賈殿勇，高世慶，段鵬飛，陳吉寶，田風霞，喻修道

1 南陽師范學院農業(yè)工程學院 河南省南水北調中線水源區(qū)水安全協(xié)同創(chuàng)新中心 河南省南水北調中線水源區(qū)生態(tài)安全重點實驗室，河南 南陽 473061

2 北京市農林科學院 北京雜交小麥工程技術研究中心，北京 100097

蚜蟲是危害農作物生產的重要害蟲，蚜蟲取食可使植株營養(yǎng)惡化，蚜蟲分泌的蜜露附著在葉片表面影響植物的光合作用，并促進霉菌的滋生，誘發(fā)植物黑霉病害[1-2]。蚜蟲還是植物病毒病的重要傳播載體，蚜蟲傳播的病毒約占所有蟲傳病毒種類的45%[3-4]。近年來因全球氣候變暖、耕作制度變化等因素影響，蚜蟲繁殖能力和適應性顯著增強，危害日趨嚴重[5-6]。據統(tǒng)計，2010–2011年間我國小麥、玉米、棉花、油菜、大豆等主要作物的蚜蟲危害面積分別占當年種植面積的 62.5%、14%、90%、32%和23%[7]。培育抗蟲品種是防治蚜蟲的最有效途徑，現有農作物種質資源中缺乏有效的抗蚜基因，常規(guī)抗蟲育種難以奏效[1,4]。因此，利用轉基因技術培育抗蚜新種質，對于保障我國糧食安全具有重要意義。

[反]-β-法尼烯 [(E)-β-farnesene，EβF]是絕大多數蚜蟲類型報警信息素的主要甚至唯一成分，可使蚜蟲產生騷動而從植株上脫落，并吸引蚜蟲天敵[8-10]。在植物中表達 EβF合成酶基因以獲得釋放EβF的轉基因植株已成為蚜蟲防治的重要策略之一。本文概述了植物抗蚜轉基因的研究現狀，并對當前EβF合成酶基因在植物抗蚜轉基因研究中的應用及存在問題進行了討論。

1 植物抗蚜轉基因研究現狀

作物抗蟲轉基因育種已持續(xù)開展20多年，多種表達蘇云金芽孢桿菌 (Bacillus thuringiensis，Bt) 毒素蛋白的轉基因作物，如棉花、玉米、大豆等已商業(yè)化種植[11]。Bt毒素蛋白對鱗翅目和鞘翅目害蟲有很強的毒殺作用，但對蚜蟲等同翅目害蟲防治效果不明顯，制約了Bt基因在植物抗蚜分子育種中的應用。目前，用于抗蚜分子育種的基因和技術主要有植物凝集素基因、植物介導的RNA干擾技術等[1-2,12]。

1.1 植物凝集素

植物凝集素是一類保守性糖結合蛋白，可與昆蟲消化道上皮細胞的糖蛋白結合，降低膜透性，并可直接降低蟲體消化酶活性，影響昆蟲對營養(yǎng)物質的吸收和消化。此外，凝集素可在昆蟲消化道內誘發(fā)病灶，促進消化道內的細菌繁殖，影響害蟲生長發(fā)育[13]。自 1988年發(fā)現蓖麻凝集素以來，人們己經從豆科、茄科、禾本科和石蒜科等眾多植物中分離鑒定出上千種植物凝集素基因，已有 10余種不同植物來源的凝集素基因用于轉基因抗蚜研究[1,13]。其中，雪花蓮凝集素(Galanthus nivalisagglutinin，gna)、半夏凝集素(Pinellia ternateagglutinin，pta) 等基因對哺乳動物的毒性較小，是抗蟲轉基因研究的熱點。轉gna基因的小麥、玉米、馬鈴薯、煙草等植物能抑制蚜蟲生長、降低蚜蟲生殖力[14-17]。pta基因與gna的序列相似性很高，轉pta基因小麥株系上蚜蟲的存活率可降為對照的54%[18]。然而，植物凝集素抗蟲具有廣譜性，對蚜蟲天敵、食草動物等可能具有毒害作用，如二星瓢蟲取食轉gna基因馬鈴薯上的蚜蟲后，其產卵力、卵的生存力和壽命明顯降低[19-20]。轉凝集素基因植物對生態(tài)環(huán)境的影響引起了人們的擔憂。

1.2 植物介導的RNA干擾

RNA干擾 (RNA interference，RNAi) 是由雙鏈RNA (Double-stranded RNA，dsRNA) 介導的一種序列特異性轉錄后基因沉默機制。dsRNA進入生物體后被宿主細胞中的 Dicer酶切割成21–23 nt的小干擾 RNA (Small interfering RNA，siRNA)；siRNA在 RNA解旋酶的作用下解鏈成正義鏈和反義鏈，反義siRNA與體內一些酶 (包括內切酶、外切酶、解旋酶等) 結合形成RNA誘導的沉默復合物 (RNA-induced silencing complex，RISC)，隨后 RISC以序列互補的方式與靶標mRNA 結合并使之降解[12,21]。研究發(fā)現，在植物中表達 dsRNA能抑制昆蟲特定基因的表達，使昆蟲生長發(fā)育受阻或致死，有效控制害蟲[12,21]。RNAi技術在蚜蟲防治方面展示出很好的應用潛力[12]，植物介導的RNAi技術已用于大麥、煙草和擬南芥等植物的抗蚜研究[4,12,22-24]。大麥中表達shp基因的 dsRNA能顯著降低麥長管蚜的繁殖力，且shp基因的沉默效應可以遺傳至第7代子蚜[22]。煙草和擬南芥中表達桃蚜MpC002、Rack-1、Mphb及MySP基因的 dsRNA，可顯著降低蚜蟲的繁殖率、減輕蚜蟲危害[4,23-24]。RNAi靶標基因的篩選是植物介導的 RNAi抗蚜應用的前提，目前鑒定出的能顯著致死或抑制蚜蟲生長的靶標基因較少。其次，植物和蚜蟲體內的核酸酶可降解外源dsRNA，降低蚜蟲目標基因的沉默效率，影響了該技術在轉基因植物抗蚜上的應用。同時，植物介導的RNAi存在潛在的安全風險，如RNAi的脫靶效應，亦有待深入研究和解決[12,25]。

此外，研究者嘗試在植物中表達抗性基因、蛋白酶抑制劑等基因來控制蚜蟲危害[26-29]?？剐曰?(R基因) 通常介導的是垂直抗性，表現出一定的物種特異性，如番茄R基因Mi-1.2轉入茄子后，不能提高茄子對蚜蟲的抗性[30]。蛋白酶抑制劑是抑制蛋白水解酶活性的一種小分子蛋白，昆蟲攝食蛋白酶抑制劑后，其腸道內的蛋白水解受阻，進而擾亂昆蟲的營養(yǎng)代謝[31]。然而，害蟲可以通過合成同工酶或直接降解的方式，快速對外源蛋白酶抑制劑產生抗性[32-34]；并且，植物中表達蛋白酶抑制劑對非靶標害蟲如蜜蜂有害[35]。因此，挖掘新的更加安全有效的抗蚜基因尤為重要。

2 蚜蟲報警信息素及EβF

蚜蟲報警信息素是蚜蟲遇到天敵等威脅時從腹管分泌的一種粘稠液滴，釋放到體外具有揮發(fā)性，能引起同類其他個體騷動并從棲息地迅速逃散或從植株上脫落，并能作為天敵捕食蚜蟲的重要線索[8]。EβF是絕大多數蚜蟲類型報警信息素的主要甚至唯一成分，包括桃蚜、玉米蚜、棉蚜、麥長管蚜、禾谷縊管蚜、麥無網長管蚜、大豆蚜等常見作物害蟲[1,7,36]。EβF利于蚜蟲防控的作用特點如下：1) 蚜蟲通過氣味結合蛋白、昆蟲化學感受蛋白等基因感應外界 EβF[37-39]，產生諸如騷動、停止取食，甚至從植物上脫落等警戒反應[7,9,36,40]。2) EβF能夠吸引多種蚜蟲天敵，如瓢蟲[10,41-42]、食蚜蠅[42-44]、草蛉[45-47]、蚜繭蜂等[42,47-48]，作為天敵的捕食信號。3) EβF能顯著提高產生有翅蚜的比率，使蚜蟲主動離開寄主植物[49-50]。4) EβF能產生類似保幼激素Ⅲ的作用，影響蚜蟲形態(tài)類型和生長發(fā)育。如一齡棉蚜受EβF誘導后，蚜蟲的發(fā)育期延長、產卵力下降、體重減輕[51]。5) EβF與殺蟲劑混用，可增加蚜蟲的活動頻率，提高殺蟲效果[52-53]。

然而，EβF在大田條件下不穩(wěn)定，易氧化分解，制約了EβF在田間的抗蚜應用[54]。隨后，為提高EβF的穩(wěn)定性，研究者嘗試了人工改造及化學合成 EβF，并取得一定進展[55-56]。蚜蟲報警信息素的專屬性表明EβF與受體之間具有特定的作用部位，受體對信息素的結構和性質有嚴格的要求，導致化學合成的EβF對蚜蟲防治效率較低。

3 植物體內EβF的代謝合成

EβF作為一種無色無味的倍半萜類化合物，還是茼蒿、野生馬鈴薯、菊花、薄荷、黃花蒿、花旗松、香橙、洋甘菊等多種植物精油的主要組分[7,57-58]。植物來源的 EβF亦可趨避蚜蟲和吸引天敵，如與栽培品種相比，野生馬鈴薯葉片揮發(fā)物中存在高量 EβF，進而對蚜蟲有很強的驅避作用[58]；?；页嵋苟耆∈澈?，玉米會釋放含有 EβF的揮發(fā)物，減少蚜蟲取食[59]。此外，菊花來源的EβF可以吸引天敵瓢蟲和蚜繭蜂減輕蚜蟲對白菜的危害[10]；洋甘菊來源的EβF可有效降低馬鈴薯和小麥的田間蚜蟲數量[42,47]。EβF合成酶是催化生成EβF的關鍵酶，但蚜蟲體內的EβF合成酶基因尚未分離鑒定[60]。

目前，研究者已對植物體內的萜類化合物及EβF生物合成機制進行了較為深入的研究。萜類化合物是植物次生代謝產物中最大的一個家族，根據所含碳原子數目不同，可分為單萜 (C10)、倍半萜 (C15) 和二萜 (C20) 等。植物萜類化合物通過兩個獨立途徑合成，即位于質體中的2-C-甲基-D-赤蘚糖醇-4-磷酸 (2-C-Methyl-D-Erythritol-4-Phosphate，MEP) 途徑和位于細胞質中的甲羥戊酸 (Mevalonate，MVA) 途徑(圖1)[61]。在植物質體中，1分子異戊烯焦磷酸 (Isopentenyl diphosphate，IPP) 和 1分子二甲丙烯焦磷酸 (Dimethylallyl diphosphate，DMAPP) 在香葉基焦磷酸合成酶(Geranyl diphosphate synthase，GPS) 的作用下，經頭尾相連生成單萜合成的底物香葉基焦磷酸 (Geranyl diphosphate，GPP)；3分子 IPP和1分子 DMAPP在香葉基香葉基焦磷酸合成酶(Geranylgeranyl diphosphate synthase，GGPS) 催化下形成二萜合成的底物香葉基香葉基焦磷酸(Geranylgeranyl diphosphate，GGPP)。在細胞質MVA途徑中，2分子IPP和1分子DMAPP在法呢基焦磷酸合成酶(Farnesyl diphosphate synthase，FPS) 催化下形成倍半萜化合物的底物法呢基焦磷酸 (Farnesyl diphosphate，FPP) (圖 1)。EβF 合成酶基因已先后從歐洲薄荷[62]、亞洲薄荷[46]、香橙[63]、花旗松[64]、黃花蒿[65]和洋甘菊[66]等植物中得到分離鑒定。其中，歐洲薄荷與亞洲薄荷來源的EβF合成酶基因僅有5個核苷酸堿基的差異，編碼的氨基酸序列完全一致[46]。植物EβF合成酶基因不含信號肽序列，主要位于細胞質中，催化MVA途徑中的FPP生成EβF，以揮發(fā)物的形式釋放到植物體外。

歐洲薄荷、黃花蒿、香橙、洋甘菊及花旗松來源的 EβF合成酶均含有 Terpene_synth (PFAM accession number: PF01397) 和Terpene_synth_C(PFAM accession number: PF03936) 結構域，這兩個結構域為植物萜類合成酶家族的典型特征(圖2)。將上述5種植物EβF合成酶與煙草表-馬兜鈴酸合成酶(5-epi-aristolochene synthase) 的氨基酸序列進行比對 (圖 2)，發(fā)現不同物種來源的EβF合成酶序列差異較大，僅存在部分保守的氨基酸殘基，如薄荷 EβF合成酶 MpβFS “DDxxD”(301?305 位) 中的 Asp301、Asp302和 Asp305?！癉DxxD”在植物萜類合成酶基因中普遍存在，參與催化反應中二價金屬離子的螯合[67]。參照煙草表-馬兜鈴酸合成酶的晶體結構，保守氨基酸殘基Arg264和 Arg266位于 A-C loop區(qū)，Asp528和 Lys537則位于J-K loop區(qū)；A-C loop與J-K loop參與表-馬兜鈴酸合成酶與底物的結合[67]。植物萜類合成酶的活性中心一般位于羧基端 (C端)，研究表明活性中心的半胱氨酸、組氨酸及精氨酸殘基是維持酶生物活性的關鍵[68-69]。序列比對發(fā)現，MpβFS有5個保守精氨酸 (Arg112,115,264,266,441) 及1個保守組氨酸殘基 (His82)，其中Arg264,266,441位于C端的Terpene_synth_C結構域內 (圖2)。

圖1 植物萜類化合物的代謝途徑Fig. 1 Terpene biosynthesis pathway in plants. MVA: the mevalonate pathway; MEP: 2-C-methyl-D-erythritol 4-phosphate pathway; IPP: isopentenyl diphosphate; DMADP: dimethylallyl diphosphate; GPP: geranyl diphosphate;FPP: farnesyl diphosphate; GGDP: geranyl geranyl diphosphate. Former researches overexpressed exogenous EβF synthase gene in the cytosol of plants, but low EβF production was observed. One strategy indicated by the gray dotted box is redirecting sesquiterpene biosynthetic pathway into plastids, that is, simultaneously overexpressing the exogenous FPP synthase and EβF synthase in the plastid of plants.

4 EβF合成酶基因在植物抗蚜分子育種中的應用

倍半萜類化合物是植物萜類化合物中最大的一類，約為單萜類化合物的7倍，常以揮發(fā)物的形式存在于植物中[70-71]。研究發(fā)現，倍半萜化合物參與植物對害蟲的直接與間接防御反應。直接防御反應中，倍半萜化合物作為毒素及害蟲取食或產卵的干擾素；間接防御反應中，植物受到害蟲取食所釋放的揮發(fā)性萜類可吸引天敵[72]。倍半萜合成酶基因在植物抗蚜分子育種中也展現出很好的應用前景，如玉米在受到?；页嵋苟耆∈澈髸吡勘磉_TPS10基因，生成 EβF、[反]-α-香柑油烯 [(E)-α-bergamotene]等多種揮發(fā)物吸引害蟲天敵；將玉米TPS10基因轉入擬南芥，轉基因株系可吸引鱗翅目害蟲天敵寄生蜂[73]。作為TPS10在水稻中的同源基因，TPS46參與EβF和檸檬烯 (Limonene) 等揮發(fā)物的生成；水稻中過表達TPS46基因可增強轉基因植株對禾谷縊管蚜的抗性[74]。

體外表達試驗表明，歐洲薄荷、花旗松、黃花蒿、洋甘菊等4種植物的EβF合成酶基因的產物均以EβF為主，純度均達到95%以上 (表1)[62,64-66]。在植物中過量表達外源EβF合成酶基因，借助細胞質中的FPP為底物，可以獲得持續(xù)釋放EβF的轉基因植株 (表1)[1,7,45-46,75-80]。如將歐洲薄荷EβF合成酶基因MpβFS轉入擬南芥，轉基因植株能夠釋放 EβF，驅避蚜蟲并吸引蚜蟲寄生性天敵——蚜繭蜂[75]。筆者等分別將黃花蒿和亞洲薄荷來源的 EβF合成酶基因 (AaβFS和MaβFS) 轉入煙草，轉基因煙草可以通過吸引大草蛉減輕蚜蟲危害，與對照植株相比，有兩個株系上的蚜蟲數量分別減少 23.6%和 29.5%[45-46]。此外，薄荷來源的EβF合成酶基因亦轉入小麥、水稻、芥菜等植物來減輕蚜蟲危害[76-79]。以上研究表明，EβF合成酶基因在作物轉基因抗蚜蟲應用上具有重要價值。蚜蟲長時間處于高濃度EβF環(huán)境中，會對EβF產生適應性[81]；蚜蟲連續(xù)取食釋放 EβF的轉基因擬南芥后，其第三代子蚜對 EβF的警戒反應降低，但顯著增強了瓢蟲對適應性子蚜的捕食[82]。

5 存在問題及展望

在植物倍半萜代謝改良過程中，目標倍半萜類化合物的生成量往往較低[1,83]，如煙草中表達紫穗槐-4,11-二烯合成酶基因 (Amorpha-4,11-diene synthase)，倍半萜化合物紫穗槐-4,11-二烯 (Amorpha-4,11-diene)的生成量只有 0.2–1.7 ng/(d·g)[84]。轉薄荷MaβFS和黃花蒿AaβFS基因煙草植株的 EβF釋放量只有1.55–4.85 ng/(d·g)[45-46]，而轉MpβFS基因水稻的EβF釋放量僅為 4.89–5.03 ng/d(d·g)[76]。600 ng/μL 以上濃度的 EβF方可顯著趨避蚜蟲[9]，EβF釋放量偏低嚴重影響了轉基因植株對蚜蟲的最佳防治效果。植物細胞質中底物FPP供應量不足是制約倍半萜化合物代謝改良的關鍵[85-86]。FPP是植物倍半萜和甾醇的共同合成底物，甾醇為植物細胞膜的組成部分，對維持細胞結構具有重要作用。鑒于甾醇對植物細胞功能的重要性，FPP優(yōu)先供應于甾醇的合成[85,87]。同時，植物中FPP的供應量因物種差異而有所不同，相比青蒿、大冷杉等植物，小麥、水稻、煙草等植物內源倍半萜的量較低，可用于生成倍半萜的FPP較少[88]。

相比倍半萜類化合物的分子代謝改良，轉基因植物單萜的生成量很高，不受底物供應的影響，表明植物質體中擁有足夠的前體 IPP和 DMAPP合成GPP (圖1)[83]。早期認為FPP合成酶僅存在于植物細胞質中，隨著研究的深入，在擬南芥線粒體及水稻、小麥、煙草的葉綠體中均發(fā)現了FPP合成酶異構體[89-90]。據此，筆者等推測這些植物葉綠體中有可能合成 FPP，隨后利用葉綠體轉導肽在煙草中表達黃花蒿EβF合成酶基因AaβFS1。轉基因植株的 EβF 釋放量達到 4.33–19.25 ng/(d·g)，與在煙草細胞質中表達AaβFS1基因的植株相比提高4?12倍[80]。盡管葉綠體擁有足夠的前體IPP和 DMAPP，但由于葉綠體中FPP合成酶異構體表達量或催化效率低，導致FPP合成量不足，轉基因煙草在溫室半自然條件下不能顯著趨避蚜蟲[80]。

筆者在前期研究的基礎上，提出通過以下兩種策略提高轉基因植株的 EβF釋放量：1) 實施EβF代謝改良過程中的多基因協(xié)同轉化，加強對FPP代謝流的調控。FPP合成酶是MVA途徑中的關鍵酶，同時表達FPP合成酶可增加植物細胞質中FPP的供應量，提高轉基因植物的EβF釋放量。2) 改變倍半萜代謝的細胞分區(qū)，即在質體中同時表達FPP合成酶和EβF合成酶基因，將合成EβF的代謝途徑由細胞質轉至質體 (圖 1)。利用質體中充足的IPP和DMAPP，催化形成足量的FPP，提高轉基因植株的EβF釋放量。Wu等[91]在煙草葉綠體中共表達 FPP合成酶及紫穗槐-4,11-二烯合成酶基因，使煙草倍半萜紫穗槐-4,11-二烯的產量提高1 000多倍。同時，為增強EβF合成酶轉基因植株的抗蚜效果，可實施EβF合成酶基因與其他抗蚜基因/技術的分子聚合育種。如在釋放EβF的轉基因植株中表達蚜蟲氣味結合蛋白基因的dsRNA，轉基因植株通過釋放EβF吸引蚜蟲天敵，而蚜蟲氣味結合蛋白基因的沉默則降低了對天敵的警戒反應，進而增強天敵對蚜蟲的捕食。

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