病毒誘導(dǎo)基因沉默技術(shù)在茄科植物功能基因組學(xué)研究中的應(yīng)用進(jìn)展

蔡 文 陳長(zhǎng)明 陳國(guó)菊 曹必好 雷建軍

（華南農(nóng)業(yè)大學(xué)園藝學(xué)院， 廣州 510642）

病毒誘導(dǎo)基因沉默技術(shù)在茄科植物功能基因組學(xué)研究中的應(yīng)用進(jìn)展

蔡 文 陳長(zhǎng)明 陳國(guó)菊 曹必好 雷建軍*

（華南農(nóng)業(yè)大學(xué)園藝學(xué)院， 廣州 510642）

病毒誘導(dǎo)基因沉默（virus-induced gene silencing，VIGS）是一種研究基因功能的反向遺傳工具，該技術(shù)具有操作簡(jiǎn)單快速、周期短、高通量、無(wú)需遺傳轉(zhuǎn)化、成本低等優(yōu)點(diǎn)，在基因探尋和功能鑒定上廣泛應(yīng)用。著重圍繞上述病毒誘導(dǎo)基因沉默技術(shù)在研究茄科（Solanaceae）植物的相關(guān)功能基因在植物次生代謝、生長(zhǎng)發(fā)育、生物和非生物脅迫幾個(gè)方面的應(yīng)用進(jìn)行了綜述和展望。

茄科植物；VIGS；功能基因組學(xué)；次生代謝；生長(zhǎng)發(fā)育；生物和非生物脅迫

隨著測(cè)序技術(shù)的進(jìn)步和成本的降低，許多重要的植物全基因組測(cè)序已經(jīng)完成，并且很多基因也被預(yù)測(cè)。但是在這些被預(yù)測(cè)到的基因中，已經(jīng)進(jìn)行了基因功能驗(yàn)證的卻鮮有報(bào)道。目前傳統(tǒng)的功能基因篩選方法主要是化學(xué)誘導(dǎo)、化學(xué)突變、插入突變以及圖位克隆等。但是這些技術(shù)操作復(fù)雜、周期長(zhǎng)、成本高、通量低，因而難以被廣泛應(yīng)用。與這些傳統(tǒng)的基因功能研究方法相比，病毒誘導(dǎo)基因沉默（Virusinduced gene silencing，VIGS）技術(shù)具有操作簡(jiǎn)單快速、周期短、成本低、高通量，且不需要進(jìn)行遺傳轉(zhuǎn)化，沉默之后表型癥狀明顯等優(yōu)勢(shì)。該技術(shù)已經(jīng)成為植物功能基因研究的一項(xiàng)重要工具，而對(duì)茄科植物進(jìn)行功能基因?qū)W研究，尤其是對(duì)茄科植物中的幾種經(jīng)濟(jì)價(jià)值高的茄科蔬菜（茄子、辣椒、番茄、馬鈴薯）和觀賞價(jià)值較高的模式植物矮牽牛重要的功能基因研究具有重要的意義，是實(shí)現(xiàn)茄科植物分子育種的重要前提。

本文從VIGS技術(shù)機(jī)制的建立及發(fā)展以及在茄科植物生長(zhǎng)發(fā)育、次生代謝、生物脅迫與非生物脅迫等幾個(gè)方面進(jìn)行了綜述和展望。

1 VIGS技術(shù)的作用機(jī)制及發(fā)展

1.1 VIGS的作用機(jī)制

病毒誘導(dǎo)基因沉默（Virus-induced gene silencing，VIGS）是一種轉(zhuǎn)錄后水平的基因沉默現(xiàn)象（post-transcriptional gene silencing，PTGS），根據(jù)植物本身存在的天然免疫機(jī)制，當(dāng)植物受攜帶目的片段的病毒侵染之后，植物會(huì)對(duì)病毒的入侵做出響應(yīng)，隨后激活其自身免疫系統(tǒng)，導(dǎo)致內(nèi)源mRNA下降從而形成的一種自我防御反應(yīng)。當(dāng)防御機(jī)制啟動(dòng)時(shí)單鏈RNA（Single-Stranded RNA，ssRNA）在RNA聚合酶的作用下形成雙鏈RNA（Double-Stranded RNA，dsRNA），之后雙鏈RNA被Dicer的核酸酶切割成21 ～ 23 nt大小的RNA片段，這些小片段被稱之為小干擾RNA。當(dāng)小干擾RNA與體內(nèi)酶結(jié)合形成RNA誘導(dǎo)的沉默復(fù)合物（RNA-induced silencing complex，RISC）之后，RNA誘導(dǎo)的沉默復(fù)合物與其同源的病毒mRNA相結(jié)合，共同切割靶片段，最后導(dǎo)致mRNA降解。

1.2 VIGS載體的發(fā)展及在茄科植物中的應(yīng)用

“VIGS”一詞最初由van Cammen[1]用來(lái)描述植物感染病毒的現(xiàn)象，如今“VIGS”已經(jīng)成為表述重組病毒敲除內(nèi)源基因表達(dá)的專業(yè)術(shù)語(yǔ)[2-3]。Kumagai等[4]以八氫番茄紅素脫氫酶（phytoene desaturase，PDS) cDNA序列為目的片段與煙草花葉病毒（Tobacco mosaic virus，TMV）重組形成重組載體沉默煙草，結(jié)果發(fā)現(xiàn)被感染的煙草葉片出現(xiàn)白化現(xiàn)象。該研究結(jié)果推測(cè)煙草葉片的白化現(xiàn)象是由于病毒載體成功侵染了煙草植株，結(jié)果導(dǎo)致其內(nèi)源mRNA水平降低。八氫番茄紅素脫氫酶（phytoene desaturase，PDS）是類胡蘿卜素合成途徑中的一個(gè)關(guān)鍵酶，在植物中具有光保護(hù)作用，當(dāng)類胡蘿卜素合成途徑被阻斷后，會(huì)導(dǎo)致植物光保護(hù)功能的喪失，最終引起白化現(xiàn)象的產(chǎn)生。

1998年Ruiz 等[3]通過(guò)構(gòu)建馬鈴薯病毒X（potato virus X，PVX）的VIGS 載體，并與PDS基因重組侵染馬鈴薯，最終在馬鈴薯葉片中也出現(xiàn)了白化現(xiàn)象，并由此推測(cè)VIGS不但可以有效抑制內(nèi)源基因的表達(dá)，而且可以用來(lái)鑒定未知基因的功能。隨后Ratcliff 等[5]以煙草脆裂病毒（Tobacco rattle virus，TRV）為載體構(gòu)建了VIGS 體系，其誘導(dǎo)基因沉默的效率在抑制轉(zhuǎn)基因和內(nèi)源基因的表達(dá)上比PVX載體和TMV載體都更具優(yōu)勢(shì)。TRV載體能夠有效克服分生組織的障礙，并且病毒載體本身對(duì)植株的傷害比PVX和TMV病毒載體更小。而Liu 等[6-7]通過(guò)改造TRV病毒載體pYL156和pYL279，使其帶上重復(fù)的啟動(dòng)子和C-端一個(gè)酶，導(dǎo)致病毒氨基酸序列發(fā)生改變，更有利于病毒RNA的產(chǎn)生，提高了內(nèi)源基因的沉默效率。目前TRV病毒載體已被廣泛應(yīng)用到矮牽牛（Petunia hybrida）、煙草（Nicotina spp.）、茄子（Solanum melongena）、辣椒（Capsicum spp.）、番茄（Solanum lycopersicum）等許多茄科植物之中。

除上述的RNA 病毒載體外，現(xiàn)在還開(kāi)發(fā)了多種DNA病毒載體。如Kjemtrup等[8]用番茄金色花葉病毒（Tomato golden mosaic virus，TGMV）的DNA-A為載體，沉默本氏煙上鎂離子螯合酶的關(guān)鍵基因Su（Sulfur）和轉(zhuǎn)熒光蛋白基因（luc），結(jié)果導(dǎo)致煙草葉片出現(xiàn)黃色突變表型，而轉(zhuǎn)luc基因植株不再發(fā)出熒光。Turnage等[9]首次用大白菜曲葉病毒（Cabbage leaf curl virus，CbLCV）沉默了擬南芥的同源基因。不久之后，Turnage等[9]與Fofana等[10]又將非洲木薯花葉病毒（African cassava mosaic virus，ACMV）載體在木薯和煙草中應(yīng)用成功。后來(lái)Pandey等[11]將番茄曲葉病毒（Tomato leaf curl virus，ToLCV）構(gòu)建的VIGS 載體成功運(yùn)用到沉默番茄和煙草中。此外，基于葡萄病毒A（Grapevine virus A，GVA）的VIGS載體在煙草和茄子中也得到了運(yùn)用[12]。

此外，Gosselé等[13]將一類較小的衛(wèi)星病毒誘導(dǎo)沉默體系（Satellite virus induce silence system，SVISS）應(yīng)用在煙草植株中。隨后通過(guò)改造中國(guó)番茄黃化曲葉病毒（Tomato yellow leaf curl China virus，TYLCCNV）和煙草曲莖病毒（Tobacco curly shoot virus，TbCSV）的衛(wèi)星DNA，分別開(kāi)發(fā)出了Betasatellite（DNAβ）-VIGS和Alphasatellite（DNA1）-VIGS 病毒衛(wèi)星沉默載體作為VIGS體系的載體被運(yùn)用在煙草、矮牽牛以及番茄之中[14-16]。表1例舉了用不同類型病毒作為沉默載體，在茄科植物不同組織部位的應(yīng)用。

表 1 茄科植物中使用的VIGS載體及接種方式

2 VIGS在茄科植物功能基因組學(xué)的應(yīng)用

隨著VIGS技術(shù)的不斷發(fā)展和完善，該技術(shù)在茄科植物中的運(yùn)用越來(lái)越廣泛，與茄科植物生長(zhǎng)發(fā)育、次生代謝、生物脅迫以及非生物脅迫相關(guān)的基因及其基因功能鑒定和分析是VIGS技術(shù)運(yùn)用的重點(diǎn)。表2例舉了運(yùn)用VIGS技術(shù)沉默茄科植物相關(guān)功能基因及沉默后的表型。

2.1 VIGS在生長(zhǎng)發(fā)育和次生代謝相關(guān)基因功能的研究

利用VIGS技術(shù)，研究者已經(jīng)發(fā)現(xiàn)番茄果實(shí)在成熟的過(guò)程中，許多基因參與調(diào)控果實(shí)的生長(zhǎng)發(fā)育，其中就包括許多與成熟相關(guān)的轉(zhuǎn)錄因子。NF-Y作為一類與成熟相關(guān)的轉(zhuǎn)錄因子，受乙烯信號(hào)的誘導(dǎo)，利用TRV病毒介導(dǎo)沉默番茄NF-Y轉(zhuǎn)錄因子后與對(duì)照植株相比，沉默植株的果實(shí)成熟延緩且顏色變?yōu)辄S色或橘紅色[24]。最近，則有研究者將VIGS技術(shù)用于研究矮牽牛的衰老，當(dāng)GRL1基因被沉默后會(huì)發(fā)現(xiàn)該植物的花衰老受到抑制，但沉默F(xiàn)BH4基因后卻會(huì)導(dǎo)致花的衰老加速，表明GRL1基因、FBH4基因?qū)Π珷颗；ǖ乃ダ暇哂姓蚧蜇?fù)向調(diào)節(jié)的功能[25]。這些基因功能的初步鑒定為研究番茄和矮牽牛的生長(zhǎng)發(fā)育提供了一定的基礎(chǔ)。

除了在生長(zhǎng)發(fā)育方面的運(yùn)用，VIGS技術(shù)在研究植物次生代謝方面也具有廣泛的應(yīng)用。Fantini等[26]通過(guò)VIGS技術(shù)研究發(fā)現(xiàn)，沉默番茄PYS1、ZDS、ZISO與CrtISO結(jié)構(gòu)基因后，與對(duì)照植株比較番茄果皮的紅色變淡，由此推測(cè)這幾個(gè)基因可能參與番茄紅素的合成。利用TRV病毒介導(dǎo)的VIGS載體沉默矮牽牛R2R3-MYB轉(zhuǎn)錄因子EOBⅡ，結(jié)果發(fā)現(xiàn)沉默植株與對(duì)照植株相比其香味變淡，并且苯丙烷類代謝香味物質(zhì)合成結(jié)構(gòu)基因的表達(dá)水平也顯著下降，表明EOII主要是通過(guò)調(diào)控這些結(jié)構(gòu)基因來(lái)實(shí)現(xiàn)的[27]。此前在煙草中運(yùn)用VIGS技術(shù)沉默煙草bHLH1，bHLH2基因，結(jié)果發(fā)現(xiàn)沉默植株與對(duì)照相比其尼古丁的含量顯著降低[28]。而在辣椒中利用瓦斯蒂克辣椒黃脈病毒（Pepper huasteco yellow veinsvirus，PHYVV）沉默辣椒素合成相關(guān)基因Pun1、Comt、Kas和AMT之后，發(fā)現(xiàn)辣椒素合成含量下降，由此推測(cè)這些基因可能在辣椒素合成中具有正向調(diào)節(jié)的功能[20,29]。Zhang等[30]通過(guò)研究辣椒CaMYB轉(zhuǎn)錄因子結(jié)果發(fā)現(xiàn)，沉默該轉(zhuǎn)錄因子能夠抑制花青素的合成，表明CaMYB參與調(diào)控花青素的生物合成。

2.2 VIGS在生物脅迫相關(guān)基因功能的研究

2.2.1 細(xì)菌病害抗性相關(guān)基因的研究 通過(guò)VIGS技術(shù)還能研究植物細(xì)菌病害相關(guān)基因的功能，植物在受到病原菌入侵的時(shí)候會(huì)通過(guò)激活體內(nèi)相關(guān)抗病基因的表達(dá)來(lái)防止病原菌的侵襲。目前，利用VIGS已經(jīng)鑒定了茄科中多種抗細(xì)菌病害的相關(guān)基因。

Oh等[31]通過(guò)對(duì)辣椒葉片噴施乙烯和水楊酸，發(fā)現(xiàn)辣椒PIF1基因的轉(zhuǎn)錄水平比對(duì)照高，說(shuō)明辣椒PIF1基因受外源乙烯和水楊酸的誘導(dǎo)，而噴施茉莉酸甲酯則對(duì)PIF1的表達(dá)沒(méi)有顯著影響，在煙草中沉默煙草PIF1基因，結(jié)果導(dǎo)致煙草對(duì)特有病原菌和非宿主性病原菌的抗性降低。由此推測(cè)受外源乙烯和水楊酸信號(hào)途徑誘導(dǎo)的PIF1基因在植物的抗病中具有重要的作用。辣椒CYP1基因?qū)儆诩?xì)胞色素P450家族基因，參與辣椒的抗病反應(yīng)。Kim等[32]沉默辣椒的CYP1基因之后對(duì)沉默植株接種地毯草黃單胞桿菌（Xanthomonas axonopodis pv， Xav），結(jié)果發(fā)現(xiàn)與對(duì)照植株相比，沉默植株對(duì)黃單胞桿菌的敏感性增加，由此說(shuō)明CYP1基因可能作為一個(gè)正向調(diào)控因子調(diào)控辣椒的抗病性。此外，有研究表明辣椒WRKY轉(zhuǎn)錄因子家族也參與辣椒的抗病反應(yīng)，沉默辣椒轉(zhuǎn)錄因子家族WRKY1基因，能夠減少地毯黃單胞桿菌的生長(zhǎng)[33]。當(dāng)WRKY40基因被沉默時(shí)，發(fā)現(xiàn)辣椒對(duì)青枯病的抗性減弱，熱休克反應(yīng)也加重[34]。Shen等[35]認(rèn)為辣椒WRKY40基因的功能同時(shí)還受堿性亮氨酸拉鏈轉(zhuǎn)錄因子家族（basic leucine zipper，bZIPs）ZIP63的調(diào)控，沉默辣椒ZIP63基因之后，發(fā)現(xiàn)辣椒對(duì)青枯病的抗性降低，同時(shí)還會(huì)降低耐熱性與耐濕性，表明ZIP63參與抗病和抗逆境。最近利用VIGS技術(shù)鑒定了LIP1、GLP1、LRR51、PTI1等基因在辣椒細(xì)菌病害中的作用，沉默辣椒中的LIP1基因后，發(fā)現(xiàn)辣椒植株對(duì)野油菜黃單胞菌辣椒致病變種（Xanthomonas campestris pv. Vesicatoria，Xcv）的抗性增強(qiáng)，當(dāng)沉默GLP1、LRR51、PTI1基因時(shí)發(fā)現(xiàn)與對(duì)照植株相比，沉默植株對(duì)細(xì)菌病抗性降低，由此推測(cè)這些基因作為負(fù)向或正向調(diào)控因子參與辣椒細(xì)菌病的抵抗過(guò)程[36-39]。

此前通過(guò)VIGS技術(shù)還鑒定了番茄抗病基因Pto介導(dǎo)的抗假單胞桿菌一系列相關(guān)基因，如NPR1、GTA、TGAla、TGA2.2、MEK1、MEK2和NTF6等[7,40-42]。之后又有研究者利用VIGS技術(shù)并結(jié)合傳統(tǒng)突變篩選技術(shù)，發(fā)現(xiàn)并鑒定了丁香假單胞桿菌（P. syringae）致病機(jī)制中茉莉酸-異亮氨酸（JA-isoleucine）類似物冠菌素（Coronatine，COR）的功能，以及水楊酸、NO合成酶基因iNOS和冠菌素/茉莉酸途徑中SGT1和SlALC1 基因在抗P. syringae中的作用。而利用VIGS沉默番茄SGT1基因，結(jié)果發(fā)現(xiàn)沉默植株葉片壞死和缺綠的癥狀和未沉默的植株相比癥狀較輕。由此說(shuō)明SGT1基因在番茄的抗病性中有著重要的作用[43-44]。利用TRVVIGS探究SISAHH基因與番茄細(xì)菌抗病性的關(guān)系，當(dāng)SISAHH 基因被沉默之后，沉默植株不僅對(duì)地毯草黃單胞桿菌（Xanthomonas axonopodis pv，Xav）的抗菌性增強(qiáng)，同時(shí)還發(fā)現(xiàn)該基因沉默還能夠增強(qiáng)對(duì)干旱的抵抗性[45]。此外，最近的一項(xiàng)研究發(fā)現(xiàn)，番茄的CDPK基因、CRK基因也參與番茄的抗病反應(yīng)。利用VIGS技術(shù)沉默這兩個(gè)基因之后，沉默植株的抗菌性減弱，揭示這兩個(gè)基因正向調(diào)控番茄對(duì)病原的入侵[46]。

2.2.2 真菌病害抗性相關(guān)基因的研究 真菌病害對(duì)于茄科植物同樣有十分重要的影響，利用VIGS技術(shù)研究真菌病害抗性相關(guān)基因近年來(lái)也取得了重大進(jìn)展。沉默辣椒的Btf3基因，導(dǎo)致接種煙草花葉病毒（Tobacco mosaic virus，TMV）的植株超敏反應(yīng)降低，在沉默植株中同時(shí)出現(xiàn)生長(zhǎng)緩慢的現(xiàn)象。Huh等[47]利用TRV介導(dǎo)沉默辣椒WRKY轉(zhuǎn)錄因子中WRKYd基因?qū)е掳l(fā)病相關(guān)基因與超敏反應(yīng)相關(guān)基因的表達(dá)降低，揭示了Btf3基因和WRKYd基因作為調(diào)控因子調(diào)控超敏反應(yīng)從而阻止病源菌的蔓延。最近有學(xué)者研究沉默番茄WRKY41與WRKY54基因，發(fā)現(xiàn)即使對(duì)沉默植株接種番茄黃化花葉病毒（Tomato yellow leaf curly virus，TYLCV），TYLCV的DNA含量也比對(duì)照下低，表明WRKY41基因、WRKY54基因與番茄的抗病性有關(guān)[48]。

2.2.3 病毒病抗性相關(guān)基因的研究 經(jīng)過(guò)長(zhǎng)期的不斷探索，利用VIGS技術(shù)找到了一系列與茄科植物病毒病抗性相關(guān)的基因。Liu等[49]通過(guò)研究發(fā)現(xiàn)，NBCLIN-1基因與N基因介導(dǎo)的程序性細(xì)胞死亡有關(guān)，沉默煙草的NBCLIN-1基因后與對(duì)照植株相比，結(jié)果卻發(fā)現(xiàn)沉默組對(duì)TMV的抗性降低。而本氏煙草RanGAP2基因也被認(rèn)為與Rx介導(dǎo)的PVX抗性相關(guān)，26S蛋白酶體亞基-RPN9是廣譜病毒系統(tǒng)運(yùn)輸所需的。Jin等[50]通過(guò)沉默煙草RPN9基因發(fā)現(xiàn)，當(dāng)RPN9基因被沉默后，煙草中TMV與蕪菁花葉病毒（Turnip mosaic vires，TuMV）的運(yùn)動(dòng)受到了明顯的抑制，并且在葉脈構(gòu)造中會(huì)導(dǎo)致木質(zhì)部增加而韌皮部減少。并由此推測(cè)RPN9基因主要通過(guò)改變維管束組織從而抑制病毒的運(yùn)動(dòng)。Tameling和Baulcombe[51]利用TRV介導(dǎo)的VIGS沉默煙草和馬鈴薯的RanGAP2基因，發(fā)現(xiàn)會(huì)破壞煙草和馬鈴薯植株對(duì)PVX的極端抗性。且RanGAP2基因作為Rx復(fù)合物的一部分，能夠與Rx發(fā)生互作。而這種互作則是Rx發(fā)揮對(duì)PVX極端抗性所必需的。

番茄LeHT1基因是一類己糖轉(zhuǎn)運(yùn)蛋白相關(guān)的基因，利用VIGS技術(shù)沉默LeHT1基因的實(shí)驗(yàn)發(fā)現(xiàn)，在沉默番茄植株中細(xì)胞壞死癥狀嚴(yán)重，病毒傳播速度加快，據(jù)此推測(cè)LeHT1基因可能通過(guò)抑制病毒的移動(dòng)減少病毒的積累從而正向發(fā)揮調(diào)控番茄抗病性的功能。Czosnek等[52]利用TRV病毒載體篩選出一批抗中國(guó)番茄黃化曲葉病毒(Tomato yellow leaf curl China virus，TYLCCNV)的基因，并且通過(guò)綠色熒光蛋白（Green f uorescent protein，GFP）實(shí)驗(yàn)，在本氏煙草驗(yàn)證了病毒的傳播時(shí)間和位置。

除了上述幾種植物之外，利用VIGS技術(shù)在辣椒中也鑒定出了與病毒病抗性相關(guān)的基因。WRKY轉(zhuǎn)錄因子家族的WRKYd基因功能的表達(dá)可能與植物防御相關(guān)激素和TMV-P0的侵染相關(guān)。Huh等[53]利用TRV病毒介導(dǎo)的VIGS載體沉默辣椒WRKYd基因，發(fā)現(xiàn)沉默會(huì)影響TMV-P0介導(dǎo)的超敏反應(yīng)，同時(shí)也會(huì)影響TMV-P0病毒蛋白外殼在植物中的積累，并最終導(dǎo)致發(fā)病相關(guān)基因與超敏反應(yīng)相關(guān)基因的減少，從而降低植物的超敏反應(yīng)。

2.3 VIGS在非生物脅迫相關(guān)基因功能的研究

VIGS還可以用于非生物脅迫相關(guān)基因的功能研究。煙草NAH20基因是一種堿性亮氨酸拉鏈蛋白編碼的基因，能夠被干旱和傷害等非生物脅迫誘導(dǎo)，同時(shí)該基因的功能還受植物激素的調(diào)控，研究者認(rèn)為脫落酸（abscisic acid，ABA）的積累能夠激活該基因發(fā)揮功能。在煙草中利用VIGS方法沉默NAH20基因能夠降低ABA的積累。證實(shí)了NAH20參與抗旱A(chǔ)BA途徑[54]程序性細(xì)胞死亡（Program cell death，PCD）。該過(guò)程是植物在逆境條件下發(fā)揮調(diào)節(jié)功能的一種自我保護(hù)機(jī)制，而ERF109作為乙烯的響應(yīng)因子，推測(cè)可能通過(guò)抑制程序性細(xì)胞死亡從而提高植株的耐鹽性。Bahieldin等[55]通過(guò)VIGS技術(shù)沉默煙草ERF109基因?qū)е聼煵莸柠}脅迫加劇，驗(yàn)證了煙草ERF109基因的功能與植物的耐鹽性相關(guān)。

表 2 利用VIGS研究的茄科植物基因及其表型

續(xù)表2

在辣椒中利用VIGS技術(shù)挖掘了許多與辣椒耐鹽性和抗旱性相關(guān)的正向或負(fù)向調(diào)控基因。如RAV1與OXR1基因，沉默辣椒這兩個(gè)基因，結(jié)果導(dǎo)致辣椒鹽脅迫與滲透脅迫加重[56]。Lim等[57]在辣椒葉綠體中發(fā)現(xiàn)了與辣椒抗旱性相關(guān)的DIN1基因，與之前的研究不同，高鹽和干旱處理能顯著誘導(dǎo)DIN1的表達(dá)。利用TRV載體介導(dǎo)沉默DIN1基因之后，卻提高了辣椒的抗旱性。屬于泛素連接酶類的AIR1基因也被鑒定出與抗旱性相關(guān)，在干旱和ABA信號(hào)途徑中起著重要的作用。當(dāng)沉默辣椒AIR1基因后，同樣沉默植株的抗旱性也被增強(qiáng)[58]。而Lim等[59]通過(guò)沉默辣椒胚胎發(fā)育晚期蛋白基因LEA1基因，發(fā)現(xiàn)沉默植株枯萎嚴(yán)重導(dǎo)致再次吸水困難且難以存活，同時(shí)對(duì)干旱脅迫和鹽脅迫抵抗減弱，由此推測(cè)LEA1基因可能參與了辣椒鹽脅迫的調(diào)控途徑。

利用VIGS技術(shù)還在番茄中找到了一系列與非生物脅迫相關(guān)的基因。沉默番茄GRX1基因，沉默植株不僅對(duì)氧脅迫更加敏感，同時(shí)也對(duì)鹽脅迫與干旱脅迫更加敏感，而且還會(huì)降低植株的相對(duì)含水量。但在擬南芥中超表達(dá)GRX1基因則能夠正向調(diào)節(jié)與抗氧化、耐鹽性和抗旱性相關(guān)基因的表達(dá)[23]。除此之外，在番茄中還存在與熱脅迫響應(yīng)相關(guān)的基因如WRKY33與ATG。Zhou 等[60]用TRV介導(dǎo)的VIGS技術(shù)沉默番茄葉中ATG基因后與對(duì)照番茄植株相比，沉默植株在45℃熱處理16 h時(shí)，ATG5基因和ATG7基因的表達(dá)量下降了70% ～ 80%，并且沉默植株再被放回室溫時(shí)不能重新恢復(fù)，而是逐漸枯萎且伴隨嚴(yán)重的電解質(zhì)滲出現(xiàn)象。而當(dāng)WRKY33基因被沉默后，沉默植株的點(diǎn)狀綠色熒光信號(hào)只有對(duì)照的25% ～ 30%。位于番茄內(nèi)質(zhì)網(wǎng)的E3泛素連接酶類基因SpRing，其表達(dá)受到溫度的影響，低溫能夠抑制該基因的表達(dá)。利用VIGS技術(shù)沉默番茄SpRing基因，與對(duì)照植株相比，沉默植株中的可溶性糖含量下降、膜脂過(guò)氧化明顯，丙二醛的含量較高且光合作用受到明顯的抑制，推測(cè)SpRing基因能夠正向響應(yīng)番茄逆性脅迫[61]。

3 問(wèn)題與展望

雖然VIGS 技術(shù)在植物基因功能鑒定方面的應(yīng)用取得了重大的進(jìn)展，但是還存在一定的局限性。主要體現(xiàn)在以下幾點(diǎn)：首先，在沉默的同一植株中容易出現(xiàn)沉默不均一的現(xiàn)象；其次，VIGS 技術(shù)無(wú)法將靶基因完全沉默，而未沉默的部分基因?qū)⒗^續(xù)發(fā)揮蛋白功能，從而帶來(lái)干擾癥狀；再次，這種瞬時(shí)沉默技術(shù)往往在沉默過(guò)程中還具有不穩(wěn)定性，且持續(xù)性較差，一般只能維持幾個(gè)月，而且無(wú)法穩(wěn)定遺傳給下一代，難以應(yīng)用在植物早期苗期表達(dá)的功能考察研究。并且在無(wú)法獲得某一特定基因的完整序列時(shí)，存在基因家族功能冗余的干擾；除此之外，環(huán)境因子對(duì)沉默效率的影響較大，例如溫度、濕度、光照等。

但是隨著分子生物學(xué)的不斷發(fā)展以及各種功能基因研究技術(shù)的開(kāi)發(fā)，VIGS技術(shù)還是取得了很大進(jìn)步，并且在功能基因組學(xué)廣受重視。更多的病毒載體被開(kāi)發(fā)而加以運(yùn)用，由此前以RNA病毒載體中TRV病毒作為主要沉默載體發(fā)展到RNA病毒載體、DNA病毒載體、衛(wèi)星病毒載體等多種病毒載體同時(shí)并存。并且在運(yùn)用的植物類型上也不斷豐富，從茄科植物的煙草、矮牽牛等模式植物再到番茄、辣椒、茄子、馬鈴薯等。對(duì)于遺傳轉(zhuǎn)化困難的植物，VIGS技術(shù)以快速、高通量、低成本等優(yōu)勢(shì)解決了這一難題。今后隨著這一技術(shù)的不斷完善和發(fā)展，特異性與穩(wěn)定性更高的病毒載體將得到開(kāi)發(fā)，以及一些制約VIGS技術(shù)的關(guān)鍵因素也將取得突破，相信在今后包括植物分子育種與植物保護(hù)在內(nèi)的領(lǐng)域都將進(jìn)一步得到廣泛的應(yīng)用。

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Application of Virus-Induced Gene Silencing Technology in Researchon Solanaceae Plants Functional Genomics

CAI Wen CHEN Changming CHEN Guoju CAO Bihao LEI Jianjun*

（College of Horticulture, South China Agricultural University, Guangzhou 510642, China）

Virus-induced gene silencing (VIGS) is areverse-genetics technology for studying gene function in plants and it’s widely used in the gene identif cation and function validation owing to simple operating, short cycle, high-throughput, transformation-free and low cost. This paper reviews and then prospects mainly the application and development of the VIGS technology in research of functional genes related with plant secondary metabolism, growth and development, biotic and abiotic stress in Solanaceae plants.

Solanaceae plants；VIGS；functional genomics；secondary metabolism；growth and development；biotic and abiotic stress

2017-02-09

國(guó)家自然科學(xué)基金項(xiàng)目（31572124）；廣東省公益研究與能力建設(shè)專項(xiàng)（2015B020202009，2014B020202005）；廣州市科創(chuàng)委項(xiàng)目（201508030021）

蔡文（1990-），男，碩士研究生，研究方向?yàn)槭卟诉z傳育種與生物技術(shù)；E-mail：136827769@qq.com

*通信作者：雷建軍（1957-），男，博士，教授；E-mail：jjlei@scau.edu.cn