昆蟲(chóng)種群遺傳控制技術(shù)中啟動(dòng)子的研究

劉桂清， 嚴(yán)　盈， 王玉生， 張桂芬， 萬(wàn)方浩,5*

1中國(guó)農(nóng)業(yè)科學(xué)院植物保護(hù)研究所，植物病蟲(chóng)害生物學(xué)國(guó)家重點(diǎn)實(shí)驗(yàn)室，北京100193； 2廣東省昆蟲(chóng)研究所，

廣東省野生動(dòng)物保護(hù)和利用公共實(shí)驗(yàn)室，廣東省農(nóng)業(yè)害蟲(chóng)綜合治理重點(diǎn)實(shí)驗(yàn)室，廣東 廣州 510260；

3Department of Entomology, North Carolina State University, Campus Box 7613, Raleigh, NC 27695-7613,

USA； 4Genetic Engineering and Society Center and W.M. Keck Center for Behavioral Biology,

North Carolina State University, Raleigh, NC 27695-7613, USA；

5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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昆蟲(chóng)種群遺傳控制技術(shù)中啟動(dòng)子的研究

劉桂清1,2， 嚴(yán)盈1,3，4， 王玉生1， 張桂芬1， 萬(wàn)方浩1,5*

1中國(guó)農(nóng)業(yè)科學(xué)院植物保護(hù)研究所，植物病蟲(chóng)害生物學(xué)國(guó)家重點(diǎn)實(shí)驗(yàn)室，北京100193；2廣東省昆蟲(chóng)研究所，

廣東省野生動(dòng)物保護(hù)和利用公共實(shí)驗(yàn)室，廣東省農(nóng)業(yè)害蟲(chóng)綜合治理重點(diǎn)實(shí)驗(yàn)室，廣東 廣州 510260；

3Department of Entomology, North Carolina State University, Campus Box 7613, Raleigh, NC 27695-7613,

USA；4Genetic Engineering and Society Center and W.M. Keck Center for Behavioral Biology,

North Carolina State University, Raleigh, NC 27695-7613, USA；

5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:利用昆蟲(chóng)遺傳轉(zhuǎn)化技術(shù)對(duì)害蟲(chóng)進(jìn)行遺傳控制是害蟲(chóng)防治研究的新方向，該技術(shù)具有物種特異、防效高且對(duì)環(huán)境友好的特點(diǎn)。啟動(dòng)子是基因表達(dá)調(diào)控的重要元件，選擇合適的啟動(dòng)子是外源基因高效、準(zhǔn)確表達(dá)的關(guān)鍵，對(duì)獲得高效、穩(wěn)定的遺傳修飾昆蟲(chóng)品系至關(guān)重要。本文簡(jiǎn)要介紹了昆蟲(chóng)基因啟動(dòng)子的結(jié)構(gòu)特征，重點(diǎn)描述了昆蟲(chóng)種群遺傳防治中組成型啟動(dòng)子、性別和組織特異型啟動(dòng)子、特定發(fā)育時(shí)期啟動(dòng)子和誘導(dǎo)型啟動(dòng)子的研究和應(yīng)用概況，并對(duì)這幾類啟動(dòng)子在害蟲(chóng)遺傳控制中的應(yīng)用前景進(jìn)行了展望。

關(guān)鍵詞:遺傳防治； 昆蟲(chóng)不育技術(shù)； 遺傳轉(zhuǎn)化； 組成型啟動(dòng)子； 特異性啟動(dòng)子； 誘導(dǎo)型啟動(dòng)子

Insect gene promoters used in insect population genetic control

Gui-qing LIU1,2, Ying YAN1,3，4, Yu-sheng WANG1, Gui-fen ZHANG1, Fang-hao WAN1,5*

1StateKeyLaboratoryforBiologyofPlantDiseasesandInsectPests,InstituteofPlantProtection,ChineseAcademyofAgricultural

Sciences,Beijing100193,China;2GuangdongKeyLaboratoryofIntegratedPestManagementinAgriculture,GuangdongPublic

基因的表達(dá)調(diào)控是分子生物學(xué)領(lǐng)域的熱門(mén)課題，可以分為轉(zhuǎn)錄水平調(diào)控、轉(zhuǎn)錄后水平調(diào)控、翻譯水平調(diào)控和蛋白質(zhì)水平調(diào)控等。在特定的時(shí)間內(nèi)，成千上萬(wàn)的真核生物基因中，只有15%左右的基因能在特定的細(xì)胞中表達(dá)有功能的RNA與蛋白質(zhì)等產(chǎn)物(Liang & Pardee,1992)。轉(zhuǎn)錄水平調(diào)控被認(rèn)為是基因表達(dá)最重要的調(diào)控方式之一?；虻霓D(zhuǎn)錄受啟動(dòng)子和增強(qiáng)子等特定的順式作用元件(Cis-acting element)和蛋白質(zhì)因子等具有可擴(kuò)散特性的反式作用因子(Trans-acting factor)的影響。

昆蟲(chóng)是地球上種類最多的物種，對(duì)昆蟲(chóng)基因表達(dá)調(diào)控機(jī)理進(jìn)行解析有助于害蟲(chóng)防治策略的發(fā)展。自首例遺傳轉(zhuǎn)化果蠅誕生(Rubin & Spradling,1982)后，昆蟲(chóng)遺傳轉(zhuǎn)化技術(shù)成為科學(xué)家研究的熱點(diǎn)，利用昆蟲(chóng)遺傳轉(zhuǎn)化技術(shù)獲得遺傳修飾品系對(duì)害蟲(chóng)進(jìn)行遺傳控制是害蟲(chóng)防治研究的新方向(Alphey & Andreasen,2002; Heinrich & Scott,2000; Horn & Wimmer,2003; Ogaugwuetal.,2013； Scheteligetal.,2009; Thomasetal.,2000)。對(duì)目標(biāo)害蟲(chóng)進(jìn)行遺傳控制，需要將遺傳修飾昆蟲(chóng)品系釋放到野外，品系中外源基因穩(wěn)定、高效且有目的性地表達(dá)至關(guān)重要，因此，選擇合適的昆蟲(chóng)啟動(dòng)子是建立遺傳修飾昆蟲(chóng)品系首先要考慮的問(wèn)題。目前，關(guān)于啟動(dòng)子的結(jié)構(gòu)及其對(duì)基因的轉(zhuǎn)錄調(diào)控研究已有一系列進(jìn)展，許多啟動(dòng)子得以鑒定并應(yīng)用到害蟲(chóng)遺傳控制中。本文將重點(diǎn)綜述啟動(dòng)子在昆蟲(chóng)遺傳控制中的研究及應(yīng)用概況。

1　昆蟲(chóng)啟動(dòng)子結(jié)構(gòu)特征

啟動(dòng)子是指RNA聚合酶及一些反式作用因子識(shí)別并與之結(jié)合從而正確有效地起始轉(zhuǎn)錄的一段特異性DNA序列。真核生物有3種RNA聚合酶，每一種都有自己特定的啟動(dòng)子類型。RNA聚合酶Ⅰ只轉(zhuǎn)錄rRNA，只有一種啟動(dòng)子類型。RNA聚合酶Ⅱ負(fù)責(zé)蛋白質(zhì)基因和部分SnRNA基因的轉(zhuǎn)錄，其啟動(dòng)子結(jié)構(gòu)最為復(fù)雜，通常所說(shuō)的核心啟動(dòng)子(Core promoter)即指RNA聚合酶Ⅱ的啟動(dòng)子。RNA聚合酶Ⅲ負(fù)責(zé)轉(zhuǎn)錄tDNA和5SrDNA，其啟動(dòng)子位于轉(zhuǎn)錄的DNA序列之內(nèi)，稱為下游啟動(dòng)子。

啟動(dòng)子是決定RNA聚合酶Ⅱ轉(zhuǎn)錄起始點(diǎn)和轉(zhuǎn)錄頻率的關(guān)鍵元件，由多個(gè)獨(dú)立的、具有特征性的核苷酸序列組成。昆蟲(chóng)啟動(dòng)子具備真核生物啟動(dòng)子的典型特征，其結(jié)構(gòu)如下：在5′端轉(zhuǎn)錄起始點(diǎn)上游約-20~-30 bp的區(qū)域存在TATA盒(TATA-box)，它對(duì)轉(zhuǎn)錄起始位點(diǎn)的定位十分重要，是絕大多數(shù)真核生物基因正確表達(dá)所必需的元件，一致序列為T(mén)ATA(A/T)A(A/T)。在不含TATA box的啟動(dòng)子中，往往由上游啟動(dòng)子元件行使TATA box的功能，主要有2個(gè)部位：GC box，分布在轉(zhuǎn)錄起始位點(diǎn)上游-128~-23 bp的區(qū)域內(nèi)，與轉(zhuǎn)錄因子的結(jié)合有關(guān)，其中常有1個(gè)以上的SP1結(jié)合位點(diǎn)，一致序列為GGCGGG；CAAT box，在轉(zhuǎn)錄起始位點(diǎn)上游 -159~-51 bp的區(qū)域內(nèi)分布比較集中，一致序列為GG(C/A)CAATCT，普遍存在于生物體中，與轉(zhuǎn)錄起始頻率有關(guān)(Weaver,2002)。

啟動(dòng)子具有如下特征：(1) 序列特異性，在啟動(dòng)子的DNA序列中，通常含有幾個(gè)保守的序列框，序列框中堿基的變化會(huì)導(dǎo)致轉(zhuǎn)錄啟動(dòng)滯后和轉(zhuǎn)錄速度減慢；(2) 方向性，啟動(dòng)子是一種有方向性的順式調(diào)控元件，在正反2種方向中只有一種具有啟動(dòng)功能；(3) 位置特性，啟動(dòng)子只能位于所啟動(dòng)轉(zhuǎn)錄基因的上游或基因內(nèi)的前端；(4) 種屬特異性，原核生物的不同種、屬，真核生物的不同組織都具有不同類型的啟動(dòng)子。但一般來(lái)說(shuō), 親緣關(guān)系越近的2種生物，其啟動(dòng)子通用的可能性也越大(吳乃虎，2001； 夏江東等，2006)。

2　昆蟲(chóng)啟動(dòng)子研究概況

啟動(dòng)子按其功能及作用方式可分為3類：組成型啟動(dòng)子、特異型啟動(dòng)子和誘導(dǎo)型啟動(dòng)子，但在某些情況下，一種類型的啟動(dòng)子會(huì)兼有其他類型啟動(dòng)子的特性。下面將舉例說(shuō)明這幾類啟動(dòng)子的特性及其在昆蟲(chóng)遺傳控制中的應(yīng)用，并對(duì)昆蟲(chóng)遺傳控制技術(shù)中應(yīng)用的啟動(dòng)子及其啟動(dòng)的外源基因表達(dá)的遺傳修飾昆蟲(chóng)進(jìn)行總結(jié)(表1)。

2.1　組成型啟動(dòng)子

組成型啟動(dòng)子控制外源基因的表達(dá)大體恒定在一定水平，該類啟動(dòng)子具有啟動(dòng)效率高、甲基化程度相對(duì)較低和遺傳性狀穩(wěn)定等特點(diǎn)，是基因工程中應(yīng)用最早、最為廣泛的一類啟動(dòng)子。組成型啟動(dòng)子在遺傳控制技術(shù)中的應(yīng)用主要體現(xiàn)在以下3方面：(1)基因防治，由家蠶核型多角體病毒(Bombyxmorinucleopolyhedrovirus,Bm-NPV)引起的家蠶血液型膿病是對(duì)養(yǎng)蠶業(yè)危害最為嚴(yán)重的病害之一，該病傳染力極強(qiáng)，難以控制。利用昆蟲(chóng)桿狀病毒極早期蛋白基因immediateearly(ie1)啟動(dòng)子介導(dǎo)對(duì)Bm-NPV具有很強(qiáng)抑制作用的家蠶脂肪酶1 (Bmlipase-1)的增量表達(dá)，獲得提高宿主對(duì)疾病抵抗力的家蠶品系(Jiangetal.,2012)；(2)遺傳轉(zhuǎn)化輔助質(zhì)粒helper的構(gòu)建，能提高轉(zhuǎn)座酶的瞬時(shí)表達(dá)水平，對(duì)提高遺傳轉(zhuǎn)化效率十分有效(Kapetanakietal.,2002)，ie1基因啟動(dòng)子在增強(qiáng)子hr5的協(xié)同作用下能高效表達(dá)轉(zhuǎn)座酶基因，提高轉(zhuǎn)座子piggyBac的轉(zhuǎn)座活性，廣泛用于遺傳轉(zhuǎn)化體系輔助質(zhì)粒的構(gòu)建(王娜等，2010)；(3)標(biāo)記遺傳修飾昆蟲(chóng)，由于組成型啟動(dòng)子高效、穩(wěn)定表達(dá)靶標(biāo)基因的特性，被廣泛用于啟動(dòng)EGFP、DsRed等熒光蛋白的表達(dá)。這里以多聚泛素基因polyubiquitin(PUb)啟動(dòng)子啟動(dòng)DsRed在遺傳修飾斑翅果蠅DrosophilasuzukiiMatsumura中表達(dá)為例，說(shuō)明組成型啟動(dòng)子的作用機(jī)制。

表1　昆蟲(chóng)遺傳控制中的啟動(dòng)子應(yīng)用概況

2.1.1PUb啟動(dòng)子作用機(jī)制泛素(Ubiquitin)是一種由76個(gè)氨基酸的氨基和羧基縮合而成的小分子蛋白，相對(duì)分子質(zhì)量8.6 kD，最早從牛胸腺中分離得到，幾乎在所有的真核生物中都極其保守。根據(jù)編碼泛素前體蛋白質(zhì)的差異可分為多聚泛素基因(polyubiquitin)和泛素延伸基因(ubiquitinextension)。泛素基因在昆蟲(chóng)中腸、脂肪體、馬氏管和飛行肌等組織中高水平表達(dá)，參與調(diào)控昆蟲(chóng)生命活動(dòng)的各個(gè)過(guò)程(Barrioetal.,1994)。泛素基因啟動(dòng)子的結(jié)構(gòu)一般包括幾個(gè)特定的基序(Sequence motifs)，+l處為轉(zhuǎn)錄起始位點(diǎn)(帽子位點(diǎn))，一般為A，兩邊通常為嘧啶堿基；以-25位為中心的區(qū)域富含AT序列，是啟動(dòng)子主要的功能性成分，可能通過(guò)一些調(diào)節(jié)蛋白直接或間接作用來(lái)控制RNA聚合酶的活性，從而特異性地轉(zhuǎn)錄出一定量的mRNA；G 框位于泛素基因啟動(dòng)子-96 bp位置 ，一致序列為CACGTG，是高度保守的轉(zhuǎn)錄元件之一。目前，研究所知的多數(shù)泛素基因的起始轉(zhuǎn)錄區(qū)上游都有一段內(nèi)含子序列，位于轉(zhuǎn)錄起始位點(diǎn)至翻譯起始密碼子之間，對(duì)目的基因的表達(dá)具有較大促進(jìn)作用(Andersonetal.,2010)。

2.1.2PUb啟動(dòng)子應(yīng)用實(shí)例斑翅果蠅，又稱鈴木氏果蠅，寄主范圍廣，危害成熟或即將成熟的櫻桃、桃、歐洲李、葡萄、草莓、樹(shù)莓、藍(lán)莓、柿子和番茄等，幼蟲(chóng)在果實(shí)內(nèi)取食，給果園造成嚴(yán)重?fù)p失。該蟲(chóng)1916年在日本山梨縣首先發(fā)現(xiàn)，20世紀(jì)80年代在美國(guó)夏威夷定殖，傳播速度非常快，目前在美洲、歐洲、非洲和亞洲等國(guó)均有發(fā)現(xiàn)，已成為一種世界性農(nóng)業(yè)害蟲(chóng)(Hauser，2011)。除加強(qiáng)對(duì)該蟲(chóng)的檢疫外，利用昆蟲(chóng)遺傳轉(zhuǎn)化技術(shù)對(duì)該蟲(chóng)進(jìn)行遺傳控制也是一種有效的預(yù)防或根除該蟲(chóng)的方法。目前，美國(guó)北卡羅萊拉州立大學(xué)已經(jīng)獲得了遺傳修飾斑翅果蠅(圖1)，Pub啟動(dòng)子調(diào)控DsRed熒光蛋白在斑翅果蠅整個(gè)蟲(chóng)體均有表達(dá)，而不是局限在某個(gè)組織表達(dá)。Schetelig & Handler (2013)利用Pub啟動(dòng)子調(diào)控EGFP熒光蛋白的表達(dá),獲得在整個(gè)蟲(chóng)體均能檢測(cè)到綠色熒光的遺傳修飾斑翅果蠅。

圖1　多聚泛素基因啟動(dòng)子Pub驅(qū)動(dòng)DsRed表達(dá)的遺傳修飾斑翅果蠅

2.2　組織特異啟動(dòng)子

在組織特異啟動(dòng)子調(diào)控下，基因的表達(dá)往往只發(fā)生在某些特定的器官或組織部位，表現(xiàn)出發(fā)育調(diào)節(jié)的特性。應(yīng)用昆蟲(chóng)遺傳轉(zhuǎn)化技術(shù)建立遺傳修飾昆蟲(chóng)品系不僅能提高現(xiàn)有的害蟲(chóng)控制方法，也有利于開(kāi)發(fā)出新的害蟲(chóng)控制方法。利用性別和組織特異啟動(dòng)子啟動(dòng)外源基因的目的性表達(dá)是這項(xiàng)技術(shù)得以成功的關(guān)鍵因素之一(Handler,2002),如昆蟲(chóng)不育技術(shù)中遺傳定性品系、雄性不育和精子熒光標(biāo)記品系的開(kāi)發(fā)。下面以精子熒光標(biāo)記品系的建立為例，說(shuō)明組織特異啟動(dòng)子的作用機(jī)制。

2.2.1精巢特異β2-tubulin啟動(dòng)子作用機(jī)制精巢特異表達(dá)基因β2-tubulin最早在黑腹果蠅Drosophilamelanogaster(Meigen) 中分離并鑒定，該基因僅在幼蟲(chóng)和成蟲(chóng)精子形成階段發(fā)生作用，特別是其基因絲功能依賴C-端尾氨基酸基序是該基因區(qū)別保守的tubulin家族其他成員的典型特征(Hoyleetal.,1995; Raffetal.,2008)。繼黑腹果蠅之后，該基因陸續(xù)在美洲煙夜蛾Heliothisvirescens(Fabricius) (Davis & Miller,1988)、斑須按蚊AnophelesstephensiListon (Catterucciaetal.,2005)、埃及伊蚊Aedesaegypti(L.) (Smithetal.,2007)、地中海實(shí)蠅Ceratitiscapitata(Wiedemann) (Scolarietal.,2008)，以及加勒比按實(shí)蠅Anastrephasuspensa(Loew)、墨西哥按實(shí)蠅Anastrephaludens(Loew)和桔小實(shí)蠅Bactroceradorsalis(Hendel) (Zimowskaetal.,2009)等昆蟲(chóng)中被分離得到。

在果蠅中，精巢特異表達(dá)基因β2-tubulin啟動(dòng)子啟動(dòng)外源基因在精巢特異表達(dá)。啟動(dòng)子刪減試驗(yàn)結(jié)果表明，該基因啟動(dòng)子轉(zhuǎn)錄位點(diǎn)上游53 bp及下游23 bp序列足以調(diào)控外源基因在精巢的準(zhǔn)確轉(zhuǎn)錄。體外突變?cè)囼?yàn)表明，一個(gè)在海德氏果蠅蠅Drosophilahydei(Sturtevant)和黑腹果蠅中保守的14 bp基序ATCGYAGTAGYCTA是控制β2-tubulin啟動(dòng)子精巢特異表達(dá)的唯一元件(Michielsetal.,1989)。

2.2.2精巢特異β2-tubulin啟動(dòng)子應(yīng)用實(shí)例近年來(lái)，利用β2-tubulin啟動(dòng)子驅(qū)動(dòng)熒光蛋白EGFP或DsRed已在斑須按蚊 (Catterucciaetal.,2005)、埃及伊蚊 (Smithetal.,2007)、地中海實(shí)蠅(Scolarietal.,2008)和加勒比按實(shí)蠅(Zimowskaetal.,2009)的精巢中表達(dá)，并成功建立了精子熒光標(biāo)記品系。以加勒比按實(shí)蠅為例，精子熒光標(biāo)記品系雄蟲(chóng)精巢和與熒光標(biāo)記雄蟲(chóng)交配后的雌蟲(chóng)受精囊中均能檢測(cè)到熒光蛋白的表達(dá)(圖2，Zimowskaetal.,2009)。精子熒光標(biāo)記品系的應(yīng)用主要體現(xiàn)在以下3個(gè)方面：(1) 性別分離，借助識(shí)別不同熒光的分揀機(jī)分離雌、雄蟲(chóng)，目前已開(kāi)發(fā)的機(jī)械光學(xué)的熒光分揀機(jī)(COPOS，Union Biometrica)可以用于分離精子熒光標(biāo)記品系的雌、雄幼蟲(chóng)，已被成功用于分揀岡比亞按蚊雄蟲(chóng)(Maroisetal., 2012)；(2)田間監(jiān)測(cè)，替代傳統(tǒng)的熒光粉標(biāo)記方法，提高了SIT監(jiān)測(cè)的準(zhǔn)確性和效果；(3)為繁殖生物學(xué)研究提供技術(shù)手段，而且精子熒光標(biāo)記有利于研究精子轉(zhuǎn)移、儲(chǔ)存、使用和競(jìng)爭(zhēng)等繁殖生物學(xué)的相關(guān)內(nèi)容，為更好地防治害蟲(chóng)提供理論依據(jù)。

圖2　β2-tubulin啟動(dòng)子驅(qū)動(dòng)德克薩斯紅色熒光在加勒比按實(shí)蠅雄蟲(chóng)精巢特異表達(dá)

Windbichleretal.(2008)利用β2-tubulin啟動(dòng)歸位核酸內(nèi)切酶I-PpoI的表達(dá)，由于I-PpoI能高度特異地靶定于X染色體連鎖的28S核糖體基因重復(fù)序列，而當(dāng)同源染色體中的一條具有歸位核酸內(nèi)切酶時(shí)，歸位核酸內(nèi)切酶將切割另一條染色體，并以前者為模板進(jìn)行復(fù)制，在岡比亞按蚊AnophelesgambiaeGiles的精子發(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(lái)源的X染色體，導(dǎo)致后代雌蟲(chóng)在胚胎期死亡，產(chǎn)生全部為攜帶歸位核酸內(nèi)切酶的雄蚊。

2.2.3組織特異啟動(dòng)子的應(yīng)用進(jìn)展近年來(lái)，組織特異啟動(dòng)子的分離與鑒定取得了一定進(jìn)展，分別體現(xiàn)在卵巢、脂肪體、中腸和唾液等組織中多種特異表達(dá)啟動(dòng)子的鑒定與應(yīng)用上。例如，黑腹果蠅中有3種卵黃蛋白基因只在雌蟲(chóng)脂肪體和卵巢濾泡細(xì)胞中表達(dá)(Brennanetal.,1982; Isaac & Bownes,1982)。S?ndergaardetal.(1995)通過(guò)缺失卵黃原蛋白Vitellogenin啟動(dòng)子(Vg)啟動(dòng)報(bào)告基因表達(dá),構(gòu)建轉(zhuǎn)基因果蠅的方法，鑒定了卵黃原蛋白基因啟動(dòng)子中與營(yíng)養(yǎng)響應(yīng)相關(guān)的調(diào)控區(qū)域。Heinrich & Scott(2000)和 Thomasetal.(2000)在黑腹果蠅中成功建立了利用卵黃蛋白yolk protein基因yp3和yp1啟動(dòng)子驅(qū)動(dòng)致死基因只在雌蟲(chóng)中條件性表達(dá)的遺傳定性系統(tǒng) 。Kokozaetal. (2000)利用埃及伊蚊的雌性特異卵黃原蛋白Vitellogenin啟動(dòng)子(Vg)調(diào)控防御素defensins A的表達(dá)，培育的埃及伊蚊通過(guò)吸血激活防御素表達(dá)，吸血24 h后防御素即在該蚊蟲(chóng)脂肪體中高水平表達(dá)，而由于防御素具有殺滅細(xì)菌及潛在的抗瘧原蟲(chóng)作用，所以培育的埃及伊蚊可以通過(guò)干擾病原體的傳播有效地阻斷蚊媒病的擴(kuò)散。繼此之后，越來(lái)越多物種的雌性特異表達(dá)基因被分離，主要包括卵黃蛋白Vitellogenins (Rina & Savakis,1991)、絨毛膜蛋白Chorion (Vlachouetal.,1997; Vlachou & Komitopoulou,2001)和抗細(xì)菌多肽Ceratotoxins (Marchinietal.,1997; Rosettoetal.,2000)，但這些基因的啟動(dòng)子和調(diào)控元件尚有待進(jìn)一步鑒定。

脂肪體是營(yíng)養(yǎng)和能量的儲(chǔ)存及供給中心，又是體內(nèi)激素作用的靶組織，同時(shí)還具有貯存排泄和解毒等生理功能。在果蠅、按蚊等重要昆蟲(chóng)類群中，有關(guān)脂肪體特異表達(dá)基因的啟動(dòng)子的研究較為深入，并在基因功能與應(yīng)用的研究中發(fā)揮了重要作用。如利用脂肪體高度特異的幼蟲(chóng)血清蛋白LSP (Larval serum protein)啟動(dòng)子和果蠅雙元表達(dá)系統(tǒng)GAL4-UAS建立的脂肪體特異表達(dá)系統(tǒng)可廣泛用于脂肪體相關(guān)基因功能研究(Lazarevaetal.,2007)。在雄蟲(chóng)脂肪體中，特異表達(dá)的雄性特異血清蛋白MSSP (Male-specific serum protein)被分離(Christophidesetal.,2000a)，地中海實(shí)蠅中有5種MSSP被分離，其中最主要的2種是MSSP-α和MSSP-β的二聚體多肽，其他的是MSSP-α和MSSP-β的均質(zhì)或異質(zhì)聚體多肽(Thymianouetal.,1995)。MSSP-α2基因的2個(gè)啟動(dòng)子片段α2PS和α2PL驅(qū)動(dòng)目的基因如半乳糖苷酶基因LacZ，僅在雄蟲(chóng)脂肪體表達(dá)，α2PL的活性強(qiáng)于α2PS，這2種啟動(dòng)子可用于構(gòu)建基于乙醇脫氫酶的遺傳性別區(qū)分品系(Christophidesetal.,2001; Komitopoulouetal.,2004)。而MSSP-β2則驅(qū)動(dòng)目的基因在雌、雄蟲(chóng)中腸組織中的特異表達(dá)(Christophidesetal.,2000b)。

中腸是昆蟲(chóng)重要的免疫組織器官，研究中腸特異啟動(dòng)子對(duì)揭示中腸特異表達(dá)基因表達(dá)調(diào)控機(jī)理、免疫應(yīng)答和應(yīng)用研究具有重要意義。Abrahametal.(2005)利用岡比亞按蚊圍食膜基質(zhì)蛋白Peritrophin-1啟動(dòng)子(Aper1)驅(qū)動(dòng)磷脂酶Phospholipase A2 (PLA2)在中腸特異表達(dá)，導(dǎo)致瘧原蟲(chóng)的卵囊形成減少約80%。Itoetal.(2002)利用斑須按蚊中腸特異的羧肽酶carboxypeptidase啟動(dòng)子(CP)驅(qū)動(dòng)唾腺和中腸結(jié)合多肽(SM1)表達(dá)，該種多肽與中腸結(jié)合，使瘧原蟲(chóng)失去生存環(huán)境，從而阻斷其傳播。

此外，一些唾液特異表達(dá)基因如maltase-likeI(malI)、mpyrase(mpy)、30Ka和30Kb的啟動(dòng)子亦被分離鑒定(Coatesetal.,1999; Mathuretal.,2010)，利用30Kb啟動(dòng)子驅(qū)動(dòng)Membranes no protein (Mnp)在中腸特異表達(dá)，能明顯降低埃及伊蚊經(jīng)唾液和唾腺感染的登革熱病毒的流行及其致病強(qiáng)度(Mathuretal.,2010)。

2.3　特定發(fā)育時(shí)期啟動(dòng)子

特定發(fā)育時(shí)期表達(dá)的啟動(dòng)子是指在昆蟲(chóng)生長(zhǎng)發(fā)育某個(gè)階段特異表達(dá)基因的啟動(dòng)子。應(yīng)用廣泛的特定發(fā)育時(shí)期啟動(dòng)子包括胚胎囊胚層細(xì)胞分化基因nullo和serendipityα(sryα)啟動(dòng)子以及生殖細(xì)胞特異表達(dá)基因nanos和vasa啟動(dòng)子。nullo和serendipityα基因是果蠅胚胎時(shí)期表達(dá)的基因(Hunteretal.,2002; Ibnsoudaetal.,1993)，這2個(gè)基因編碼的微絲網(wǎng)絡(luò)結(jié)構(gòu)成分能促進(jìn)胚胎囊胚層細(xì)胞的分化(Ibnsoudaetal.,1993)。Horn & Wimmer(2003)利用這2個(gè)基因的啟動(dòng)子驅(qū)動(dòng)促細(xì)胞凋亡基因hid(Headinvolutiondefective)在胚胎期大量表達(dá)，通過(guò)四環(huán)素抑制致死系統(tǒng)控制hid蛋白條件性地表達(dá)，建立了果蠅胚胎致死系統(tǒng)。Scheteligetal.(2009)通過(guò)分離地中海實(shí)蠅內(nèi)源sryα啟動(dòng)子，該系統(tǒng)被成功轉(zhuǎn)移到地中海實(shí)蠅，建立地中海實(shí)蠅胚胎致死系統(tǒng)。之后，Ogauguwuetal.(2013)和 Schetelig & Handler(2012)通過(guò)將該系統(tǒng)與性別特異剪切系統(tǒng)結(jié)合，成功建立了地中海實(shí)蠅和加勒比按實(shí)蠅雌性胚胎期特異致死系統(tǒng)。而生殖細(xì)胞特異表達(dá)基因nanos和vasa啟動(dòng)子被認(rèn)為在生殖不育品系的建立中有望得到應(yīng)用(Adelmanetal.,2007; Papathanosetal.,2009)。

2.3.1Nanos基因啟動(dòng)子作用機(jī)制Nanos基因最早在果蠅中發(fā)現(xiàn)，是一種母性效應(yīng)基因。該基因編碼一種RNA結(jié)合蛋白，這種蛋白與pumilioRNA結(jié)合蛋白相互作用形成一種核糖核蛋白復(fù)合物，一起抑制母源hunchbackmRNA的翻譯，從而調(diào)控果蠅胚胎后腹部細(xì)胞的分化。進(jìn)一步的研究表明,nanos基因的缺失將使果蠅不能形成性腺，從而產(chǎn)生異常生殖細(xì)胞。Nanos啟動(dòng)子轉(zhuǎn)錄起始位點(diǎn)上游 -108 bp~+97 bp為增強(qiáng)子區(qū)，-108 bp~+97 bp間的啟動(dòng)子片段足以調(diào)控外源基因GFP在果蠅生殖細(xì)胞中的表達(dá)(Alietal.,2010)。

2.3.2Nanos基因啟動(dòng)子應(yīng)用實(shí)例Nanos基因在地中海實(shí)蠅、岡比亞按蚊、斑須按蚊和埃及伊蚊中的表達(dá)模式與果蠅相同，主要在胚胎早期及雌蟲(chóng)發(fā)育的卵母細(xì)胞中積累(Calvoetal.,2005; Ogaugwuetal.,2013)。Adelmanetal.(2007)利用nanos基因的啟動(dòng)子區(qū)域和非編碼區(qū)驅(qū)動(dòng)外源mariner轉(zhuǎn)座酶MosI編碼DNA在埃及伊蚊雌蟲(chóng)生殖細(xì)胞特異轉(zhuǎn)錄，MosI mRNA在發(fā)育中的卵母細(xì)胞中積累并定位于早期發(fā)育胚胎的后極孔，并能再次將MosI整合到埃及伊蚊基因組，表明nanos啟動(dòng)子有望成為基于轉(zhuǎn)座元件的基因驅(qū)動(dòng)系統(tǒng)(TE-based gene driver system)，利用轉(zhuǎn)座子技術(shù)建立抗登革熱病毒的遺傳修飾蚊并用于蚊蟲(chóng)種群替代防控策略具有較大的應(yīng)用潛力。最新的基因組編輯技術(shù)CRISPR/CAS 9，利用nanos啟動(dòng)子在生殖細(xì)胞中過(guò)表達(dá)了cas9酶，提高和優(yōu)化了果蠅中基因組編輯的效率(Kondoetal.,2013)。

2.4　誘導(dǎo)型啟動(dòng)子

誘導(dǎo)型啟動(dòng)子通常僅在特定環(huán)境條件下表現(xiàn)活性，這些條件可以是物理的、化學(xué)的或生物的。誘導(dǎo)型啟動(dòng)子往往具有增強(qiáng)子、沉默子或類似功能的序列結(jié)構(gòu)，感受誘導(dǎo)的序列都具有明顯的專一性。誘導(dǎo)型啟動(dòng)子通常在某些特定的物理或化學(xué)因素的刺激下，可大幅度提高基因的轉(zhuǎn)錄水平，如果蠅熱激蛋白啟動(dòng)子能夠進(jìn)行熱激調(diào)節(jié)，其中hsp70啟動(dòng)子的熱激活性最強(qiáng)，使用也最為廣泛。

2.4.1熱激蛋白基因啟動(dòng)子作用機(jī)制Nover(1987)、 Pelham & Bienz(1982)和 Topoletal.(1985)通過(guò)比較果蠅的hsp83、hsp70、hsp68、hsp27、hsp26、hsp23和hsp22的啟動(dòng)子序列，發(fā)現(xiàn)有一個(gè)14 bp的回文序列，其中10 bp為高度保守，其序列為CTnGAAnnTTCnAG，稱為HSE (Heat shock element)元件，能夠進(jìn)行熱激調(diào)控，如果只配對(duì)4或5個(gè)堿基，則序列沒(méi)有熱激活性，而l或2個(gè)堿基的錯(cuò)配可以被容忍，沒(méi)有特定的堿基是非必需的。HSE中存在HSF1 (Heat shock factor l) (Morimoto,1993)、CHBF (constitutive HSE-binding factor) (Mosseretal.,1988)和Ku因子(Turturici,2009)等的結(jié)合位點(diǎn)，這也是HSE序列能夠?qū)崿F(xiàn)熱激調(diào)節(jié)的原因。

2.4.2Hsp70啟動(dòng)子應(yīng)用實(shí)例果蠅hsp70啟動(dòng)子應(yīng)用非常廣泛，常被用于驅(qū)動(dòng)外源基因在其他昆蟲(chóng)中的條件性表達(dá)(Fuetal.,2007； Gongetal.,2005)，也用于構(gòu)建遺傳轉(zhuǎn)化體系的輔助質(zhì)粒，驅(qū)動(dòng)Minos和piggyBac等轉(zhuǎn)座酶基因的表達(dá)，提高轉(zhuǎn)座活性，其有效性已在蠅類和蚊蟲(chóng)等的轉(zhuǎn)化中得以證明(Catterucciaetal.,2000; Ogaugwuetal.,2013)。研究表明，該啟動(dòng)子在異質(zhì)系統(tǒng)中能發(fā)揮作用(Atkinson & O′Brochta,1992; Bergeetal.,1985; Bienz & Pelham,1982; Voellmy & Rungger,1982)，但在非果蠅昆蟲(chóng)中的表達(dá)活性相對(duì)較低(Atkinson & O′Brochta,1992; Bergeetal.,1985)，若需要獲得該基因的高表達(dá)，則需要分離hsp70的同源類似物啟動(dòng)子。Conchaetal.(2012)分離并鑒定銅綠蠅hsp83、hsp70、hsp23和hsp24等基因，hsp83在銅綠蠅各個(gè)發(fā)育階段均高水平表達(dá)，熱激后，其表達(dá)水平提高2～10倍，而hsp70的表達(dá)水平相對(duì)較低，但熱激后，其表達(dá)水平有很大的提高，約230～770倍，hsp23和hsp24的表達(dá)水平則在各個(gè)發(fā)育階段有較大差異，該研究結(jié)果為組成型或條件性誘導(dǎo)基因的表達(dá)，建立遺傳修飾銅綠蠅奠定基礎(chǔ)。

3　展望

啟動(dòng)子是基因轉(zhuǎn)錄調(diào)控的重要元件，決定了mRNA的時(shí)空轉(zhuǎn)錄，從而實(shí)現(xiàn)生物的有序分化發(fā)育過(guò)程。研究啟動(dòng)子的結(jié)構(gòu)和功能，對(duì)于基因表達(dá)模式和基因調(diào)控網(wǎng)絡(luò)等方面十分重要。根據(jù)組成型啟動(dòng)子、組織特異性啟動(dòng)子和誘導(dǎo)型啟動(dòng)子的特性和作用機(jī)制的不同，各類啟動(dòng)子在昆蟲(chóng)遺傳控制技術(shù)中的應(yīng)用也有所差別。組成型基因多為管家基因，該類啟動(dòng)子已被證明能有效地用于驅(qū)動(dòng)標(biāo)記基因如熒光蛋白的表達(dá)，該類啟動(dòng)子持續(xù)穩(wěn)定地高水平表達(dá)使遺傳修飾昆蟲(chóng)的篩選工作更加切實(shí)可行。性別、組織特異性啟動(dòng)子能實(shí)現(xiàn)外源效應(yīng)基因僅在單一性別或目的組織中表達(dá)，實(shí)現(xiàn)遺傳控制害蟲(chóng)特異性致死或?qū)⒅滤阑蛱禺愋詡鬟f。誘導(dǎo)型啟動(dòng)子可以條件性地控制外源效應(yīng)基因的表達(dá)。根據(jù)昆蟲(chóng)遺傳控制策略的不同，選擇性地選擇合適的啟動(dòng)子構(gòu)建外源基因驅(qū)動(dòng)系統(tǒng)是非常重要的。隨著昆蟲(chóng)啟動(dòng)子分離、鑒定工作的進(jìn)一步完善發(fā)展，更多適合的啟動(dòng)子將會(huì)被應(yīng)用到昆蟲(chóng)遺傳控制技術(shù)中的驅(qū)動(dòng)系統(tǒng)，促進(jìn)害蟲(chóng)遺傳防治技術(shù)的發(fā)展。

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(責(zé)任編輯:郭瑩)

LaboratoryofWildAnimalConservationandUtilization,GuangdongEntomologicalInstitute,Guangzhou,Guangdong510260,

China;3DepartmentofEntomology,NorthCarolinaStateUniversity,CampusBox7613,Raleigh,NC27695-7613,USA；

4GeneticEngineeringandSocietyCenterandW.M.KeckCenterforBehavioralBiology,NorthCarolinaStateUniversity,

Raleigh,NC27695-7613,USA；5CollegeofAgricultureandPlantProtection,

QingdaoAgriculturalUniversity,Qingdao,Shandong266109,China

Abstract:Genetic control using germ-line transformation is the new trend in the research of insect pest control. It is a species-specific, highly efficient and environmental friendly pest control strategy. Gene promoter, one of the key component for gene expression regulation, is requisite for the construction of high-efficient and stable genetic modified strains for insect population genetic control. In this review, the basic structural characteristics of insect gene promoters are briefly described. The progress and application of different type promoters such as constitutive promoter, sex- and tissue-specific promoter, age-specific promoter and inducible promoter, are also introduced. Finally, the future prospect of different types of promoters used in insect population genetic control is disccused.

Key words:insect population genetic control; sterile insect technique; genetic transformation; constitutive promoter; specific promoter; inducible promoter

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

作者簡(jiǎn)介:呂志創(chuàng)， 女， 助理研究員。 研究方向： 入侵昆蟲(chóng)分子生態(tài)學(xué)。 E-mail： grasslzc@163.com

基金項(xiàng)目:國(guó)家“973”計(jì)劃項(xiàng)目(2009CB119200)； 國(guó)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(chóng)害疫情監(jiān)測(cè)與防治項(xiàng)目(2003-2015)； 中國(guó)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)： 2014-12-16接受日期(Accepted)： 2015-01-07

DOI:10.3969/j.issn.2095-1787.2015.02.005