RNAi技术在农业害虫防治中的应用研究进展

RNA干扰(RNA interference,RNAi)是指由内源或外源的双链RNA(double-stranded RNA,dsRNA)引发的mRNA降解,导致特异性阻碍靶标基因表达的现象,在昆虫学研究领域中得到广泛应用。如功能基因研究、基因表达调控及信号传导通路、益虫保护、新型农药的开发、害虫防治等。本文总结了RNAi技术应用于害虫防治上的原理,即以miRNA、siRNA及piRNA等小分子RNA介导的基因沉默过程,分析了影响RNAi技术防治效率的因素,如dsRNA导入方式、导入剂量和靶标基因的选择等,旨在进一步探讨RNAi应用于害虫防治的机理和存在问题,为运用RNAi技术防治害虫新思路奠定基础。     

草地贪夜蛾潜在RNAi靶标致死基因及纳米载体介导RNAi技术的应用和展望

草地贪夜蛾Spodoptera frugiperda是一种世界性重大农业害虫,在全球多个国家普遍发生,其幼虫可为害玉米、水稻等多种农作物。该虫于2019年初入侵我国,对我国农业生产构成了严重的威胁,防控形势严峻。为寻求一种草地贪夜蛾的绿色防控方法,本文对草地贪夜蛾潜在RNA干扰(RNA interference,RNAi)靶标致死基因、RNAi传统双链RNA(double-stranded RNA,dsRNA)递送技术的瓶颈以及纳米载体介导的RNAi技术应用进行概括,并对纳米载体介导的RNAi技术应用前景进行展望。     

RNA杀虫剂的研究进展

以害虫关键基因为靶向的RNA干扰技术在害虫可持续治理领域具有良好的应用前景.近年来,随着核酸纳米递送平台和dsRNA的高效合成技术日渐成熟,RNA杀虫剂的研发速度加快.本文结合最新的研究进展,总结了 RNA杀虫剂研发的关键技术问题,对纳米载体高效递送dsRNA平台及纳米载体介导的核酸体壁渗透系统、工程菌批量合成发夹结构...     

RNA干扰在鳞翅目昆虫中的应用研究进展

鳞翅目(Lepidoptera)是昆虫纲中的第二大目,现已经记载的鳞翅目昆虫多达18万个种.鳞翅目昆虫中的许多成员是重要的全球性农业害虫.多数鳞翅目害虫具有繁殖快、危害重、抗药性强及长距离迁飞等特性,对农业生产构成巨大威胁.RNA干扰(RNAi)技术是指通过将目的基因特异性同源双链RNA(dsRNA)导入到细胞内,引起与其同源的mRNA特异性降解,从而达成目标基因表达沉默的一种分子技术.目前该技术已被广泛应用于鳞翅目昆虫的基因功能研究和绿色害虫防治策略探索,并在近年来取得了显著成效和进展.基于此,对RNAi在昆虫中的作用机理进行了归纳和概括,并重点总结和探讨了近年来RNAi技术在鳞翅目昆虫基因功能研究以及鳞翅目害虫防治新方法探索方面取得的新进展,以期为鳞翅目昆虫相关科学研究和生产实践提供参考.     

RNAi在农业病虫害防控中的应用研究进展

RNAi (RNA干扰) 是指由诱导分子siRNA (小干扰 RNA)、miRNA (微小RNA)或piRNA (P 转座子诱导互作 RNA) 特异性降解或者抑制同源mRNA,引起靶标基因沉默的现象。RNAi技术具有操作简便、特异性和选择性强等显著特点,是目前农业生命科学领域最有可能应用于病虫害防控的新技术之一。本文通过综述近年来RNAi在农业病虫害防控领域应用的最新研究成果,并对RNAi技术在新靶标基因筛选、高效dsRNA载体开发、与传统农药相结合以及拓宽应用范围等诸多方面的发展前景进行了展望,同时还针对RNAi干扰效率、稳定性、成本控制、抗性发展及抗性治理等方面所面临的挑战进行了深入探讨,提出了合理建议。基于RNAi技术的病虫害防控策略将继续焕发新的活力,为综合防控提供新理念。     

“RNAi农药”防治植物病毒病的研究进展

病毒病一直是农作物重要病害,目前为止还没有一种有效的方法能够防治植物病毒病。"RNAi农药"是指体外制备的能够诱发病原菌和昆虫某基因沉默从而达到防治目的的dsRNA或者siRNA产品。本文总结了喷施诱导的基因沉默(Spray-induced gene silencing,SIGS)过程,"RNAi农药"的研究历程及其在防治植物病毒病中的应用进展,分析了存在的问题并对其未来发展进行了展望。     

纳米材料结合的dsRNA对烟草花叶病毒侵染的抑制

基于病毒基因的外源双链RNA（dsRNA）可以在植物体内诱导RNAi,但dsRNA易降解、作用时间有限,限制了这项技术的应用。本研究以TMV衣壳蛋白（CP）基因为目的片段,体外合成dsRNA,分别与2种纳米材料壳聚糖（chitosan,CS）和层状双氢氧化物（layered double hydroxide,LDH）融合,合成CS-dsRNA和LDH-dsRNA 2种制剂。测试了2种制剂的稳定性及其对TMV的预防和治疗效果。结果表明,在4、25和37℃,30 d的稳定性测定试验中,dsRNA的量较初始值分别减少了80.01%、58.89%和59.76%,降解比较明显,dsRNA在4℃的降解速率略高于在25和37℃的降解速率;CS-dsRNA制剂中dsRNA的量较初始值分别增加了65.98%、103.82%和157%,LDH-dsRNA制剂中dsRNA的量较初始值分别增加了54.30%、58.4%和66.42%,dsRNA被CS与LDH 2种纳米材料包裹后降解率明显下降,而且随着温度的升高dsRNA的量有所增加,说明纳米材料融合的dsRNA有缓释效应。半叶法试验表明,dsRNA、CS-dsRNA和LDH-dsRNA对TMV的预防效果分别为60.12%、57.79%和55.18%,治疗效果分别为65.01%、58.40%和59.08%。本研究表明,纳米材料CS与LDH对dsRNA起到了良好的保护、缓释及提高稳定性的作用,在利用RNA干扰防治植物病毒病害中具有良好的应用前景。     

RNAi基因沉默技术及其在植物抗病性改良中应用的策略

RNAi (RNA interference) 是一种由dsRNA参与、对靶基因表达进行干扰或沉默的现象。由此发展起来的RNAi基因沉默技术已成为当今植物基因功能研究和遗传改良的一个重要手段。该技术已经在靶向病原物(真菌、细菌、病毒和线虫)基因沉默方面得到了广泛的应用,并且产生了一批抗病性增强的转基因植物。人工设计和合成的amiRNAs和ata siRNAs的成功研发加快了RNAi技术的应用。本文对RNAi基因沉默机制、RNAi技术研发进展及其在植物抗病性遗传改良中的应用进行综述,并对其应用策略进行探讨。     

RNAi在昆虫控制领域的研究进展

RNAi技术在研究昆虫功能基因组学方面已经越来越成熟,基本涉及了昆虫的所有目,并为害虫的控制和益虫种群保护筛选出许多合适的基因,越来越多的研究结果证明了这一方法的可行性。为了在这一领域更好地运用RNAi技术就必须了解干扰片段的不同递呈方式及其在昆虫体内的吸收机制。因此,就目前在昆虫中常用的干扰片段递呈方法和吸收途径进行了简要介绍,对近年RNAi在害虫控制和昆虫种群保护等方面的研究进展进行了综述与展望。     

两种杀虫剂复配的固体纳米分散体的增效研究

为延缓害虫对生物农药的抗性,显著提高生物农药有效利用率,本研究在排除剂型组成及制备工艺对杀虫活性影响的条件下,采用自乳化-载体固化法制备了具有生物农药特性的甲氨基阿维菌素苯甲酸盐和仿生型农药高效氯氟氰菊酯的复配固体纳米分散体。纳米制剂呈现出了良好的叶面浸润与滞留特性。高效氯氟氰菊酯和甲氨基阿维菌素苯甲酸盐质量比为3:1、2:1、1:1、1:2和1:3的复配纳米制剂处理棉蚜24 h后的LC50分别为24.0、21.71、33.26、17.96和16.09μg/m L,共毒系数分别为264.74、229.59、106.82、158.72和162.78,显著增效配比为3:1和2:1的组合。研究结果表明滞留量为影响制剂生物活性的关键指标。该杀虫剂复配纳米剂型的创制及增效效应的研究为延长生物农药使用寿命,解决害虫抗性问题提供了新的理论依据与技术途径,具有广阔的应用前景。     

Ingested RNA interference for managing the populations of the Colorado potato beetle, Leptinotarsa decemlineata

BACKGROUND: RNA interference (RNAi) is a breakthrough technology for conducting functional genomics studies and also as a potential tool for crop protection against insect pests. The major challenge for efficient pest control using RNAi in the field is the development of efficient and reliable methods for production and delivery of double‐stranded RNA (dsRNA). In this paper, the potential of feeding dsRNA expressed in bacteria or synthesized in vitro to manage populations of Colorado potato beetle, Leptinotarsa decemlineata (Say) (CPB), was investigated. RESULTS: Feeding RNAi successfully triggered the silencing of all five target genes tested and caused significant mortality and reduced body weight gain in the treated beetles. This study provides the first example of an effective RNAi response in insects after feeding dsRNA produced in bacteria. CONCLUSION: These results suggest that the efficient induction of RNAi using bacteria to deliver dsRNA is a possible method for management of CPB. This could be also a promising bioassay approach for genome‐wide screens to identify effective target genes for use as novel RNAi‐based insecticides. Copyright © 2010 Society of Chemical Industry     

RNAi‐mediated plant protection against aphids

Aphids (Aphididae) are major agricultural pests that cause significant yield losses of crop plants each year by inflicting damage both through the direct effects of feeding and by vectoring harmful plant viruses. Expression of double‐stranded RNA (dsRNA) directed against suitable insect target genes in transgenic plants has been shown to give protection against pests through plant‐mediated RNA interference (RNAi). Thus, as a potential alternative and effective strategy for insect pest management in agricultural practice, plant‐mediated RNAi for aphid control has received close attention in recent years. In this review, the mechanism of RNAi in insects and the so far explored effective RNAi target genes in aphids, their potential applications in the development of transgenic plants for aphid control and the major challenges in this regard are reviewed, and the future prospects of using plant‐mediated RNAi for aphid control are discussed. This review is intended to be a helpful insight into the generation of aphid‐resistant plants through plant‐mediated RNAi strategy. © 2016 Society of Chemical Industry     

RNA interference technology in crop protection against arthropod pests,pathogens and nematodes

Scientists have made significant progress in understanding and unraveling several aspects of double‐stranded RNA (dsRNA)‐mediated gene silencing during the last two decades. Now that the RNA interference (RNAi) mechanism is well understood, it is time to consider how to apply the acquired knowledge to agriculture and crop protection. Some RNAi‐based products are already available for farmers and more are expected to reach the market soon. Tailor‐made dsRNA as an active ingredient for biopesticide formulations is considered a raw material that can be used for diverse purposes, from pest control and bee protection against viruses to pesticide resistance management. The RNAi mechanism works at the messenger RNA (mRNA) level, exploiting a sequence‐dependent mode of action, which makes it unique in potency and selectivity compared with conventional agrochemicals. Furthermore, the use of RNAi in crop protection can be achieved by employing plant‐incorporated protectants through plant transformation, but also by non‐transformative strategies such as the use of formulations of sprayable RNAs as direct control agents, resistance factor repressors or developmental disruptors. In this review, RNAi is presented in an agricultural context (discussing products that have been launched on the market or will soon be available), and we go beyond the classical presentation of successful examples of RNAi in pest‐insect control and comprehensively explore its potential for the control of plant pathogens, nematodes and mites, and to fight against diseases and parasites in beneficial insects. Moreover, we also discuss its use as a repressor for the management of pesticide‐resistant weeds and insects. Finally, this review reports on the advances in non‐transformative dsRNA delivery and the production costs of dsRNA, and discusses environmental considerations. © 2017 Society of Chemical Industry     

喷施诱导的基因沉默(SIGS)技术控制植物病害研究进展

喷施诱导的基因沉默(spray-induced gene silencing, SIGS)技术是一种新型基因沉默技术, 其以病原菌生长发育和致病相关基因为靶标, 将体外合成的针对靶基因的dsRNA喷施于植物表面, 抑制靶基因的表达?在病原菌侵染寄主的过程中, 病原菌可直接从寄主植物表面摄取dsRNA, 也可由植物吸收dsRNA后, 直接以dsRNA的形式或将dsRNA剪切成sRNAs转运至病原菌体内诱导病原菌相关基因沉默, 从而抑制病菌的侵染和扩展?由于SIGS技术不需要转基因, 再加上其具有高效性?特异性和环境友好性, 故显示出巨大的应用潜力?本文综述了SIGS技术的最新研究进展, 总结了影响SIGS控制植物病害的因素及未来的研究方向, 展望了其应用前景?     

Cloning arginine kinase gene and its RNAi in Bursaphelenchus xylophilus causing pine wilt disease

Since Bursaphelenchus xylophilus causes serious losses in pine forestry, new ways of controlling this nematode damage are urgently needed. Arginine kinase (AK) is a phosphotransferase, which plays a critical role in cellular energy metabolism in invertebrates. It is only present in invertebrates and may be a suitable chemotherapeutic target in the control of this pest. RNA interference (RNAi) technology has been developed in biological science in recent decades as a powerful tool to silence the target gene function in the post-translational status. In this study, one AK gene, BxAK1 (GeneBank accession No. EU853862) was firstly cloned, and then its functions were identified by RNAi technology. Results show that the full-length cDNA of the BxAK1 gene contains 1206 base pairs and an 1086?bp open reading frame encoding 361 amino acids. The length of the BxAK1 genomic coding region contains 2430?bp consisting of four introns of 421, 117, 475, and 268?bp respectively, and five exons of 57, 207, 309, 360, and 215?bp respectively. A dsRNA targeting BxAK1 was constructed and tested for its RNAi effect on B. xylophilus by soaking bioassays. RNAi not only significantly increased the mortality of B. xylophilus, but also greatly reduced its fecundity and fertility. These results suggest that RNAi targeting BxAK1 may be an effective approach for controlling nematode pests.     

Molecular mechanisms influencing efficiency of RNA interference in insects

RNA interference (RNAi) is an endogenous, sequence‐specific gene‐silencing mechanism elicited by small RNA molecules. RNAi is a powerful reverse genetic tool, and is currently being utilized for managing insects and viruses. Widespread implementation of RNAi‐based pest management strategies is currently hindered by inefficient and highly variable results when different insect species, strains, developmental stages, tissues, and genes are targeted. Mechanistic studies have shown that double‐stranded ribonucleases (dsRNases), endosomal entrapment, deficient function of the core machinery, and inadequate immune stimulation contribute to limited RNAi efficiency. However, a comprehensive understanding of the molecular mechanisms limiting RNAi efficiency remains elusive. Recent advances in dsRNA stability in physiological tissues, dsRNA internalization into cells, the composition and function of the core RNAi machinery, as well as small‐interfering RNA/double‐stranded RNA amplification and spreading mechanisms are reviewed to establish a global understanding of the obstacles impeding wider understanding of RNAi mechanisms in insects. © 2018 Society of Chemical Industry     

Prospects,challenges and current status of RNAi through insect feeding

RNA interference is a phenomenon in which the introduction of double‐stranded RNA (dsRNA) into cells triggers the degradation of the complementary messenger RNA in a sequence‐specific manner. Suppressing expression of vital genes could lead to insect death, therefore this technology has been considered as a potential strategy for insect pest control. There are three main routes of dsRNA administration into insects: (i) injections to the hemolymph, (ii) topical, and (iii) feeding. In this review, we focus on dsRNA administration through feeding. We summarize novel strategies that have been developed to improve the efficacy of this method, such as the use of nano‐based formulations, engineered microorganisms, and transgenic plants. We also expose the hurdles that have to be overcome in order to use this technique as a reliable pest management method. © 2019 Society of Chemical Industry