何康來, 王振營, 沈 萍

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; 2中華人民共和國農(nóng)業(yè)部科技發(fā)展中心，北京 100122

轉(zhuǎn)蘇云金芽孢桿菌Bacillusthuringiensis(Bt)殺蟲蛋白基因抗蟲作物已在世界許多國家大面積應(yīng)用，以防治鱗翅目和鞘翅目害蟲(James,2014； Vaughnetal.,2005)。Bt殺蟲蛋白被靶標(biāo)昆蟲攝入后在中腸酶的作用下，經(jīng)一系列構(gòu)型變化，插入中腸細(xì)胞的原生質(zhì)膜，從而形成孔洞，最終殺死害蟲(Rajamohanetal.,1998)。然而，室內(nèi)和田間的大量研究結(jié)果表明，大面積使用單一和具有相同作用模式的Bt殺蟲蛋白，會(huì)導(dǎo)致靶標(biāo)害蟲產(chǎn)生抗性，從而使轉(zhuǎn)Bt基因抗蟲作物防治害蟲的功能失效(Tabashniketal.,2008)。因此，害蟲防治策略中包含不同殺蟲作用機(jī)理的轉(zhuǎn)基因作物，對于預(yù)防和治理害蟲抗性，使轉(zhuǎn)基因抗蟲作物在害蟲防治中得以可持續(xù)利用具有重要意義。

RNA干涉(RNA interference,RNAi)在基因功能研究方面顯示了重要作用(Bucheretal.,2002)，同時(shí)在臨床醫(yī)學(xué)(Huvenn & Smagghe,2010)和害蟲防治領(lǐng)域(Gatehouse & Price,2011)有巨大的潛力。由于RNAi的殺蟲機(jī)理與Bt蛋白完全不同，其可作為害蟲Bt抗性治理的又一潛在新途徑。有研究表明，基于RNAi的轉(zhuǎn)基因抗蟲作物可用于害蟲防治(Baumetal.,2007; Maoetal.,2007)。但是，有關(guān)RNAi轉(zhuǎn)基因抗蟲作物的環(huán)境安全性也受到科學(xué)家的關(guān)注。因此，本文就目前RNAi生物技術(shù)作物及其環(huán)境風(fēng)險(xiǎn)評(píng)價(jià)研究進(jìn)行簡要綜述。

1 RNAi生物技術(shù)作物研發(fā)進(jìn)展

1.1 dsRNA施用方法的研究

在真核生物(包括昆蟲)中普遍存在由雙鏈RNA(double-stranded RNA，dsRNA)引發(fā)特異序列的基因沉默現(xiàn)象(Hannon,2002)。在植物中被稱之為轉(zhuǎn)錄后基因沉默(post-transcriptional gene silencing)(Baulcombe,2004)，在動(dòng)物中則被稱之為RNAi(Hannon,2002)。通過取食或注射大量dsRNA引發(fā)重要基因沉默，可導(dǎo)致昆蟲停止取食，進(jìn)而死亡。在傳統(tǒng)的遺傳模式生物中應(yīng)用RNAi開展基因功能研究已有十多年的歷史。在昆蟲方面也有許多報(bào)道，如通過微量注射沉默基因以探索基因功能(Amdametal.,2003; Brownetal.,2009; Bucheretal.,2002; Suazoetal.,2009; Tomoyasu & Denell,2004)。由于基因沉默僅發(fā)生于“感染”了dsRNA的細(xì)胞，選擇適宜的施用方法是RNAi成功的重要一步。已報(bào)道的方法包括注射法(Bettencourtetal.,2002; Quanetal.,2002)、攝食法(Turneretal.,2006)、浸泡法(Aronsteinetal.,2006； Eatonetal.,2002； Rajagopaletal.,2002; Timmons & Fire,1998)和基因槍法(Yuenetal.,2008)等。與此同時(shí)，科學(xué)家在積極探討和研發(fā)應(yīng)用RNAi防治害蟲的新方法。例如，直接噴霧dsRNA后，亞洲玉米螟Ostriniafurnacalis(Guenée)幼蟲的死亡率可達(dá)到50%，同時(shí)噴霧處理幼蟲和人工飼料，死亡率可達(dá)73%～100%(Wangetal.,2011)；點(diǎn)滴法施用丙酮稀釋的AaeIAP1 dsRNA能殺死埃及伊蚊Aedesaegypti(L.)雌蚊(Pridgeonetal.,2008)；將幾丁聚糖基因AgCHS1 dsRNA制成納米微膠囊喂食非洲瘧蚊Anophelesgambiae(Zhangetal.,2010)及以表達(dá)dsRNA的轉(zhuǎn)基因工程菌喂食實(shí)蠅等都能取得一定的殺蟲效果(Gura,2000； Lietal.,2011; Timmons & Fire,1998)。在實(shí)驗(yàn)室，喂食dsRNA實(shí)現(xiàn)RNAi具有簡單、無需特殊設(shè)備(注射法需要精密的微量注射儀)、易操作、經(jīng)濟(jì)、省時(shí)、無入侵性傷口(相比注射法)等特點(diǎn)，且是客觀自然性的攝入，因此實(shí)用性強(qiáng)；同時(shí)，符合田間應(yīng)用技術(shù)的開發(fā)，如dsRNA制劑、RNAi生物技術(shù)作物等。

1.2 RNAi生物技術(shù)作物的研發(fā)

隨著轉(zhuǎn)Bt基因抗蟲作物的應(yīng)用，主要靶標(biāo)鱗翅目害蟲得到了有效控制(Wuetal.,2008),而一些次要害蟲如盲蝽、蚜蟲、飛虱等刺吸式害蟲上升為主要害蟲(Luetal.,2010)，目前還未發(fā)現(xiàn)對這類昆蟲有殺蟲活性的Bt殺蟲蛋白。因此，RNAi生物技術(shù)作物將為包括這類昆蟲在內(nèi)的害蟲防治提供了新途徑(Gatehouse & Price,2011)。此外，由于Bt作物的大面積應(yīng)用，某些靶標(biāo)害蟲已在一些地區(qū)對Bt作物產(chǎn)生了抗性(Alietal.,2006; Lietal.,2004、2007; Mattenetal.,2008; Tabashniketal.,2008; van Rensburg,2007)，使Bt作物的持續(xù)應(yīng)用受到嚴(yán)重的威脅。研發(fā)RNAi生物技術(shù)作物對害蟲Bt抗性治理具有重要意義。如有研究表明，喂食dsRNA沉默小菜蛾P(guān)lutellaxylostellaL.氯菊酯抗性品系過表達(dá)的細(xì)胞色素P450基因CYP6BG1，可顯著提高其對殺蟲劑氯菊酯的敏感性(Bautistaetal.,2009)。

早期的研究表明，經(jīng)口攝食的dsRNA對不同害蟲的作用效果不一致。如對斜紋夜蛾Spodopteralitura(Fabricius)幼蟲注射dsRNA能引發(fā)RNAi反應(yīng)，而攝食dsRNA不能引起RNAi反應(yīng)(Rajagopaletal.,2002)；而蘋淺褐卷蛾Epiphyaspostvittana(Walker)攝食大量的dsRNA能引發(fā)目標(biāo)基因轉(zhuǎn)錄水平(mRNA)下降，但未引起死亡(Turneretal.,2006)。近年來，許多研究表明，添加了dsRNA的人工飼料或表達(dá)dsRNA的轉(zhuǎn)基因植物均能成功殺死靶標(biāo)害蟲(Whyardetal.,2009)，如鞘翅目昆蟲玉米根葉甲(WCR)DiabroticavirgiferavirgiferaLeConte、南方玉米根葉甲(SCR)DiabroticaundecimpunctatahowardiBarber和馬鈴薯甲蟲Leptinotarsadecemlineata(Say)(Baumetal.,2007)，鱗翅目昆蟲棉鈴蟲HelicoverpaarmigeraHübner(Maoetal.,2007)和甜菜夜蛾SpodopteraexiguaHübner(Zhuetal.,2012)，半翅目昆蟲褐飛虱Nilaparvatalugens(Stal)(Zhaetal.,2011)，疾病媒介昆蟲采采蠅Glossina和埃及伊蚊(Coyetal.,2012; Walsheetal.,2009)，以及筑巢昆蟲白蟻Reticulitermesflavipes(Kollar)(Zhouetal.,2008)等。

1.3 dsRNA作用機(jī)理的研究

dsRNA隨昆蟲攝食進(jìn)入中腸，被中腸細(xì)胞“吞食”，在RNaseⅢ核酶家族的Dicer作用下加工成小干涉(si)RNA(21 bp+每條鏈3′ 2個(gè)延長堿基)而啟動(dòng)RNAi信號(hào)通路，即siRNA結(jié)合到一個(gè)被稱作RNA誘導(dǎo)沉默復(fù)合體(RISC)的蛋白復(fù)合體上，由RISC啟動(dòng)降解特異性的目標(biāo)mRNA(Fireetal.,1998)。RNA依賴的RNA聚合酶(RdRp)利用siRNA為引物、目標(biāo)基因?yàn)槟０妫铣尚碌膁sRNA，進(jìn)而引起RNAi效應(yīng)在蟲體內(nèi)擴(kuò)散(Price & Gatehouse,2008)。如果目標(biāo)mRNA在昆蟲體內(nèi)編碼的是一個(gè)基本功能性蛋白，其表達(dá)受阻將導(dǎo)致昆蟲死亡。在昆蟲方面，經(jīng)口攝食dsRNA的RNAi系統(tǒng)作用機(jī)理已有報(bào)道(Bolognesietal.,2012)，其整個(gè)作用時(shí)序過程一般包括：dsRNA攝食；1 d后，靶標(biāo)mRNA在中腸和體組織顯著下降，此時(shí)目標(biāo)蛋白水平還未受到影響，沒有出現(xiàn)死亡；3 d后，靶標(biāo)mRNA在中腸和體組織持續(xù)減少，并在組織中持續(xù)蔓延；5 d后靶標(biāo)蛋白顯著降低；隨分子水平靶標(biāo)基因表達(dá)的時(shí)序性降低及系統(tǒng)性擴(kuò)散，生物測定的幼蟲表現(xiàn)為生長受到抑制，繼而死亡。

在人工飼料中添加目標(biāo)dsRNA飼喂靶標(biāo)昆蟲的致死(或生長顯著受抑制)時(shí)間在12 d以上，125種dsRNA在52 ng·cm-2劑量下有顯著的殺蟲活性，其中14種dsRNA對WCR的LC50≤ 5.20 ng·cm-2，殺蟲效果最好的LC50達(dá)到0.57 ng·cm-2(Baumetal.,2007)。以能沉默WCR β-微管蛋白、V-ATPase A亞基和V-ATPase E亞基同源基因的dsRNA飼喂SCR，具有顯著的殺蟲活性；以能沉默WCR V-ATPase A亞基和V-ATPase E亞基同源基因的dsRNA飼喂馬鈴薯甲蟲，具有顯著的殺蟲活性；表達(dá)WCR V-ATPase A 亞基等目標(biāo)基因的dsRNA的轉(zhuǎn)基因玉米能顯著降低根的受害(Baumetal.,2007)。

2 RNAi生物技術(shù)作物的環(huán)境風(fēng)險(xiǎn)

與轉(zhuǎn)Bt基因生物技術(shù)作物相似，RNAi生物技術(shù)作物的環(huán)境風(fēng)險(xiǎn)主要包括遺傳穩(wěn)定性、生存競爭能力與雜草性、花粉介導(dǎo)的基因飄逸、潛在的接觸途徑和生態(tài)殘留、對非靶標(biāo)節(jié)肢動(dòng)物(包括天敵及有益昆蟲)的影響?；赗NAi生物技術(shù)作物的特異性，環(huán)境風(fēng)險(xiǎn)尤其要考察以下幾個(gè)方面。

2.1 非期望的基因沉默(unintended gene silencing)

基于RNAi的生物技術(shù)作物對生活和棲息在其植株、組織器官或殘留秸稈等中的非靶標(biāo)生物可能存在風(fēng)險(xiǎn)，即RNAi有時(shí)錯(cuò)誤沉默了其他生物的基因。據(jù)報(bào)道，取食了siRNA的昆蟲由于液泡膜上ATP酶看家基因(housekeeping gene)的mRNA被切割而使其生長受抑制或死亡(Baumetal.,2007)。如果靶標(biāo)害蟲的看家基因和其他非靶標(biāo)如有益昆蟲等的同源性足夠高，就有可能引起非期望的基因沉默負(fù)效應(yīng)(Bachmanetal.,2013)。基因組數(shù)據(jù)庫和科學(xué)設(shè)計(jì)的室內(nèi)喂食生測試驗(yàn)為確定非期望基因沉默的影響提供了依據(jù)。但是，目前許多非靶標(biāo)生物基因組數(shù)據(jù)相對短缺。

可遺傳的基因突變(即堿基替換、刪除、插入)發(fā)生在包括作物及其害蟲等在內(nèi)的所有生物中。同時(shí)，在種群內(nèi)個(gè)體間存在遺傳多態(tài)性(即DNA序列的微小變異)(Gordon & Waterhouse,2007; Whangbo & Hunter,2008)。因此，非靶標(biāo)生物如發(fā)生突變，就可能對RNAi生物技術(shù)作物產(chǎn)生敏感。

2.2 靶外結(jié)合(off-target binding)

大量文獻(xiàn)報(bào)道了siRNA引發(fā)的各種基因沉默現(xiàn)象(Hammondetal.,2001)。由于RNAi可能沉默完全錯(cuò)誤的基因(Jacksonetal.,2003; Jarosch & Moritz,2012； Scacherietal.,2004)，siRNA介導(dǎo)的基因沉默的特異性是一個(gè)在RNAi應(yīng)用中必須要考慮的關(guān)鍵因素。研究表明，由于靶標(biāo)位點(diǎn)單核苷酸錯(cuò)配和G∶U間的搖擺配對，可能引起潛在的靶外結(jié)合(Saxenaetal.,2003)，導(dǎo)致非靶標(biāo)內(nèi)源基因表達(dá)的沉默。RNAi生物技術(shù)作物也可能發(fā)生變異，改變siRNA分子的堿基序列和基因沉默模式，進(jìn)而產(chǎn)生靶外結(jié)合效應(yīng)。有學(xué)者認(rèn)為，siRNA介導(dǎo)的基因沉默的特異性是siRNA特異，而不是靶標(biāo)基因特異，即使siRNA和mRNA之間的部分互補(bǔ)(僅11個(gè)連續(xù)的核苷酸)可改變非特異性mRNA的轉(zhuǎn)錄水平，siRNA也可能交互沉默序列或相似度較低的非靶標(biāo)mRNA(Birminghametal.,2006; Haley & Zamore,2004; Jacksonetal.,2003)。靶外結(jié)合沉默能引起明顯表型效應(yīng)(Fedorovetal.,2006; Linetal.,2005),且其主要是由于siRNA的RISC-entering strand存在一個(gè)4個(gè)堿基的基序UGCC (Fedorovetal.,2006)。

為避免靶外結(jié)合引起的沉默效應(yīng)，簡單的預(yù)防方法是比對生物基因組數(shù)據(jù)庫是否存在與siRNA靶標(biāo)基因同源的基因。此外，通過化學(xué)修飾siRNA，特別是先導(dǎo)鏈第2位進(jìn)行2′-O-甲基核糖取代(Jacksonetal.,2006),可降低或消除非期望的靶外結(jié)合沉默效應(yīng)。

2.3 靶標(biāo)害蟲的抗性

由于害蟲種群內(nèi)某些個(gè)體靶標(biāo)mRNA突變和多態(tài)性可能引起其對某一特定的dsRNA序列的基因沉默產(chǎn)生抗性，從而導(dǎo)致RNAi生物技術(shù)作物防治效果下降。目前，有關(guān)靶標(biāo)害蟲對于RNAi產(chǎn)生抗性的模式缺乏研究。如果這一問題發(fā)生，可選擇或針對同一基因的其他部位或某一新基因設(shè)計(jì)一種新的dsRNA來治理(Yuetal.,2013)。

2.4 siRNA的環(huán)境持久性

實(shí)驗(yàn)室土壤微環(huán)境中Bt棉花和Bt玉米表達(dá)的Cry1Ac和Cry1Ab殺蟲蛋白會(huì)很快降解，半衰期為16 d或更短(Badeaetal.,2010; Sims & Holden,1996; Sims & Ream,1997)；在連續(xù)種植Cry1Ac棉或Cry1Ab玉米多年的農(nóng)田土壤中也未檢測到相應(yīng)Bt殺蟲蛋白的殘留或富集(Headetal.,2002)。據(jù)此，美國環(huán)保署發(fā)布，連續(xù)種植Bt作物，在土壤中不會(huì)出現(xiàn)Bt殺蟲蛋白富集現(xiàn)象(Kough & Edelstein,2012)。有關(guān)植物組織或胞外DNA在微生態(tài)土壤環(huán)境中的降解動(dòng)態(tài)已有報(bào)道。如轉(zhuǎn)基因和非轉(zhuǎn)基因大豆冷凍干燥葉子DNA在土壤中的半衰期僅1.4 d(Levy-Boothetal.,2008)；轉(zhuǎn)基因和非轉(zhuǎn)基因玉米和大豆的DNA于室溫下在土壤滲濾液中的半衰期分別不到2和4 h(Guldenetal.,2005)。在一項(xiàng)土壤中添加干燥的DNA或RNA，通過分析氮的釋放情況衡量核酸的降解，結(jié)果表明，RNA和DNA在各種土壤中的降解速度相同(Keownetal.,2004)。體外轉(zhuǎn)錄的純WCR的空泡排序蛋白基因DvSnf7的dsRNA或來自抗WCR的轉(zhuǎn)基因玉米殘?bào)w的DvSnf7 dsRNA在不同土壤中(包括不同土壤結(jié)構(gòu)、pH、黏性等)的半衰期為15～28 h，2 d內(nèi)檢測不到生物活性(Dubelmanetal.,2014)。由此可見，裸露的核酸會(huì)在土壤中很快分解，為界定基于RNAi的農(nóng)業(yè)生物技術(shù)產(chǎn)品在環(huán)境中殘留的潛在生態(tài)風(fēng)險(xiǎn)提供了依據(jù)。

2.5 不確定性

在任何生態(tài)風(fēng)險(xiǎn)評(píng)估中，認(rèn)知的不確定性是固有的，評(píng)估者應(yīng)了解不確定性的范圍(Suter,2007)?；诘鞍椎纳锛夹g(shù)作物已商業(yè)化種植20年，對其風(fēng)險(xiǎn)評(píng)估的研究時(shí)間可能更長。這不僅明確了外源基因表達(dá)的蛋白尤其是Bt蛋白的殺蟲作用模式，而且回答了許多生態(tài)風(fēng)險(xiǎn)問題(Conneretal.,2003; Sanvidoetal.,2007)。von Kraussetal.(2008)對不確定性進(jìn)行了評(píng)估，認(rèn)為在田間多變的條件下和隨時(shí)間推移基因沉默表現(xiàn)原理不確定，專家之間對于基因沉默的因果關(guān)系看法不盡相同。在風(fēng)險(xiǎn)評(píng)估基礎(chǔ)上做決策時(shí)，監(jiān)管者和利益相關(guān)方需要找到風(fēng)險(xiǎn)和不確定性相關(guān)的風(fēng)險(xiǎn)評(píng)估之間的平衡點(diǎn)。RNAi技術(shù)整體上呈現(xiàn)低環(huán)境風(fēng)險(xiǎn)，但如果了解到這些低風(fēng)險(xiǎn)的高度不確定性，在商業(yè)化之前就必須進(jìn)行大量的試驗(yàn)和管理方法的研究。

3 RNAi生物技術(shù)作物的環(huán)境風(fēng)險(xiǎn)評(píng)估

RNAi和Bt生物技術(shù)作物都是通過作物系統(tǒng)表達(dá)能啟動(dòng)RNAi的dsRNA或具有殺蟲作用的Bt殺蟲蛋白，而棲息于作物田的生物種類不會(huì)因此而改變。因此，暴露于二者的生物種類理論上相同或相似；為使作物整個(gè)生育期都能受到保護(hù)，目的基因在整個(gè)生育期都能表達(dá)。但是，RNAi生物技術(shù)作物的靶標(biāo)對象更多，殺蟲劑量閾值較高，致死或生長顯著受抑制的時(shí)間較長，需要特殊的生測方法測定致死效應(yīng)。因此，RNAi與轉(zhuǎn)Bt基因等生物技術(shù)作物的環(huán)境風(fēng)險(xiǎn)評(píng)估既有相同的框架，亦有不同的內(nèi)容。

3.1 功能基因及其表達(dá)特征

Bt作物外源目的基因編碼Bt殺蟲蛋白，與害蟲中腸緣膜上的受體結(jié)合使細(xì)胞溶解，作用位點(diǎn)在昆蟲中腸上(Bravoetal.,2007)。目的基因表達(dá)的穩(wěn)定性通過特定的免疫試紙條進(jìn)行定性檢測，ELISA進(jìn)行蛋白定量檢測。RNAi作物外源目的基因編碼20～24 nt siRNA，siRNA與昆蟲體內(nèi)蛋白形成RISC，導(dǎo)致靶標(biāo)mRNA降解，引起目的基因沉默(Fireetal.,1998; Ghildiyal & Zamore,2009; Obbardetal.,2009; Price & Gatehouse,2008)。雖然在模式植物擬南芥Arabidopsis中已明確多種作用模式，但在多數(shù)農(nóng)作物中還缺乏相應(yīng)的數(shù)據(jù)。影響RNAi效果的因素主要有以下幾個(gè)方面。

(1)dsRNA序列。Tereniusetal.(2011)系統(tǒng)分析總結(jié)了鱗翅目中不同種類昆蟲和功能基因與RNAi效果的關(guān)系。除靶標(biāo)基因沉默效果外，還與靶外結(jié)合和非期望的非靶標(biāo)生物影響有關(guān)。Whyardetal.(2009)認(rèn)為，dsRNA可作為種特異性的“胃毒”殺蟲劑，如利用種特異性的dsRNA可分別沉默果蠅DrosophilamelanogasterMeigen、赤擬谷盜Triboliumcastaneum(Herbst)、桃蚜Acyrthosiphonpisum(Harris)和煙草天蛾Manducasexta(L.) 4種害蟲的V-ATP酶基因，針對靶標(biāo)γ微管蛋白基因3′ UTR區(qū)(不存在19～21 nt序列片段的匹配)設(shè)計(jì)短(<40 nt)的dsRNA也可選擇性殺死果蠅屬Drosophila的4種果蠅。

(2)dsRNA長度。不同長度的dsRNA引起RNAi的效果不同(Whyardetal.,2009)。WCR和馬鈴薯甲蟲的DvSnf7 dsRNA與目標(biāo)基因最少有21 nt的連續(xù)匹配才能有顯著的RNAi生物活性；對于相似種，DvSnf7同源序列上最少有3個(gè)21 nt的匹配才能在甄別高劑量下產(chǎn)生顯著的活性(Bachmanetal.,2013)。多數(shù)喂食試驗(yàn)中獲得較好效果的dsRNA長度為300～600 nt。dsRNA長度影響昆蟲細(xì)胞系(Salehetal.,2006)以及昆蟲(Maoetal.,2007)的攝取和沉默效果。研究表明，較長dsRNA的沉默效果較好，可能與其在環(huán)境中存留時(shí)間更長有關(guān)(Baumetal.,2007)。

(3)dsRNA濃度。取得最佳沉默效果的dsRNA濃度因靶標(biāo)基因和生物種類而異，并不是dsRNA的濃度越高越好(Meyering-Vos & Muller,2007; Shakesbyetal.,2009)。

(4)沉默效果的持續(xù)性。給橘小實(shí)蠅Bactroceradorsalis(Hendel)分別喂食沉默核糖體蛋白R(shí)pl19、V-ATPaseD亞基、脂肪酸碳鏈延長酶 Noa 和一種小型GTP酶 Rab11基因的4種dsRNAds-rpl19、ds-v-ATP-d、ds-noa、ds-rab11溶液和表達(dá)dsRNA的轉(zhuǎn)基因大腸桿菌(Escherichiacolistrain HT115)，可使目標(biāo)基因沉默，引起20%蟲體死亡或雌蠅產(chǎn)卵量下降，然而，當(dāng)持續(xù)飼喂14 d后，目標(biāo)基因的表達(dá)反而上調(diào)(Lietal.,2011)。

3.2 殺蟲譜及非靶標(biāo)生物的影響

RNAi生物技術(shù)作物的作用方式是通過攝食進(jìn)入生物體內(nèi)。因此，應(yīng)該建立農(nóng)業(yè)生態(tài)系統(tǒng)中生物多樣性數(shù)據(jù)，明確接觸RNAi生物技術(shù)作物的種類，且建立這些種類的基因組數(shù)據(jù)庫，清楚與siRNA具有同源性序列基因的表現(xiàn)型，就能確定RNAi生物技術(shù)作物的殺蟲活性譜。

有關(guān)Bt作物對非靶標(biāo)生物的影響評(píng)價(jià)，各國要求有異，根據(jù)Bt蛋白殺蟲作用譜的特異性，通常在各生態(tài)功能團(tuán)內(nèi)選擇一定的相關(guān)指示性生物進(jìn)行生物測定和分級(jí)啟動(dòng)測試(Romeisetal.,2008)。

通過靶外結(jié)合和非期望的基因沉默的毒理基因組學(xué)分析RNAi生物技術(shù)作物防治害蟲的靶標(biāo)，可能是分類學(xué)上特異性的一種(Whyardetal.,2009)。靶標(biāo)害蟲分類學(xué)上高度特異性的優(yōu)點(diǎn)在于僅有靶標(biāo)害蟲的近似種受到dsRNA的影響(Romiesetal.,2008)。因此，要確定某一dsRNA的殺蟲譜，必須評(píng)估其對與靶標(biāo)害蟲親緣相近種類的潛在影響。

3.3 環(huán)境殘留

明確生物技術(shù)作物外源性狀表達(dá)產(chǎn)物(無論是殺蟲蛋白還是dsRNA)在環(huán)境中的時(shí)空分布是生態(tài)安全評(píng)價(jià)的重要內(nèi)容之一，同時(shí)可界定環(huán)境中非靶標(biāo)生物接觸的客觀途徑和劑量。因此，應(yīng)加強(qiáng)對sRNA在環(huán)境中的持久性和位移趨勢，如基因飄逸、sRNA在土壤和水體中的存留時(shí)間、被非靶標(biāo)生物攝入的可能性，以及發(fā)生系統(tǒng)性基因沉默后的表型特征等方面的研究。生態(tài)毒理學(xué)模式生物的基因文庫比對和DNA芯片法是開展該項(xiàng)評(píng)價(jià)的重要方法(Robbensetal.,2007)。

3.4 功能性狀的持續(xù)穩(wěn)定性

在評(píng)價(jià)RNAi生物技術(shù)作物功能性狀的遺傳穩(wěn)定性時(shí)，編碼siRNA基因比編碼蛋白基因的變異率高(Obbardetal.,2009)。因此，應(yīng)分析害蟲的抗性發(fā)展，評(píng)估其發(fā)生時(shí)間、影響規(guī)模(局部的、區(qū)域的或全國性的)，以及嚴(yán)重程度，并建立預(yù)測模型。此外，只有建立統(tǒng)一的sRNA活性標(biāo)準(zhǔn)，才能開展可比性評(píng)價(jià)(von Kraussetal.,2008)。

4 結(jié)束語

具有植物保護(hù)性狀的RNAi作物是繼Bt作物之后推動(dòng)害蟲防治技術(shù)提升的又一重要成就。同時(shí)，為研發(fā)對重大害蟲，如蚜蟲(Muttietal.,2006; Whyardetal.,2009)、飛虱(Chenetal.,2010; Upadhyayetal.,2011)、蝽(Araujoetal.,2006)具有殺蟲譜專一(Alsfordetal.,2011; Haas & Zody,2010)的新一代生物技術(shù)作物提供了廣闊的前景。此外，dsRNA制劑的研發(fā)和應(yīng)用將極大地豐富農(nóng)作物病蟲害防治技術(shù)(Burand & Hunter,2013; Huvenne & Smagghe,2010)。如噴施dsRNA粗提液可有效控制植物病毒病的發(fā)生，且目的dsRNA可在葉面存留數(shù)天(Tenlladoetal.,2004)。納米包裹(Zhangetal.,2010)、土壤處理和拌種等方法可用于防治地下害蟲。工程菌是大量生產(chǎn)dsRNA的重要途徑之一(Timmons & Fire,1998)。

現(xiàn)行的轉(zhuǎn)基因生物環(huán)境風(fēng)險(xiǎn)評(píng)估程序需要進(jìn)一步完善，以適應(yīng)新的RNAi生物技術(shù)作物研發(fā)和應(yīng)用的需求。建立專家意見庫，進(jìn)而準(zhǔn)確提出潛在風(fēng)險(xiǎn)問題，以確保收集有價(jià)值的科學(xué)數(shù)據(jù)，甄別風(fēng)險(xiǎn)評(píng)估中的不確定性；確立科學(xué)合理的室內(nèi)和田間可控條件下的評(píng)價(jià)試驗(yàn)設(shè)計(jì)和實(shí)施方案；提供有關(guān)遺傳穩(wěn)定性、靶外結(jié)合(或脫靶效應(yīng))、非期望的基因沉默(包括非靶標(biāo)的影響)，以及sRNA在各種生境中殘留時(shí)間的科學(xué)評(píng)估數(shù)據(jù)。

生物信息學(xué)方法將是風(fēng)險(xiǎn)評(píng)估的最基本的方法。因此，需要解決環(huán)境監(jiān)測中sRNA的提取和鑒定方法；建立標(biāo)準(zhǔn)化的生態(tài)毒理學(xué)模式生物的基因文庫和DNA芯片(Robbensetal.,2007)；研究RNAi作物身份驗(yàn)證、監(jiān)測等的定量和定性檢測方法。從科學(xué)的角度來看，具有作物保護(hù)性狀的RNAi作物的環(huán)境風(fēng)險(xiǎn)是可預(yù)測的，且在科學(xué)管理?xiàng)l件下是可避免或可控的。

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