許　軍， 張宏波， 韓民錦， 譚安江， 黃勇平， 張　澤*

1中國(guó)科學(xué)院上海生命科學(xué)研究院植物生理生態(tài)研究所，昆蟲發(fā)育與進(jìn)化生物學(xué)重點(diǎn)實(shí)驗(yàn)室，

上海 200032； 2中國(guó)科學(xué)院大學(xué)，北京 100049； 3重慶大學(xué)生命科學(xué)學(xué)院，重慶 400044

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昆蟲轉(zhuǎn)座子在轉(zhuǎn)基因技術(shù)中的應(yīng)用

許軍1,2， 張宏波3， 韓民錦3， 譚安江1， 黃勇平1， 張澤3*

1中國(guó)科學(xué)院上海生命科學(xué)研究院植物生理生態(tài)研究所，昆蟲發(fā)育與進(jìn)化生物學(xué)重點(diǎn)實(shí)驗(yàn)室，

上海 200032；2中國(guó)科學(xué)院大學(xué)，北京 100049；3重慶大學(xué)生命科學(xué)學(xué)院，重慶 400044

摘要:轉(zhuǎn)座子是基因組中一段可移動(dòng)的DNA重復(fù)片段。越來(lái)越多的研究表明，轉(zhuǎn)座子是真核生物基因組的主要組成成分，是基因組和表型進(jìn)化的主要?jiǎng)恿χ?，并且?duì)基因表達(dá)調(diào)控網(wǎng)絡(luò)的進(jìn)化具有重要的貢獻(xiàn)。由于轉(zhuǎn)座子在基因組內(nèi)具有可移動(dòng)性，使其在生物技術(shù)和分子生物學(xué)領(lǐng)域備受重視，尤其在轉(zhuǎn)基因技術(shù)上得到了廣泛應(yīng)用。本文綜述了轉(zhuǎn)座子在昆蟲中的分布、類型及功能，重點(diǎn)闡述不同昆蟲轉(zhuǎn)座子在轉(zhuǎn)基因技術(shù)中的應(yīng)用，并對(duì)轉(zhuǎn)基因安全性和轉(zhuǎn)座子穩(wěn)定性進(jìn)行了討論。

關(guān)鍵詞:轉(zhuǎn)座子； 昆蟲； 轉(zhuǎn)基因； 安全性

1956年McClintock在玉米中發(fā)現(xiàn)了控制元件，這種控制元件是基因組中一段可移動(dòng)的DNA序列，可以通過(guò)不同轉(zhuǎn)座機(jī)制從基因組的一個(gè)位置“跳躍”到另一個(gè)位置，后來(lái)這種控制元件被命名為轉(zhuǎn)座元件或轉(zhuǎn)座子(Transposon)(McClintock,1956)。根據(jù)其轉(zhuǎn)座機(jī)制的不同主要分為DNA轉(zhuǎn)座子(DNA transposon,Class 2)和反轉(zhuǎn)錄轉(zhuǎn)座子(Retrotransposon,Class 1)(Wickeretal.,2007)。DNA轉(zhuǎn)座子以“剪切和粘貼”(Cut and paste)機(jī)制進(jìn)行轉(zhuǎn)座，在其過(guò)程中轉(zhuǎn)座酶將供體位點(diǎn)的DNA轉(zhuǎn)座子以雙鏈DNA的形式切割并整合到靶位點(diǎn)完成轉(zhuǎn)座。反轉(zhuǎn)錄轉(zhuǎn)座子以“拷貝和粘貼”(Copy and paste)機(jī)制轉(zhuǎn)座，其先以雙鏈DNA為模板轉(zhuǎn)錄成RNA，再逆轉(zhuǎn)錄合成cDNA，最后整合到基因組中完成轉(zhuǎn)座(圖1)。反轉(zhuǎn)錄轉(zhuǎn)座子包括長(zhǎng)末端重復(fù)元件(Long terminal repeats,LTRs)和非長(zhǎng)末端重復(fù)元件(Non-long terminal repeats,Non-LTRs);非長(zhǎng)末端重復(fù)元件又可分為自主的長(zhǎng)散布元件(Long interspersed elements,LINEs)和非自主的短散布元件(Short interspersed elements,SINEs)。

轉(zhuǎn)座子是真核生物基因組的主要組成成分，如轉(zhuǎn)座子約占人類基因組序列的45%、玉米基因組序列的75%、果蠅Drosophilamelanogaster基因組序列的15%~22% 和飛蝗Locustamigratoria基因組的41%等(Ashburner & Bergman,2005; Kazazian，2004; Quesnevilleetal.,2005; Wangetal.,2014)。轉(zhuǎn)座子具有轉(zhuǎn)座能力，并且可以通過(guò)轉(zhuǎn)座增加其在基因組中的拷貝，因此，轉(zhuǎn)座子是基因組進(jìn)化的主要?jiǎng)恿χ?。另外，無(wú)論是在轉(zhuǎn)錄水平還是轉(zhuǎn)錄后水平，轉(zhuǎn)座子均可以通過(guò)多種機(jī)制改變鄰近基因的結(jié)構(gòu)和表達(dá)(圖2)。某些轉(zhuǎn)座子內(nèi)部可能包含一些順式調(diào)節(jié)元件，當(dāng)其發(fā)生轉(zhuǎn)座插入到功能基因附近時(shí)就有可能改變鄰近基因的表達(dá)(Feschotte,2008)。例如，人類基因組中大約有22%的啟動(dòng)子序列來(lái)自轉(zhuǎn)座子 (Jordanetal.,2003; van de Lagemaatetal.,2003)。當(dāng)轉(zhuǎn)座子插入到一些基因上游的順式調(diào)控元件并且破壞了原有的順式調(diào)節(jié)元件時(shí)，有可能導(dǎo)致其鄰近基因表達(dá)的關(guān)閉，還有一些轉(zhuǎn)座子可能會(huì)作為表觀沉默的靶位點(diǎn)影響鄰近基因的表達(dá)(Girard & Freeling,1999; Grewal & Jia,2007; Slotkin & Martienssen,2007； Wallaceetal.,1991)。此外，轉(zhuǎn)座子可以通過(guò)外顯子化改變鄰近基因的結(jié)構(gòu)和表達(dá)(Nietal.,2007; Piriyapongsaetal.,2007)。

圖1　3類主要轉(zhuǎn)座子的轉(zhuǎn)座機(jī)制圖Fig.1　Translocation mechanisms of three types of transposable elements

轉(zhuǎn)座子可以通過(guò)自身或其他自主轉(zhuǎn)座子編碼的轉(zhuǎn)座酶完成其在基因組中的轉(zhuǎn)座，近年利用轉(zhuǎn)座子開發(fā)的轉(zhuǎn)基因載體已成為功能基因組學(xué)研究的重要工具，并且極大地推動(dòng)了基礎(chǔ)理論和應(yīng)用生物學(xué)的發(fā)展。Nelson & Klein (1984)用轉(zhuǎn)座子標(biāo)簽法克隆了玉米bronze基因。Khillanetal.(1985)將目的基因置于P因子中，在轉(zhuǎn)座酶的幫助下，將攜帶目的基因的P因子從質(zhì)粒轉(zhuǎn)到老鼠的染色體上，從而達(dá)到轉(zhuǎn)基因的目的。Couplandetal.(1988)在煙草中證明了負(fù)責(zé)玉米Ac轉(zhuǎn)座子轉(zhuǎn)座的核心序列。目前，研究人員已經(jīng)開發(fā)出了適用于多種生物體的轉(zhuǎn)基因載體，如應(yīng)用piggyBac轉(zhuǎn)座子在包括小鼠在內(nèi)的許多物種中成功獲得了轉(zhuǎn)基因個(gè)體。同時(shí)，由于轉(zhuǎn)座子在物種內(nèi)或不同物種基因組間具有可移動(dòng)性或不穩(wěn)定性，給轉(zhuǎn)基因過(guò)程中的生物安全帶來(lái)了潛在風(fēng)險(xiǎn)。

1　昆蟲中轉(zhuǎn)座子的分布

隨著大規(guī)?；蚪M測(cè)序技術(shù)的發(fā)展和5000個(gè)昆蟲基因組計(jì)劃(i5k)的開展，目前已有近123個(gè)昆蟲基因組被測(cè)序(http:∥www.ncbi.nlm.nih.gov/genome/browse/)，對(duì)已測(cè)序昆蟲基因組進(jìn)行注釋后發(fā)現(xiàn)，轉(zhuǎn)座子廣泛分布于昆蟲基因組中,并且不同昆蟲基因組中轉(zhuǎn)座子含量存在很大的差別。如家蠶Bombyxmori基因組中已鑒定了1308個(gè)轉(zhuǎn)座子家族，約占整個(gè)基因組序列的45%，僅次于埃及伊蚊Aedesaegypti基因組中的轉(zhuǎn)座子含量(47%) (Neneetal.,2007; Osanai-Futahashietal.,2008)。家蠶轉(zhuǎn)座子多數(shù)集中于LTR、LINE和TIR等3類，其中，LINE類轉(zhuǎn)座子含量最多，TIR 和LTR類轉(zhuǎn)座子次之(圖3)(Xuetal.,2013)。在雙翅目模式昆蟲果蠅屬中轉(zhuǎn)座子含量相對(duì)較低，約占整個(gè)果蠅屬基因組序列的15%~22%，其中，LTR類反轉(zhuǎn)錄轉(zhuǎn)座子最多，LINE類反轉(zhuǎn)錄轉(zhuǎn)座子和TIR類轉(zhuǎn)座子次之。另外，在已測(cè)序果蠅屬的12 個(gè)種中存在一種特異的轉(zhuǎn)座子家族DINE-1，該轉(zhuǎn)座子家族僅分布于雙翅目昆蟲中(Clarketal.,2007)。親緣關(guān)系較近的物種間轉(zhuǎn)座子種類也存在巨大差異。如埃及伊蚊基因組中只含有一種高度退化的Mariner 元件，而這種元件在岡比亞按蚊Anophelesgambiae中達(dá)到了20種以上；在埃及伊蚊中發(fā)現(xiàn)岡比亞按蚊中沒(méi)有的3種特異元件，即Non-LTR的LOA 元件、LTR的Osvaldo元件和Penelope家族(Holtetal.,2002; Neneetal.,2007)。這一結(jié)果證實(shí)轉(zhuǎn)座子在不同系統(tǒng)中進(jìn)化方式不同。

圖2　轉(zhuǎn)座子對(duì)基因結(jié)構(gòu)和表達(dá)的影響Fig.2　The influence of transposable elements on structures and expression of genes

圖3　家蠶基因組中不同轉(zhuǎn)座子的種類Fig.3　The classification of transposable elements in the silkworm, Bombyx mori

2　昆蟲轉(zhuǎn)座子的應(yīng)用

轉(zhuǎn)座元件的發(fā)現(xiàn)為遺傳學(xué)研究提供了一種分子操作手段。如轉(zhuǎn)座子插入位點(diǎn)的多樣性可以作為遺傳標(biāo)記用于連鎖和進(jìn)化分析，還可以開發(fā)出轉(zhuǎn)基因載體。轉(zhuǎn)基因昆蟲是被認(rèn)為繼轉(zhuǎn)基因微生物和轉(zhuǎn)基因植物之后又一項(xiàng)可以帶動(dòng)工業(yè)生產(chǎn)的分子生物學(xué)技術(shù)。通過(guò)該技術(shù)對(duì)經(jīng)濟(jì)昆蟲進(jìn)行改良，可以阻斷媒介昆蟲對(duì)疾病的傳播和農(nóng)林業(yè)害蟲的危害。昆蟲轉(zhuǎn)基因系統(tǒng)中的轉(zhuǎn)座子主要有5種(圖4；Handler,2001)。

2.1　Minos轉(zhuǎn)座子

Minos轉(zhuǎn)座子是從海德爾果蠅Drosophilahydei中分離得到，其長(zhǎng)度為1.4 kb，具有100 bp的末端反向重復(fù)序列(Franz & Savakis,1991)。Minos轉(zhuǎn)座子在地中海實(shí)蠅Ceratitiscapitata中的轉(zhuǎn)座效率為1%~3%(Loukerisetal.,1995)。Minos轉(zhuǎn)座子還可以在雙翅目和鱗翅目的細(xì)胞系中發(fā)生轉(zhuǎn)座，并且已應(yīng)用該轉(zhuǎn)座子成功獲得斯氏按蚊Anophelesstephensi和黑果蠅Drosophilavirilis轉(zhuǎn)基因個(gè)體(Catterucciaetal.,2000a、2000b; Klinakisetal.,2000)。

2.2　Mos1 (Mariner)轉(zhuǎn)座子

Mos1轉(zhuǎn)座子是從馬里塔尼亞果蠅Drosophilamauritiana中發(fā)現(xiàn)，并且與體細(xì)胞不穩(wěn)定性等位基因whitepeach相關(guān)聯(lián)(Haymer & Marsh,1986; Jacobsonetal.,1986)。其長(zhǎng)度為1286 bp，具有28 bp的末端反向重復(fù)序列，其中有4個(gè)堿基錯(cuò)配。與其他TC1轉(zhuǎn)座子相似，其在插入位點(diǎn)兩端通常會(huì)形成2 bp(TA)的靶位點(diǎn)正向重復(fù)序列。利用Mariner轉(zhuǎn)座子作為載體已經(jīng)成功獲得果蠅和埃及伊蚊轉(zhuǎn)基因個(gè)體(Coatesetal.,1998; Garzaetal.,1991; Lidholmetal.,1993; Lohe & Hartl,1996)。

圖4　昆蟲轉(zhuǎn)基因系統(tǒng)中的轉(zhuǎn)座子Fig.4　Transposable elements in insect transgenic vector systems

2.3　P因子

由于P因子能引起黑腹果蠅Drosophilamelanogaster生殖障礙而被鑒定出來(lái)，目前，P因子已經(jīng)廣泛應(yīng)用于果蠅遺傳學(xué)研究，是目前研究最詳細(xì)的一類昆蟲轉(zhuǎn)座子(Kidwell,1977)。P轉(zhuǎn)座子長(zhǎng)度為2.9 kb，具有31 bp的末端反向重復(fù)序列。中間含有可編碼轉(zhuǎn)座酶的序列，已經(jīng)建立了成熟的P轉(zhuǎn)座子和轉(zhuǎn)座酶輔助系統(tǒng)(Rubin & Spradling,1982)。但是，該轉(zhuǎn)座子在除果蠅以外的昆蟲中沒(méi)有轉(zhuǎn)座活性，因此，其在應(yīng)用上受到了極大限制。

2.4　hobo轉(zhuǎn)座子

系統(tǒng)發(fā)生分析表明，hobo和植物中的Ac和Tam3轉(zhuǎn)座子具有同源性(Calvietal.,1991)。這暗示hobo轉(zhuǎn)座子作為轉(zhuǎn)基因載體可能具有更為廣泛的應(yīng)用前景。昆蟲中的hobo轉(zhuǎn)座子包括家蠅Muscadomestica中的Hermer、果實(shí)蠅中的Homer、橘小實(shí)蠅Bactroceradorsalis中的hopper等(Handler & Gomez,1997; Pinkertonetal.,1999; Warrenetal.,1994)。目前，已應(yīng)用這些轉(zhuǎn)座子成功獲得埃及伊蚊、赤擬谷盜Triboliumcastaneum、廄螫蠅Stomoxyscalcitrans、致倦庫(kù)蚊Culexquinquefasciatus和地中海實(shí)蠅轉(zhuǎn)基因個(gè)體(Berghammeretal.,1999; Jasinskieneetal.,1998; O′Brochtaetal.,2000)。

2.5　piggyBac轉(zhuǎn)座子

piggyBac是來(lái)源于鱗翅目昆蟲的DNA轉(zhuǎn)座子，最初從桿狀病毒侵染粉紋夜蛾Trichoplusiani的TN-368細(xì)胞中分離得到(Fraseretal.,1983)。其全長(zhǎng)2472 bp, 并且具有一對(duì)13 bp末端反向重復(fù)序列和一對(duì)19 bp副末端重復(fù)序列，在副末端重復(fù)序列之間是2.1 kb轉(zhuǎn)錄單元，包含一個(gè)高頻率切除和轉(zhuǎn)座必需的1.8 kb編碼轉(zhuǎn)座酶的開放閱讀框。piggyBac轉(zhuǎn)座子常在TTAA目標(biāo)位點(diǎn)插入，因此，也被歸納為TTAA特殊的可移動(dòng)因子家族(Caryetal.,1989)。目前，該轉(zhuǎn)座子系統(tǒng)已經(jīng)在地中海實(shí)蠅、果蠅、赤擬谷盜、加勒比按實(shí)蠅Anastrephasuspensa、橘小實(shí)蠅、家蠶、棉紅鈴蟲Pectinophoragossypiella、家蠅、淡色按蚊Anophelesalbimanus、埃及伊蚊中成功實(shí)現(xiàn)了轉(zhuǎn)基因(Berghammeretal.,1999； Handleretal.,1998; Handler & Harrell,1999; Handler & McCombs,2000; Peloquinetal.,2000; Tamuraetal.,2000; Thibaultetal.,1999)。

3　轉(zhuǎn)座子穩(wěn)定性與轉(zhuǎn)基因生物安全

轉(zhuǎn)基因安全涉及2個(gè)方面：(1)轉(zhuǎn)入的基因是否會(huì)對(duì)其他生物造成危害，包括對(duì)環(huán)境造成不利影響或影響取食者的發(fā)育及繁殖；(2)轉(zhuǎn)基因是否會(huì)發(fā)生水平轉(zhuǎn)移，即轉(zhuǎn)座元件是否會(huì)發(fā)生二次轉(zhuǎn)座。在轉(zhuǎn)座酶的作用下轉(zhuǎn)座子只發(fā)生一次移動(dòng)，固定于轉(zhuǎn)入生物的基因組內(nèi)，而通常這種轉(zhuǎn)座酶不存在于轉(zhuǎn)入生物體內(nèi)，所以不會(huì)因發(fā)生跳動(dòng)而侵入其他生物體。但是，在與環(huán)境互作的過(guò)程中是否會(huì)發(fā)生再次轉(zhuǎn)座或跳動(dòng)尚不清楚。

例如，棉紅鈴蟲是世界上最具破壞性的棉花害蟲。據(jù)美國(guó)棉花協(xié)會(huì)統(tǒng)計(jì)，其每年造成將近2400萬(wàn)美元的經(jīng)濟(jì)損失。美國(guó)農(nóng)業(yè)部在加州圣華金河谷選擇了25000 hm2田塊開展了攜帶綠色熒光蛋白基因篩選標(biāo)記的轉(zhuǎn)基因棉紅鈴蟲的小規(guī)模試驗(yàn)，遺傳轉(zhuǎn)化式是由piggyBac轉(zhuǎn)座子介導(dǎo)的轉(zhuǎn)基因。野外釋放試驗(yàn)充分考慮了多重物理和生物學(xué)因素，包括地理隔離、籠子隔離、生殖不育、雄性信息素陷進(jìn)、移除可能含有野生型棉紅鈴蟲的棉花、釋放大量不育的棉紅鈴蟲、必要時(shí)殺蟲劑處理等。檢測(cè)發(fā)現(xiàn)，與野生型相比，在實(shí)驗(yàn)室飼養(yǎng)20代左右的能穩(wěn)定遺傳綠色熒光蛋白的棉紅鈴蟲的產(chǎn)卵量下降了19.8%，而piggyBac轉(zhuǎn)座子的基因組整合位點(diǎn)沒(méi)有發(fā)生任何變化，即轉(zhuǎn)座子可以穩(wěn)定地固定在基因組中而不消失或發(fā)生二次轉(zhuǎn)座(Milleretal.,2001)。但是，由于試驗(yàn)的短期性和局限性，并不能夠證實(shí)轉(zhuǎn)座子可以一直穩(wěn)定地遺傳下去。

4　結(jié)語(yǔ)

在轉(zhuǎn)基因昆蟲中，載體基因是隨目的基因一同導(dǎo)入的非必需元素。用于昆蟲轉(zhuǎn)基因的載體主要是轉(zhuǎn)座子。目前,piggyBac轉(zhuǎn)座子在昆蟲遺傳轉(zhuǎn)化中應(yīng)用最廣泛，穩(wěn)定性相對(duì)較高，應(yīng)用該轉(zhuǎn)座子已成功轉(zhuǎn)化了多種昆蟲。但是，還需要進(jìn)一步評(píng)估這些轉(zhuǎn)基因昆蟲的遺傳穩(wěn)定性、各個(gè)世代基因表達(dá)的一致性，以及是否會(huì)發(fā)生基因水平轉(zhuǎn)移等。因此，開發(fā)高效穩(wěn)定的轉(zhuǎn)座子系統(tǒng)對(duì)于昆蟲遺傳轉(zhuǎn)化體系的建立和生物安全具有十分重要的意義。

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(責(zé)任編輯:楊郁霞)

The application of insect transposons in transgenic technology

Jun XU1,2, Hong-bo ZHANG3, Min-jin HAN3, An-jiang TAN1, Yong-ping HUANG1, Ze ZHANG3*

1Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes

forBiologicalSciences,ChineseAcademyofSciences,Shanghai200032,China;2University of Chinese Academy of Sciences,

Beijing100049,China;3School of Life Sciences, Chongqing University, Chongqing 400044, China

Abstract:Transposon is a class of mobile repetitive DNA segments in the genome. Many studies found that transposons constitute a significant component of eukaryotic genomes. They are the main driving force of genomic and phenotypic evolution, and have an important contribution to the evolution of gene regulatory networks. Transposon mobility in the genome makes them an attractive tool in the field of biotechnology and molecular biology, especially in transgenic technology. In this review, we introduced the distribution, types and functions of transposons in the insects, reviewed the application and examples of insect transposons in transgenic techniques, and discussed the transgenic security and stability of transposon.

Key words:transposon; insect; transgene; security

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

作者簡(jiǎn)介:劉桂清， 女， 助理研究員。 研究方向： 昆蟲分子生物學(xué)與外來(lái)生物入侵。 E-mail： pepsiliu81@163.com

基金項(xiàng)目: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)： 2015-01-12接受日期(Accepted)： 2015-02-09

DOI:10. 3969/j.issn.2095-1787.2015.02.004