王玉生， 李建偉， 張桂芬， 嚴(yán)　盈,2，3， 李昕玥， 萬(wàn)方浩,4*

1中國(guó)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(guó)家重點(diǎn)實(shí)驗(yàn)室，北京 100193； 2Department of Entomology,

North Carolina State University, Campus Box 7613, Raleigh, NC 27695-7613, USA； 3Genetic Engineering

and Society Center and W. M. Keck Center for Behavioral Biology, North Carolina State University,

Raleigh, NC 27695-7613, USA; 4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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遺傳不育技術(shù)在蚊媒疾病防控中的應(yīng)用

王玉生1， 李建偉1， 張桂芬1， 嚴(yán)盈1,2，3， 李昕玥1， 萬(wàn)方浩1,4*

1中國(guó)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(guó)家重點(diǎn)實(shí)驗(yàn)室，北京 100193；2Department of Entomology,

North Carolina State University, Campus Box 7613, Raleigh, NC 27695-7613, USA；3Genetic Engineering

and Society Center and W. M. Keck Center for Behavioral Biology, North Carolina State University,

Raleigh, NC 27695-7613, USA;4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:瘧疾、登革熱等重大傳染性蚊媒疾病嚴(yán)重危害人類健康,且目前缺乏有效的藥物和疫苗，防治埃及伊蚊、岡比亞按蚊等媒介昆蟲是控制和消除這些疾病的有效手段?；瘜W(xué)殺蟲劑的大規(guī)模使用在一定程度上控制了疾病的傳播，但其抗藥性和環(huán)境污染等問(wèn)題也隨之而來(lái)。分子生物學(xué)的飛速發(fā)展為昆蟲不育技術(shù)(SIT)的更新及害蟲防治提供了新的策略，由此發(fā)展起來(lái)的以釋放攜帶顯性致死基因昆蟲(RIDL)為代表的一系列遺傳不育技術(shù)為蚊蟲種群防控提供了更加有效的選擇。本文概述了遺傳技術(shù)在蚊蟲防控中的應(yīng)用進(jìn)展，包括蚊蟲遺傳防治的歷史和策略，闡述了RIDL技術(shù)體系的原理，同時(shí)介紹了相關(guān)遺傳控制品系和已經(jīng)開展的田間釋放研究，展示了遺傳修飾不育技術(shù)在蚊媒疾病防治中的巨大潛力。

關(guān)鍵詞:蚊媒昆蟲； 遺傳防治； 昆蟲不育技術(shù)； 釋放攜帶顯性致死基因昆蟲的技術(shù)

Application of genetic pest management in the control

of mosquito-borne diseases

Yu-sheng WANG1, Jian-wei LI1, Gui-fen ZHANG1, Ying YAN1,2，3, Xin-yue LI1, Fang-hao WAN1,4*

瘧疾、登革熱、絲蟲病、黃熱病等以蚊蟲為媒介的重大傳染疾病嚴(yán)重威脅人類的健康，蚊媒防治是控制和消除這些疾病的有效手段。傳統(tǒng)的蚊媒防治以化學(xué)藥劑為主，但抗藥性以及化學(xué)藥劑對(duì)環(huán)境的污染和生態(tài)破壞等問(wèn)題日益嚴(yán)重。分子生物學(xué)的發(fā)展為蚊媒的防治提供了新的途徑，其中以昆蟲遺傳修飾技術(shù)與昆蟲不育技術(shù)(Sterile insect technique,SIT)(Knipling,1970)相結(jié)合發(fā)展起來(lái)的昆蟲遺傳修飾不育技術(shù)為害蟲防治提供了新的思路。通過(guò)在媒介種群中引入攜帶顯性致死基因或病原體抗性基因等害蟲控制效應(yīng)基因的人工品系，能夠有效降低目標(biāo)種群的數(shù)量或進(jìn)行種群替代，從而阻斷蚊媒對(duì)病原微生物的傳播(周秀娟等,2008; Catterucciaetal.,2009; Klassen,2009; Wilke & Marrelli,2012)。近年來(lái)，蚊媒昆蟲的遺傳修飾不育技術(shù)研究發(fā)展迅速，相關(guān)品系在世界各地被廣泛使用并取得了較好的效果，表明其在蚊媒疾病的防控中具有巨大的應(yīng)用潛力(Catterucciaetal.,2009; Wilke & Marrelli,2012)。

1　蚊蟲遺傳防治的歷史

SIT是指通過(guò)釋放輻照(Irradiation)等處理的雄蟲與野生型雌蟲交配，使其不育從而降低目標(biāo)昆蟲種群數(shù)量的一種害蟲控制技術(shù)(Knipling,1970)，其具有物種特異、環(huán)境友好、可工廠化生產(chǎn)、大面積控制等特點(diǎn)(Hendrichsetal.,2002)。SIT在蚊媒防治中的應(yīng)用已有較長(zhǎng)的歷史(表1； Benedict & Robinson,2003)。獲得不育昆蟲的手段除了輻照不育以外，還包括化學(xué)不育(Chemosterilization,Ch)、胞質(zhì)不親和性(Cytoplasmic incompatibility,CI)、雜交不育(Hybrid male sterility,Hy)、減數(shù)分裂驅(qū)動(dòng)(Meiotic drive)、染色體移位和重排(Chromosomal translocation and rearrangements,Tr)等，其中以輻照不育應(yīng)用最為廣泛(Benedict & Robinson,2003)。

表1　傳統(tǒng)SIT技術(shù)在蚊媒防治中的應(yīng)用(Benedict & Robinson,2003)

續(xù)表1

續(xù)表1

Ch.化學(xué)不育法；CI.細(xì)胞質(zhì)不親和性；Ga.γ射線；Hy.雜交不育；Ma.僅標(biāo)記(無(wú)不育處理)；Sg.分離失調(diào)；Tr.染色體易位和重排。

Ch. Chemosterilization; CI. Cytoplasmic incompatibility; Ga. Gamma irradiation; Hy. Hyhybrid male sterility; Ma. Marker only; Sg. Segregation distorter; Tr. Translocation and other chromosomal rearrangements.

2　蚊子遺傳防治的主要策略

2.1　種群壓制

種群壓制是通過(guò)降低一定區(qū)域內(nèi)目標(biāo)媒介蚊蟲種群數(shù)量來(lái)控制蚊媒疾病的傳播，該策略與化學(xué)藥劑的目的類似，但是避免了殺蟲劑抗性、殺傷非目標(biāo)昆蟲、環(huán)境污染等問(wèn)題，這一策略的主要代表手段包括傳統(tǒng)SIT、釋放攜帶顯性致死基因昆蟲的技術(shù)(Release of insects carrying a dominant lethal,RIDL)、X或常染色體連鎖的歸巢內(nèi)切酶基因(Homingendonucleasegene,HEG)系統(tǒng)、不相容昆蟲技術(shù)(Incompatible insect technique,IIT)、致死—拯救(Killer-Rescue)系統(tǒng)、多位點(diǎn)混合(Multi-locus assortment,MLA)等。在種群壓制策略中，必須通過(guò)周期性釋放來(lái)保證效應(yīng)基因在目標(biāo)種群中的擴(kuò)散和基因頻率的提高。SIT是蚊蟲種群壓制策略中應(yīng)用最廣泛的是害蟲防治手段之一(Alphey,2014)，但該技術(shù)也存在一些難以克服的缺點(diǎn)，特別是γ射線等在誘導(dǎo)雄蚊不育的同時(shí)降低了其野外適合度(Scolarietal.,2008; Thomasetal.,2000)。而基于轉(zhuǎn)座子活性和性別決定系統(tǒng)發(fā)展起來(lái)的RIDL和fsRIDL(female-specific RIDL)(Alphey,2014; Heinrich & Scott,2000; Thomasetal.,2000),能夠釋放攜帶條件致死基因純合子品系(Homozygous strains)的雄蚊，該純合子品系與野生型雌蚊交配后，雌性后代在特異致死基因作用下死亡，而雄性后代繼續(xù)攜帶致死基因與野生型雌蟲交配，引起目標(biāo)種群數(shù)量的減少，連續(xù)釋放后甚至能根除種群，從而阻斷蚊媒對(duì)病原微生物的傳播(Catterucciaetal.,2009; Klassen,2009; Wilke & Marrelli,2012)。與傳統(tǒng)SIT相比，RIDL對(duì)蚊蟲交配和野外生存適合度的損傷低，且免去了不育處理的環(huán)節(jié)，fsRIDL甚至不需要性別篩選，這為致死基因作用時(shí)間的選擇提供了更大的靈活性(Phucetal.,2007; Thomasetal.,2000)，大大節(jié)省了人力和物力，具有更高的遺傳控制效率(Alpheyetal.,2011; Blacketal.,2011)。

HEG驅(qū)動(dòng)系統(tǒng)主要利用HEG酶能夠定向識(shí)別插入在染色體上特定兩段DNA序列之間的特點(diǎn)，當(dāng)兩條同源染色體中一條具有HEG基因時(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，即homing現(xiàn)象(Sinkins & Gould,2006)。由于歸巢內(nèi)切酶I-PpoI對(duì)與X染色體連鎖的28S核糖體基因的重復(fù)序列高度特異性靶定(Nolanetal.,2011)，當(dāng)其與雄蟲X染色體靶定時(shí)，可以在精子發(fā)生過(guò)程中切割X染色體導(dǎo)致后代雌性不存活或不產(chǎn)生X型精子；與常染色體靶定時(shí)會(huì)導(dǎo)致fsRIDL；而當(dāng)其與Y連鎖則后代所有雄蚊均帶有該基因，從而在減數(shù)分裂驅(qū)動(dòng)下擴(kuò)散(Burt,2003; Catterucciaetal.,2005; Deredecetal.,2008; Windbichleretal.,2011)。 而IIT則利用了昆蟲內(nèi)共生菌Wolbachia的胞質(zhì)不相容性(Cytoplasmic incompatibility,CI)，將攜帶Wolbachia的雄蚊與不攜帶或攜帶不同類型Wolbachia的雌蚊交配，誘導(dǎo)產(chǎn)生胞質(zhì)不相容性，其后代在胚胎期死亡；而含同種類型Wolbachia的雌雄蚊交配產(chǎn)生的后代可正常發(fā)育并感染沃爾巴克氏體，從而使攜帶該Wolbachia的品系在野生種群中迅速擴(kuò)散，最終降低靶標(biāo)昆蟲的數(shù)量(Alphey,2014; Hancock & Godfray,2012; Laven,1967; O′Connoretal.,2012; Werrenetal.,2008; Zabalouetal.,2004、2009)。

2.2　種群替代

種群替代是指將能夠傳播病原物的蚊蟲品系替換為無(wú)法致病的品系(Alphey,2014)，其主要代表技術(shù)包括顯性不足(Underdominance,UD)技術(shù)、Wolbachia驅(qū)動(dòng)系統(tǒng)、Medea元件驅(qū)動(dòng)系統(tǒng)、轉(zhuǎn)座子(Transposons)轉(zhuǎn)化系統(tǒng)、Y染色體連鎖的HEG驅(qū)動(dòng)系統(tǒng)等。在種群替代策略中，效應(yīng)基因能夠在目標(biāo)種群中自主擴(kuò)散。蚊媒、病原微生物、抗性基因和基因驅(qū)動(dòng)系統(tǒng)之間復(fù)雜的進(jìn)化關(guān)系是種群替代策略研究的核心環(huán)節(jié)。目前已知的Wolbachia驅(qū)動(dòng)系統(tǒng)、Medea驅(qū)動(dòng)系統(tǒng)、轉(zhuǎn)座子轉(zhuǎn)化系統(tǒng)、顯性不足技術(shù)、HEG等均能促進(jìn)蚊媒抗性的產(chǎn)生(Alphey,2014; Chenetal.,2007; Moreiraetal.,2009)。

轉(zhuǎn)座子是基因組中能自主復(fù)制和移位的DNA區(qū)段，廣泛存在于昆蟲基因組，不過(guò)大多數(shù)轉(zhuǎn)座子已發(fā)生突變不表現(xiàn)活性。目前，轉(zhuǎn)座子已被廣泛應(yīng)用于分子生物學(xué)研究。轉(zhuǎn)座子在染色體不同位點(diǎn)的插入有可能導(dǎo)致外源基因失活或染色體重排，進(jìn)而使蚊蟲的適應(yīng)性下降。來(lái)自粉紋夜蛾Trichoplusiani的piggyBac轉(zhuǎn)座子能特異識(shí)別TTAA位點(diǎn)，并準(zhǔn)確切除與插入外源基因，可轉(zhuǎn)入的外源基因的大小幾乎不受限制，也無(wú)物種限制，是遺傳修飾系統(tǒng)中應(yīng)用最廣泛的轉(zhuǎn)座子之一(Fraseretal.,1983; Handler,2002)，目前應(yīng)用piggyBac轉(zhuǎn)座子已成功獲得了蚊媒的多個(gè)遺傳轉(zhuǎn)化品系(Fuetal.,2010; Phucetal.,2007; Wiseetal.,2011)。此外，研究者也將Hermes(Jasinskieneetal.,1998; Zhao & Eggleston,1998)、Minos(Catterucciaetal.,2000a、2000b)、Mariner(Coatesetal.,1998)等轉(zhuǎn)座子抗性基因成功轉(zhuǎn)入蚊蟲的細(xì)胞系或得到遺傳修飾品系。由于大部分非蚊蟲來(lái)源的轉(zhuǎn)座子在蚊蟲中的遺傳轉(zhuǎn)化成功率較低，給其應(yīng)用帶來(lái)了一定的局限性(O′Brochtaetal.,2003)，因此需要挖掘蚊媒自身位點(diǎn)特異且非連鎖的轉(zhuǎn)座子(Rasgon & Gould,2005)。Arensburgeretal. (2005)已在岡比亞按蚊Anophelesgambiae中發(fā)現(xiàn)了Herves轉(zhuǎn)座子，但其調(diào)控機(jī)制尚不明晰。

內(nèi)生菌Wolbachia能夠通過(guò)增強(qiáng)蚊蟲的自身免疫力或改變蚊蟲的代謝通路等方式(Brennanetal.,2008; Panetal.,2012)，誘導(dǎo)蚊媒對(duì)病原微生物的抗性(Bianetal.,2010、2013; Moreiraetal.,2009)，抑制甚至清除病原微生物的感染(Walkeretal.,2011)。將抗病的蚊蟲釋放于靶標(biāo)蚊媒種群中引起胞質(zhì)不相容性，抗性Wolbachia逐步擴(kuò)散到靶標(biāo)種群中，進(jìn)而取代易感靶標(biāo)蚊媒，從根源上控制了蚊媒病的傳播?；诔鄶M谷盜Triboliumcastaneum的Medea元件也能輔助蚊媒對(duì)病原微生物抗性的產(chǎn)生(Chenetal.,2007)， Medea元件由母系特異啟動(dòng)子驅(qū)動(dòng)的對(duì)胚胎有毒性的RNA或蛋白和受精卵特異的啟動(dòng)子驅(qū)動(dòng)解毒蛋白組合在一起。由于雌性雜合子后代均具有母系遺傳的毒素基因，當(dāng)后代未遺傳到解毒基因時(shí)將死亡，當(dāng)遺傳到母/父系來(lái)源的解毒基因時(shí)將存活(Alphey,2014)。通過(guò)染色體易位等遺傳操作產(chǎn)生的顯性不足(Curtis,1968; Davisetal.,2001; Magori & Gould,2006)和Y染色體連鎖的HEG(Alphey,2014; Burt,2003; Catterucciaetal.,2005; Deredecetal.,2008)也能驅(qū)動(dòng)種群替代。不同種群替代技術(shù)的驅(qū)動(dòng)效率不同，驅(qū)動(dòng)效率較低的Wolbachia系統(tǒng)和顯性不足系統(tǒng)需要更多的初始釋放數(shù)量，而驅(qū)動(dòng)效率較高的轉(zhuǎn)座子系統(tǒng)和HEG需要的初始釋放數(shù)量則相對(duì)較低(Alphey,2014)。

3　RIDL技術(shù)

3.1RIDL技術(shù)原理和現(xiàn)有品系

基于tet-off系統(tǒng)調(diào)控效應(yīng)基因的表達(dá)是目前RIDL技術(shù)實(shí)現(xiàn)蚊蟲特異性致死的最主要方式。在tet-off系統(tǒng)中,當(dāng)大腸桿菌Escherichcoli的轉(zhuǎn)座子Tn10的四環(huán)素阻遏因子(tetracycline repressor,tetR)與四環(huán)素結(jié)合時(shí)，tetR不能阻抑四環(huán)素抗性操縱子(tetracycline-resistance operon,tetO)，因此下游轉(zhuǎn)錄不受抑制。將tetR的部分序列與單純皰疹病毒VP16的轉(zhuǎn)錄活性區(qū)段組合為四環(huán)素轉(zhuǎn)錄激活因子(tetracycline transcriptional activator,tTA)，tTA與性別/組織/發(fā)育階段特異性啟動(dòng)子構(gòu)建為tet-off驅(qū)動(dòng)載體，tetO與CMV啟動(dòng)子構(gòu)成四環(huán)素響應(yīng)元件(Tetracycline response element,TRE)，TRE與效應(yīng)基因組合成效應(yīng)載體，進(jìn)而組建為完整的tet-off表達(dá)系統(tǒng)(Gossen & Bujard,1992)。在缺乏四環(huán)素時(shí)，tTA與tetO結(jié)合引發(fā)效應(yīng)基因表達(dá)；但在飼養(yǎng)條件存在四環(huán)素時(shí)，tTA與四環(huán)素結(jié)合而不與tetO結(jié)合，無(wú)法激活下游效應(yīng)基因的表達(dá)。

通過(guò)特定遺傳標(biāo)記篩選轉(zhuǎn)化品系是RIDL構(gòu)建過(guò)程中的關(guān)鍵步驟，利用不同啟動(dòng)子驅(qū)動(dòng)熒光標(biāo)記是目前的常用手段。優(yōu)良的啟動(dòng)子—熒光基因表達(dá)模式能夠大大降低轉(zhuǎn)化篩選的難度，在提高RIDL品系構(gòu)建效率的同時(shí)，有助于釋放品系的后期監(jiān)測(cè)。組成型啟動(dòng)子通常具有表達(dá)強(qiáng)度高、表達(dá)周期長(zhǎng)和表達(dá)面積大的優(yōu)點(diǎn)，英國(guó)Oxitec公司采用組成型啟動(dòng)子Hr5-IE1構(gòu)建了性能良好的遺傳熒光標(biāo)記，全身表達(dá)DsRed2或GFP的埃及伊蚊Aedesaegypti幼蟲在熒光濾鏡下清晰可見(圖1A)；而表達(dá)Hr5IE1-DsRed2的幼蟲肛乳頭呈現(xiàn)出斑點(diǎn)熒光模式，主要是來(lái)自核位點(diǎn)的熒光信號(hào)(圖1B)。此外，3xP3啟動(dòng)子也常常用于構(gòu)建蚊子的遺傳熒光標(biāo)記，以驅(qū)動(dòng)如AmCyan(圖1C)或DsRed(圖1D)熒光基因在埃及伊蚊光學(xué)神經(jīng)中的表達(dá)。

目前開發(fā)的蚊子RIDL品系主要來(lái)自O(shè)xitec公司，目標(biāo)物種包括埃及伊蚊、白紋伊蚊Aedesalbopictus和岡比亞按蚊等，有的品系已經(jīng)進(jìn)入田間釋放階段(表2)。Phucetal.(2007)通過(guò)模型研究發(fā)現(xiàn),由于存在密度依賴效應(yīng)，攜帶晚期表達(dá)效應(yīng)基因的RIDL品系與早期品系相比，不僅能夠大大減少釋放的初始蟲源數(shù)量，而且能更快地達(dá)到控制種群的目的，具有更好的防治效果；通過(guò)構(gòu)建埃及伊蚊RIDL品系LA513A(OX513A)進(jìn)行驗(yàn)證，在此系統(tǒng)中tTA通過(guò)反復(fù)結(jié)合tetO而不斷積累，最終達(dá)到致死劑量;由于tTA既是轉(zhuǎn)錄激活因子，也是效應(yīng)基因，該體系被稱作單元件系統(tǒng)(Gongetal.,2005)。此外，F(xiàn)uetal.(2010)將雌蚊飛行肌特異性啟動(dòng)子AeAct-4(Muozetal.,2004)與tTA元件連接構(gòu)建tet-off驅(qū)動(dòng)載體，將細(xì)胞凋亡基因Nipp1Dm和michelob_x與TRE連接構(gòu)建效應(yīng)載體，將驅(qū)動(dòng)品系OX3545分別與效應(yīng)品系 OX3547和OX3582雜交，獲得的雌性后代在四環(huán)素缺乏時(shí)引發(fā)效應(yīng)基因在飛行肌的特異性表達(dá)，從而喪失了飛行能力成為無(wú)翅型，比例高達(dá)65.8%~98.3%，而雄蚊則不受影響；該體系既需要表達(dá)tTA的驅(qū)動(dòng)載體，也需要表達(dá)致死基因的效應(yīng)載體，因此又稱作雙元件系統(tǒng)(Alpheyetal.,2008)。同時(shí)，F(xiàn)uetal.(2010)利用AeAct-4啟動(dòng)子構(gòu)建了單元件系統(tǒng)并得到了OX3604C品系，當(dāng)不存在四環(huán)素時(shí)，過(guò)量表達(dá)的tTA導(dǎo)致飛行肌細(xì)胞凋亡，后代雌蚊幾乎全部無(wú)翅，而雄蚊則不受影響。飛行能力對(duì)蚊子營(yíng)養(yǎng)獲取、交配及逃生等至關(guān)重要(Labbéetal.,2012)，因此OX3604C實(shí)質(zhì)上等同于基于tet-off調(diào)控下的雌性特異致死品系。Wiseetal.(2011)調(diào)查了埃及伊蚊OX3604C品系雄蚊用于SIT的潛力，實(shí)驗(yàn)室條件下當(dāng)以遺傳修飾雄蚊∶野生型雄蚊=(8.5~10)∶1，每周釋放1次時(shí)，10~20周便可壓制野生型蚊蟲。Labbéetal.(2012)采用白紋伊蚊的AealbAct-4啟動(dòng)子構(gòu)建了RIDL品系OX4358并取得了與OX3604C近似的結(jié)果，表明Actin-4啟動(dòng)子的雌性飛行肌特異活性可能在不同蚊子種類中保守。

圖1　埃及伊蚊遺傳修飾品系中不同遺傳標(biāo)記的熒光模式(圖片由英國(guó)Oxitec公司的Luke Alphey博士提供)

種類Species表型Phenotype品系Strains標(biāo)記Marker轉(zhuǎn)座子Transposons啟動(dòng)子Promoter致死基因Lethalgene參考文獻(xiàn)References埃及伊蚊Aedesaegypti條件致死RepressiblelethalityOX513A(LA513)DsRedpiggyBacAct5CtTAPhucetal．，2007LA882IE-2雌蚊特異致死Female-specificflightlessOX3545OX3547DsRedpiggyBacAeAct-4Nipp1DmFuetal．，2010；Wiseetal．，2011OX3545OX3582michelob＿xOX3604CtTAOX3688AmCyanpiggyBacAeAct-4tTALabbéetal．，2012OX4358AealbAct-4白紋伊蚊Aedesalbopictus雌蚊特異致死Female-specificflightlessOX3688AmCyanpiggyBacAeAct-4tTALabbéetal．，2012OX4358AealbAct-4岡比亞按蚊Anophelesgambiae雌蚊特異胚胎期致死Female-specificembryoniclethalityβ2PpoeGFPDsRedpiggyBacβ2tubulinHEGGalizietal．，2014；Windbichleretal．，2008

此外，RIDL品系在岡比亞按蚊的種群遺傳控制中也取得了理想進(jìn)展。Windbichleretal.(2008)采用在雄蚊睪丸精子發(fā)生時(shí)特異表達(dá)的β2tubulin(Catterucciaetal.,2005)啟動(dòng)子驅(qū)動(dòng)效應(yīng)基因HEG的表達(dá)，導(dǎo)致后代雌蚊在胚胎期死亡，大幅度改變了后代的性別構(gòu)成，使目標(biāo)種群無(wú)法繼續(xù)繁衍。由于HEG蛋白與X染色體連鎖的28S核糖體基因的重復(fù)序列高度特異靶定，在β2tubulin驅(qū)動(dòng)下在雄蚊精子發(fā)生時(shí)切割X染色體，當(dāng)導(dǎo)入胚胎時(shí)還能切割母系來(lái)源的X染色體，這種雄蚊與野生型雌蚊交配后導(dǎo)致后代雌蚊在胚胎期死亡，產(chǎn)生的后代幾乎全為攜帶效應(yīng)基因的雄蚊(Windbichleretal.,2008)。Galizietal.(2014)發(fā)現(xiàn)應(yīng)用該技術(shù)的雄蚊與野生型雌蚊交配后產(chǎn)生的子代95%以上為雄性，且雄蚊的生育能力未受明顯影響，到第6代時(shí)蚊蟲種群因缺少雌性而無(wú)法繁衍，為有效控制瘧疾等傳染病的傳播提供了更為高效、經(jīng)濟(jì)的方法。

3.2　田間試驗(yàn)

研究遺傳修飾不育技術(shù)的最終目標(biāo)是將培育的RIDL品系釋放于野外替代自然界中如埃及伊蚊、岡比亞按蚊等重大傳染性疾病的傳播媒介，從而從根本上阻斷蚊媒疾病的傳播，而RIDL品系能否成功壓制野外種群的一個(gè)重要特征在于其將攜帶的顯性效應(yīng)基因向目標(biāo)種群傳遞擴(kuò)散的能力及穩(wěn)定性(Scottetal.,2002)。RIDL品系在自然界中的生存(如存活率、壽命、羽化率等)、繁殖(交配競(jìng)爭(zhēng)力等)、擴(kuò)散(飛行能力等)的適應(yīng)性和競(jìng)爭(zhēng)力是反映效應(yīng)基因傳遞能力(Massonnet-Bruneeletal.,2013)的重要指標(biāo)。研究表明，遺傳操作中轉(zhuǎn)座子的遺傳轉(zhuǎn)化、啟動(dòng)子和標(biāo)記基因的表達(dá)以及在飼養(yǎng)時(shí)為獲得純合品系采用的近親交配等帶來(lái)的自然選擇壓力，有可能對(duì)遺傳修飾品系的適應(yīng)性產(chǎn)生影響(Catterucciaetal.,2003; Irvinetal.,2004; Marrellietal.,2006; Massonnet-Bruneeletal.,2013)。目前應(yīng)用最多的OX513A品系，采用的是組成型啟動(dòng)子Act5C，其對(duì)適合度的影響可能比組織特異性啟動(dòng)子更大(Massonnet-Bruneeletal.,2013)。因此，必須在實(shí)驗(yàn)室和田間開放條件下對(duì)RIDL品系的適合度及其釋放策略進(jìn)行詳細(xì)調(diào)查。已有多個(gè)國(guó)家圍繞該方面開展了廣泛研究，并提出了RIDL品系的釋放前標(biāo)準(zhǔn)(Benedict & Robinson,2003; Yakobetal.,2008)。已經(jīng)報(bào)道的室內(nèi)研究結(jié)果并不完全一致，但基本可以得出如下結(jié)論：RIDL品系在生活史特性、繁殖和擴(kuò)散能力等方面有一定的下降，但是與野生型相比不存在較大的差異；這可以通過(guò)進(jìn)一步優(yōu)化飼養(yǎng)體系和方法得到改善，最優(yōu)釋放策略的制定也有助于獲得理想的控制效果(表3)。

目前田間開放條件的釋放研究主要集中于埃及伊蚊的OX513A品系。Harrisetal.(2011)于2009年在Grand Cayman地區(qū)首次進(jìn)行了OX513A雄蚊的田間釋放，結(jié)果表明，釋放的雄蚊能成功與野外雌蚊交配并使其受精，具有與野生雄蚊相當(dāng)?shù)姆敝衬芰Γ尫藕笫占穆逊趸龅挠紫x能檢測(cè)到熒光，且隨時(shí)間推移比率逐漸提高，反映了RIDL在野外逐步降低野生型種群的過(guò)程。此外，模型分析發(fā)現(xiàn)，當(dāng)田間野生型和釋放RIDL品系交配比率達(dá)13%~57%時(shí)才能成功抑制目標(biāo)種群。因此，Harrisetal.(2012)于2010進(jìn)行了第2次釋放研究，釋放4~6 周后野生型種群受到抑制，11 周時(shí)RIDL∶野生雄蚊高達(dá)25.2∶1，卵帶熒光比率為88%，表明OX513A品系成功實(shí)現(xiàn)了對(duì)野生型的壓制。Lacroixetal.(2012)在馬來(lái)西亞Pahang地區(qū)也進(jìn)行了OX513A和野生型實(shí)驗(yàn)室品系的釋放，結(jié)果表明,RIDL品系不會(huì)對(duì)人類健康和環(huán)境產(chǎn)生不利影響，OX513A和野生型壽命相當(dāng)，雖然飛行能力有一定減弱但釋放措施的改善能提高其應(yīng)用效果。此外，Alphey (2014)在巴西應(yīng)用OX513A品系也成功地實(shí)現(xiàn)了對(duì)2個(gè)目標(biāo)種群的壓制，并且后續(xù)的大規(guī)模釋放研究還在繼續(xù)開展。

4　總結(jié)

遺傳不育技術(shù)成功地將昆蟲不育技術(shù)與新興的遺傳修飾技術(shù)相結(jié)合，成為物種特異、環(huán)境安全、科學(xué)高效的有害生物治理手段，具有傳統(tǒng)防治方法難以比擬的優(yōu)勢(shì)。目前，已經(jīng)開發(fā)了包括埃及伊蚊、白紋伊蚊、岡比亞按蚊在內(nèi)的眾多主要媒介蚊蟲的特異性致死或無(wú)翅型RIDL品系，并進(jìn)行了一系列適應(yīng)性測(cè)試和安全性評(píng)估，針對(duì)埃及伊蚊的OX513A品系已經(jīng)實(shí)現(xiàn)了田間釋放，驗(yàn)證了RIDL技術(shù)在防控蚊媒疾病中的可行性。種群的釋放策略對(duì)蚊媒種群的遺傳控制效果具有重要的影響，合理評(píng)估遺傳修飾品系的適應(yīng)性、擴(kuò)散能力和生殖能力，構(gòu)建基于昆蟲學(xué)、流行病學(xué)、生物經(jīng)濟(jì)學(xué)的數(shù)學(xué)模型，進(jìn)而優(yōu)化釋放比例，合理布局釋放點(diǎn)、釋放頻率、持續(xù)時(shí)間等是達(dá)到最優(yōu)釋放策略的必要程序(Alpheyetal.,2011； Atkinsonetal.，2007)。此外，遺傳控制技術(shù)與化學(xué)防治、生物防治等多種控制措施聯(lián)合應(yīng)用，將能更加有效地控制和阻斷瘧疾、登革熱等重大蚊媒疾病的發(fā)生和傳播。

表3　RIDL品系適應(yīng)性的室內(nèi)研究結(jié)果

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(責(zé)任編輯:楊郁霞)

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

Sciences,Beijing100193,China;2Department of Entomology, North Carolina State University, Campus Box 7613, Raleigh,

NC27695-7613,USA；3Genetic Engineering and Society Center and W. M. Keck Center for Behavioral Biology,

NorthCarolinaStateUniversity,Raleigh,NC27695-7613,USA;4College of Agriculture and Plant Protection,

QingdaoAgriculturalUniversity,Qingdao,Shandong266109,China

Abstract:Mosquito-borne diseases, such as dengue fever and malaria, are global problems and pose a serious threat to public health. An estimated 2.5 billion people live in areas at the risk of epidemic transmission. For now, no vaccines are available against the pathogens respnsible for these diseases, and the mosquito control is considered as one of the most effective ways to reduce transmission. Mass application of pesticides could reduce the mosquito population but it also brings problems like insect resistance and environmental pollution. The release of insects with dominant lethality (RIDL) technology and other genetic control systems based on the traditional sterile insect technique (SIT) provide new strategies to control disease vector mosquitos, such as Aedes aegypti and Anopheles gambiae. Those new version of genetic control methods are species-specific and environment-friendly, and now being developed and tested worldwide. Here the principle and recent progress of mosquito genetic control are reviewed. The history of mosquito SIT is introduced, and the genetic control strategies including self-limiting and self-sustaining populations are also illustrated. The development, as well as laboratory and field trials of RIDL strains are described. It is suggested that genetic control strategies such as RIDL are promising methods to fight against mosquitoes carrying human diseases.

Key words:disease vector mosquito; genetic control; sterile insect technique; release of insects carrying a dominant lethal

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

作者簡(jiǎn)介:武強(qiáng)， 男， 博士研究生。 研究方向: 昆蟲生物化學(xué)與分子生物學(xué)。 E-mail： wuqiang8510@163.com

基金項(xiàng)目:科技導(dǎo)報(bào)社博士生創(chuàng)新研究資助計(jì)劃(kjdb201001-3)； 中德合作科研項(xiàng)目[PPP項(xiàng)目、留金歐(2012) 6014、留金歐(2014 )6013]； 環(huán)保公益性行業(yè)科研專項(xiàng)(201409061)； 農(nóng)業(yè)部2014年農(nóng)作物病蟲鼠害疫情監(jiān)測(cè)與防治(外來(lái)入侵生物防治)項(xiàng)目； 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)： 2014-12-20接受日期(Accepted)： 2015-01-17

DOI:10. 3969/j.issn.2095-1787.2015.02.008