申建茹， 嚴(yán)　盈,2,3， 武　強(qiáng)， 李建偉， 張桂芬， 萬方浩,4*

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； 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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昆蟲遺傳轉(zhuǎn)化品系的常用標(biāo)記

申建茹1， 嚴(yán)盈1,2,3， 武強(qiáng)1， 李建偉1， 張桂芬1， 萬方浩1,4*

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；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:遺傳轉(zhuǎn)化標(biāo)記是將遺傳修飾昆蟲從野生型種群中分辨出來的根據(jù)，遺傳轉(zhuǎn)化昆蟲的鑒定、轉(zhuǎn)化品系的維持及其遺傳穩(wěn)定性的監(jiān)測(cè)都依賴于可靠的標(biāo)記系統(tǒng)，發(fā)展易于應(yīng)用和監(jiān)測(cè)的轉(zhuǎn)化標(biāo)記能夠極大地促進(jìn)害蟲遺傳防治的相關(guān)研究。用于遺傳修飾昆蟲的轉(zhuǎn)化標(biāo)記主要有昆蟲眼睛顏色標(biāo)記基因、抗藥性標(biāo)記基因和熒光蛋白標(biāo)記基因等。非果蠅類昆蟲首個(gè)遺傳轉(zhuǎn)化品系的鑒定是通過眼睛顏色突變而實(shí)現(xiàn)，但大多數(shù)昆蟲物種沒有可用的突變體或缺少相應(yīng)基因的信息，從而限制了眼睛顏色標(biāo)記的應(yīng)用?？顾幮曰驑?biāo)記雖然能夠通過對(duì)轉(zhuǎn)化昆蟲進(jìn)行集體選擇而大幅度提高篩選轉(zhuǎn)化體的效率，但由于其鑒定的準(zhǔn)確性不高且存在安全性問題，未得到廣泛應(yīng)用。熒光蛋白標(biāo)記基因的發(fā)展則顯著拓寬了能夠轉(zhuǎn)化的昆蟲種類。從水母分離的綠色熒光蛋白(GFP)經(jīng)突變方法獲得了多種不同熒光性質(zhì)的突變體，經(jīng)人為修飾后與適宜的強(qiáng)啟動(dòng)子構(gòu)成轉(zhuǎn)化標(biāo)記載體，能夠有效鑒定更多昆蟲物種的遺傳轉(zhuǎn)化個(gè)體，其中應(yīng)用較多的是增強(qiáng)型綠色熒光蛋白(EGFP)。此外，從珊瑚屬海葵中分離得到的紅色DsRed標(biāo)記基因提供了多樣化的紅色熒光蛋白選擇，在某些生物中DsRed與GFP聯(lián)合應(yīng)用的表現(xiàn)明顯優(yōu)于GFP突變體，所以其應(yīng)用前景也非常廣泛。本文著重從眼睛顏色、抗藥性和熒光蛋白等3個(gè)方面闡述了標(biāo)記基因的發(fā)展歷史與現(xiàn)狀，并對(duì)其今后的發(fā)展方向進(jìn)行了展望。

關(guān)鍵詞:遺傳修飾昆蟲； 轉(zhuǎn)化標(biāo)記; 眼睛顏色標(biāo)記; 抗藥性標(biāo)記; 熒光蛋白標(biāo)記

Commonly used transformation markers in genetically modified insects

Jian-ru SHEN1, Ying YAN1,2,3, Qiang WU1, Jian-wei LI1, Gui-fen ZHANG1, Fang-hao WAN1,4*

昆蟲遺傳轉(zhuǎn)化技術(shù)是將攜帶外源基因的轉(zhuǎn)座子導(dǎo)入到目標(biāo)昆蟲的基因組，使其獲得特定表型的分子生物學(xué)操縱手段。昆蟲遺傳轉(zhuǎn)化研究對(duì)于深入了解昆蟲生理和行為意義重大，同時(shí)已成為一種新型有效的害蟲控制策略。自1982年首次在黑腹果蠅Drosophilamelanogaster中應(yīng)用P-元件實(shí)現(xiàn)胚胎轉(zhuǎn)化(Rubin & Spradling,1982)之后，昆蟲遺傳轉(zhuǎn)化研究逐漸興起并得到廣泛重視，隨后開發(fā)了各種轉(zhuǎn)座元件如Minos、Mariner、Hermes和piggyBac等，并被成功應(yīng)用于重要醫(yī)學(xué)和農(nóng)業(yè)害蟲的遺傳防治研究中。各種轉(zhuǎn)化標(biāo)記的發(fā)展顯著促進(jìn)了昆蟲遺傳修飾的研究，大大拓寬了能夠被轉(zhuǎn)化的昆蟲種類(Atkinsonetal.,2001； Handler,2001a; Handler & James,2000)。遺傳轉(zhuǎn)化標(biāo)記是將遺傳修飾昆蟲從野生型種群中分辨出來的根據(jù)，遺傳轉(zhuǎn)化昆蟲的鑒定、轉(zhuǎn)化品系的維持及其遺傳穩(wěn)定性的監(jiān)測(cè)都依賴于可靠的標(biāo)記系統(tǒng)，發(fā)展易于應(yīng)用和監(jiān)測(cè)的轉(zhuǎn)化標(biāo)記能夠極大地促進(jìn)害蟲遺傳防治的相關(guān)研究。用于遺傳修飾昆蟲的轉(zhuǎn)化標(biāo)記主要有昆蟲眼睛顏色標(biāo)記基因、抗藥性標(biāo)記基因和熒光蛋白標(biāo)記基因等(Alphey,2002)。

1　眼睛顏色基因轉(zhuǎn)化標(biāo)記

各種控制眼睛顏色基因的發(fā)掘，豐富了昆蟲遺傳修飾研究的眼睛顏色標(biāo)記。早期對(duì)果蠅眼睛顏色突變的研究揭示了編碼色氨酸加氧酶的vermilion基因(Searlesetal.,1990; Whiteetal.,1996)和編碼犬尿氨酸—單加氧酶的cinnabar基因(Corneletal.,1997； Warrenetal.，1996)均參與色素產(chǎn)生的過程。在黑腹果蠅和地中海實(shí)蠅Ceratitiscapitata中，white基因負(fù)責(zé)編碼昆蟲復(fù)眼中色素引入和組裝的ABC轉(zhuǎn)運(yùn)蛋白(Bhalla,1968； Ewartetal.,1994)。通常，這些在復(fù)眼中能產(chǎn)生色素的基因如white(w)、vermilion(v)和cinnabar(cn)等均可用作遺傳修飾昆蟲研究的眼睛顏色標(biāo)記基因。野生型基因突變的等位基因會(huì)影響昆蟲復(fù)眼的顏色，將這些基因引入適宜的野生昆蟲中，即可產(chǎn)生可見的復(fù)眼表現(xiàn)型差異(Rubin & Spradling,1982)。這些基因大多為2~3 kb，其突變基本不會(huì)造成昆蟲適合度的降低，過量表達(dá)對(duì)生物體也無害。同時(shí)，對(duì)其檢測(cè)無需特殊的檢測(cè)系統(tǒng)，所以眼睛顏色標(biāo)記基因更易于被接受，從而得到廣泛應(yīng)用。

眼睛顏色突變體及其相應(yīng)基因用作評(píng)價(jià)性標(biāo)記體系，促進(jìn)了果蠅和其他昆蟲遺傳修飾技術(shù)的發(fā)展(Lorenzenetal.,2002)，黑腹果蠅的首次胚胎轉(zhuǎn)化和黑果蠅Drosophilavirilis轉(zhuǎn)化品系的獲得均依賴于可見眼睛顏色標(biāo)記系統(tǒng)的應(yīng)用(Gomez & Handler,1997； Rubin & Spradling,1982)。地中海實(shí)蠅(Handleretal.,1998; Loukerisetal.,1995; Micheletal.,2001)和埃及伊蚊Aedesaegypti(Coatesetal.,1998； Jasinskieneetal.,1998)的首次成功轉(zhuǎn)化很大程度上得益于眼睛顏色突變體的存在和用于突變—拯救選擇的野生型基因的克隆及可用性。在地中海實(shí)蠅中，白色眼睛基因座中的一個(gè)無效突變可被克隆的野生型拷貝所補(bǔ)充(Zwiebeletal.,1995)，隨后相似的基因也用于轉(zhuǎn)化同樣存在白色眼睛品系的橘小實(shí)蠅Bactroceradorsalis(Handler & McCombs,2000)。黑腹果蠅cn基因可以拯救埃及伊蚊突變品系的白色眼睛表型品系。對(duì)于赤擬谷盜Triboliumcastaneum，通過克隆其v和c基因建立了基于攜帶眼睛顏色突變vermillionwhite拯救的轉(zhuǎn)化體系。應(yīng)用眼睛顏色標(biāo)記基因進(jìn)行遺傳修飾的昆蟲物種如表1所示。

表1　采用眼睛顏色標(biāo)記的遺傳修飾昆蟲

2　抗藥性基因轉(zhuǎn)化標(biāo)記

最初對(duì)單獨(dú)發(fā)揮作用的顯性選擇標(biāo)記的研究主要集中于抗藥性基因，如對(duì)新霉素類似物有抗性的磷酸轉(zhuǎn)移酶基因NPTII(Steller & Pirrotta,1985)、對(duì)對(duì)硫磷有抗性的有機(jī)磷脫氫酶基因opd(Benedictetal.,1995; Phillipsetal.,1990)，以及對(duì)有機(jī)氯殺蟲劑狄氏劑dieldrin有抗性的Rdl基因(Ffrench-Constantetal.,1991)等?？顾幮詷?biāo)記基因最早在岡比亞按蚊Anophelesgambiae中得以應(yīng)用，岡比亞按蚊的第一個(gè)轉(zhuǎn)化品系是應(yīng)用編碼新霉素羧酸酯酶的neo基因作為選擇標(biāo)記而建立起來(Milleretal.,1987)，擁有neo基因的轉(zhuǎn)化品系可以獲得對(duì)氨基糖苷類抗生素G418的抗性。然而，由于在黑腹果蠅中能夠確定基于G418抗性篩選的較佳條件，該基因標(biāo)記只在黑腹果蠅中成功應(yīng)用(Steller & Pirrotta,1985)。

對(duì)于大部分昆蟲來說，篩選到適宜的抗藥性基因遺傳轉(zhuǎn)化標(biāo)記，可以對(duì)試驗(yàn)昆蟲進(jìn)行集體選擇，從而大幅度提高篩選轉(zhuǎn)化體的效率，這種優(yōu)勢(shì)使其成為可見眼睛顏色標(biāo)記之外的另一個(gè)重要轉(zhuǎn)化標(biāo)記。然而，抗藥性標(biāo)記的廣泛應(yīng)用還存在諸多問題。首先，轉(zhuǎn)化體篩選的準(zhǔn)確性。野生型昆蟲種群對(duì)某些藥物或抗生素的抗性具有波動(dòng)性；同時(shí)，轉(zhuǎn)座子介導(dǎo)的遺傳修飾昆蟲并不能將特定的靶基因轉(zhuǎn)化到特定的基因組位置上，所以轉(zhuǎn)化試驗(yàn)將會(huì)得到不同數(shù)量的插入子插入到不同位點(diǎn)的多種轉(zhuǎn)化體；此外，由于位置抑制效應(yīng)的差異，不同轉(zhuǎn)化體之間轉(zhuǎn)化標(biāo)記的表達(dá)水平也存在明顯差異。因此，在沒有其他可用標(biāo)記的昆蟲物種中應(yīng)用抗藥性標(biāo)記篩選轉(zhuǎn)化體，易篩選出未轉(zhuǎn)化成功的假陽性個(gè)體，或誤殺大多數(shù)轉(zhuǎn)化成功的假陰性個(gè)體。其次，安全性。很多藥物都具有毒性，且操作過程需要研究人員暴露于藥物中，所以該技術(shù)不被廣泛接受。同時(shí)，轉(zhuǎn)化品系的維系傳代需要依靠抗藥性的選擇，轉(zhuǎn)化品系的天然抗性選擇機(jī)制會(huì)隨世代的增加而加強(qiáng)，而抗藥性標(biāo)記可能使連鎖的轉(zhuǎn)化基因具有選擇性優(yōu)勢(shì)，因此對(duì)以釋放遺傳修飾昆蟲為最終目的的害蟲治理項(xiàng)目而言，其將面臨更大的抗性問題。目前，殺蟲劑抗性(Hemingway & Ranson,2000)和抗生素抗性(Monroe & Polk,2000)已成為威脅人類健康的嚴(yán)重問題，而抗藥性標(biāo)記的使用將會(huì)使現(xiàn)有的局勢(shì)變得更為嚴(yán)峻。

3　熒光蛋白轉(zhuǎn)化標(biāo)記

轉(zhuǎn)座子介導(dǎo)的昆蟲遺傳修飾研究方法具有隨機(jī)插入的特性，所以要想對(duì)轉(zhuǎn)化個(gè)體進(jìn)行準(zhǔn)確檢測(cè)，就需要應(yīng)用在不同表達(dá)水平均能被穩(wěn)定監(jiān)測(cè)的遺傳轉(zhuǎn)化標(biāo)記。該種標(biāo)記基因應(yīng)具有顯性表達(dá)、非破壞性、野生型背景中可見等特性。從水母Aequoreavictoria(Prasheretal.,1992)中分離得到的編碼綠色熒光蛋白(green fluorescent protein，GFP)基因具備轉(zhuǎn)化標(biāo)記的基本特性，GFP在多種不同的有機(jī)體中均可顯示出亮綠色的熒光，且在有機(jī)體不同組織中表達(dá)的綠色熒光易于被監(jiān)測(cè)(Tsien,1998)。GFP自被發(fā)現(xiàn)以來，以其良好的熒光特性成為被廣泛使用的報(bào)告基因或體內(nèi)蛋白定位的融合標(biāo)簽(Brand,1999； Chalfieetal.,1994; Cubittetal.,1995； Plautzetal.,1996)。然而，由于野生型GFP的相對(duì)不可溶性和位于紫外光譜內(nèi)激發(fā)峰的限制，尤其是長(zhǎng)時(shí)間暴露在紫外光條件下不適宜篩選活體生物等因素，限制了其在遺傳修飾昆蟲鑒定和篩選中的應(yīng)用。

隨著更可溶性GFP突變品系如增強(qiáng)型GFP (EGFP) (Cormacketal.,1996; Yangetal.,1996)的發(fā)展，上述問題基本得以解決。EGFP激發(fā)峰為488 nm，能夠在更無害的藍(lán)光下被激發(fā)，強(qiáng)度比野生型GFP提高35倍，適合快捷無損傷檢測(cè)。在黑腹果蠅中，EGFP標(biāo)記與眼睛顏色基因標(biāo)記聯(lián)合應(yīng)用，驗(yàn)證了EGFP對(duì)該物種的適用性(Handler & Harrell,1999; Hornetal.,2000)，并證實(shí)EGFP遺傳轉(zhuǎn)化標(biāo)記比其常規(guī)轉(zhuǎn)化標(biāo)記即眼睛顏色基因標(biāo)記“mini”-white更加靈敏、可靠。以埃及伊蚊為靶標(biāo)的驗(yàn)證結(jié)果與黑腹果蠅相似(Pinkertonetal.,2000)，可能與“mini”-white基因受位置抑制效應(yīng)更強(qiáng)有關(guān)。此外，由于啟動(dòng)子的不同，即使基因連鎖插入到相同的染色體位置上，不同基因受位置抑制的效應(yīng)也可能存在明顯差異(Bhadraetal.,1998)。通常，EGFP基因標(biāo)記比眼睛顏色基因標(biāo)記受到完全性抑制的可能性更小(Handler & Harrell,1999; Hornetal.,2000)。EGFP具有可溶性更佳、受藍(lán)光激發(fā)、不易受完全性位置抑制等特性，是首個(gè)被廣泛應(yīng)用的熒光變體，也是目前昆蟲遺傳修飾研究的主要轉(zhuǎn)化標(biāo)記。Higgs & Lewis (2000)詳細(xì)綜述了GFP突變品系作為遺傳修飾昆蟲標(biāo)記的優(yōu)勢(shì)，Hornetal.(2002)也指出其優(yōu)勢(shì)之一就是能應(yīng)用野生型生物體，這對(duì)于缺少可見型突變品系或突變品系很弱的昆蟲物種至關(guān)重要。雙翅目、鱗翅目和鞘翅目等3個(gè)目不同物種的成功轉(zhuǎn)化，表明EGFP可以被用作昆蟲遺傳修飾的轉(zhuǎn)化標(biāo)記(表2~4)。

然而，熒光標(biāo)記在昆蟲遺傳修飾研究中仍存在一些問題。首先，篩選遺傳修飾昆蟲過程中長(zhǎng)時(shí)間的強(qiáng)光照射可能會(huì)導(dǎo)致昆蟲死亡；其次，很多組織器官如馬氏管、幾丁質(zhì)外骨骼或壞死組織的自發(fā)光可能會(huì)干擾轉(zhuǎn)化體的檢測(cè)；再次，成蟲表皮高強(qiáng)度的黑化會(huì)阻礙對(duì)其內(nèi)部組織表達(dá)的EGFP的監(jiān)測(cè)。很多昆蟲的胚胎、幼蟲或蛹期階段比較透明，根據(jù)胚胎的發(fā)育歷期以及遺傳轉(zhuǎn)化標(biāo)記經(jīng)過內(nèi)部環(huán)化和氧化達(dá)到成熟所需的時(shí)間(Davisetal.,1995)推測(cè)，幼蟲孵化之前的階段可能是篩選遺傳修飾昆蟲的最佳時(shí)期。在該階段進(jìn)行熒光篩選不僅能夠達(dá)到快速檢測(cè)的目的，而且避免了飼養(yǎng)全部G1代遺傳修飾昆蟲，這對(duì)幼蟲食材珍貴但食量大或世代周期很長(zhǎng)的物種而言非常重要。為了更準(zhǔn)確地監(jiān)測(cè)單拷貝插入的轉(zhuǎn)化基因，可以借助強(qiáng)啟動(dòng)子驅(qū)動(dòng)EGFP的高效表達(dá)。同時(shí)，根據(jù)研究的具體需求，組成型和組織特異性的啟動(dòng)子都可用來構(gòu)建EGFP的獨(dú)立標(biāo)記系統(tǒng)。

表2　采用熒光蛋白基因作為轉(zhuǎn)化標(biāo)記的雙翅目昆蟲

表3　采用熒光蛋白基因作為轉(zhuǎn)化標(biāo)記的鱗翅目昆蟲

表4　采用熒光蛋白基因作為轉(zhuǎn)化標(biāo)記的鞘翅目昆蟲

3.1　組成型啟動(dòng)子驅(qū)動(dòng)的EGFP

遺傳修飾昆蟲轉(zhuǎn)化載體構(gòu)建程序中應(yīng)用強(qiáng)啟動(dòng)子驅(qū)動(dòng)EGFP的表達(dá)，有利于準(zhǔn)確檢測(cè)單拷貝插入子。組成型啟動(dòng)子在所有細(xì)胞中都有活性，所以能夠在昆蟲發(fā)育的所有階段(包括胚胎、幼蟲和成蟲)篩選轉(zhuǎn)化體。Handler & Harrell (1999、2001a)成功地應(yīng)用黑腹果蠅polyubiquitin啟動(dòng)子驅(qū)動(dòng)EGFP的表達(dá)，構(gòu)建了PUbnlsEGFP轉(zhuǎn)化標(biāo)記，在黑腹果蠅和加勒比按實(shí)蠅Anastrephasuspensa整個(gè)發(fā)育階段中實(shí)現(xiàn)了熒光的表達(dá)。該標(biāo)記載體的EGFP被融合到一核定位信號(hào)上，熒光蛋白的亞細(xì)胞定位利于準(zhǔn)確地從非核定位的自發(fā)熒光背景中鑒定轉(zhuǎn)化體。這對(duì)由位置效應(yīng)而導(dǎo)致EGFP低表達(dá)水平的轉(zhuǎn)化體的鑒定尤為重要。

另一種常用的驅(qū)動(dòng)EGFP的組成型啟動(dòng)子來自黑腹果蠅actin5C基因。轉(zhuǎn)化標(biāo)記actin5C:EGFP在黑腹果蠅、埃及伊蚊和斯氏按蚊Anophelesstephensi各發(fā)育階段的表現(xiàn)均很好(Catterucciaetal.,2000； Pinkertonetal.,2000)，但只能介導(dǎo)廄螫蠅Stomoxyscalcitrans低水平非均質(zhì)性的EGFP表達(dá)(O′Brochtaetal.,2000)，表明actin5C啟動(dòng)子可能并非應(yīng)用于各物種的最佳啟動(dòng)子。鱗翅目的家蠶Bombyxmori(Tamuraetal.,2000)和棉紅鈴蟲Pectinophoragossypiella(Peloquinetal.,2000)的第一次系統(tǒng)的胚胎轉(zhuǎn)化，是選用家蠶actinBmA3作為啟動(dòng)子驅(qū)動(dòng)EGFP的表達(dá)。雖然通過EGFP的表達(dá)成功鑒定了這2個(gè)物種的轉(zhuǎn)化體，但在其胚胎期并未檢測(cè)到BmA3:EGFP標(biāo)記的表達(dá)。此外，盡管BmA3啟動(dòng)子在中腸的活性比較明顯(Mangeetal.,1997)，但很多昆蟲食物的自發(fā)光現(xiàn)象導(dǎo)致只能檢測(cè)到轉(zhuǎn)化基因多重插入的個(gè)體中強(qiáng)烈表達(dá)的EGFP，因此中腸是轉(zhuǎn)化體難以有效鑒定的組織之一。而其他的熒光標(biāo)記，如DsRed造成生物組織自發(fā)光的現(xiàn)象則較少(Handler & Harrell,2001b)。

3.2驅(qū)動(dòng)眼睛特異性熒光表達(dá)的通用轉(zhuǎn)化標(biāo)記3xP3-EGFP

多細(xì)胞動(dòng)物的眼睛發(fā)育都受到進(jìn)化保守遺傳通路的控制，而這個(gè)通路受轉(zhuǎn)錄激活因子Pax-6/Eyeless的調(diào)控(Callaertsetal.,1997)，Pax-6結(jié)合位點(diǎn)P3調(diào)節(jié)光受體特異性基因的表達(dá)(Shengetal.,1997)?；诖耍珺erghammeretal.(1999)在單轉(zhuǎn)錄因子激活的人工啟動(dòng)子的基礎(chǔ)上發(fā)展了一個(gè)通用轉(zhuǎn)化標(biāo)記，即將3個(gè)P3位點(diǎn)的串聯(lián)重復(fù)序列置于TATA同源物(3xP3)的前邊，驅(qū)動(dòng)眼睛特異性EGFP的強(qiáng)表達(dá)(Hornetal.,2000)。3xP3與EGFP聯(lián)合，最初在赤擬谷盜和果蠅中應(yīng)用成功(Berghammeretal.,1999)。3xP3-EGFP標(biāo)記載體主要在赤擬谷盜的眼睛和腦中表達(dá)，并且在整個(gè)生活周期均能表達(dá)EGFP和DsRed(圖1；Lorenzenetal.,2007)。 Shengetal.(1997)應(yīng)用人工3xP3啟動(dòng)子構(gòu)建的載體也能夠介導(dǎo)EGFP在其受測(cè)昆蟲的幼蟲、蛹和成蟲眼睛中表達(dá)，這與Pax-6常規(guī)功能相一致，所以該組織特異性啟動(dòng)子與組成型啟動(dòng)子相似，可用于鑒定轉(zhuǎn)化昆蟲的所有發(fā)育階段(Hornetal.,2000)。3xP3-EGFP只有1.3 kb，而較小的轉(zhuǎn)座載體通常能產(chǎn)生更高的轉(zhuǎn)化效率。值得一提的是，3xP3-EGFP標(biāo)記能夠在G1代轉(zhuǎn)化昆蟲的胚胎發(fā)育末期產(chǎn)生可檢測(cè)到的表達(dá)(圖1A)，從而實(shí)現(xiàn)轉(zhuǎn)化個(gè)體的鑒定，省卻了將所有實(shí)驗(yàn)昆蟲飼養(yǎng)至成蟲的繁瑣工序，該標(biāo)記對(duì)幼蟲食量較大或人工飼料成本較高的昆蟲具有重要價(jià)值。

圖1　3xP3驅(qū)動(dòng)EGFP和DsRed在赤擬谷盜中表達(dá)

多細(xì)胞動(dòng)物眼睛發(fā)育中Pax-6的“主調(diào)節(jié)器”功能，揭示3xP3-EGFP標(biāo)記可以應(yīng)用到所有具有眼睛的動(dòng)物中。野生型昆蟲復(fù)眼的小眼通常通過眼睛色素相互隔離，所以只能在朝向觀察器的小眼中檢測(cè)到熒光(圖1C)。這對(duì)于鑒定野生型黑腹果蠅、斯氏按蚊(Itoetal.,2002)、家蠶(Thomasetal.,2002)和赤擬谷盜的轉(zhuǎn)化成蟲難度不大(Berghammeretal.,1999)；但其他物種如家蠅Muscadomestica或埃及伊蚊成蟲眼睛的色素會(huì)將熒光完全屏蔽或猝滅，從而導(dǎo)致鑒定的失敗(Hedigeretal.,2001; Kokozaetal.,2001)。然而，在野生型家蠅和埃及伊蚊的幼蟲和蛹階段，能夠檢測(cè)到3xP3-EGFP介導(dǎo)的眼睛熒光的表達(dá)(Hedigeretal.,2001; Kokozaetal.,2001)，表明3xP3-EGFP的轉(zhuǎn)化標(biāo)記體系既能用于野生型品系，也能用于突變品系。

熒光標(biāo)記在視覺系統(tǒng)如眼睛中的表達(dá)，使得其在具有很厚或黑化表皮的動(dòng)物中也能被檢測(cè)到(圖1B)。熒光標(biāo)記的選擇和轉(zhuǎn)化個(gè)體鑒定的最佳發(fā)育階段的確定，很大程度上依賴于昆蟲外表皮的形成和黑化以及眼睛發(fā)育和色素形成的時(shí)間與程度。對(duì)于大多數(shù)昆蟲而言，程序操作和熒光檢測(cè)的最佳時(shí)期可能都是胚胎末期和幼蟲期，這限制了3xP3-EGFP標(biāo)記在該階段視覺系統(tǒng)不發(fā)達(dá)的昆蟲中的應(yīng)用。然而，研究證實(shí)3xP3-EGFP標(biāo)記能夠介導(dǎo)熒光在黑腹果蠅胚胎末期或幼蟲期中樞神經(jīng)系統(tǒng)、部分外周神經(jīng)系統(tǒng)、肛板和后腸中的表達(dá)(Hornetal.,2000)，在鞘翅目和鱗翅目昆蟲中也觀察到中樞神經(jīng)系統(tǒng)中熒光的表達(dá)(Thomasetal.,2002)。這拓展了3xP3-EGFP標(biāo)記在幼蟲階段沒有眼睛或視覺系統(tǒng)不發(fā)達(dá)昆蟲中的應(yīng)用。迄今為止，以3xP3-EGFP為基礎(chǔ)的轉(zhuǎn)化系統(tǒng)已用于3個(gè)目昆蟲轉(zhuǎn)化個(gè)體的生產(chǎn)和鑒定，這充分表明人工構(gòu)建的3xP3-EGFP標(biāo)記與轉(zhuǎn)座子聯(lián)合具有廣泛的適用性(Horn & Wimmer,2000; Hornetal.,2002)。

3.3　熒光蛋白的毒性

哺乳動(dòng)物細(xì)胞培養(yǎng)試驗(yàn)結(jié)果表明，水母GFP及其突變體的高水平表達(dá)能夠造成對(duì)細(xì)胞的毒性(Hanazonoetal.,1997)，但毒性問題對(duì)GFP作為昆蟲轉(zhuǎn)化標(biāo)記應(yīng)用的影響并非特別嚴(yán)重,僅以polyubiquitin或actin5C驅(qū)動(dòng)的EGFP標(biāo)記轉(zhuǎn)化埃及伊蚊RED品系時(shí)表現(xiàn)出了毒性，因此只能建立EGFP低表達(dá)品系，所有高表達(dá)的轉(zhuǎn)化G1后代在蛹期全部死亡。該種效應(yīng)是由高水平表達(dá)的EGFP造成還是由針對(duì)特定品系轉(zhuǎn)化方法中的不同參數(shù)造成尚不明確。在黑腹果蠅和野生型埃及伊蚊中，actin5C:EGFP的表達(dá)均未對(duì)其生育力造成明顯不利影響(Pinkertonetal.,2000)。同時(shí)，3xP3-EGFP標(biāo)記即使在眼睛和中樞神經(jīng)系統(tǒng)中高水平表達(dá)并產(chǎn)生強(qiáng)烈的熒光，也未發(fā)現(xiàn)其對(duì)轉(zhuǎn)化昆蟲的存活率存在顯著性影響(Berghammeretal.,1999)。

對(duì)于遺傳不育釋放項(xiàng)目而言，不僅要考慮遺傳修飾昆蟲的生育能力，而且要考慮釋放昆蟲與野生型昆蟲的競(jìng)爭(zhēng)力以及轉(zhuǎn)化品系的穩(wěn)定性。通常，熒光轉(zhuǎn)化標(biāo)記是否對(duì)轉(zhuǎn)化昆蟲的壽命、繁殖力、生育力或適合度造成一定的影響，對(duì)評(píng)估項(xiàng)目的效益具有決定性意義。鑒于GFP的潛在毒性，組織特異性啟動(dòng)子驅(qū)動(dòng)的熒光轉(zhuǎn)化標(biāo)記可能更適于遺傳不育釋放項(xiàng)目的研究。因?yàn)榻M成型啟動(dòng)子介導(dǎo)的熒光在轉(zhuǎn)化昆蟲毒性敏感組織中表達(dá)的可能性更大，而組織特異性啟動(dòng)子驅(qū)動(dòng)的熒光在限定空間或組織內(nèi)表達(dá)，可以避免對(duì)遺傳修飾昆蟲關(guān)鍵敏感組織的不利影響。如從海洋珊瑚蟲海鰓Renillareniformis中克隆的另一綠色熒光蛋白基因(Ward & Cormier,1979)經(jīng)人為修飾(hrGFP; Stratagene)后，在哺乳動(dòng)物培養(yǎng)試驗(yàn)中的毒性低于水母GFP突變體(Feltsetal.,2000)。天然珊瑚蟲GFP作為生物學(xué)標(biāo)記比水母GFP具有更大的優(yōu)勢(shì)和更廣闊的應(yīng)用前景。在光吸收方面，珊瑚蟲GFP的消光系數(shù)比野生型水母GFP高5倍，比人源化紅移轉(zhuǎn)變的水母蛋白高2.5倍。然而，有關(guān)hrGFP在昆蟲轉(zhuǎn)化中的應(yīng)用還未見報(bào)道。

3.4　EBFP、ECFP和EYFP轉(zhuǎn)化標(biāo)記

在模式生物中，GFP和EGFP通常用作分析增強(qiáng)子或啟動(dòng)子的報(bào)告基因，以標(biāo)記特定的組織或細(xì)胞，或作為體內(nèi)亞細(xì)胞蛋白定位的融合標(biāo)簽(Tsien,1998)。非模式昆蟲的深入研究也迫切需要GFP或EGFP的表達(dá)載體。然而，這些表達(dá)載體與EGFP轉(zhuǎn)化標(biāo)記聯(lián)合應(yīng)用可能會(huì)產(chǎn)生一些干擾問題，所以報(bào)告基因和轉(zhuǎn)化標(biāo)記的研究仍需發(fā)展多樣化、可區(qū)分的熒光分子。

EBFP是GFP的一個(gè)藍(lán)光突變系，其熒光的激發(fā)峰和發(fā)射峰分別為383和445 nm (Pattersonetal.,1997)?；贓BFP與EGFP的光譜差異，足以應(yīng)用特異性過濾裝置清晰地將EBFP從EGFP中區(qū)分出來。然而，EBFP的量子產(chǎn)率低,光褪色較快，所以當(dāng)需要鑒定的個(gè)體數(shù)量很多或照射時(shí)間較長(zhǎng)時(shí)，EBFP并不適宜用作轉(zhuǎn)化標(biāo)記。GFP的另一個(gè)更穩(wěn)定的突變系為青色熒光突變系ECFP，其激發(fā)峰和發(fā)射峰分別為434和477 nm (Pattersonetal.,2001)。該突變品系能夠用更無害的藍(lán)光進(jìn)行激發(fā)，且穩(wěn)定性強(qiáng)，適宜用作轉(zhuǎn)化標(biāo)記(Horn & Wimmer,2000)。但ECFP的光譜不能與EGFP完全分開，所以限制了其與帶有GFP和EGFP載體的聯(lián)合應(yīng)用。應(yīng)用特異性的過濾裝置能夠?qū)GFP從GFP的黃色突變品系EYFP中完全區(qū)分出來，EYFP的激發(fā)峰和發(fā)射峰分別為514和527 nm (Cubittetal.,1995)。ECFP和EYFP的量子產(chǎn)率和光褪色時(shí)間特性較佳(Pattersonetal.,2001)，可用作獨(dú)立的遺傳修飾昆蟲轉(zhuǎn)化標(biāo)記(Horn & Wimmer,2000)。各種熒光蛋白及突變體的激發(fā)峰和發(fā)射峰值如表5所示,GFP突變體及DsRed表達(dá)的熒光如圖2所示。

表5　用于遺傳修飾昆蟲的熒光蛋白特性

圖2　熒光突變體的熒光顏色及激發(fā)峰值(Patterson et al.,2001)Fig.2　Fluorescent color of GFP variants and Ds-Red and their excitation max. (Patterson et al.,2001)

3.5　紅色熒光DsRed轉(zhuǎn)化標(biāo)記

從珊瑚屬?？鸇iscosomastriata中分離的紅色熒光蛋白DsRed (drFP583)，是另一種可用的熒光標(biāo)記(Matzetal.,1999)。DsRed與水母GFP熒光發(fā)色團(tuán)附近的保守性氨基酸序列具有23%的相似性(Walletal.,2000; Yarbroughetal.,2001)。DsRed的激發(fā)峰和發(fā)射峰分別為558和583 nm。較高的光褪色抗性、高量子產(chǎn)率以及較長(zhǎng)的壽命是其作為轉(zhuǎn)化標(biāo)記的理想特性。更為重要的是，DsRed在多數(shù)生物組織中表達(dá)的熒光都在自發(fā)光范圍以外，更利于轉(zhuǎn)化體的準(zhǔn)確鑒定。但是，DsRed的成熟時(shí)間較長(zhǎng)，在遺傳修飾轉(zhuǎn)化昆蟲的鑒定過程中不能像EGFP一樣在胚胎發(fā)育期就能被檢測(cè)到(Bairdetal.,2000； Hornetal.,2000)。

人工修飾過的突變系DsRed1與DsRed具有相似的熒光特性(Matzetal.,1999)。Handler & Harrell (2001b)采用果蠅polyubiquitin啟動(dòng)子驅(qū)動(dòng)DsRed1的表達(dá)以鑒定遺傳修飾的黑腹果蠅幼蟲和成蟲，結(jié)果顯示，PUbDsRed1介導(dǎo)表達(dá)的紅色熒光比較明亮，并且與EGFP相比，更低數(shù)量級(jí)的DsRed1表達(dá)量也能被監(jiān)測(cè)，而較高的信噪比有利于轉(zhuǎn)化體的鑒定。Horn & Wimmer (2000)利用人工3xP3眼睛啟動(dòng)子驅(qū)動(dòng)DsRed1的表達(dá)，檢測(cè)其在黑腹果蠅中作為轉(zhuǎn)化標(biāo)記的適用性，結(jié)果表明，在成蟲白色突變品系和野生型黑腹果蠅的復(fù)眼和單眼中均能輕易地檢測(cè)到強(qiáng)烈表達(dá)的紅色熒光，且透過輕微黑化的頭殼也能在成蟲腦中檢測(cè)到DsRed1的表達(dá)，而EGFP的綠色熒光則被阻斷。在澳大利亞銅綠蠅Luciliacuprina雙元件系統(tǒng)中，通過雜交雙雜合子品系(Double heterozygous line)篩選雙純合子品系(Double homozygous line)，由于親代的雄蟲和雌蟲分別含有一個(gè)拷貝的ZsGreen和DsRed，經(jīng)過減數(shù)分裂后子代可能含有不同熒光蛋白類型和拷貝數(shù)(圖3)。ZsGreen和DsRed均由強(qiáng)組成型啟動(dòng)子Lchsp83驅(qū)動(dòng)，因此雙拷貝Lchsp83-DsRed幼蟲即使在白光照下也能被看出DsRed的表達(dá)(圖3A)；在GFP2濾鏡下ZsGreen綠色熒光會(huì)受到紅色熒光的干擾(圖3B)；而GFP-NB(Narrow broad)濾鏡則屏蔽了紅色熒光，更容易篩選出雙拷貝Lchsp83-ZsGreen的幼蟲(圖3C)；再結(jié)合DsRed濾鏡篩選雙拷貝Lchsp83-DsRed的幼蟲(圖3D)。

圖3　澳大利亞銅綠蠅3齡幼蟲的雙元件系統(tǒng)熒光圖片

另一突變品系DsRed2具有與DsRed1相似的熒光特性，且可溶性更好，成熟更快，形成多聚物的可能性更低，甚至毒性更低。然而，作為適宜的報(bào)告基因，24 h左右的成熟時(shí)間依然較長(zhǎng)。DsRed的另一突變系E5，也稱作“熒光計(jì)時(shí)器”(Terskikhetal.,2000)，能夠在幾小時(shí)后檢測(cè)到熒光信號(hào)，成熟之前由最初的綠色熒光變?yōu)榧t光熒光。該標(biāo)記目前的功能是用作內(nèi)部熒光時(shí)鐘的報(bào)告基因，可以檢測(cè)基因表達(dá)的時(shí)空動(dòng)態(tài)。熒光顯示的綠色、黃色(綠色和紅色疊加)或紅色狀態(tài)，表明基因的活化和下調(diào)表達(dá)的情況(Terskikhetal.,2000)。綠色—紅色熒光計(jì)時(shí)器，作為報(bào)告基因可與ECFP聯(lián)合應(yīng)用，并作為昆蟲遺傳修飾研究的可辨認(rèn)標(biāo)記，但是目前還沒有成功應(yīng)用的報(bào)道。

4　結(jié)語

自1982年科學(xué)家成功轉(zhuǎn)化出首例遺傳修飾的果蠅以來，昆蟲遺傳修飾技術(shù)因其潛在的廣泛應(yīng)用前景而成為研究熱點(diǎn)。昆蟲遺傳修飾技術(shù)的開發(fā)與應(yīng)用離不開性狀優(yōu)良的標(biāo)記基因。作為遺傳修飾轉(zhuǎn)化載體構(gòu)建的關(guān)鍵組成部分之一，標(biāo)記基因?qū)τ谶z傳修飾昆蟲轉(zhuǎn)化體的準(zhǔn)確鑒定和轉(zhuǎn)化昆蟲穩(wěn)定性的監(jiān)測(cè)具有重要意義，開發(fā)可靠性高、穩(wěn)定性好、應(yīng)用面廣的轉(zhuǎn)化標(biāo)記基因，對(duì)于充分挖掘遺傳修飾技術(shù)的潛力非常重要。眼睛顏色基因轉(zhuǎn)化標(biāo)記的多數(shù)特征雖然比較理想(Sarkar & Collins,2000)，但多數(shù)重要的衛(wèi)生害蟲和農(nóng)業(yè)害蟲缺少適宜的受體突變品系，從而限制了該標(biāo)記的應(yīng)用。盡管理論上各物種都能產(chǎn)生突變—恢復(fù)轉(zhuǎn)化標(biāo)記，但突變株的獲得、相應(yīng)基因的克隆、突變表型的最終恢復(fù)等一系列步驟往往需要耗費(fèi)大量的時(shí)間和人力物力。抗藥性基因標(biāo)記不易獲得，且在轉(zhuǎn)化昆蟲的鑒定過程中存在諸多準(zhǔn)確性和安全性方面的問題。因此，要對(duì)更多的昆蟲物種進(jìn)行廣泛而深入的遺傳修飾研究，就需要開發(fā)性能更佳的適宜野生型背景使用的標(biāo)記系統(tǒng)。

熒光蛋白基因能夠在野生型背景轉(zhuǎn)化后代中起作用(Tsien,1998)，通過突變方法獲得的多種不同熒光性質(zhì)的突變體，因具有快速、簡(jiǎn)便、低毒等特點(diǎn)而得以廣泛應(yīng)用，其中應(yīng)用較多的是EGFP和DsRed標(biāo)記基因。組成型和組織特異性的啟動(dòng)子都可用來構(gòu)建EGFP的獨(dú)立標(biāo)記系統(tǒng)以驅(qū)動(dòng)EGFP的高效表達(dá)，但由于天然啟動(dòng)子均來源于特定的物種而具有物種特異性，因此，每個(gè)組成型啟動(dòng)子的熒光轉(zhuǎn)化標(biāo)記只能應(yīng)用到近緣物種。 此外，綠色熒光蛋白的自發(fā)光現(xiàn)象也限制了其在某些物種中的應(yīng)用。紅色熒光蛋白DsRed造成生物組織自發(fā)光的現(xiàn)象則較少(Handler & Harrell,2001b)，更利于轉(zhuǎn)化體的準(zhǔn)確鑒定;在某些生物中與GFP聯(lián)合應(yīng)用的表現(xiàn)優(yōu)于GFP突變體，所以應(yīng)用前景很廣泛。DsRed熒光在生物組織中長(zhǎng)達(dá)數(shù)周的壽命(Matzetal.,1999)和光褪色的抗性，也是不育昆蟲釋放技術(shù)在田間應(yīng)用的理想特性(Peloquinetal.,2000)，能用于穩(wěn)定監(jiān)測(cè)野生型種群的擴(kuò)散和其在野外環(huán)境中與其他物種間的水平傳播。然而，DsRed較長(zhǎng)的成熟時(shí)間限定了轉(zhuǎn)化體鑒定的階段，阻礙了DsRed作為報(bào)告基因在短期基因表達(dá)研究中的應(yīng)用(Bairdetal.,2000; Handler & Harrell,2001b)。鑒于大量不同的GFP/EGFP報(bào)告基因和融合標(biāo)簽載體都已經(jīng)可用，針對(duì)具體的轉(zhuǎn)化物種，需要根據(jù)物種的具體情況選擇適合的熒光轉(zhuǎn)化標(biāo)記，避免假陽性或假陰性現(xiàn)象，或通過更換標(biāo)記逐一將其解決。目前規(guī)避干擾的最好方法就是聯(lián)合應(yīng)用以GFP為基礎(chǔ)的體內(nèi)報(bào)告基因與以DsRed1或DsRed2為基礎(chǔ)的轉(zhuǎn)化標(biāo)記。即使EGFP和DsRed在相同的組織中同時(shí)表達(dá)，應(yīng)用特異性的過濾裝置也能夠?qū)⑵渫耆珔^(qū)分開，從而進(jìn)行獨(dú)立的鑒定和監(jiān)測(cè)。

除了眼睛顏色標(biāo)記基因、抗藥性標(biāo)記基因和綠色熒光蛋白及上文中提到的突變體外，還有ZsGreen等其他的熒光蛋白標(biāo)記和蛹顏色標(biāo)記(McCombs & Saul,1995; Wappneretal.,1995)。基于水母GFP的開發(fā)，在其他生物如珊瑚、?？⑺?、甲殼類動(dòng)物甚至低等脊索動(dòng)物中相繼發(fā)現(xiàn)了GFP樣蛋白(Wiedenmannetal.,2009)，熒光光譜覆蓋藍(lán)色到遠(yuǎn)紅光，使熒光蛋白的適用范圍不斷擴(kuò)大。更多更有效的熒光蛋白和其他標(biāo)記基因的獲得，以及更適宜特定物種的轉(zhuǎn)化系統(tǒng)和檢測(cè)技術(shù)的發(fā)展，大大提高了對(duì)任何一種昆蟲進(jìn)行遺傳修飾改造的可能性。

昆蟲遺傳修飾技術(shù)為基因表達(dá)調(diào)控、生物大分子相互作用、胚胎發(fā)育以及發(fā)展生物傳感器等研究創(chuàng)造了條件，同時(shí)為農(nóng)林害蟲和媒介害蟲的防治提供了新的思路。應(yīng)用遺傳修飾手段獲得的不育昆蟲釋放技術(shù)是一種可控制甚至根除靶標(biāo)害蟲的環(huán)境友好型防控措施。為了保障釋放昆蟲的最佳防控效果，要求遺傳修飾轉(zhuǎn)化昆蟲中的轉(zhuǎn)化標(biāo)記除不影響靶標(biāo)物種的競(jìng)爭(zhēng)性和適合度之外，還需要具有良好的遺傳穩(wěn)定性，以便于對(duì)其長(zhǎng)期監(jiān)測(cè)，達(dá)到靈活調(diào)控釋放不育昆蟲與野生昆蟲的比例，獲取最佳防控效果的目標(biāo)。然而，遺傳修飾昆蟲的釋放尤其是攜帶致死基因的昆蟲的釋放還存在一定的風(fēng)險(xiǎn)，所以在監(jiān)測(cè)釋放昆蟲環(huán)境穩(wěn)定性的同時(shí)，需要監(jiān)控其在物種間的水平傳播，避免對(duì)生物多樣性、生態(tài)環(huán)境和人體健康產(chǎn)生潛在的不利影響。

致謝：赤擬谷盜與澳大利亞銅綠蠅熒光圖片分別來自北卡羅來納州立大學(xué)Dr. Marce Lorenzen與Dr. Max Scott實(shí)驗(yàn)室，在此表示衷心的感謝。

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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；4Department of Agronomy and Plant

Protection,QingdaoAgriculturalUniversity,Qingdao,Shandong266109,China

Abstract:Transformation markers offer a tool to distinguish the genetically modified insects from wild types. Both the identification of transformants and the maintenance of transformed lines depend on reliable transformation makers. In addition, the evaluation of the genetic stability of released genetically modified insects needs strong and stable markers. Thus the development of broadly applicable, easily detectable and reliable transformation markers will facilitate the study of genetic pest management. In general, eye color genes, drug resistance genes and fluorescent protein genes can be used as markers in genetically modified insects. The first efficient identification of a non-drosophilid insect transformation line was based on the rescue of eye color mutant phenotypes. However, for most insect species, the application of eye color markers is limited because of the lack of suitable recipient mutant strains and less information on related genes. Markers based on drug resistance genes can improve the screening efficiency of transformants, but the selection for drug resistance is problematic and prone to have false positives or negatives with potential biosecurity problems. Fluorescent protein gene markers significantly facilitate the development of stable insect transformation lines. The green fluorescent protein (GFP, isolated from the jellyfish Aequorea victoria) and its variants with various fluorescent characteristics can be combined with suitable, strong promoters to serve as transformation markers for a wide range of insect species and guarantee the reliable screening of the transformants. In this category, the enhanced green fluorescent protein (EGFP) was mostly used. Besides, the red fluorescent protein (DsRed), isolated from the mushroom coral, Discosoma striata, provides a selection of red fluorescent proteins with better performance than GFP mutants. This paper reviews the history and status of transformation markers including eye color genes, drug resistance genes and the fluorescent protein genes. The potential roles of transformation markers in genetic pest management are also discussed.

Key words:genetically modified insect; transformation marker; eye color gene; drug resistance gene; fluorescent protein gene

通訊作者*(Author for correspondence), E-mail: zezhang@cqu.edu.cn

作者簡(jiǎn)介:許軍， 男， 博士研究生。 研究方向： 昆蟲生殖生物學(xué)。 E-mail: xzgxcxj@163.com

基金項(xiàng)目:國家自然科學(xué)基金國際合作項(xiàng)目(31420103918)

收稿日期(Received)： 2014-11-14接受日期(Accepted)： 2015-03-09

DOI:10. 3969/j.issn.2095-1787.2015.02.003