王玉生， 蔡玉音， 武　強(qiáng) ，嚴(yán)　盈,3,4， 張桂芬， 劉桂清,5， 萬方浩,6*

1中國農(nóng)業(yè)科學(xué)院植物保護(hù)研究所，植物病蟲害生物學(xué)國家重點(diǎn)實(shí)驗(yàn)室，北京 100193； 2天津市寶坻區(qū)

大白街道辦事處農(nóng)業(yè)辦公室，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廣東省昆蟲研究所，廣東 廣州 510260； 6青島農(nóng)業(yè)大學(xué)

農(nóng)學(xué)與植物保護(hù)學(xué)院，山東 青島 266109

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害蟲遺傳不育技術(shù)中致死基因的研究與應(yīng)用

王玉生1+， 蔡玉音1,2+， 武強(qiáng)1，嚴(yán)盈1,3,4， 張桂芬1， 劉桂清1,5， 萬方浩1,6*

1中國農(nóng)業(yè)科學(xué)院植物保護(hù)研究所，植物病蟲害生物學(xué)國家重點(diǎn)實(shí)驗(yàn)室，北京 100193；2天津市寶坻區(qū)

大白街道辦事處農(nóng)業(yè)辦公室，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廣東省昆蟲研究所，廣東 廣州 510260；6青島農(nóng)業(yè)大學(xué)

農(nóng)學(xué)與植物保護(hù)學(xué)院，山東 青島 266109

摘要:基于遺傳修飾手段的昆蟲不育技術(shù)(SIT)作為一類物種特異、環(huán)境友好、科學(xué)高效的新興策略，在害蟲防治中具有廣闊的應(yīng)用前景。釋放攜帶顯性致死基因昆蟲的技術(shù)(RIDL)是改進(jìn)傳統(tǒng)SIT的重要手段之一，主要包括四環(huán)素調(diào)控系統(tǒng)、特異性啟動(dòng)子、性別特異剪接系統(tǒng)和特異性致死基因等重要元件，其中根據(jù)不同昆蟲的特點(diǎn)選擇合適的特異性致死基因?qū)τ跇?gòu)建遺傳不育品系至關(guān)重要。這些致死基因或受到阻遏調(diào)控系統(tǒng)的控制、或特異的在雌蟲中表達(dá)、亦或直接作用于X染色體，導(dǎo)致后代在特定發(fā)育階段或特定性別中條件致死。本文綜述了RHG家族(reapr、hid、grim、michelobx)細(xì)胞凋亡基因、轉(zhuǎn)錄激活因子tTA及Nipp1Dm、歸巢內(nèi)切酶基因等在害蟲遺傳不育技術(shù)中的研究和應(yīng)用，討論了特定致死基因的效應(yīng)機(jī)理和應(yīng)用特點(diǎn)，并對(duì)其可能的發(fā)展方向進(jìn)行了展望。由于不同效應(yīng)基因的致死作用和調(diào)控機(jī)理尚未完全明晰，因此深入研究特異致死基因的凋亡機(jī)制和在不同物種中的兼容作用，將為害蟲遺傳防控提供更多的研究思路和手段。

關(guān)鍵詞:昆蟲不育技術(shù); 致死基因; 細(xì)胞凋亡基因; 轉(zhuǎn)錄激活因子; 歸巢內(nèi)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

近年來，昆蟲遺傳修飾技術(shù)的飛速發(fā)展為農(nóng)林害蟲和衛(wèi)生害蟲的防治提供了更為新穎有效的思路，特別是與昆蟲不育技術(shù)(Sterile insect technique,SIT)相結(jié)合,具有環(huán)境友好、物種特異和防治高效等優(yōu)點(diǎn)，彌補(bǔ)了傳統(tǒng)SIT技術(shù)的缺點(diǎn)，在害蟲防治方面取得了一系列進(jìn)展，形成了以釋放攜帶顯性致死基因昆蟲(Release of insects carrying a dominant lethal,RIDL)技術(shù)為代表的害蟲遺傳控制新策略。RIDL通過向害蟲種群中引入攜帶致死基因純合子的昆蟲，在與野生型昆蟲交配后產(chǎn)生的后代中，這些致死基因或受到阻遏調(diào)控系統(tǒng)的控制、或特異地在雌蟲中表達(dá)、亦或直接作用于X染色體，導(dǎo)致后代在特定條件/發(fā)育階段/性別中致死，從而引起害蟲種群瓦解，達(dá)到高效控制重大農(nóng)林害蟲的目的。構(gòu)建一個(gè)成功的RIDL品系需要特異的調(diào)控元件、特異的致死基因和高效的遺傳轉(zhuǎn)化系統(tǒng)等關(guān)鍵因子。

基于四環(huán)素系統(tǒng)(tet-off)調(diào)控致死基因的表達(dá)以實(shí)現(xiàn)對(duì)靶標(biāo)昆蟲的特異性致死，是目前RIDL品系構(gòu)建中最常用的昆蟲遺傳調(diào)控系統(tǒng)。tet-off依據(jù)的原理是：大腸桿菌E.coli轉(zhuǎn)座子Tn10的四環(huán)素阻遏因子(Tetracycline repressor,tetR)與四環(huán)素結(jié)合，導(dǎo)致其不能阻抑四環(huán)素抗性操縱子(Tetracycline-resistance operon,tetO)，下游轉(zhuǎn)錄隨即啟動(dòng)(廖偉,1998)。Gossen & Bujard(1992)將tetR的部分序列與單純皰疹病毒VP16的轉(zhuǎn)錄活化區(qū)域組成四環(huán)素轉(zhuǎn)錄激活因子(Tetracycline transcriptional activator,tTA)，tTA與特異性啟動(dòng)子構(gòu)建為tet-off驅(qū)動(dòng)載體，將tetO與CMV啟動(dòng)子構(gòu)成四環(huán)素響應(yīng)元件(Tetracycline response element,TRE)，TRE與致死基因組合成效應(yīng)載體，由此便得到了tet-off雙元件系統(tǒng)。缺乏四環(huán)素時(shí)，tTA與tetO結(jié)合引發(fā)致死基因表達(dá)；四環(huán)素存在時(shí)，tTA與四環(huán)素結(jié)合而不與tetO結(jié)合，進(jìn)而無法激活下游基因的表達(dá)。

特異致死基因的篩選對(duì)于構(gòu)建昆蟲種群遺傳調(diào)控系統(tǒng)至關(guān)重要，這些基因通常需要滿足以下幾個(gè)特征：能被阻抑調(diào)控、能在特異性啟動(dòng)子作用下在特異性的組織/發(fā)育時(shí)期/性別中表達(dá)、表現(xiàn)為顯性致死。目前在害蟲遺傳調(diào)控技術(shù)中最常用的致死基因主要包括細(xì)胞凋亡基因(如reapr、hid、grim、michelob_x等RHG家族基因)、轉(zhuǎn)錄激活因子tTA、Nipp1Dm和歸巢內(nèi)切酶基因等，本文將分別討論這幾類致死基因的特點(diǎn)及其在不同物種中的應(yīng)用情況(表1)。

1　細(xì)胞凋亡相關(guān)基因

細(xì)胞凋亡(Apoptosis)即I型細(xì)胞程序性死亡(Programmed cell death,PCD)是由多層次嚴(yán)格調(diào)控的細(xì)胞自主有序死亡的過程，在機(jī)體的生命活動(dòng)中具有重要的生物學(xué)意義(Bakeevaetal.,2007; Kerretal.,1972; Wyllie,1980)，與多種疾病的發(fā)生密切相關(guān)(張金葉,2011; Vila & Przedborski,2003)。分子生物學(xué)的飛速發(fā)展使人們對(duì)細(xì)胞凋亡有了深入理解，細(xì)胞凋亡涉及一系列基因的激活、表達(dá)和調(diào)控等(Liuetal.,1996; Morizaneetal.,2005)，迄今其復(fù)雜的分子機(jī)理尚不完全明晰。

半胱氨酸天冬氨酸特異性蛋白酶(Cysteinyl asparate specific proteinase,Caspase)是細(xì)胞凋亡調(diào)控網(wǎng)絡(luò)的核心，分為起始和效應(yīng)2類。死亡受體和線粒體通路是依賴Caspase細(xì)胞凋亡的主要途徑(Juoetal.,1998; Lavriketal.,2005; Li & Yuan,2008)。哺乳動(dòng)物中線粒體釋放的細(xì)胞色素C(Cytochrome,CytC)與凋亡蛋白酶活化因子(Apoptosis protease activating factor 1,Apaf-1)結(jié)合，形成凋亡體，激活起始Caspase-9，進(jìn)而活化效應(yīng)Caspase-3和Caspase-7，啟動(dòng)Caspase級(jí)聯(lián)反應(yīng)(圖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，能阻斷CytC釋放，其baculovirus IAP repeat(BIR)結(jié)構(gòu)域與凋亡體小亞基結(jié)合從而抑制Caspase-9(LaCasseetal.,1998; Ryooetal.,2004; Yanetal.,2004)。但是當(dāng)細(xì)胞接受凋亡信號(hào)時(shí)，線粒體釋放的Smac等引起IAPs的泛素化，進(jìn)而阻抑IAPs對(duì)Caspase-9的活性抑制(Cashioetal.,2005)。模式生物黑腹果蠅Drosophilamelanogaster(Meigen)在細(xì)胞凋亡時(shí)，促凋亡基因RHG家族轉(zhuǎn)錄激活，引起凋亡蛋白抑制因子DIAP1的自我泛素化，解除其對(duì)Caspase-9同源物Dronc酶原的抑制作用，釋放的Dronc則與Apaf-1的同源物Dark(Hawkinsetal., 2000; Lee & Baehrecke, 2000)結(jié)合活化，進(jìn)而激活與Caspase-3，7功能相似的Drice和Dcp-1,細(xì)胞開始凋亡(Aramaetal.,2006; Salvesen & Duckett,2002; Scheteligetal.,2011b; Steller,2008; Taitetal.,2004)。該過程與哺乳動(dòng)物Smac等阻抑IAPs對(duì)Caspase-9的活性抑制類似，與哺乳動(dòng)物不同的是，果蠅中Apaf-1的同源物Dark的活化不需要CytC參與(Dorstynetal.,2002; Jiang & Wang,2000; Lietal.,1997; Varkeyetal.,1999)。RHG家族的reaper、hid和grim基因分別控制不同的信號(hào)通路(Danial & Korsmeyer,2004)，是果蠅細(xì)胞凋亡最主要的調(diào)控因子，與凋亡抑制蛋白協(xié)調(diào)作用共同決定細(xì)胞凋亡(圖1)。

表1　昆蟲遺傳控制中的致死基因

1.1　hid基因

頭部退化缺陷基因(Head involution defective,hid)，又稱wrinkled(W)基因。果蠅的reaper、hid、grim基因均位于其3號(hào)染色體的75CI1, 2基因座中(Whiteetal.,1994)。Gretheretal.(1995)通過克隆得到了果蠅hid基因，并對(duì)hid的生物信息學(xué)及其在細(xì)胞凋亡過程中的功能等進(jìn)行了詳細(xì)研究。結(jié)果表明，果蠅hid蛋白由410個(gè)氨基酸殘基組成，其N-末端有一段RHG家族蛋白相對(duì)保守的IAP-binding-motif(IBM)結(jié)構(gòu)域，能夠與果蠅凋亡蛋白抑制因子DIAP1等中的BIR結(jié)構(gòu)域特異結(jié)合，解除其對(duì)Caspase的阻抑(Yin & Thummel,2004; Zachariouetal.,2003)。Caspase誘導(dǎo)的細(xì)胞開始凋亡，是hid特異性誘導(dǎo)凋亡的必要條件(Scheteligetal.,2011b)；同時(shí)N-末端存在grim helix 3(GH3)基序，有助于hid在線粒體的定位，使其在細(xì)胞凋亡中發(fā)揮輔助作用，與IBM協(xié)同促進(jìn)細(xì)胞凋亡；IBM和GH3的共同存在使細(xì)胞凋亡效果明顯提高(Bryantetal.,2009)。研究證實(shí)，加勒比按實(shí)蠅Anastrephasuspensa(Loew)和果蠅的IBM、GH3缺失，能大幅降低細(xì)胞凋亡效果(Scheteligetal.,2011b)。hid蛋白還是絲裂原激活的蛋白激酶信號(hào)通路(Mitogen-activated protein kinase,MAPK)的靶分子，果蠅和加勒比按實(shí)蠅的hid蛋白有若干個(gè)MAPK磷酸化活性位點(diǎn)，MAPK可能通過yan和pnt等轉(zhuǎn)錄調(diào)節(jié)因子抑制hid的轉(zhuǎn)錄，阻斷下游細(xì)胞凋亡(Bergmannetal.,1998、 2002)。將果蠅hid基因的5個(gè)磷酸化位點(diǎn)突變后，DmhidAla5比沒有突變的hid基因的致死效率高(Horn & Wimmer,2002)。此外，microRNA和轉(zhuǎn)錄因子E2F能分別與果蠅hid的3′UTR和5′增強(qiáng)子區(qū)域結(jié)合，調(diào)節(jié)其活性(Brenneckeetal.,2003; Tanaka-Matakatsuetal.,2009)。

Gretheretal.(1995)研究發(fā)現(xiàn)，hid突變體胚胎中的細(xì)胞凋亡明顯減少，導(dǎo)致中樞神經(jīng)系統(tǒng)產(chǎn)生大量額外細(xì)胞；用hsp70的啟動(dòng)子表達(dá)hid時(shí)發(fā)現(xiàn)，hid能誘導(dǎo)野生型和hid缺陷的H99果蠅的胚胎細(xì)胞凋亡，且在胚胎細(xì)胞凋亡處能檢測(cè)到其mRNA。在眼部特異性啟動(dòng)子的作用下，pGMR-hid在果蠅視網(wǎng)膜處能異位表達(dá)并導(dǎo)致眼睛消融，而凋亡抑制蛋白p-35的共表達(dá)則抑制hid基因誘導(dǎo)的凋亡。hid基因表達(dá)分析表明，在果蠅幼蟲成熟過程中hid表達(dá)上調(diào)，使唾液腺等組織細(xì)胞迅速凋亡，形態(tài)發(fā)生變化(Yin & Thummel,2004)；然而，由于細(xì)胞凋亡在昆蟲生命活動(dòng)中起重要作用，因此hid基因在成蟲期也會(huì)有一定水平的穩(wěn)定表達(dá)；而在高日齡的果蠅成蟲中hid基因表達(dá)量的上升，可能與hid基因在果蠅衰老中發(fā)揮作用有關(guān)(Zhengetal.,2005)。此外，Bergmannetal.(1998)發(fā)現(xiàn)，hid不僅在果蠅注定死亡的細(xì)胞中表達(dá)，在部分正常存活的細(xì)胞中也有表達(dá)，而同屬RHG家族的reaper和grim則僅在將要死亡的細(xì)胞中表達(dá)，這可能與RHG家族的不同基因控制不同的信號(hào)通路有關(guān)(Danial & Korsmeyer,2004)。

圖1　哺乳動(dòng)物和果蠅細(xì)胞凋亡途徑(Cashio et al.,2005)

除果蠅外，hid基因已在加勒比按實(shí)蠅(Scheteligetal.,2011b)和橘小實(shí)蠅BactroceradorsalisHendel(蔡玉音等, 2014)等昆蟲中克隆得到，并進(jìn)行了生物信息學(xué)與表達(dá)分析，推導(dǎo)的氨基酸序列與果蠅hid蛋白有較高的相似性，這表明昆蟲hid蛋白存在一定的保守性，且均具有RHG蛋白家族特異的IBM和GH3結(jié)構(gòu)域。Scheteligetal.(2011b)和蔡玉音等(2014)研究發(fā)現(xiàn)，加勒比按實(shí)蠅和橘小實(shí)蠅幼蟲化蛹過程中，在蛻皮激素誘導(dǎo)下hid基因的表達(dá)迅速上調(diào)，這與果蠅相關(guān)研究結(jié)果類似，即在此階段hid基因誘導(dǎo)細(xì)胞迅速更新，進(jìn)而完成“變態(tài)”發(fā)育。此外，Scheteligetal.(2011b)還驗(yàn)證了加勒比按實(shí)蠅As-hid基因?qū)永毡劝磳?shí)蠅胚胎AsE01和果蠅S2細(xì)胞系的促凋亡能力，并發(fā)現(xiàn)，同物種來源的hid基因在細(xì)胞系水平的致死效果優(yōu)于異種hid基因。

目前，應(yīng)用hid基因進(jìn)行RIDL品系的構(gòu)建已經(jīng)取得了長足進(jìn)展。Horn & Wimmer(2002)基于tet-off調(diào)控系統(tǒng)和piggyBac轉(zhuǎn)座子基因驅(qū)動(dòng)系統(tǒng)，通過驅(qū)動(dòng)載體中細(xì)胞囊胚期特異性表達(dá)的serendipityα(sryα)及nullo的啟動(dòng)子/增強(qiáng)子，調(diào)控效應(yīng)載體中磷酸化位點(diǎn)突變的DmhidAla5基因表達(dá)，得到了果蠅胚胎條件致死品系，該品系在地中海實(shí)蠅Ceratitiscapitata(Wiedenmann)和加勒比按實(shí)蠅上(Scheteligetal.,2009a; Schetelig & Handler,2012a)也成功構(gòu)建。實(shí)驗(yàn)室飼養(yǎng)時(shí)，添加四環(huán)素能阻抑致死基因的表達(dá)，釋放的不育雄蟲與野生雌蟲交配后產(chǎn)生的子代由于缺乏四環(huán)素，均在胚胎期死亡。在此基礎(chǔ)上，若將雌性特異剪接的內(nèi)含子片段插入到效應(yīng)載體以驅(qū)動(dòng)hid在雌蟲的特異表達(dá)，將獲得雌性胚胎特異致死品系。目前，已在加勒比按實(shí)蠅(Schetelig & Handler,2012b)和地中海實(shí)蠅(Ogaugwuetal.,2013)中獲得成功，其中地中海實(shí)蠅純合子品系與野生型交配后的雌性后代全部在卵期死亡，獲得的單一雄性RIDL品系與SIT結(jié)合將有助于實(shí)現(xiàn)目標(biāo)害蟲的有效防控。

1.2　reaper基因

果蠅3號(hào)染色體的75CI1,2區(qū)段為其胚胎凋亡所必需，該區(qū)段的缺失將抑制細(xì)胞凋亡的產(chǎn)生，reaper基因是在該區(qū)段克隆的第一個(gè)細(xì)胞凋亡調(diào)控基因(Whiteetal.,1994)。目前，reaper基因已在黑腹果蠅(Whiteetal.,1994)、家蠶BombyxmoriL.(Bryantetal.,2009)、加勒比按實(shí)蠅(Scheteligetal.,2011b)和銅綠蠅Luciliacuprina(Gmelin.)(Chenetal.,2004)等昆蟲中克隆得到，并對(duì)其進(jìn)行了功能研究。reaper基因缺失的果蠅胚胎與hid基因缺陷時(shí)類似，細(xì)胞凋亡明顯受抑，中樞神經(jīng)系統(tǒng)也有大量額外細(xì)胞產(chǎn)生，reaper基因表達(dá)則恢復(fù)細(xì)胞凋亡(Whiteetal.,1994)。在野生型胚胎中，reaper基因在注定死亡的細(xì)胞中特異表達(dá)，與hid基因在部分正常細(xì)胞中也有所表達(dá)不同(Bergmannetal.,1998)。此外，reaper基因在眼部的異位表達(dá)也具有與hid基因類似的結(jié)果，能導(dǎo)致眼睛消融。凋亡抑制蛋白p-35蛋白的共表達(dá)則抑制reaper基因的活性(Chenetal.,1996; Gretheretal.,1995)。同時(shí)，reaper基因還參與外界因素誘導(dǎo)的細(xì)胞凋亡，X射線、紫外線和蛻皮激素等都能刺激果蠅中reaper基因的表達(dá)(Liuetal.,2011; Zhangetal.,2008)。

reaper蛋白不僅能阻抑果蠅DIAP1等凋亡抑制蛋白對(duì)細(xì)胞凋亡的抑制能力，還可激活DIAP1的泛素化，降解或抑制DIAP1的翻譯(Holleyetal.,2002)，使DIAP1不能阻抑Caspase凋亡。據(jù)報(bào)道，果蠅的Morgue、UbcD1和UbcD2等泛素連接酶與reaper介導(dǎo)的DIAP1等的泛素化有關(guān)(Ryooetal.,2002; Wingetal.,2002)。也有研究發(fā)現(xiàn)，由于物種間的reaper存在一定保守性，果蠅的reaper能導(dǎo)致哺乳動(dòng)物細(xì)胞凋亡(Abrams,1999)，也能與非洲爪蟾屬Xenopus的Scythe蛋白作用，而果蠅體內(nèi)也存在Scythe類似物，表明reaper可能介導(dǎo)Scythe參與的CytC凋亡途徑(Thressetal.,1998)。

果蠅reaper基因編碼的蛋白質(zhì)包括65個(gè)氨基酸殘基(Whiteetal.,1996)，與hid蛋白的氨基酸序列相似性較低，但其N-末端也具有RHG家族蛋白相對(duì)保守的IBM結(jié)構(gòu)域，該結(jié)構(gòu)域與reaper蛋白對(duì)IAP活性的抑制密切相關(guān)(Yin & Thummel,2004; Zachariouetal.,2003)，果蠅、加勒比按實(shí)蠅和銅綠蠅reaper蛋白IBM結(jié)構(gòu)域的缺失能大幅影響reaper的促凋亡活性(Chenetal.,2004; Scheteligetal.,2011b; Zhouetal.,2005)。reaper的GH3結(jié)構(gòu)域?yàn)槠湓诰€粒體中定位所必需，在DIAP1的泛素化降解中也發(fā)揮重要作用(Olsonetal.,2003)，保證了IBM缺失時(shí)對(duì)細(xì)胞凋亡的誘導(dǎo)，也能誘導(dǎo)CytC釋放并促使細(xì)胞凋亡(Freeletal.,2008)。此外，Sanduetal.(2010)和Scheteligetal.(2011b)研究發(fā)現(xiàn)，果蠅和加勒比按實(shí)蠅的reaper蛋白均具有一個(gè)中央螺旋結(jié)構(gòu)域，該結(jié)構(gòu)域在reaper與hid的互作過程中發(fā)揮重要作用，hid和reaper的結(jié)合使得reaper能更穩(wěn)固于線粒體上，而hid與grim則不具有類似結(jié)構(gòu)(蔡玉音等,2014; Scheteligetal.,2011b)，reaper與hid的另一區(qū)別在于reaper的活性不受MAPK途徑的抑制。

reaper與hid基因的功能還具有諸多相似之處，在幼蟲化蛹時(shí)reaper的表達(dá)上調(diào)，促使組織細(xì)胞迅速更新?lián)Q代，完成“幼蟲—蛹”的變化。reaper基因的表達(dá)也能誘導(dǎo)正常細(xì)胞的凋亡，如Scheteligetal.(2011b)研究發(fā)現(xiàn)，果蠅和加勒比按實(shí)蠅的reaper基因能誘導(dǎo)加勒比按實(shí)蠅胚胎AsE01和果蠅S2細(xì)胞系的凋亡，且與hid基因作用結(jié)果相似，即同源物種來源的reaper基因的致死效果優(yōu)于異種reaper基因。進(jìn)一步研究還發(fā)現(xiàn)，reaper和hid共表達(dá)促凋亡的作用更明顯(Scheteligetal.,2011b; Yooetal.,2002)，表明reaper可能與hid控制不同的凋亡途徑，二者之間協(xié)同作用，共同調(diào)控細(xì)胞凋亡，進(jìn)而推測(cè)reaper和hid的聯(lián)合應(yīng)用可能會(huì)取得更好的遺傳調(diào)控效果。

1.3　grim基因

grim基因同樣位于果蠅3號(hào)染色體75C1,2這一橫跨約300 kb的區(qū)段上，grim位于reaper和hid之間。grim基因與RHG家族的hid、reaper基因功能類似，其表達(dá)也能恢復(fù)H99缺陷型胚胎細(xì)胞凋亡，且凋亡處能明顯檢測(cè)到該基因的表達(dá)(Chenetal.,1996)。grim在果蠅胚胎中的表達(dá)模式與reaper、hid的表達(dá)相似，與果蠅胚胎發(fā)育過程中細(xì)胞凋亡的進(jìn)程統(tǒng)一，不過在早期胚胎細(xì)胞凋亡時(shí)能檢測(cè)到grim表達(dá)，但reaper則無；此外，grim與reaper一樣僅在將要死亡的細(xì)胞中表達(dá)(Bergmannetal.,1998; Chenetal.,1996)。grim基因在眼部特異性啟動(dòng)子的誘導(dǎo)下的異位表達(dá)也能導(dǎo)致果蠅眼睛的消融，在細(xì)胞系水平的研究也證實(shí)了grim基因的功能，其對(duì)細(xì)胞凋亡的誘導(dǎo)不需要reaper和hid基因的參與，且對(duì)細(xì)胞凋亡的誘導(dǎo)受到共表達(dá)的p-35蛋白的抑制(Chenetal.,1996; Clem,2007)。據(jù)Chenetal.(1996)推測(cè)，grim同hid和reaper基因一樣，對(duì)凋亡的調(diào)控位于p-35上游，grim、reaper和hid可能分別參與多個(gè)調(diào)控途徑，以及同一下游凋亡途徑，下游途徑受到p-35蛋白的抑制。

果蠅grim編碼的蛋白質(zhì)序列有138個(gè)氨基酸殘基，具有RHG蛋白家族保守的IBM和GH3結(jié)構(gòu)域，其N-末端IBM結(jié)構(gòu)域發(fā)揮保守的抑制凋亡蛋白抑制因子DIAP1等活性的功能。其GH3結(jié)構(gòu)域與reaper蛋白的相似性較高，功能也與reaper的GH3類似：輔助其在線粒體的定位，激活凋亡蛋白抑制因子DIAP1等的降解(Olsonetal.,2003)，從而解除DIAP1對(duì)Dronc等Caspase的抑制，導(dǎo)致細(xì)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，其推導(dǎo)的氨基酸序列與果蠅RHG家族蛋白質(zhì)整體序列相似度較低，但是已報(bào)道的岡比亞按蚊Anophelesgambiae(L.)(Zhouetal.,2005)、埃及伊蚊Aedesaegypti(L.)(Zhouetal.,2005)、致倦庫蚊CulexquinquefasciatusSay(Liuetal.,2011)和三帶喙庫蚊CulextritaeniorhynchusGiles(徐瑤,2012)的michelob_x編碼的蛋白同RHG蛋白一樣，具有較為保守的N-端IBM結(jié)構(gòu)域并可歸入RHG家族，為reaper在蚊蟲中的同源基因，不過michelob_x蛋白不具有GH3結(jié)構(gòu)域(徐瑤,2012; Liuetal.,2011; Zhouetal.,2005)。

岡比亞按蚊、埃及伊蚊、致倦庫蚊和三帶喙庫蚊中的michelob_x基因在果蠅的S2細(xì)胞或白紋伊蚊Aedesalbopictus(Skuse)的C6/36細(xì)胞中的表達(dá)都能誘導(dǎo)細(xì)胞快速調(diào)亡(徐瑤,2012; Liuetal.,2011; Zhouetal.,2005)。埃及伊蚊michelob_x蛋白的促凋亡活性依賴其N-末端的IBM結(jié)構(gòu)域，IBM結(jié)構(gòu)域的功能及促凋亡機(jī)制類似于果蠅RHG蛋白家族(Bryantetal.,2008; Liuetal.,2011; Wangetal.,2008)，能夠移除凋亡抑制蛋白AeIAP1對(duì)Caspase-9同源的AeDronc的阻抑，活化的AeDronc與AeArk結(jié)合，激活Caspase-16，引發(fā)凋亡(圖2A； Ocampoetal.,2013)。當(dāng)IBM缺失時(shí)，michelob_x蛋白的促凋亡活性也喪失，這可能與michelob_x蛋白不具有GH3結(jié)構(gòu)域的凋亡輔助作用有關(guān)(徐瑤,2012; Liuetal.,2011; Zhouetal.,2005)。此外，michelob_x在蚊蟲發(fā)育和紫外照射(Zhouetal.,2005)、病毒感染(Liuetal.,2011; Wangetal.,2008)等外因?qū)е碌募?xì)胞凋亡中起中樞調(diào)節(jié)作用。將含有埃及伊蚊michelob_x的重組SINVs病毒感染埃及伊蚊C6/36細(xì)胞系時(shí)，michelob_x能引起細(xì)胞大量凋亡，阻抑病毒持續(xù)性裂解導(dǎo)致的感染(Wangetal.,2008)。michelob_x在桿狀病毒CuniNPV引起的致倦庫蚊和埃及伊蚊細(xì)胞凋亡中也發(fā)揮重要作用，其表達(dá)與病毒感染導(dǎo)致的細(xì)胞凋亡進(jìn)程協(xié)調(diào)(Liuetal.,2011)。michelob_x參與蚊蟲病毒感染誘導(dǎo)的免疫反應(yīng)，與凋亡抑制蛋白p-35或IAP的共表達(dá)則抑制michelob_x引起的Caspase反應(yīng)(Liuetal.,2011; Wangetal.,2008; Zhouetal.,2005)。

細(xì)胞凋亡過程涉及一系列機(jī)理尚未完全明晰的基因的相互作用，并與其他代謝途徑交互作用形成復(fù)雜的代謝調(diào)控網(wǎng)絡(luò)，以維持機(jī)體組織穩(wěn)態(tài)及正常生命活動(dòng)。研究發(fā)現(xiàn)，細(xì)胞凋亡在昆蟲防御病毒等病原體感染過程中發(fā)揮重要作用(Clarke & Clem,2003; Zhouetal.,2005)。除michelob_x外，昆蟲體內(nèi)的另外一種起始Caspase Dredd也參與此過程(圖2B； Ocampoetal.,2013)，果蠅和埃及伊蚊的Dredd均具有2個(gè)死亡結(jié)構(gòu)域(Death domain，DD)，當(dāng)接受死亡信號(hào)后與其配體FADD(Fas associated death domain containing protein)通過DD結(jié)構(gòu)域結(jié)合，多聚化形成死亡誘導(dǎo)信號(hào)復(fù)合體(Death-inducing signaling complexes，DISCs)，活化Dredd，進(jìn)而開啟下游細(xì)胞的凋亡。雖然果蠅和埃及伊蚊的Dredd均因誘導(dǎo)細(xì)胞凋亡作用而被發(fā)現(xiàn)，但近年來的研究表明，Dredd主要在機(jī)體免疫途徑發(fā)揮作用，但其機(jī)理尚未完全明晰。

圖2　埃及伊蚊細(xì)胞凋亡途徑及其參與免疫反應(yīng)(Ocampo et al.,2013)

michelob_x基因在登革熱的主要媒介昆蟲埃及伊蚊的遺傳控制研究中已經(jīng)取得了顯著進(jìn)展。Fuetal.(2010)采用基于tet-off調(diào)控系統(tǒng)的昆蟲遺傳控制技術(shù)，利用在雌蚊間接飛行肌特異性表達(dá)的肌動(dòng)蛋白AeAct-4基因的啟動(dòng)子調(diào)控促凋亡基因michelob_x的表達(dá)，獲得了無翅型不育雌蚊，后代雌蚊中65.8%~98.3%的個(gè)體為無翅型，雄蚊則無該表型，這一研究為醫(yī)學(xué)媒介蚊蟲的遺傳控制提供了新思路。

2　轉(zhuǎn)錄激活因子tTA的致死效應(yīng)

研究表明，低水平表達(dá)的tTA蛋白對(duì)細(xì)胞無害，高水平表達(dá)的tTA對(duì)細(xì)胞則有毒害作用，該毒害作用可能是由于高水平的tTA干擾依賴泛素的蛋白質(zhì)降解所致。Gongetal.(2005)利用高表達(dá)的tTA對(duì)細(xì)胞的毒性作用，將tet-off簡(jiǎn)化為單元件系統(tǒng)，tTA除發(fā)揮轉(zhuǎn)錄激活因子功能外，還發(fā)揮致死基因功能：存在四環(huán)素時(shí)，tTA的表達(dá)被抑制，低水平的tTA對(duì)昆蟲無害；不存在四環(huán)素時(shí)，tTA與tetO結(jié)合，進(jìn)一步促進(jìn)tTA的表達(dá)，高水平累積的tTA導(dǎo)致昆蟲死亡。利用該單元件系統(tǒng)，目前已構(gòu)建了地中海實(shí)蠅Ceratitiscapitata(Wiedenmann)(Gongetal.,2005)、埃及伊蚊(Phucetal.,2007)和棉紅鈴蟲Pectinophoragossypiella(Saunders)(Morrisonetal.,2012)等害蟲的胚胎條件致死品系。其中Phucetal.(2007)構(gòu)建的埃及伊蚊RIDL品系OX513A已經(jīng)實(shí)現(xiàn)了田間開放條件的釋放研究(Harrisetal.,2011、 2012)。Fuetal.(2007)基于tet-off單元件系統(tǒng)，將地中海實(shí)蠅雌性特異內(nèi)含子片段插入tTA編碼區(qū)，構(gòu)建了雌性特異致死品系。不含四環(huán)素時(shí)，選擇性剪切內(nèi)含子在雌蟲體內(nèi)調(diào)控下游tTA蛋白高表達(dá)，雄蟲則不表達(dá)，從而導(dǎo)致雌蟲死亡，由此獲得的雄蟲可以與SIT技術(shù)相結(jié)合，并進(jìn)行野外釋放。transformer和doublesex等雌性特異剪切基因的深入研究，使得雌性特異致死品系取得了廣泛成功，目前，小菜蛾P(guān)lutellaxylostellaL.、棉紅鈴蟲(Jinetal.,2013)、橄欖實(shí)蠅Bactroceraoleae(Gmelin.)(Antetal.,2012)、家蠶(Tanetal.,2013)和銅綠蠅(Lietal.,2014)的相關(guān)品系已被成功構(gòu)建。此外，F(xiàn)uetal.(2010)和Labbéetal.(2012)分別用雌蚊飛行肌特異性啟動(dòng)子Act-4驅(qū)動(dòng)單元件系統(tǒng)中tTA的高表達(dá)，得到了埃及伊蚊和白紋伊蚊的雌蚊無翅型品系。

3　其他致死基因

3.1　Nipp1Dm基因

Nipp1(Nuclear inhibitor of protein phosphatase type 1)是細(xì)胞核內(nèi)Ⅰ型(絲氨酸/蘇氨酸型)蛋白磷酸酶(Protein phosphatase type 1,PP1)的抑制劑(Parkeretal.,2002)。PP1能與許多蛋白質(zhì)形成復(fù)合體輔助其進(jìn)行亞細(xì)胞定位，調(diào)控蛋白在特異性部位發(fā)揮功能，而且與細(xì)胞周期、糖原代謝和RNA合成等功能密切相關(guān)(Linetal.,1999)。果蠅Nipp1Dm基因位于果蠅2號(hào)染色體53F4-5區(qū)段，Nipp1Dm蛋白具有多個(gè)功能性結(jié)構(gòu)域，其保守的中心RVXF結(jié)構(gòu)域能專一性地結(jié)合PP1，抑制PP1的活性，導(dǎo)致細(xì)胞死亡，C-端結(jié)構(gòu)域有RNA結(jié)合功能，且Nipp1定位于核小點(diǎn)(Nuclear speckles)(Parkeretal.,2002)。

Parkeretal.(2002)分析了Nipp1Dm在果蠅胚胎、幼蟲頭部和成蟲器官芽的表達(dá)模式，發(fā)現(xiàn)其在各發(fā)育階段均表達(dá)，異位表達(dá)將導(dǎo)致有絲分裂異常、細(xì)胞凋亡、肌肉不能正常發(fā)育、翅發(fā)育異常和子代不育等(Bennettetal.,2003; Parkeretal.,2002)，上述現(xiàn)象同時(shí)也受到了PP1共表達(dá)的抑制。

Nipp1Dm基因在蚊媒昆蟲的遺傳控制中也有一定的應(yīng)用，F(xiàn)uetal.(2010)和Marinottietal.(2013)分別在埃及伊蚊和斯氏按蚊Anophelesstephensi(Liston)中用同源的AeAct-4和AsAct-4啟動(dòng)子，以Nipp1Dm為致死基因，獲得了雌性后代無翅的RIDL品系。

3.2　homing endonuclease genes

對(duì)于XY染色體決定型的雙翅目昆蟲，歸巢內(nèi)切酶(Homingendonucleasegenes,HEG)也是其遺傳控制品系構(gòu)建的一個(gè)有應(yīng)用前景的致死基因。HEG屬于自私基因，能特異地識(shí)別染色體上的2段特定序列，并定向插入在這2段序列之間，當(dāng)同源染色體中的一條具有HEG時(shí)，HEG酶將切割另一條染色體，并以前者為模板進(jìn)行復(fù)制(Sinkins & Gould,2006)。由于homing endonuclease Ⅰ-PpoⅠ能高度特異的靶定與X染色體連鎖的28S核糖體基因的重復(fù)序列(Nolanetal.,2011)，如將HEG基因置于雄蟲精子發(fā)生時(shí)特異表達(dá)的β2tubulin控制下，在精子發(fā)生時(shí)切割X染色體，當(dāng)其轉(zhuǎn)入胚胎時(shí)還能切割母系來源的X染色體，導(dǎo)致后代雌蟲在胚胎期死亡，產(chǎn)生全為攜帶HEG的雄蟲(Windbichleretal.,2008)，從而大幅度改變后代的性別構(gòu)成，致使蚊群無法繼續(xù)繁衍。Windbichleretal.(2008)和Galizietal.(2014)利用HEG基因分別成功構(gòu)建了瘧疾的蚊媒岡比亞按蚊的RIDL不育品系。

4　總結(jié)與展望

目前，害蟲種群遺傳調(diào)控研究中，常用的致死基因中促凋亡基因占據(jù)了很大比例，但在細(xì)胞凋亡過程中，凋亡抑制因子和促凋亡基因協(xié)同作用，多層次共同調(diào)控細(xì)胞凋亡通路，迄今為止其復(fù)雜的分子機(jī)理尚不完全明晰。因此，需要加大對(duì)昆蟲特別是非模式物種的細(xì)胞凋亡基因研究，解析其細(xì)胞凋亡途徑及機(jī)理，該研究結(jié)果將為條件致死品系的獲得奠定基礎(chǔ)。此外，近年來歸巢內(nèi)切酶等的發(fā)現(xiàn)擴(kuò)展了對(duì)雙翅目昆蟲遺傳控制致死基因的理解，然而其機(jī)理尚需透徹剖析，在害蟲控制中的效果也需要更多的實(shí)踐研究。同時(shí)，遺傳控制品系在工廠大規(guī)模飼養(yǎng)時(shí)，致死基因效應(yīng)的阻抑物四環(huán)素或阿霉素的添加與否及其添加濃度也是應(yīng)用遺傳控制技術(shù)防治害蟲需要考慮的重要因素(Scheteligetal.,2009a; Schetelig & Handler,2012a)。果蠅等昆蟲由于母體傳遞效應(yīng)，幼蟲期不添加四環(huán)素，致死基因也不會(huì)發(fā)揮致死作用，但地中海實(shí)蠅中母體的殘余四環(huán)素則不起阻抑作用。四環(huán)素和阿霉素的濃度對(duì)昆蟲生長發(fā)育等也有重要影響，而且也是飼養(yǎng)成本的重要指標(biāo)。因此，優(yōu)化飼養(yǎng)條件對(duì)致死基因發(fā)揮效應(yīng)十分重要。

啟動(dòng)子區(qū)段的完整與否對(duì)tTA表達(dá)的驅(qū)動(dòng)及致死效率有顯著影響，而且同物種來源的啟動(dòng)子驅(qū)動(dòng)效率更高，環(huán)境安全性也更高(Scheteligetal.,2009a; Schetelig & Handler,2012a)。外源基因在基因組的轉(zhuǎn)化插入位點(diǎn)同樣影響致死效率，并且在不同物種表現(xiàn)的作用規(guī)律也不相同(Horn & Wimmer,2002; Scheteligetal.,2009a)。因此，近年來產(chǎn)生的位點(diǎn)特異的遺傳修飾技術(shù)的應(yīng)用可能會(huì)收到更好的控制效率，其安全性也更高(Scheteligetal.,2009b、2011a)。此外，致死基因的表達(dá)及作用發(fā)揮受到各因素的多層次調(diào)控，僅僅依靠生物信息學(xué)的分析和現(xiàn)有研究無法做出完整的判斷。一方面，如果蠅、加勒比按實(shí)蠅和橘小實(shí)蠅的hid蛋白均存在若干個(gè)MAPK磷酸化活性位點(diǎn)，其活性會(huì)受到多種轉(zhuǎn)錄因子和microRNA的調(diào)控(Bergmannetal.,2002; Brenneckeetal.,2003; Tanaka-Matakatsuetal.,2009)；另一方面，高水平表達(dá)的tTA則可能干擾依賴泛素的蛋白質(zhì)降解，導(dǎo)致細(xì)胞凋亡，也被廣泛用作害蟲遺傳控制的致死基因。但其物種專一性不高，有可能通過載體轉(zhuǎn)移影響其他物種(曾保勝等，2013)。所以，確保致死基因的物種特異性是害蟲遺傳控制必須考慮的前提。由于不同效應(yīng)基因的致死作用和調(diào)控機(jī)理尚未完全明晰，因此深入研究特異性致死基因的凋亡機(jī)制和在不同物種中的兼容作用，將為害蟲遺傳防控提供更多的研究思路和手段。

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(責(zé)任編輯:郭瑩)

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

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

作者簡(jiǎn)介:王玉生, 男, 碩士研究生。 研究方向： 入侵昆蟲遺傳控制。 E-mail: yushengwang01@163.com

基金項(xiàng)目:國家“973”計(jì)劃項(xiàng)目(2009CB119200)； 國家“十一五”科技支撐計(jì)劃課題(2006BAD08A18)； 農(nóng)業(yè)部農(nóng)作物病蟲害疫情監(jiān)測(cè)與防治項(xiàng)目(2003-2015)； 中國農(nóng)科院科技創(chuàng)新工程(2013-2015)； 人力資源社會(huì)保障部2014年度留學(xué)人員科技活動(dòng)擇優(yōu)資助項(xiàng)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