RNAi生物技术作物环境风险评估研究进展

何康来, 王振营, 沈 萍

1中国农业科学院植物保护研究所，植物病虫害生物学国家重点实验室，北京 100193; 2中华人民共和国农业部科技发展中心，北京 100122

转苏云金芽孢杆菌Bacillusthuringiensis(Bt)杀虫蛋白基因抗虫作物已在世界许多国家大面积应用，以防治鳞翅目和鞘翅目害虫(James,2014； Vaughnetal.,2005)。Bt杀虫蛋白被靶标昆虫摄入后在中肠酶的作用下，经一系列构型变化，插入中肠细胞的原生质膜，从而形成孔洞，最终杀死害虫(Rajamohanetal.,1998)。然而，室内和田间的大量研究结果表明，大面积使用单一和具有相同作用模式的Bt杀虫蛋白，会导致靶标害虫产生抗性，从而使转Bt基因抗虫作物防治害虫的功能失效(Tabashniketal.,2008)。因此，害虫防治策略中包含不同杀虫作用机理的转基因作物，对于预防和治理害虫抗性，使转基因抗虫作物在害虫防治中得以可持续利用具有重要意义。

RNA干涉(RNA interference,RNAi)在基因功能研究方面显示了重要作用(Bucheretal.,2002)，同时在临床医学(Huvenn & Smagghe,2010)和害虫防治领域(Gatehouse & Price,2011)有巨大的潜力。由于RNAi的杀虫机理与Bt蛋白完全不同，其可作为害虫Bt抗性治理的又一潜在新途径。有研究表明，基于RNAi的转基因抗虫作物可用于害虫防治(Baumetal.,2007; Maoetal.,2007)。但是，有关RNAi转基因抗虫作物的环境安全性也受到科学家的关注。因此，本文就目前RNAi生物技术作物及其环境风险评价研究进行简要综述。

1 RNAi生物技术作物研发进展

1.1 dsRNA施用方法的研究

在真核生物(包括昆虫)中普遍存在由双链RNA(double-stranded RNA，dsRNA)引发特异序列的基因沉默现象(Hannon,2002)。在植物中被称之为转录后基因沉默(post-transcriptional gene silencing)(Baulcombe,2004)，在动物中则被称之为RNAi(Hannon,2002)。通过取食或注射大量dsRNA引发重要基因沉默，可导致昆虫停止取食，进而死亡。在传统的遗传模式生物中应用RNAi开展基因功能研究已有十多年的历史。在昆虫方面也有许多报道，如通过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)。由于基因沉默仅发生于“感染”了dsRNA的细胞，选择适宜的施用方法是RNAi成功的重要一步。已报道的方法包括注射法(Bettencourtetal.,2002; Quanetal.,2002)、摄食法(Turneretal.,2006)、浸泡法(Aronsteinetal.,2006； Eatonetal.,2002； Rajagopaletal.,2002; Timmons & Fire,1998)和基因枪法(Yuenetal.,2008)等。与此同时，科学家在积极探讨和研发应用RNAi防治害虫的新方法。例如，直接喷雾dsRNA后，亚洲玉米螟Ostriniafurnacalis(Guenée)幼虫的死亡率可达到50%，同时喷雾处理幼虫和人工饲料，死亡率可达73%～100%(Wangetal.,2011)；点滴法施用丙酮稀释的AaeIAP1 dsRNA能杀死埃及伊蚊Aedesaegypti(L.)雌蚊(Pridgeonetal.,2008)；将几丁聚糖基因AgCHS1 dsRNA制成纳米微胶囊喂食非洲疟蚊Anophelesgambiae(Zhangetal.,2010)及以表达dsRNA的转基因工程菌喂食实蝇等都能取得一定的杀虫效果(Gura,2000； Lietal.,2011; Timmons & Fire,1998)。在实验室，喂食dsRNA实现RNAi具有简单、无需特殊设备(注射法需要精密的微量注射仪)、易操作、经济、省时、无入侵性伤口(相比注射法)等特点，且是客观自然性的摄入，因此实用性强；同时，符合田间应用技术的开发，如dsRNA制剂、RNAi生物技术作物等。

1.2 RNAi生物技术作物的研发

随着转Bt基因抗虫作物的应用，主要靶标鳞翅目害虫得到了有效控制(Wuetal.,2008),而一些次要害虫如盲蝽、蚜虫、飞虱等刺吸式害虫上升为主要害虫(Luetal.,2010)，目前还未发现对这类昆虫有杀虫活性的Bt杀虫蛋白。因此，RNAi生物技术作物将为包括这类昆虫在内的害虫防治提供了新途径(Gatehouse & Price,2011)。此外，由于Bt作物的大面积应用，某些靶标害虫已在一些地区对Bt作物产生了抗性(Alietal.,2006; Lietal.,2004、2007; Mattenetal.,2008; Tabashniketal.,2008; van Rensburg,2007)，使Bt作物的持续应用受到严重的威胁。研发RNAi生物技术作物对害虫Bt抗性治理具有重要意义。如有研究表明，喂食dsRNA沉默小菜蛾PlutellaxylostellaL.氯菊酯抗性品系过表达的细胞色素P450基因CYP6BG1，可显著提高其对杀虫剂氯菊酯的敏感性(Bautistaetal.,2009)。

早期的研究表明，经口摄食的dsRNA对不同害虫的作用效果不一致。如对斜纹夜蛾Spodopteralitura(Fabricius)幼虫注射dsRNA能引发RNAi反应，而摄食dsRNA不能引起RNAi反应(Rajagopaletal.,2002)；而苹浅褐卷蛾Epiphyaspostvittana(Walker)摄食大量的dsRNA能引发目标基因转录水平(mRNA)下降，但未引起死亡(Turneretal.,2006)。近年来，许多研究表明，添加了dsRNA的人工饲料或表达dsRNA的转基因植物均能成功杀死靶标害虫(Whyardetal.,2009)，如鞘翅目昆虫玉米根叶甲(WCR)DiabroticavirgiferavirgiferaLeConte、南方玉米根叶甲(SCR)DiabroticaundecimpunctatahowardiBarber和马铃薯甲虫Leptinotarsadecemlineata(Say)(Baumetal.,2007)，鳞翅目昆虫棉铃虫HelicoverpaarmigeraHübner(Maoetal.,2007)和甜菜夜蛾SpodopteraexiguaHübner(Zhuetal.,2012)，半翅目昆虫褐飞虱Nilaparvatalugens(Stal)(Zhaetal.,2011)，疾病媒介昆虫采采蝇Glossina和埃及伊蚊(Coyetal.,2012; Walsheetal.,2009)，以及筑巢昆虫白蚁Reticulitermesflavipes(Kollar)(Zhouetal.,2008)等。

1.3 dsRNA作用机理的研究

dsRNA随昆虫摄食进入中肠，被中肠细胞“吞食”，在RNaseⅢ核酶家族的Dicer作用下加工成小干涉(si)RNA(21 bp+每条链3′ 2个延长碱基)而启动RNAi信号通路，即siRNA结合到一个被称作RNA诱导沉默复合体(RISC)的蛋白复合体上，由RISC启动降解特异性的目标mRNA(Fireetal.,1998)。RNA依赖的RNA聚合酶(RdRp)利用siRNA为引物、目标基因为模版，合成新的dsRNA，进而引起RNAi效应在虫体内扩散(Price & Gatehouse,2008)。如果目标mRNA在昆虫体内编码的是一个基本功能性蛋白，其表达受阻将导致昆虫死亡。在昆虫方面，经口摄食dsRNA的RNAi系统作用机理已有报道(Bolognesietal.,2012)，其整个作用时序过程一般包括：dsRNA摄食；1 d后，靶标mRNA在中肠和体组织显著下降，此时目标蛋白水平还未受到影响，没有出现死亡；3 d后，靶标mRNA在中肠和体组织持续减少，并在组织中持续蔓延；5 d后靶标蛋白显著降低；随分子水平靶标基因表达的时序性降低及系统性扩散，生物测定的幼虫表现为生长受到抑制，继而死亡。

在人工饲料中添加目标dsRNA饲喂靶标昆虫的致死(或生长显著受抑制)时间在12 d以上，125种dsRNA在52 ng·cm-2剂量下有显著的杀虫活性，其中14种dsRNA对WCR的LC50≤ 5.20 ng·cm-2，杀虫效果最好的LC50达到0.57 ng·cm-2(Baumetal.,2007)。以能沉默WCR β-微管蛋白、V-ATPase A亚基和V-ATPase E亚基同源基因的dsRNA饲喂SCR，具有显著的杀虫活性；以能沉默WCR V-ATPase A亚基和V-ATPase E亚基同源基因的dsRNA饲喂马铃薯甲虫，具有显著的杀虫活性；表达WCR V-ATPase A 亚基等目标基因的dsRNA的转基因玉米能显著降低根的受害(Baumetal.,2007)。

2 RNAi生物技术作物的环境风险

与转Bt基因生物技术作物相似，RNAi生物技术作物的环境风险主要包括遗传稳定性、生存竞争能力与杂草性、花粉介导的基因飘逸、潜在的接触途径和生态残留、对非靶标节肢动物(包括天敌及有益昆虫)的影响。基于RNAi生物技术作物的特异性，环境风险尤其要考察以下几个方面。

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

基于RNAi的生物技术作物对生活和栖息在其植株、组织器官或残留秸秆等中的非靶标生物可能存在风险，即RNAi有时错误沉默了其他生物的基因。据报道，取食了siRNA的昆虫由于液泡膜上ATP酶看家基因(housekeeping gene)的mRNA被切割而使其生长受抑制或死亡(Baumetal.,2007)。如果靶标害虫的看家基因和其他非靶标如有益昆虫等的同源性足够高，就有可能引起非期望的基因沉默负效应(Bachmanetal.,2013)。基因组数据库和科学设计的室内喂食生测试验为确定非期望基因沉默的影响提供了依据。但是，目前许多非靶标生物基因组数据相对短缺。

可遗传的基因突变(即碱基替换、删除、插入)发生在包括作物及其害虫等在内的所有生物中。同时，在种群内个体间存在遗传多态性(即DNA序列的微小变异)(Gordon & Waterhouse,2007; Whangbo & Hunter,2008)。因此，非靶标生物如发生突变，就可能对RNAi生物技术作物产生敏感。

2.2 靶外结合(off-target binding)

大量文献报道了siRNA引发的各种基因沉默现象(Hammondetal.,2001)。由于RNAi可能沉默完全错误的基因(Jacksonetal.,2003; Jarosch & Moritz,2012； Scacherietal.,2004)，siRNA介导的基因沉默的特异性是一个在RNAi应用中必须要考虑的关键因素。研究表明，由于靶标位点单核苷酸错配和G∶U间的摇摆配对，可能引起潜在的靶外结合(Saxenaetal.,2003)，导致非靶标内源基因表达的沉默。RNAi生物技术作物也可能发生变异，改变siRNA分子的碱基序列和基因沉默模式，进而产生靶外结合效应。有学者认为，siRNA介导的基因沉默的特异性是siRNA特异，而不是靶标基因特异，即使siRNA和mRNA之间的部分互补(仅11个连续的核苷酸)可改变非特异性mRNA的转录水平，siRNA也可能交互沉默序列或相似度较低的非靶标mRNA(Birminghametal.,2006; Haley & Zamore,2004; Jacksonetal.,2003)。靶外结合沉默能引起明显表型效应(Fedorovetal.,2006; Linetal.,2005),且其主要是由于siRNA的RISC-entering strand存在一个4个碱基的基序UGCC (Fedorovetal.,2006)。

为避免靶外结合引起的沉默效应，简单的预防方法是比对生物基因组数据库是否存在与siRNA靶标基因同源的基因。此外，通过化学修饰siRNA，特别是先导链第2位进行2′-O-甲基核糖取代(Jacksonetal.,2006),可降低或消除非期望的靶外结合沉默效应。

2.3 靶标害虫的抗性

由于害虫种群内某些个体靶标mRNA突变和多态性可能引起其对某一特定的dsRNA序列的基因沉默产生抗性，从而导致RNAi生物技术作物防治效果下降。目前，有关靶标害虫对于RNAi产生抗性的模式缺乏研究。如果这一问题发生，可选择或针对同一基因的其他部位或某一新基因设计一种新的dsRNA来治理(Yuetal.,2013)。

2.4 siRNA的环境持久性

实验室土壤微环境中Bt棉花和Bt玉米表达的Cry1Ac和Cry1Ab杀虫蛋白会很快降解，半衰期为16 d或更短(Badeaetal.,2010; Sims & Holden,1996; Sims & Ream,1997)；在连续种植Cry1Ac棉或Cry1Ab玉米多年的农田土壤中也未检测到相应Bt杀虫蛋白的残留或富集(Headetal.,2002)。据此，美国环保署发布，连续种植Bt作物，在土壤中不会出现Bt杀虫蛋白富集现象(Kough & Edelstein,2012)。有关植物组织或胞外DNA在微生态土壤环境中的降解动态已有报道。如转基因和非转基因大豆冷冻干燥叶子DNA在土壤中的半衰期仅1.4 d(Levy-Boothetal.,2008)；转基因和非转基因玉米和大豆的DNA于室温下在土壤渗滤液中的半衰期分别不到2和4 h(Guldenetal.,2005)。在一项土壤中添加干燥的DNA或RNA，通过分析氮的释放情况衡量核酸的降解，结果表明，RNA和DNA在各种土壤中的降解速度相同(Keownetal.,2004)。体外转录的纯WCR的空泡排序蛋白基因DvSnf7的dsRNA或来自抗WCR的转基因玉米残体的DvSnf7 dsRNA在不同土壤中(包括不同土壤结构、pH、黏性等)的半衰期为15～28 h，2 d内检测不到生物活性(Dubelmanetal.,2014)。由此可见，裸露的核酸会在土壤中很快分解，为界定基于RNAi的农业生物技术产品在环境中残留的潜在生态风险提供了依据。

2.5 不确定性

在任何生态风险评估中，认知的不确定性是固有的，评估者应了解不确定性的范围(Suter,2007)。基于蛋白的生物技术作物已商业化种植20年，对其风险评估的研究时间可能更长。这不仅明确了外源基因表达的蛋白尤其是Bt蛋白的杀虫作用模式，而且回答了许多生态风险问题(Conneretal.,2003; Sanvidoetal.,2007)。von Kraussetal.(2008)对不确定性进行了评估，认为在田间多变的条件下和随时间推移基因沉默表现原理不确定，专家之间对于基因沉默的因果关系看法不尽相同。在风险评估基础上做决策时，监管者和利益相关方需要找到风险和不确定性相关的风险评估之间的平衡点。RNAi技术整体上呈现低环境风险，但如果了解到这些低风险的高度不确定性，在商业化之前就必须进行大量的试验和管理方法的研究。

3 RNAi生物技术作物的环境风险评估

RNAi和Bt生物技术作物都是通过作物系统表达能启动RNAi的dsRNA或具有杀虫作用的Bt杀虫蛋白，而栖息于作物田的生物种类不会因此而改变。因此，暴露于二者的生物种类理论上相同或相似；为使作物整个生育期都能受到保护，目的基因在整个生育期都能表达。但是，RNAi生物技术作物的靶标对象更多，杀虫剂量阈值较高，致死或生长显著受抑制的时间较长，需要特殊的生测方法测定致死效应。因此，RNAi与转Bt基因等生物技术作物的环境风险评估既有相同的框架，亦有不同的内容。

3.1 功能基因及其表达特征

Bt作物外源目的基因编码Bt杀虫蛋白，与害虫中肠缘膜上的受体结合使细胞溶解，作用位点在昆虫中肠上(Bravoetal.,2007)。目的基因表达的稳定性通过特定的免疫试纸条进行定性检测，ELISA进行蛋白定量检测。RNAi作物外源目的基因编码20～24 nt siRNA，siRNA与昆虫体内蛋白形成RISC，导致靶标mRNA降解，引起目的基因沉默(Fireetal.,1998; Ghildiyal & Zamore,2009; Obbardetal.,2009; Price & Gatehouse,2008)。虽然在模式植物拟南芥Arabidopsis中已明确多种作用模式，但在多数农作物中还缺乏相应的数据。影响RNAi效果的因素主要有以下几个方面。

(1)dsRNA序列。Tereniusetal.(2011)系统分析总结了鳞翅目中不同种类昆虫和功能基因与RNAi效果的关系。除靶标基因沉默效果外，还与靶外结合和非期望的非靶标生物影响有关。Whyardetal.(2009)认为，dsRNA可作为种特异性的“胃毒”杀虫剂，如利用种特异性的dsRNA可分别沉默果蝇DrosophilamelanogasterMeigen、赤拟谷盗Triboliumcastaneum(Herbst)、桃蚜Acyrthosiphonpisum(Harris)和烟草天蛾Manducasexta(L.) 4种害虫的V-ATP酶基因，针对靶标γ微管蛋白基因3′ UTR区(不存在19～21 nt序列片段的匹配)设计短(<40 nt)的dsRNA也可选择性杀死果蝇属Drosophila的4种果蝇。

(2)dsRNA长度。不同长度的dsRNA引起RNAi的效果不同(Whyardetal.,2009)。WCR和马铃薯甲虫的DvSnf7 dsRNA与目标基因最少有21 nt的连续匹配才能有显著的RNAi生物活性；对于相似种，DvSnf7同源序列上最少有3个21 nt的匹配才能在甄别高剂量下产生显著的活性(Bachmanetal.,2013)。多数喂食试验中获得较好效果的dsRNA长度为300～600 nt。dsRNA长度影响昆虫细胞系(Salehetal.,2006)以及昆虫(Maoetal.,2007)的摄取和沉默效果。研究表明，较长dsRNA的沉默效果较好，可能与其在环境中存留时间更长有关(Baumetal.,2007)。

(3)dsRNA浓度。取得最佳沉默效果的dsRNA浓度因靶标基因和生物种类而异，并不是dsRNA的浓度越高越好(Meyering-Vos & Muller,2007; Shakesbyetal.,2009)。

(4)沉默效果的持续性。给橘小实蝇Bactroceradorsalis(Hendel)分别喂食沉默核糖体蛋白Rpl19、V-ATPaseD亚基、脂肪酸碳链延长酶 Noa 和一种小型GTP酶 Rab11基因的4种dsRNAds-rpl19、ds-v-ATP-d、ds-noa、ds-rab11溶液和表达dsRNA的转基因大肠杆菌(Escherichiacolistrain HT115)，可使目标基因沉默，引起20%虫体死亡或雌蝇产卵量下降，然而，当持续饲喂14 d后，目标基因的表达反而上调(Lietal.,2011)。

3.2 杀虫谱及非靶标生物的影响

RNAi生物技术作物的作用方式是通过摄食进入生物体内。因此，应该建立农业生态系统中生物多样性数据，明确接触RNAi生物技术作物的种类，且建立这些种类的基因组数据库，清楚与siRNA具有同源性序列基因的表现型，就能确定RNAi生物技术作物的杀虫活性谱。

有关Bt作物对非靶标生物的影响评价，各国要求有异，根据Bt蛋白杀虫作用谱的特异性，通常在各生态功能团内选择一定的相关指示性生物进行生物测定和分级启动测试(Romeisetal.,2008)。

通过靶外结合和非期望的基因沉默的毒理基因组学分析RNAi生物技术作物防治害虫的靶标，可能是分类学上特异性的一种(Whyardetal.,2009)。靶标害虫分类学上高度特异性的优点在于仅有靶标害虫的近似种受到dsRNA的影响(Romiesetal.,2008)。因此，要确定某一dsRNA的杀虫谱，必须评估其对与靶标害虫亲缘相近种类的潜在影响。

3.3 环境残留

明确生物技术作物外源性状表达产物(无论是杀虫蛋白还是dsRNA)在环境中的时空分布是生态安全评价的重要内容之一，同时可界定环境中非靶标生物接触的客观途径和剂量。因此，应加强对sRNA在环境中的持久性和位移趋势，如基因飘逸、sRNA在土壤和水体中的存留时间、被非靶标生物摄入的可能性，以及发生系统性基因沉默后的表型特征等方面的研究。生态毒理学模式生物的基因文库比对和DNA芯片法是开展该项评价的重要方法(Robbensetal.,2007)。

3.4 功能性状的持续稳定性

在评价RNAi生物技术作物功能性状的遗传稳定性时，编码siRNA基因比编码蛋白基因的变异率高(Obbardetal.,2009)。因此，应分析害虫的抗性发展，评估其发生时间、影响规模(局部的、区域的或全国性的)，以及严重程度，并建立预测模型。此外，只有建立统一的sRNA活性标准，才能开展可比性评价(von Kraussetal.,2008)。

4 结束语

具有植物保护性状的RNAi作物是继Bt作物之后推动害虫防治技术提升的又一重要成就。同时，为研发对重大害虫，如蚜虫(Muttietal.,2006; Whyardetal.,2009)、飞虱(Chenetal.,2010; Upadhyayetal.,2011)、蝽(Araujoetal.,2006)具有杀虫谱专一(Alsfordetal.,2011; Haas & Zody,2010)的新一代生物技术作物提供了广阔的前景。此外，dsRNA制剂的研发和应用将极大地丰富农作物病虫害防治技术(Burand & Hunter,2013; Huvenne & Smagghe,2010)。如喷施dsRNA粗提液可有效控制植物病毒病的发生，且目的dsRNA可在叶面存留数天(Tenlladoetal.,2004)。纳米包裹(Zhangetal.,2010)、土壤处理和拌种等方法可用于防治地下害虫。工程菌是大量生产dsRNA的重要途径之一(Timmons & Fire,1998)。

现行的转基因生物环境风险评估程序需要进一步完善，以适应新的RNAi生物技术作物研发和应用的需求。建立专家意见库，进而准确提出潜在风险问题，以确保收集有价值的科学数据，甄别风险评估中的不确定性；确立科学合理的室内和田间可控条件下的评价试验设计和实施方案；提供有关遗传稳定性、靶外结合(或脱靶效应)、非期望的基因沉默(包括非靶标的影响)，以及sRNA在各种生境中残留时间的科学评估数据。

生物信息学方法将是风险评估的最基本的方法。因此，需要解决环境监测中sRNA的提取和鉴定方法；建立标准化的生态毒理学模式生物的基因文库和DNA芯片(Robbensetal.,2007)；研究RNAi作物身份验证、监测等的定量和定性检测方法。从科学的角度来看，具有作物保护性状的RNAi作物的环境风险是可预测的，且在科学管理条件下是可避免或可控的。

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