在胚胎期死亡,产生全为携带HEG的雄虫(Windbichleretal.,2008),从而大幅度改变后代的性别构成,致使蚊群无法继续繁衍。Windbichleretal.(2008)和Galizietal.(2014)利用HEG基因分别成功构建了疟疾的蚊媒冈比亚按蚊的RIDL不育品系。

4 总结与展望

目前,害虫种群遗传调控研究中,常用的致死基因中促凋亡基因占据了很大比例,但在细胞凋亡过程中,凋亡抑制因子和促凋亡基因协同作用,多层次共同调控细胞凋亡通路,迄今为止其复杂的分子机理尚不完全明晰。因此,需要加大对昆虫特别是非模式物种的细胞凋亡基因研究,解析其细胞凋亡途径及机理,该研究结果将为条件致死品系的获得奠定基础。此外,近年来归巢内切酶等的发现扩展了对双翅目昆虫遗传控制致死基因的理解,然而其机理尚需透彻剖析,在害虫控制中的效果也需要更多的实践研究。同时,遗传控制品系在工厂大规模饲养时,致死基因效应的阻抑物四环素或阿霉素的添加与否及其添加浓度也是应用遗传控制技术防治害虫需要考虑的重要因素(Scheteligetal.,2009a; Schetelig & Handler,2012a)。果蝇等昆虫由于母体传递效应,幼虫期不添加四环素,致死基因也不会发挥致死作用,但地中海实蝇中母体的残余四环素则不起阻抑作用。四环素和阿霉素的浓度对昆虫生长发育等也有重要影响,而且也是饲养成本的重要指标。因此,优化饲养条件对致死基因发挥效应十分重要。

启动子区段的完整与否对tTA表达的驱动及致死效率有显著影响,而且同物种来源的启动子驱动效率更高,环境安全性也更高(Scheteligetal.,2009a; Schetelig & Handler,2012a)。外源基因在基因组的转化插入位点同样影响致死效率,并且在不同物种表现的作用规律也不相同(Horn & Wimmer,2002; Scheteligetal.,2009a)。因此,近年来产生的位点特异的遗传修饰技术的应用可能会收到更好的控制效率,其安全性也更高(Scheteligetal.,2009b、2011a)。此外,致死基因的表达及作用发挥受到各因素的多层次调控,仅仅依靠生物信息学的分析和现有研究无法做出完整的判断。一方面,如果蝇、加勒比按实蝇和橘小实蝇的hid蛋白均存在若干个MAPK磷酸化活性位点,其活性会受到多种转录因子和microRNA的调控(Bergmannetal.,2002; Brenneckeetal.,2003; Tanaka-Matakatsuetal.,2009);另一方面,高水平表达的tTA则可能干扰依赖泛素的蛋白质降解,导致细胞凋亡,也被广泛用作害虫遗传控制的致死基因。但其物种专一性不高,有可能通过载体转移影响其他物种(曾保胜等,2013)。所以,确保致死基因的物种特异性是害虫遗传控制必须考虑的前提。由于不同效应基因的致死作用和调控机理尚未完全明晰,因此深入研究特异性致死基因的凋亡机制和在不同物种中的兼容作用,将为害虫遗传防控提供更多的研究思路和手段。

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(责任编辑:郭莹)

Sciences,Beijing100193,China;2Agricultural Office of Dabai Sub-district Administration, Tianjin 301802, China;3Department

ofEntomology,NorthCarolinaStateUniversity,Raleigh,NC,USA, 27606;4Genetic Engineering and Society Center

andW.M.KeckCenterforBehavioralBiology,NorthCarolinaStateUniversity,Raleigh,NC,USA, 27606;

5Guangdong Entomological Institute, Guangzhou, Guangdong 510260, China;6College of Agriculture

andPlantProtection,QingdaoAgriculturalUniversity,Qingdao,Shandong266109,China

Abstract:The sterile insect technique (SIT) is a species-specific, environment-friendly, and highly effective tool for insect pest control. Currently, release of insects carrying a dominant lethal gene (RIDL) is one of the most studied techniques that were developed to enhance traditional SIT. A standard RIDL system includes a tet-off system, gene specific promoters, components involved in sex determination and the effective lethal genes. Specifically, selecting the proper lethal genes is a very important step that determines both efficiency and stability of RIDL strain. The lethal genes can be repressible, or specifically expressed in females, or directly cut the X chromosome, all of which way lead to repressible/embryo-specific/female-specific lethality of offspring. Here the studies and applications of cell death genes such as RHG family (reapr, hid, grim, michelobx), tetracycline transcriptional activator (tTA), Nipp1Dm and homing endonuclease genes (HEG) are reviewed. The function mechanism and application feature of certain lethal genes are discussed. More research on the structural character and regulation pathway of important lethal genes are needed to further understand cell apoptosis, as well as the development of new tools for genetic pest management.

Key words:sterile insect technique; lethal genes; cell death genes; tetracycline transcriptional activator; homing endonuclease genes

通讯作者*(Author for correspondence), E-mail: wanfanghao@caas.cn

作者简介:王玉生, 男, 硕士研究生。 研究方向: 入侵昆虫遗传控制。 E-mail: yushengwang01@163.com

基金项目:国家“973”计划项目(2009CB119200); 国家“十一五”科技支撑计划课题(2006BAD08A18); 农业部农作物病虫害疫情监测与防治项目(2003-2015); 中国农科院科技创新工程(2013-2015); 人力资源社会保障部2014年度留学人员科技活动择优资助项目

收稿日期(Received): 2014-12-10接受日期(Accepted): 2015-02-27

DOI:10. 3969/j.issn.2095-1787.2015.02.007