RNA干扰在植物功能基因组学和遗传改良中的应用研究进展

doi:10.13560/j.cnki.biotech.bull.1985.2025-0645

摘要/Abstract

摘要：

RNA干扰（RNA interference, RNAi）是一种由双链RNA（double-stranded RNA, dsRNA）介导，具有序列特异性的基因沉默机制，广泛存在于真核生物中。其分子机制主要包括dsRNA的加工、小干扰RNA（small interfering RNA, siRNA）或微RNA（microRNA, miRNA）的生成，以及RNA诱导沉默复合体介导的靶mRNA的特异性降解或翻译抑制。基于该机制的RNAi技术已成为植物功能基因组学研究的强大工具，并在作物抗病虫遗传改良与绿色防控领域展现出巨大潜力。本文系统综述了RNAi的发现历程、分子机制及其应用，重点探讨其三大核心应用：病毒诱导基因沉默（virus-induced gene silencing, VIGS）利用工程化病毒载体实现寄主基因的瞬时沉默，为植物基因功能研究提供了高效手段；寄主诱导基因沉默（host-induced gene silencing, HIGS）通过植物体内表达的RNAi分子靶向沉默病原体或者害虫的关键基因，旨在培育具有对真菌、病毒及害虫持久抗性的转基因作物；喷雾诱导基因沉默（spray-induced gene silencing, SIGS）作为一种非转基因策略，通过喷施设计的靶向dsRNA，经靶标病原体或害虫摄取后抑制其关键基因表达，从而干扰其侵染/为害过程，为病虫害防控提供了一种环境友好的新型策略。未来，RNAi技术的进一步发展有望得益于合成生物学、人工智能与纳米递送系统的深度融合，以优化靶标设计、提升沉默效率并降低脱靶风险。随着关键技术的突破与应用瓶颈的克服，RNAi在推动植物科学研究和实现可持续农业发展方面具有广阔的应用前景。

关键词: RNA干扰, 病毒诱导基因沉默, 寄主诱导基因沉默, 喷雾诱导基因沉默, 跨界RNAi, 基因沉默, 双链RNA, 病虫害防治

Abstract:

RNA interference (RNAi) is a sequence-specific gene silencing mechanism mediated by double-stranded RNA (dsRNA) and conserved in eukaryotes. Its molecular mechanism primarily involves the processing of dsRNA, the generation of small interfering RNA (siRNA) or microRNA (miRNA), and the sequence-specific degradation or translational inhibition of target mRNA mediated by the RNA-induced silencing complex (RISC). Based on this mechanism, RNAi technology has become a powerful tool for plant functional genomics research and demonstrates immense potential in the genetic improvement of crops for resistance to diseases and pests, as well as in green and sustainable pest control. This review systematically summarizes the discovery history and molecular mechanism of RNAi and focuses on its three core applications: Virus-induced gene silencing (VIGS) utilizes engineered viral vectors to achieve transient silencing of host genes, providing an efficient approach for plant gene functional studies. Host-induced gene silencing (HIGS) aims to breed transgenic crops with durable resistance to fungi, viruses, and pests by expressing RNAi molecules within the plant to target and silence key genes in pathogens or pests. Spray-induced gene silencing (SIGS), as a non-transgenic strategy, involves spraying designed target-specific dsRNA. Upon uptake by the target pathogen or pest, this dsRNA inhibits the expressions of their key genes, thereby interfering with their infection/infestation process, offering a novel, environmentally friendly strategy for disease and pest control. In the future, further advancements in RNAi technology are expected to benefit from the deep integration of synthetic biology, artificial intelligence, and nano-delivery systems to optimize target design, enhance silencing efficiency, and reduce off-target risks. With breakthroughs in key technologies and the overcoming of application bottlenecks, RNAi holds exceptionally broad application prospects for advancing plant science research and achieving sustainable agriculture.

Key words: RNA interference, virus-induced gene silencing, host-induced gene silencing, spray-induced gene silencing, cross-kingdom RNAi, gene silencing, double-stranded RNA, disease and pest control

黄雯婧, 任思潮, 林俐, 王幼平, 吴健. RNA干扰在植物功能基因组学和遗传改良中的应用研究进展[J]. 生物技术通报, 2025, 41(9): 1-21.

HUANG Wen-jing, REN Si-chao, LIN Li, WANG You-ping, WU Jian. Advances in RNA Interference Technology for Plant Functional Genomics and Crop Improvement[J]. Biotechnology Bulletin, 2025, 41(9): 1-21.

图/表 5

图1 RNA 干扰发现历程呈现1989‒2006年RNA干扰技术发现和理论基础形成的关键节点：从核酸序列同源性调控基因表达的发现，到双链RNA介导基因沉默机制的阐明，最终以诺贝尔奖确认其重要性。本文图片均使用Biorender.com绘制，下同

Fig. 1 Discovery history of RNA interferenceFrom left to right, Figure 1 presents key milestones in the discovery of RNA interference technology and the formation of its theoretical basis from 1989 to 2006: ranging from the discovery that nucleic acid sequence homology regulates gene expression, to clarification of dsRNA-mediated gene silencing, and finally to the recognition of its significance by the Nobel Prize. All figures were created in https://Biorender.com. The same below

图2 RNA干扰机制A：外源性小干扰RNA（small interfering RNA, siRNA）介导的RNA干扰（RNA interference, RNAi）机制；Dicer-like（DCL）酶切割外源dsRNA为siRNA，组装入含Argonaute（AGO）蛋白的RNA诱导沉默复合物（RNA-induced silencing complex, RISC）；胞质中RISC通过siRNA互补结合并切割靶mRNA（转录后沉默）；核内siRNA-AGO结合RNA聚合酶Ⅴ转录的lncRNA，介导RNA指导的DNA或组蛋白甲基化（转录水平沉默）；B：内源性微RNA（microRNA, miRNA）介导的RNAi机制；核内DCL1复合体将pri-miRNA加工为pre-miRNA及miRNA双链，经HASTY转运至胞质；成熟miRNA装载RISC，AGO介导靶mRNA切割（植物中以切割为主）

Fig. 2 Mechanism of RNA interferenceA: Mechanism of RNA interference (RNAi) mediated by exogenous small interfering RNA (siRNA). Dicer-like (DCL) cleaves exogenous dsRNA into siRNAs, which are then assembled into Argonaute (AGO)-containing RNA-induced silencing complex (RISC). In the cytoplasm, RISC binds to target mRNA through siRNA complementarity and cleaves it (post-transcriptional silencing). In the nucleus, siRNA-AGO complexes bind to lncRNAs transcribed by RNA polymerase V, mediating RNA-directed DNA or histone methylation (transcriptional silencing). B: Mechanism of RNAi mediated by endogenous microRNA (miRNA) in the nucleus, the DCL1 complex processes pri-miRNA into pre-miRNA and miRNA duplexes, which are then transported to the cytoplasm via HASTY. The mature miRNA is loaded into RISC, and AGO mediates the cleavage of target mRNA (with cleavage being the primary mechanism in plants)

图3 不同病毒介导的病毒诱导基因沉默（VIGS）机制左图展示了由单链DNA病毒介导的病毒诱导基因沉默（virus-induced gene silencing， VIGS）机制，病毒经受体介导进入细胞、脱壳后，单链DNA进入细胞核，借寄主酶滚环复制成环状双链DNA，随后被DCL3加工为24 nt siRNA，引导RISC-AGO4介导DNA甲基化抑制转录。右图展示了由正义单链RNA病毒介导的VIGS机制，病毒进入细胞后，正义单链RNA作mRNA翻译复制酶，复制中形成的dsRNA及局部双链，被DCL4/DCL2加工为21/22 nt siRNA，经RISC介导mRNA降解、抑制翻译

Fig. 3 Mechanisms of virus-induced gene silencing (VIGS) mediated by different virusesThe left figure illustrates the mechanism of virus-induced gene silencing (VIGS) mediated by single-stranded DNA (ssDNA) viruses: Following receptor-mediated entry into the cell and uncoating, the viral ssDNA enters the nucleus, where it is converted into circular double-stranded DNA (dsDNA) through rolling-circle replication using host enzymes. This dsDNA is subsequently processed by DCL3 into 24-nucleotide (nt) siRNAs, which guide the RISC-AGO4 complex to mediate DNA methylation and thereby suppress transcription. The right figure depicts the VIGS mechanism mediated by positive-sense single-stranded RNA (+ssRNA) viruses: After entering the cell, the viral +ssRNA functions as mRNA to translate replicases. During replication, the formed dsRNA and local double-stranded regions are processed by DCL4/DCL2 into 21/22-nt siRNAs, which then mediate mRNA degradation and translation inhibition via RISC

图4 寄主诱导基因沉默（HIGS）和喷雾诱导基因沉默（SIGS）的RNAi机制A：寄主诱导基因沉默（host-induced gene silencing, HIGS）机制，从上到下依次是：咀嚼式昆虫取食植物时，摄入叶绿体中未被DCL切割的长链dsRNA，经内吞、SID蛋白等途径入细胞，触发RNAi沉默靶基因；刺吸式昆虫取食韧皮部（含siRNA、dsRNA），触发RNAi沉默靶基因；真菌/病毒侵染时，植物通过囊泡传递sRNA，或自身加工hpRNA为dsRNA/siRNA，跨界沉默病原基因（传递细节待明确）；B：喷雾诱导基因沉默（spray-induced gene silencing, SIGS）技术防治病害的2种途径：第1种是病原细胞直接吸收环境中裸露dsRNA；第2种是植物介导转运，dsRNA被植物吸收后，经维管系统长距离运输，经DCL加工成siRNA或dsRNA，通过细胞外囊泡（extracellular vesicles， EVs）传递给病原体，触发基因沉默。问号表示尚未阐明的小RNA摄取机制

Fig. 4 Mechanisms of RNAi in host-induced gene silencing (HIGS) and spray-induced gene silencing (SIGS)A: The mechanism of host-induced gene silencing (HIGS) is presented from top to bottom as follows: When chewing insects feed on plants, they ingest long dsRNAs in chloroplasts that have not been cleaved by DCL. These dsRNAs enter cells through endocytosis, SID protein-mediated pathways, etc., triggering RNAi to silence target genes. When piercing-sucking insects feed on phloem (containing siRNAs and dsRNAs), RNAi is triggered to silence target genes. During fungal/viral infection, plants deliver sRNAs through vesicles, or process hpRNAs into dsRNAs/siRNAs themselves, thereby trans-kingdom silencing pathogenic genes (the details of delivery remain to be clarified). B: Two pathways of spray-induced gene silencing (SIGS) technology for disease control: Firstly, pathogenic cells directly absorb naked dsRNAs in the environment; secondly, plant-mediated transport: after being absorbed by plants, dsRNAs are transported over long distances through the vascular system, processed into siRNAs or retained as dsRNAs by DCL, and then delivered to pathogens via extracellular vesicles (EVs) to trigger gene silencing. Question marks indicate unelucidated small RNA uptake mechanisms

表1 HIGS技术在病害防控领域的研究进展

Table 1 Advances in research on HIGS technology in disease control and prevention

病原体 Pathogen	寄主植物 Host plant	靶标基因 Target gene	结果 Results	参考文献 Reference
大麦白粉病菌 Puccinia spp.	大麦	1，3-β-葡聚糖转移酶（GTF1/2）效应子Avral0	白粉病菌吸器减少并抑制菌丝生长，大麦抗性提高	［81］
大麦白粉病菌 Puccinia spp.	大麦	效应子（BEC101、C1054等8个基因）	干扰真菌分泌蛋白，阻断致病信号传导，抑制孢子萌发和侵染	［96］
小麦叶锈病菌 P. triticina	小麦	促分裂素原活化蛋白激酶（PtMAPK1）、亲环蛋白（PtCYC1）、钙依赖磷酸酶B基因（PtCNB）	病原菌中靶基因转录水平降低59%-70%，菌丝生长受限、吸器发育异常，夏孢子堆密度减少51%-68%	［97］
小麦叶锈病菌 P. triticina	小麦	丝裂原活化蛋白激酶1基因（PtMAPK1）、亲环蛋白基因（PtCYC1）	干扰真菌细胞周期和应激反应，限制菌丝扩展，真菌生物量减少超50%	［98］
小麦条锈病菌 P. striiformis	小麦	蛋白激酶A催化亚基基因（PsCPK1）	阻断cAMP信号通路，抑制菌丝极性生长和吸器形成，稳定抗病性持续4代	［99］
小麦条锈病菌 P. striiformis	小麦	丝裂原活化蛋白激酶激酶基因（PsFuz7）	抑制MAPK信号传导，显著限制病原菌菌丝发育	［100］
禾谷镰刀菌 Fusarium graminearum	大麦	麦角固醇合成相关的甾醇-14a-去甲基化酶基因（CYP51-A、CYP51-B和CYP51-C）	靶基因表达抑制率77%-92%，真菌菌丝生长被限制在接种部位	［101］
禾谷镰刀菌 Fusarium graminearum	小麦	丁质合成酶3b基因（Chs3b）	阻断细胞壁几丁质合成，抑制分生孢子萌发和侵染，降低DON毒素产生	［102］
核盘菌 Sclerotinia sclerotiorum	油菜	内切多聚半乳糖醛酸酶基因（SsPG1）、纤维二糖水解酶基因（SsCBH）和草酰乙酸乙酰水解酶基因（SsOAH1）	单靶标HIGS：病斑面积减少20.8%-38.7% 三靶标共沉默HIGS：病斑面积减少36.8%-43.7%，抗性显著增强	［104］
核盘菌 Sclerotinia sclerotiorum	油菜	内切多聚半乳糖醛酸酶基因（SsPG1）	子叶病斑面积减少51.8%-58.2%，叶片病斑面积减少20.1%-26.2%	［105］
灰霉菌 Botrytis cinerea	番茄	灰霉病菌致病基因Bc-DCL1和Bc-DCL2	靶基因表达量下降70%-90%，病斑面积减少50%-80%，孢子萌发率下降30%-60%	［92］
灰霉菌 Botrytis cinerea	马铃薯番茄	雷帕霉素靶蛋白基因（BcTOR）	靶基因表达下调60%-90%，病斑面积减少50%- 90%，真菌生物量降低60%-85%，抑制灰霉病发展	［103］
辣椒疫霉 Phytophthora capsici	烟草	纤维素合成酶基因（PcCesA3）、固醇结合蛋白基因（PcOSBP1）	病斑面积减少17.19%-33.34%，病原菌生物量降低59.64%-77.65%	［106］
立枯丝核菌 Rhizoctonia solani	水稻	效应子基因AGLIP1	抑制菌丝生长和致病力，使接种后病斑面积减少50%	［107］
葡萄座腔菌 Botryosphaeria dothidea	湖北海棠	真菌糖转运蛋白基因（BdSTP）、乙酰乳酸合成酶基因（BdALS）	病斑面积减少超50%，真菌生物量降低约60%-70%	［108］
棉铃虫 Helicoverpa armigera	烟草	棉蜕皮激素受体基因（EcR）	EcR基因表达显著下调，出现蜕皮缺陷和致死表型；对甜菜夜蛾也有抗性	［109］
二斑叶螨Tetranychus urticae	烟草	保幼激素受体基因Met	二斑叶螨死亡率达48%	［110］
桃蚜Myzus persicae	烟草	V-ATPase亚基E、微管蛋白折叠辅助因子D（TBCD）或乙酰胆碱酯酶2（MpAchE2）	单只成虫产生的若虫数量减少约30%	［112］
短体线虫Pratylenchus	小麦	肌钙蛋白C（pat-10）、钙调蛋白（unc-87）	线虫瘫痪和不协调运动，减少繁殖	［113］
南方根结线虫Meloidogyne incognita	番茄	组织蛋白酶L半胱氨酸蛋白酶（Mi-cpl-1）	线虫感染和增殖减少60%-80%	［114］
西方玉米根虫Diabrotica virgifera virgifera	玉米	V-ATPase亚基A或C	减少对根系的损害	［115］
粉虱Aleyrodidae	烟草	粉虱V-ATPaseA基因	植物定殖减少，粉虱死亡率增加	［116］
麦长管蚜Sitobion avenae	小麦	羧酸酯酶基因（CbEE4）	基因表达量降低30%-60%，羧酸酯酶活性下降，对辛硫磷农药水解能力降至20%-30%，蚜虫繁殖减少	［111］
香蕉束顶病毒Banana bunchy top virus	香蕉	复制酶基因（rep）	转基因植株对BBTV完全免疫、病毒复制几乎完全被抑制	［117］
玉米矮花叶病毒Maize dwarf mosaic virus	玉米	蛋白酶基因（P1）	15个T₂代株系均增强抗病性，其中6个株系病斑指数低于25%，病毒P1基因相对复制水平显著降低	［118］
水稻黑条矮缩病毒Rice black-streaked dwarf virus	水稻	水稻黑条矮缩病毒的P7-2和P8基因	转基因后代表现出较强抗病毒性	［119］
小麦条纹花叶病毒Wheat streak mosaic virus	小麦	小麦条纹花叶病毒外壳蛋白基因	通过RNAi抑制病毒衣壳组装、阻止病毒颗粒形成，持续抗性至T₅代	［120］
烟草条纹病毒Tobacco streak virus	烟草	烟草条纹病毒复制酶部分序列	诱导病毒基因沉默，使其获得抗TSV特性	［121］
柑橘衰退病毒Citrus tristeza virus	柑橘	沉默抑制蛋白基因p25、p20和p23	3个转基因株系对CTV-T36完全抗病，嫁接接种后无症状且无病毒	［122］
李痘病毒Plum pox virus	欧洲李、烟草	外壳蛋白基因（CP）	对PPV主要株系（D、M、Rec、EA）表现出完全抗性，病毒未发生系统性扩散	［123］

表1 HIGS技术在病害防控领域的研究进展

Table 1 Advances in research on HIGS technology in disease control and prevention

病原体 Pathogen	寄主植物 Host plant	靶标基因 Target gene	结果 Results	参考文献 Reference
大麦白粉病菌 Puccinia spp.	大麦	1，3-β-葡聚糖转移酶（GTF1/2）效应子Avral0	白粉病菌吸器减少并抑制菌丝生长，大麦抗性提高	［81］
大麦白粉病菌 Puccinia spp.	大麦	效应子（BEC101、C1054等8个基因）	干扰真菌分泌蛋白，阻断致病信号传导，抑制孢子萌发和侵染	［96］
小麦叶锈病菌 P. triticina	小麦	促分裂素原活化蛋白激酶（PtMAPK1）、亲环蛋白（PtCYC1）、钙依赖磷酸酶B基因（PtCNB）	病原菌中靶基因转录水平降低59%-70%，菌丝生长受限、吸器发育异常，夏孢子堆密度减少51%-68%	［97］
小麦叶锈病菌 P. triticina	小麦	丝裂原活化蛋白激酶1基因（PtMAPK1）、亲环蛋白基因（PtCYC1）	干扰真菌细胞周期和应激反应，限制菌丝扩展，真菌生物量减少超50%	［98］
小麦条锈病菌 P. striiformis	小麦	蛋白激酶A催化亚基基因（PsCPK1）	阻断cAMP信号通路，抑制菌丝极性生长和吸器形成，稳定抗病性持续4代	［99］
小麦条锈病菌 P. striiformis	小麦	丝裂原活化蛋白激酶激酶基因（PsFuz7）	抑制MAPK信号传导，显著限制病原菌菌丝发育	［100］
禾谷镰刀菌 Fusarium graminearum	大麦	麦角固醇合成相关的甾醇-14a-去甲基化酶基因（CYP51-A、CYP51-B和CYP51-C）	靶基因表达抑制率77%-92%，真菌菌丝生长被限制在接种部位	［101］
禾谷镰刀菌 Fusarium graminearum	小麦	丁质合成酶3b基因（Chs3b）	阻断细胞壁几丁质合成，抑制分生孢子萌发和侵染，降低DON毒素产生	［102］
核盘菌 Sclerotinia sclerotiorum	油菜	内切多聚半乳糖醛酸酶基因（SsPG1）、纤维二糖水解酶基因（SsCBH）和草酰乙酸乙酰水解酶基因（SsOAH1）	单靶标HIGS：病斑面积减少20.8%-38.7% 三靶标共沉默HIGS：病斑面积减少36.8%-43.7%，抗性显著增强	［104］
核盘菌 Sclerotinia sclerotiorum	油菜	内切多聚半乳糖醛酸酶基因（SsPG1）	子叶病斑面积减少51.8%-58.2%，叶片病斑面积减少20.1%-26.2%	［105］
灰霉菌 Botrytis cinerea	番茄	灰霉病菌致病基因Bc-DCL1和Bc-DCL2	靶基因表达量下降70%-90%，病斑面积减少50%-80%，孢子萌发率下降30%-60%	［92］
灰霉菌 Botrytis cinerea	马铃薯番茄	雷帕霉素靶蛋白基因（BcTOR）	靶基因表达下调60%-90%，病斑面积减少50%- 90%，真菌生物量降低60%-85%，抑制灰霉病发展	［103］
辣椒疫霉 Phytophthora capsici	烟草	纤维素合成酶基因（PcCesA3）、固醇结合蛋白基因（PcOSBP1）	病斑面积减少17.19%-33.34%，病原菌生物量降低59.64%-77.65%	［106］
立枯丝核菌 Rhizoctonia solani	水稻	效应子基因AGLIP1	抑制菌丝生长和致病力，使接种后病斑面积减少50%	［107］
葡萄座腔菌 Botryosphaeria dothidea	湖北海棠	真菌糖转运蛋白基因（BdSTP）、乙酰乳酸合成酶基因（BdALS）	病斑面积减少超50%，真菌生物量降低约60%-70%	［108］
棉铃虫 Helicoverpa armigera	烟草	棉蜕皮激素受体基因（EcR）	EcR基因表达显著下调，出现蜕皮缺陷和致死表型；对甜菜夜蛾也有抗性	［109］
二斑叶螨Tetranychus urticae	烟草	保幼激素受体基因Met	二斑叶螨死亡率达48%	［110］
桃蚜Myzus persicae	烟草	V-ATPase亚基E、微管蛋白折叠辅助因子D（TBCD）或乙酰胆碱酯酶2（MpAchE2）	单只成虫产生的若虫数量减少约30%	［112］
短体线虫Pratylenchus	小麦	肌钙蛋白C（pat-10）、钙调蛋白（unc-87）	线虫瘫痪和不协调运动，减少繁殖	［113］
南方根结线虫Meloidogyne incognita	番茄	组织蛋白酶L半胱氨酸蛋白酶（Mi-cpl-1）	线虫感染和增殖减少60%-80%	［114］
西方玉米根虫Diabrotica virgifera virgifera	玉米	V-ATPase亚基A或C	减少对根系的损害	［115］
粉虱Aleyrodidae	烟草	粉虱V-ATPaseA基因	植物定殖减少，粉虱死亡率增加	［116］
麦长管蚜Sitobion avenae	小麦	羧酸酯酶基因（CbEE4）	基因表达量降低30%-60%，羧酸酯酶活性下降，对辛硫磷农药水解能力降至20%-30%，蚜虫繁殖减少	［111］
香蕉束顶病毒Banana bunchy top virus	香蕉	复制酶基因（rep）	转基因植株对BBTV完全免疫、病毒复制几乎完全被抑制	［117］
玉米矮花叶病毒Maize dwarf mosaic virus	玉米	蛋白酶基因（P1）	15个T₂代株系均增强抗病性，其中6个株系病斑指数低于25%，病毒P1基因相对复制水平显著降低	［118］
水稻黑条矮缩病毒Rice black-streaked dwarf virus	水稻	水稻黑条矮缩病毒的P7-2和P8基因	转基因后代表现出较强抗病毒性	［119］
小麦条纹花叶病毒Wheat streak mosaic virus	小麦	小麦条纹花叶病毒外壳蛋白基因	通过RNAi抑制病毒衣壳组装、阻止病毒颗粒形成，持续抗性至T₅代	［120］
烟草条纹病毒Tobacco streak virus	烟草	烟草条纹病毒复制酶部分序列	诱导病毒基因沉默，使其获得抗TSV特性	［121］
柑橘衰退病毒Citrus tristeza virus	柑橘	沉默抑制蛋白基因p25、p20和p23	3个转基因株系对CTV-T36完全抗病，嫁接接种后无症状且无病毒	［122］
李痘病毒Plum pox virus	欧洲李、烟草	外壳蛋白基因（CP）	对PPV主要株系（D、M、Rec、EA）表现出完全抗性，病毒未发生系统性扩散	［123］

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