Strategies for exogenous RNA delivery in RNAi
龚流娥, 应淑敏, 张雅芬, 王教瑜, 孙国昌. 病虫害RNAi技术中的外源RNA递送策略[J]. 生物工程学报, 2023, 39(2): 459-471.
GONG Liu'e, YING Shumin, ZHANG Yafen, WANG Jiaoyu, SUN Guochang. Strategies for exogenous RNA delivery in RNAi-mediated pest management[J]. Chinese Journal of Biotechnology, 2023, 39(2): 459-471.
病虫害RNAi技术中的外源RNA递送策略
, 应淑敏2,3 , 张雅芬1 , 王教瑜2 , 孙国昌2
1. 中国计量大学生命科学学院, 浙江 杭州 3100182;
2. 浙江省农业科学院植物保护与微生物研究所 农产品质量安全危害因子与风险防控国家重点实验室, 浙江 杭州 3100213;
3. 浙江师范大学化学与生命科学学院, 浙江 金华 321004
收稿日期：2022-08-04；接收日期：2022-10-05；网络出版时间：2022-10-11
基金项目：浙江省自然科学基金(LZ20C140001)；浙江省重点研发计划(2021C02010)；浙江省农业科学院“生物健康”融通计划
摘要：病虫害严重威胁着作物安全生产。近年来，在RNA干扰(RNA interference, RNAi)基础上开发病虫害防控策略的研究得到越来越多的关注。RNAi是真核生物体内的一种基因调控过程，如何将外源RNA有效地递送到靶标生物体内，是病虫害RNAi技术能否成功的关键之一。国内外学者进行了大量研究和实践，探究影响病虫害吸收和传递外源双链RNA (double-stranded RNA, dsRNA)的因素，探索提高dsRNA递送效率的方法，取得了重要的进展。本文对相关研究进行了梳理，简述了影响病虫害对dsRNA吸收和递送的因素，对外源RNA的递送策略进行了综述，讨论了纳米颗粒复合物在dsRNA递送中的应用前景，以期为相关研究提供参考。
关键词：RNA干扰 病虫害防控 dsRNA递送 纳米复合物
Strategies for exogenous RNA delivery in RNAi-mediated pest management
GONG Liu'e1,2 , YING Shumin2,3 , ZHANG Yafen1 , WANG Jiaoyu2 , SUN Guochang2
1. College of Life Science, China Jiliang University, Hangzhou 310018, Zhejiang, China;
2. State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products, Institute of Plant Protection and Microbiology, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, Zhejiang, China;
3. College of Chemistry and Life Science, Zhejiang Normal University, Jinhua 321004, Zhejiang, China
Received: August 4, 2022; Accepted: October 5, 2022; Published: October 11, 2022
Supported by: This work was supported by the Zhejiang Provincial Natural Science Foundation (LZ20C140001), the Zhejiang Provincial Key Research and Development Program (2021C02010), and the Bio-Health Inner-Cooperation Plan in Zhejiang Academy of Agricultural Sciences
Abstract: Plant diseases and insect pests threaten the safety of crop production greatly. Traditional methods for pest management are challenged by the problems such as environmental pollution, off-target effects, and resistance of pathogens and insects. New biotechnology-based strategies for pest control are expected to be developed. RNA interference (RNAi) is an endogenous process of gene regulation, which has been widely used to study the gene functions in various organisms. In recent years, RNAi-based pest management has received increasing attention. The effective delivery of the exogenous interference RNA into the targets is a key step in RNAi-mediated plant diseases and pest control. Considerable advances were made on the mechanism of RNAi, and various RNA delivery systems were developed for efficient pest control. Here we review the latest advances on mechanisms and influencing factors of RNA delivery, summarize the strategies of exogenous RNA delivery in RNAi-mediated pest control, and highlight the advantages of nanoparticle complexes in dsRNA delivery.
Keywords: RNA interference pest management dsRNA delivery nanoparticle complex
病虫害严重威胁着作物安全生产。气候变化和农业环境变化又不断改变病虫害的发生规律，加剧病虫害的发生。传统的病虫害防控主要依赖化学农药，但化学农药的长期大量使用，导致了病虫害抗药性、环境污染和非靶标病虫害的暴发等一系列问题。利用现代生物技术寻求绿色、安全、高效的病害虫防控策略成为农业可持续发展的趋势。
RNA干扰(RNA interference, RNAi)是指在真核生物中由双链RNA诱发的同源信使RNA (messenger RNA, mRNA)降解，从而抑制靶标基因表达的现象[1]。近年来，在RNAi基础建立新型病虫害防控策略的研究，得到越来越多的关注。由于RNAi依赖于靶标生物的特异基因序列发挥作用，因此病虫害RNAi技术具有高度的靶标特异性，能有效减小对非目标生物的不良影响[2]，具有可持续和生态友好的特征。近十年来，RNAi技术对靶标害虫和病原菌的防控潜力在研究和实践中不断得到证实。
然而在农业生产中，病虫害种类多样，形态、结构各不相同，如何有效地将外源dsRNA递送到靶标致病菌或害虫体内是RNAi技术防治病虫害面临的重要挑战。世界各国的科学家们通过不断尝试和探索，不断提高外源双链RNA (double-stranded RNA, dsRNA)递送的效率，取得了一系列重要的进展。本文对相关研究进行了梳理，简述了影响病虫害dsRNA吸收和RNAi信号传递的因素，对常用的外源RNA递送技术进行了综述，并对RNAi技术应用前景和趋势进行了探论，以期为病虫害RNAi技术的研究提供参考。
1 dsRNA的摄取与体内转运机制
细胞可以从环境中吸收dsRNA，同时dsRNA或小干扰RNA (small interfering RNA, siRNA)触发的RNAi信号还可在细胞间传递。根据dsRNA是否在细胞间的传递，真核细胞中RNAi反应分为细胞自主性RNAi和非细胞自主性RNAi两大类[3]。在前者中，沉默效应仅限于吸收dsRNA的细胞；而在后者中，沉默信号能传递到其他细胞或组织，在所有能够结合dsRNA的细胞中产生沉默效应[4]。
1.1 SID蛋白的作用
dsRNA摄入和细胞间传递机制的研究是优化基于RNAi病虫害防控策略的基础[5]。目前，系统RNAi缺陷蛋白(systemic RNA interference deficiency, SID)和内吞作用在dsRNA摄入和传递中的作用研究较为深入[6]。秀丽隐杆线虫的SID蛋白一共有5个，分别是SID-1至SID-5。研究表明，SID对外源dsRNA的摄入和RNAi信号在组织间的传递起着重要作用[7]。SID-1作为dsRNA进入细胞的通道，使dsRNA能够穿过细胞膜进入细胞质；同时参与RNAi信号的胞间传递，在系统RNAi中起作用[8-9]。SID-2位于肠腔膜上，是外源dsRNA摄取所必需的，但不影响RNAi信号的细胞间传递[10]。SID-3是一个保守的酪氨酸激酶，也为dsRNA摄取所必需[11]。SID-5是一种内体相关蛋白，可与分泌蛋白Sec-22相互作用，该蛋白属于SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)类膜泡运输蛋白，负责分泌蛋白从内质网到高尔基体的膜泡转运，促进细胞间RNAi信号的传递[12]。研究表明，Sec-22参与晚期内体/多泡体和溶酶体的融合[13-14]。但截至目前，晚期内体和外泌体如何调控系统RNAi沉默信号的传递，尚不够明确。马铃薯甲虫(Leptinotarsa decemlineata) SID-1的同源蛋白silA和silC在dsRNA摄取和RNAi过程中都有重要作用[15]，但在玉米根萤叶甲(Diabrotica virgifera)中，SID类似蛋白并不影响系统RNAi[16]。
1.2 内吞作用
受体介导的内吞作用是生物体摄取生物大分子的重要途径，其中网格蛋白介导的内吞是最主要的受体介导方式。在赤拟谷盗(Tribolium castaneum)中，沉默与网格蛋白介导的内吞途径相关的基因Arf72A、Rab7、AP50和VhaSFD可以阻断RNAi反应[17]。沙漠蝗(Schistocerca gregaria)、桔小实蝇(Bactrocera dorsalis)、十字甲虫(Leptinotarsa decemlineata)、飞蝗(Diabrotica virgifera)和东亚飞蝗(Locusta migratoria)等昆虫中也有类似现象，表明内吞途径与dsRNA摄取紧密相关[18-19]。dsRNA的摄取及细胞内转运是影响RNAi效率的重要因素[20]，进一步研究内吞途径和dsRNA与靶基因之间的关系，对于提高对dsRNA摄取机制的认识和建立高效的RNAi体系具有重要意义。
2 影响dsRNA递送的因素2.1 靶基因与dsRNA长度
靶基因的选择是RNAi效率的关键因素。选择靶基因可以通过筛选已知功能的病原菌和害虫生存的关键基因，或者通过分析不同发育阶段的基因转录水平筛选特异表达的基因[21-22]。多数病原菌和害虫并非模式生物，基因组信息尚不完备，因此利用转录分析筛选靶基因更为适用[23-24]。dsRNA长度也是dsRNA摄入和RNAi效率的决定因素。在对赤拟谷盗的研究中，发现dsRNA长度对细胞摄入影响较大，dsRNA越长效果越好[25]。在马铃薯甲虫中，200 bp以上的dsRNA比60 bp的dsRNA能更有效地抑制其生长和导致死亡[26]。对西部玉米根萤叶甲的研究认为，60 bp以上的dsRNA能够有效发挥RNAi效果；此外，含有21 bp的目标匹配序列但总长为240 bp的dsRNA相较于单独的21 bp siRNA更易被西部玉米根萤叶甲中肠细胞摄入，RNAi效果更显著[27]。
病原真菌RNAi对dsRNA长度的要求相对宽泛，在已有报道中，dsRNA长度范围在20–1 000 bp左右。通过荧光素标记，研究人员发现21 bp的siRNA和800 bp以上的dsRNA分子都能被灰葡萄孢菌(Botrytis cinerea)和禾谷镰刀菌快速吸收[3, 28]。因此，关于dsRNA的有效长度尚无定论，要根据具体情况选择最适长度。
此外，对白蜡窄吉丁(Agrilus planipennis)的研究发现，多个dsRNA组合对RNAi具有增效作用[25]。但也有相反的例子，赤拟谷盗中多个dsRNA靶标导致dsRNA摄入的竞争，反而降低了RNAi的效果[29]。
2.2 核酸酶
昆虫的唾液、肠道和血淋巴中含有多种核酸酶，可降解dsRNA，影响RNAi效率。研究表明，对dsRNA降解能力的不同是昆虫间RNAi效率差异的重要因素之一[30]。例如，通过口服和注射的方式将不同的dsRNA送入豌豆蚜(Acyrthosiphon pisum)体内，发现其唾液分泌物和血淋巴都能降解dsRNA，导致不能产生有效的RNAi反应[31]。Ghodke等[32]报道，桃蚜(Myzus persicae)的中肠分泌物含有胞外核酸酶，能快速降解dsRNA，因此外源dsRNA无法有效抑制靶基因表达。此外，苜蓿叶象甲(Anthonomus grandis)消化道液中含有3种不同的核酸酶，均能降解核酸，影响RNAi效率[33]；沙漠蝗肠道中4种Sg-dsRNA酶的表达水平与RNAi的不敏感性相关[34]。
昆虫消化系统酸碱度不同，鳞翅目昆虫较鞘翅目昆虫的消化道碱性高，碱性条件下dsRNA不稳定，鳞翅目昆虫对RNAi敏感性较低，而鞘翅目昆虫对RNAi敏感性较高[35]。进一步加强核酸酶的研究，有助于更好地揭示和解决靶标病虫害对RNAi敏感性不高的问题。
2.3 dsRNA结合蛋白
dsRNA结合蛋白含有dsRNA结合域，可识别RNA二级结构。研究表明，dsRNA结合蛋白是影响RNAi的重要因素之一。真核生物、细菌和病毒中均发现多种dsRNA结合蛋白，包括dsRNA依赖的蛋白激酶、RNA特异性腺苷脱氨酶、RNA解旋酶A、双链RNA结合蛋白Staufen抗体(double stranded RNA binding protein Staufen homolog, STAU)、TAR RNA结合蛋白(TAR RNA-binding protein, TRBP)、核糖核酸酶Ⅲ、PKR活化蛋白等[36]。果蝇STAU是最典型的dsRNA结合蛋白，在昆虫、秀丽隐杆线虫和人类细胞中都有同源物[37]。对灰飞虱(Laodelphax striatellus)的研究表明，STAU同源物和TRBP影响RNAi效率[38]。马铃薯甲虫STAU (STAU-C)也与RNAi效果相关[39]。鳞翅目昆虫中尚未发现STAU-C同源物。目前，有关dsRNA结合蛋白对病虫害RNAi影响的研究还不多，dsRNA结合蛋白和RNAi的关系有待于进一步研究。
3 dsRNA的递送策略
dsRNA的高效递送是RNAi防控病虫害技术的关键。目前，病虫害防控相关的dsRNA递送策略大体分两类：转化的递送策略和非转化的递送策略。转化递送包括病毒介导的瞬时转化、核基因转化和叶绿体转基因；非转化递送包括dsRNA局部施用、纳米颗粒辅助递送以及dsRNA/纳米颗粒复合物递送。
3.1 病毒介导的瞬时转化
利用病毒载体进行外源dsRNA的瞬时递送，需将靶基因的正义链、反义链或反向重复序列构建入病毒载体，通过转染在宿主作物中产生siRNA，同时病毒不断复制实现dsRNA快速积累。基于病毒载体的RNAi (virus induced gene silencing, VIGS)具有快速、高通量、无需体外转录等多种优点，在靶基因研究、筛选和鉴定中应用较多。研究表明，无论RNA病毒还是DNA病毒，VIGS系统都能有效抑制靶基因的转录。Zhang等[40]通过VIGS系统干扰大麦条锈菌(Puccinia glumarum)钙依赖磷酸酶亚基基因PsCNA1和PsCNB1的表达，有效抑制了病原菌的产孢和菌丝扩展。通过烟草脆裂病毒载体将烟草天蛾(Manduca sexta) CYP6B46基因dsRNA导入渐狭叶烟草(Nicotiana attenuata)，烟草天蛾取食后，其肠道中的CYP6B46转录本显著减少[41]。利用烟草脆裂病毒载体在烟草中瞬时表达与烟粉虱(Bemisia tabaci)乙酰胆碱酯酶和蜕皮素受体基因同源的dsRNA，烟粉虱取食后发生白化死亡[42]。利用烟草花叶病毒在烟草中表达柑橘粉蚧(Pseudococcus citri)几丁质合酶和V-ATP酶的dsRNA，造成靶标害虫繁殖力降低、死亡率增加[43]。同样地，用马铃薯X病毒在烟草中表达扶桑绵粉蚧(Phenacoccus solenopsis)甲壳质合酶的dsRNA，导致幼虫畸形和成虫数量减少[44]。
3.2 核基因转化
通过核基因转化的方式递送dsRNA，即利用靶向病原菌或害虫基因的RNAi载体进行植物转基因。害虫取食或病原菌侵染转基因植物后，生长发育受到影响。Tian等[45]通过转基因在棉花中表达棉铃虫HMGR基因的dsRNA，有效抑制了棉铃虫HMGR基因的转录和表达水平，抑制了害虫生长。褐飞虱(Nilaparvata lugens)取食表达其蜕皮激素受体dsRNA的转基因水稻后，后代数量明显减少[46]。表达棉铃虫HaAK基因的dsRNA的转基因拟南芥，能够有效抑制棉铃虫的生长并导致幼虫死亡[47]。Jahan等[48]针对致病疫霉(Phytophthora infestans)的4个致病基因进行马铃薯转基因，发现靶向PiGPB1的转基因植株抗病性明显增强，转基因叶片中可检测到24–25 nt的小RNA分子。
利用RNAi机制与其他抗虫转基因协同作用，可以有效提高虫害防控效果。孟山都公司和陶氏农业科学公司联合发明的SmartStax PRO系统，同时表达Bt蛋白Cry 3Bb1、Cry34Ab1/35Ab1和靶向玉米根萤叶甲Snf7基因的dsRNA，显著增强了转基因玉米对玉米根萤叶甲的抑制效果，增加了Bt蛋白持效性，降低了对玉米根部的危害[49]。
3.3 叶绿体转基因
叶绿体基因组可产生大量转录本，比核基因组能更稳定地表达外源基因。由于叶绿体内缺少RNAi机制，不能将外源dsRNA切割成siRNA，更有利于dsRNA的积累，因此通过叶绿体递送dsRNA，对于提高RNAi效率更具优势。Zhang等[50]将靶向马铃薯甲虫β-actin和SHR的dsRNA分别转化烟草叶绿体，结果显示，表达β-actin dsRNA的转基因烟草对马铃薯甲虫幼虫的致死率为100%，表达SHR dsRNA的植株对幼虫致死率为70%，证明了利用叶绿体表达dsRNA的可行性。通过在烟草叶绿体中表达靶向棉铃虫几丁质合酶、细胞色素p450单加氧酶和V-ATP酶的dsRNA，棉铃虫取食转基因烟草后，体内靶基因的转录水平降到极低，幼虫的体重、生长和化蛹率显著降低[51]。Bally等[52]将棉铃虫乙酰胆碱酯酶的发夹RNA (hairpin RNA, hpRNA)整合到烟草叶绿体基因组中，实现了对靶基因的有效沉默。上述研究表明了叶绿体转基因在表达RNAi防控病虫害方面的优势。此外，叶绿体母系遗传的特性更能防止外源基因通过花粉逃逸，降低了基因漂移的风险。
3.4 dsRNA的局部递送
植物上直接施用的dsRNA能被病菌或害虫摄入并导致相应基因的沉默，因此dsRNA的局部递送也可实现病虫害的防控[53]，施用方法包括叶面喷洒、根部浸泡、树干或藤蔓注射等[54]。Koch等[55]在大麦叶片上喷施可同时靶向禾谷镰刀菌(Fusarium graminearum)麦角甾醇合成3个必需基因(CYP51A、CYP51B及CYP51C)的dsRNA，显著提高了大麦叶片对禾谷镰刀菌的抗性。Gogoi等在番茄叶片上直接施用dsRNA防治蚜虫(Aphidoidea)、粉虱(Aleyrodidae)和螨等半翅目害虫[56-57]。田间条件下，在柑橘树和葡萄藤表面施用的dsRNA可以从根部进入整株植物，有效地沉默害虫的靶标基因[58]。
核苷酸修饰可以提高dsRNA对核酸酶的耐受性，提升RNAi效率，将dsRNA持效性延长到几周甚至几个月[59]。San Miguel等[60]在靶向马铃薯甲虫actin基因的dsRNA片段上增加非天然核苷酸(noncanonical nucleotide)，增加了dsRNA对核酸酶的抗性，喷施到马铃薯叶面上，在温室条件下，植株对马铃薯甲虫的抑制效果可持续约28 d。研究还发现，喷施在叶片上的dsRNA干燥后，不再会受水冲刷的影响，增加了RNAi稳定性和应用潜力。
体外化学合成的siRNA或dsRNA、细菌或酵母表达的dsRNA以及表达dsRNA的细菌或酵母菌株都可以用于喷洒[61-62]。因此，研发可喷施的核酸农药，进行病虫害防控，具有较好的应用前景[63]。
3.5 纳米颗粒辅助递送
近年来，纳米技术的研究促进了RNA递送策略的发展。与传统的dsRNA递送相比，借助纳米颗粒进行dsRNA递送，在提高RNAi效率方面具有优势[64]。纳米颗粒包括壳聚糖、脂质体配合物、共轭聚合物和阳离子聚合物等有机复合物；磁性纳米颗粒、量子点和金纳米颗粒等无机复合物[65]。壳聚糖或壳聚糖衍生物能有效地包裹核酸，通过离子相互作用形成低毒、可降解和生物相容的纳米聚合物，在体内和体外的害虫RNAi防治实验中都有效[66-67]。壳聚糖介导的dsRNA对冈比亚按蚊(Anopheles gambiae)具有良好的RNAi效果，2个靶标基因的干扰率分别是62.8%和33.8%，显著提高了对害虫的致死率[68]。支链双亲肽胶囊(branched amphiphilic peptide capsules, BAPC)进入细胞后，能在核周积累且持续存在而不被降解。作为纳米载体，BAPC已用于将CRISPR/Cas9、dsRNA和siRNA递送到特定的组织和器官[69]。据报道，用BAPC-dsRNA复合物喂食赤拟谷盗和豌豆蚜，可有效提高RNAi效率，增加害虫死亡率[70]。
研究表明，纳米颗粒递送dsRNA可以克服多种dsRNA递送的局限，促进dsRNA转运到靶细胞，防止dsRNA被核酸酶降解，提高dsRNA在肠道中的稳定性，增强肠道细胞对dsRNA的摄取等，极大地提高dsRNA被靶标生物吸收摄取的效率[71-73]。纳米颗粒辅助递送在控制病虫害方面已显示出较好的潜力，但依然存在多种限制因素，如生理pH条件下稳定性差、无法避免被内体吞噬，以及细胞特异性差、无法高效进入靶细胞等[74-75]。
3.6 dsRNA/纳米颗粒复合物的修饰
对dsRNA/纳米颗粒复合物进行修饰，可以有效提升dsRNA的递送效率[76]。三聚磷酸钠等交联剂能提高壳聚糖纳米粒子的稳定性，与单独的壳聚糖相比，壳聚糖与三聚磷酸钠交联产生的纳米聚电解质/dsRNA复合物在dsRNA递送中更加高效[77-79]。Parsons等[80]利用聚-[N-(3-胍基丙基)甲基丙烯酰胺]-dsRNA复合物在果蝇体内的有效吸附，显著提升了RNAi效率。Christiaens等[81]设计了一种含有胍基的纳米聚合物，可耐受分解，能在高pH环境中与dsRNA形成稳定的复合物，有效克服dsRNA的降解。此外，Zheng等[82]设计了一种能够穿透蚜虫体壁进入血腔并扩散到各组织中发挥RNAi作用的新型纳米载体/dsRNA复合制剂；Yan等[83]研发了dsRNA/纳米载体/去污剂，有效提高了对蚜虫体壁的穿透能力，使dsRNA在4 h内穿透蚜虫体壁，沉默靶基因，蚜虫死亡率高达81.67%。Ma等[84]合成了一种亲水性星形纳米聚合物(star polymer, SPc)，可以通过静电力、氢键和范德华力与dsRNA自发结合，并保护dsRNA免受降解，提高dsRNA的稳定性，提升RNAi效果。在此基础上Li等[85]研发了苦参碱/SPc/dsRNA纳米复合体，通过局部应用实现了对桃蚜的抑制，提高了桃蚜死亡率，并且在野外的起效时间和持效性均显著提高。Ma等[86]建立了大肠杆菌表达系统大规模生产hpRNA，将表达的hpRNA/SPc/去污剂混合制成的RNA制剂，对桃蚜进行喷雾，显示出一定的杀虫活性。Thairu等[87]研发了一种纳米乳剂，能穿透蚜虫胸部和腹部的气孔，通过气管进入呼吸系统进而发挥RNAi作用。
上述研究表明，利用纳米颗粒和修饰的纳米颗粒复合物递送dsRNA，能显著提升RNAi的效率和病虫害抑制效果[73]，尽管目前仍处于起步阶段，但该策略在病虫害控制中具有广阔的应用前景。
4 结论与展望
近年来，RNAi技术的发展极大推动了病虫害生物学和基因功能的研究，同时使利用RNAi技术沉默病原菌和害虫的基因从而防控病虫害成为可能。由于RNAi具有基因序列特异性，基于RNAi的防控策略，能有效减小对非靶标生物和环境的影响[2]，具有广阔的发展前景。本团队主要围绕稻瘟病菌、灰霉病菌和镰刀菌等重要的植物病原真菌开展致病机理研究，鉴定了大量参与病菌生长发育和致病过程的关键基因[88-90]。目前，针对上述基因，我们通过合成siRNA和dsRNA以及创制RNAi转基因水稻等方式，开展RNAi技术防控病害的研究，获得了多个具有开发前景的基因靶点、RNA干扰片段和抗性水稻材料[91-92]。
作为一种新兴技术，RNAi同样面临着许多挑战，包括dsRNA或RNAi的非靶效应、病虫害的抗RNAi机制、靶基因的有效性和dsRNA递送效率等，都影响着RNAi防控病虫害的研究和发展。由于dsRNA/siRNA不翻译蛋白，无论单独的还是吸附在纳米颗粒表面的dsRNA都会快速降解，不易在环境中长时间积累和持续发挥作用，提高dsRNA/siRNA的环境稳定性是目前急需解决的技术难点之一[87]。从目前研究来看，利用纳米粒子复合物递送dsRNA的策略具有出巨大的发展潜力，尤其是对RNAi敏感度低的病虫害，优势更加明显。通过对dsRNA和dsRNA/纳米颗粒复合物的修饰，可进一步提高RNAi效率，在未来RNAi的应用中需重点研究[93]。此外，当前对dsRNA产品的安全性仍采用对转基因生物的监管方式，亟待建立新的适用于RNAi递送产品和技术的风险评估体系[94]。
相对于作物虫害，目前针对病害的RNAi技术研究发展偏慢。我们研究发现，不同的RNA片段对同一靶基因的干扰效果存在差异，干扰不同的基因对同一病原菌的抑制效果不同，同源基因的RNAi对不同病原菌的抑制效果也各不相同[92]。同时，由于病原菌种类多样，致病机制复杂，病原菌对外源RNA摄取能力具有较大差异。因此，病害RNAi技术的研究任务也更加艰巨，需要进一步地探索和研究。
随着环境变化和农业生产模式的变革，农作物病虫害的发生频率逐渐增加，对我国的农业发展造成严重威胁，寻求绿色、安全、高效的病害虫防控技术成为农业发展的必然需求。我们相信，在研究者的不断努力下，RNAi技术将会蓬勃发展，基于RNAi的病虫害防控策略将会不断完善，在减少化学农药使用、降低环境污染和保护生物多样性等方面发挥重要作用。
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