昆虫遗传转化品系的常用标记

遗传转化标记是将遗传修饰昆虫从野生型种群中分辨出来的根据,遗传转化昆虫的鉴定、转化品系的维持及其遗传稳定性的监测都依赖于可靠的标记系统,发展易于应用和监测的转化标记能够极大地促进害虫遗传防治的相关研究。用于遗传修饰昆虫的转化标记主要有昆虫眼睛颜色标记基因、抗药性标记基因和荧光蛋白标记基因等。非果蝇类昆虫首个遗传转化品系的鉴定是通过眼睛颜色突变而实现,但大多数昆虫物种没有可用的突变体或缺少相应基因的信息,从而限制了眼睛颜色标记的应用。抗药性基因标记虽然能够通过对转化昆虫进行集体选择而大幅度提高筛选转化体的效率,但由于其鉴定的准确性不高且存在安全性问题,未得到广泛应用。荧光蛋白标记基因的发展则显著拓宽了能够转化的昆虫种类。从水母分离的绿色荧光蛋白(GFP)经突变方法获得了多种不同荧光性质的突变体,经人为修饰后与适宜的强启动子构成转化标记载体,能够有效鉴定更多昆虫物种的遗传转化个体,其中应用较多的是增强型绿色荧光蛋白(EGFP)。此外,从珊瑚属海葵中分离得到的红色DsRed标记基因提供了多样化的红色荧光蛋白选择,在某些生物中DsRed与GFP联合应用的表现明显优于GFP突变体,所以其应用前景也非常广泛。本文着重从眼睛颜色、抗药性和荧光蛋白等3个方面阐述了标记基因的发展历史与现状,并对其今后的发展方向进行了展望。     

RNA干扰技术在昆虫学领域研究进展

RNA干扰(RNAi)是生物体内源基因发生转录后特异性降解的一种生理现象,广泛存在于生物体内。RNAi主要由小干扰RNA诱发阻碍目的基因的翻译或转录,造成目标信使RNA沉默。RNAi具有高效、特异性强等优点,被广泛应用于昆虫基因功能研究,并显示出了开发新型病虫害管理策略的巨大潜力。主要阐述了RNAi的沉默机制,双链RNA转入昆虫体内的几种方式,以及RNAi技术在不同目昆虫中研究的最新进展。最后,对RNAi技术存在的不足之处进行了简单总结,还对RNAi技术在害虫防治中的应用进行了展望,以期为该技术广泛应用于农业害虫防治提供理论支持。     

High mortality caused by high dose of dsRNA in the green rice leafhopper Nephotettix cincticeps (Hemiptera: Cicadellidae)

RNA interference (RNAi) is a useful tool for gene functional studies in non-model insects and pest insects. Establishment of experimental conditions for RNAi, which differ from insect to insect, is important for evaluating the effect of dsRNA injection of relevant genes. When injecting dsRNA into the green rice leafhopper Nephotettix cincticeps (Uhler), high mortality was observed. Therefore, the adverse effects of injection of dsRNA on leafhopper development were examined to assess the suitable conditions for RNAi in this species. Injection manipulation by using a glass capillary did not affect leafhopper survival but delayed the molt of the corresponding instar. High mortality was observed when large amounts of dsRNA were administered. This adverse effect of dsRNA was examined in 2 genes, exogenous EGFP gene and endogenous peptidoglycan recognition protein gene (NcPGRP12). Injection of a high dose (60 ng/insect) caused high mortality in all stages tested: 4th instar, 5th instar, and female adult. A relatively low dose (6 ng/insect) did not cause high mortality, retaining a high potential for gene silencing. Since RNAi is highly effective in this species, the deleterious effect of large amounts of dsRNA could be avoided by administering a low dsRNA dose.     

Feasibility,limitation and possible solutions of RNAi‐based technology for insect pest control

Abstract Numerous studies indicate that target gene silencing by RNA interference (RNAi) could lead to insect death. This phenomenon has been considered as a potential strategy for insect pest control, and it is termed RNAi‐mediated crop protection. However, there are many limitations using RNAi‐based technology for pest control, with the effectiveness target gene selection and reliable double‐strand RNA (dsRNA) delivery being two of the major challenges. With respect to target gene selection, at present, the use of homologous genes and genome‐scale high‐throughput screening are the main strategies adopted by researchers. Once the target gene is identified, dsRNA can be delivered by micro‐injection or by feeding as a dietary component. However, micro‐injection, which is the most common method, can only be used in laboratory experiments. Expression of dsRNAs directed against insect genes in transgenic plants and spraying dsRNA reagents have been shown to induce RNAi effects on target insects. Hence, RNAi‐mediated crop protection has been considered as a potential new‐generation technology for pest control, or as a complementary method of existing pest control strategies; however, further development to improve the efficacy of protection and range of species affected is necessary. In this review, we have summarized current research on RNAi‐based technology for pest insect management. Current progress has proven that RNAi technology has the potential to be a tool for designing a new generation of insect control measures. To accelerate its practical application in crop protection, further study on dsRNA uptake mechanisms based on the knowledge of insect physiology and biochemistry is needed.     

昆虫RNAi通路及其核心元件的研究综述

RNAi作为分子生物学的一种重要技术,在昆虫基因功能和功能基因组研究中得到广泛应用,同时,有关昆虫RNAi的机制也受到了大家的关注。近年来的研究结果表明,昆虫RNAi的通路与其他动物相同,根据引起基因沉默的RNA分子的类型,可以分为siRNA、miRNA和piRNA 3种不同的通路。昆虫RNAi通路中的核心元件包括了:(1)行使切割作用的RNaseⅢ家族成员Drosha和Dicer;(2)用来降解目的 mRNA的Argonaute蛋白;(3)dsRNA结合蛋白Pasha、R2D2和Loquacious。了解昆虫RNAi的通路及其核心元件,有助于我们更好地理解昆虫RNAi的分子机制和改进实现RNAi的方法,对促进昆虫RNAi技术的研究及其在害虫防控中的应用具有指导意义。     

Fluorescent transformation markers for insect transgenesis

The first effectively achieved germ-line transformations of non-drosophilid insects were based on mutant rescue of eye color phenotypes. However, for most insect species neither visible mutants nor corresponding cloned genes are available. Therefore, the development of broadly applicable and reliable transformation markers will be of great importance to fully exploit the enormous potential transgenic insect technology has to offer. Here we review transposon-mediated germ-line transformation approaches that employ green fluorescent protein (GFP) variants to identify successful gene transfer. Furthermore, we provide novel data on the use of DsRed as an additional red fluorescent transformation marker for insect transgenesis. In conclusion, fluorescent proteins controlled by suitable strong promoters possess ideal characteristics to serve as transformation markers for a wide range of insect species.     

A current perspective on insect gene transformation

The genetic transformation of non-drosophilid insects is now possible with several systems, with germ-line transformation reported in published and unpublished accounts for about 12 species using four different transposon vectors. For some of these species, transformation can now be considered routine. Other vector systems include viruses and bacterial symbionts that have demonstrated utility in species and applications requiring transient expression, and for some, the potential exists for genomic integration. Many of these findings are quite recent, presenting a dramatic turning point in our ability to study and manipulate agriculturally and medically important insects. This review discusses these findings from the perspective of all the contributions that has made this technology a reality, the research that has yet to be done for its safe and efficient use in a broader range of species, and an overview of the available methodology to effectively utilize these systems.     

RNA interference in infectious tropical diseases

Introduction of double-stranded RNA (dsRNA) into some cells or organisms results in degradation of its homologous mRNA, a process called RNA interference (RNAi). The dsRNAs are processed into short interfering RNAs (siRNAs) that subsequently bind to the RNA-induced silencing complex (RISC), causing degradation of target mRNAs. Because of this sequence-specific ability to silence target genes, RNAi has been extensively used to study gene functions and has the potential to control disease pathogens or vectors. With this promise of RNAi to control pathogens and vectors, this paper reviews the current status of RNAi in protozoans, animal parasitic helminths and disease-transmitting vectors, such as insects. Many pathogens and vectors cause severe parasitic diseases in tropical regions and it is difficult to control once the host has been invaded. Intracellularly, RNAi can be highly effective in impeding parasitic development and proliferation within the host. To fully realize its potential as a means to control tropical diseases, appropriate delivery methods for RNAi should be developed, and possible off-target effects should be minimized for specific gene suppression. RNAi can also be utilized to reduce vector competence to interfere with disease transmission, as genes critical for pathogenesis of tropical diseases are knockdowned via RNAi.     

Lipids help double-stranded RNA in endosomal escape and improve RNA interference in the fall armyworm,Spodoptera frugiperda

RNA interference (RNAi) is a valuable method for understanding the gene function and holds great potential for insect pest management. While RNAi is efficient and systemic in coleopteran insects, RNAi is inefficient in lepidopteran insects. In this study, we explored the possibility of improving RNAi in the fall armyworm (FAW), Spodoptera frugiperda cells by formulating dsRNA with Cellfectin II (CFII) transfection reagent. The CFII formulated dsRNA was protected from degradation by endonucleases present in Sf9 cells conditioned medium, hemolymph and midgut lumen contents collected from the FAW larvae. Lipid formulated dsRNA also showed reduced accumulation in the endosomes of Sf9 cells and FAW tissues. Exposing Sf9 cells and tissues to CFII formulated dsRNA caused a significant knockdown of endogenous genes. CFII formulated dsIAP fed to FAW larvae induced knockdown of iap gene, growth retardation and mortality. Processing of dsRNA into siRNA was detected in Sf9 cells and Spodoptera frugiperda larvae treated with CFII conjugated ³²P-UTP labeled dsGFP. Overall, the present study concluded that delivering dsRNA formulated with CFII transfection reagent helps dsRNA escapes from the endosomal accumulation and improved RNAi efficiency in the FAW cells and tissues.     

Mechanisms of dsRNA uptake in insects and potential of RNAi for pest control: A review

RNA interference already proved its usefulness in functional genomic research on insects, but it also has considerable potential for the control of pest insects. For this purpose, the insect should be able to autonomously take up the dsRNA, for example through feeding and digestion in its midgut. In this review we bring together current knowledge on the uptake mechanisms of dsRNA in insects and the potential of RNAi to affect pest insects. At least two pathways for dsRNA uptake in insects are described: the transmembrane channel-mediated uptake mechanism based on Caenorhabditis elegans’ SID-1 protein and an ‘alternative’ endocytosis-mediated uptake mechanism. In the second part of the review dsRNA feeding experiments on insects are brought together for the first time, highlighting the achievement of implementing RNAi in insect control with the first successful experiments in transgenic plants and the diversity of successfully tested insect orders/species and target genes. We conclude with points of discussion and concerns regarding further research on dsRNA uptake mechanisms and the promising application possibilities for RNAi in insect control.     

昆虫的RNA干扰

RNA干扰(RNAi)是一种强有力的分子生物学技术, 在昆虫研究中得到了较多的应用。目前, RNAi技术主要应用于昆虫功能基因和功能基因组研究, 已在多个目的19种昆虫上实现了RNAi。在昆虫上实现RNAi的方法主要有注射、浸泡、喂食、转基因和病毒介导等方法, 这些方法各有特点, 其中喂食法因其简单而最有应用前景。昆虫RNAi的系统性较为复杂, 只有部分昆虫具有RNAi的系统性。昆虫中RNAi信号传导的基因可能是sid-1, 但昆虫RNAi的系统性机理还不是很清楚。转基因植物产生的dsRNA实现了对作物的保护, 证实了RNAi技术可用于害虫控制, 为害虫控制开辟了新领域。昆虫的RNAi研究处在起步阶段, 研究昆虫RNAi的机理, 特别是RNAi在昆虫体内的系统性扩散机理, 改进实现RNAi的方法, 提高RNAi技术在昆虫研究中的应用, 有利于昆虫基因功能鉴定和害虫控制, 促进昆虫学科的发展。     

RNAi技术在昆虫学中的研究进展及展望

在昆虫中,RNAi是一种对抗外源病毒的天然免疫方式,基于生物体中的这种内在机制而建立的RNAi技术已经被广泛用来研究多种昆虫基因的功能。近年的研究结果表明RNAi技术在抵御害虫和防治益虫疾病方面具有潜在的应用价值,有可能对农业有害生物的控制起到巨大的推动作用。本文综述了RNAi与昆虫免疫、及其在昆虫基因功能研究、害虫控制、益虫疾病控制和昆虫系统生物学方面的最新研究进展,并展望了RNAi在昆虫学研究中的发展趋势。     

The Development and Application of a Multiple Gene Co-Silencing System Using Endogenous URA3 as a Reporter Gene in Ganoderma lucidum

Ganoderma lucidum is one of the most important medicinal mushrooms; however, molecular genetics research on this species has been limited due to a lack of reliable reverse genetic tools. In this study, the endogenous orotidine 5'-monophosphate decarboxylase gene (URA3) was cloned as a silencing reporter, and four gene-silencing methods using hairpin, sense, antisense, and dual promoter constructs, were introduced into G. lucidum through a simple electroporation procedure. A comparison and evaluation of silencing efficiency demonstrated that all of the four methods differentially suppressed the expression of URA3. Our data unequivocally indicate that the dual promoter silencing vector yields the highest rate of URA3 silencing compared with other vectors (up to 81.9%). To highlight the advantages of the dual promoter system, we constructed a co-silencing system based on the dual promoter method and succeeded in co-silencing URA3 and laccase in G. lucidum. The reduction of the mRNA levels of the two genes were correlated. Thus, the screening efficiency for RNAi knockdown of multiple genes may be improved by the co-silencing of an endogenous reporter gene. The molecular tools developed in this study should facilitate the isolation of genes and the characterization of the functions of multiple genes in this pharmaceutically important species, and these tools should be highly useful for the study of other basidiomycetes.     

Pupal RNA interference methods for analyzing adult development in stag beetles

Holometabolous insects dramatically change their morphology via molt, both from larva to pupa and from pupa to adult. In nonmodel insects, RNA interference (RNAi) is a strong tool for analyzing gene function during postembryonic development. In many cases, larval RNAi is effective for analyzing genes involved in morphogenesis via metamorphosis. However, RNAi of genes involved in development sometimes results in lethality before animals metamorphose to pupae and/or adults, making it impossible to analyze their function during the pupal period. In this study, we establish a pupal RNAi system in the stag beetle Dorcus rectus. We selected the genes white and scarlet for RNAi knockdown to investigate appropriate injection timing and position. Both genes are known to be involved in eye pigmentation. By using these candidate genes, we demonstrate the potential of pupal RNAi in this experimental system. This method will be useful for analyzing pupal-specific morphogenesis including fine-shaping of the enlarged male mandible in this species.     

Insect transgenesis and its potential role in agriculture and human health

The ability to genetically engineer insects other than Drosophila melanogaster has further extended modern genetic techniques into important insect pest species ranging from fruit fly pests of horticulture to mosquito vectors of human disease. In only a relatively short period of time, a range of transgenes have been inserted into more than 10 insect pest species. Genetic transformation of these pest species has proven to be a very important laboratory tool in analyzing gene function and effects on phenotype however the full extension of this technology into the field is yet to be realized. Here we briefly review the development of transgenic technology in pest insect species and discuss the challenges that remain in this applied area of insect genetics and entomology.     

象甲科昆虫转录组研究进展

转录组是细胞在特定发育阶段或生理条件下的全部转录本及其数量总和,并能揭示特定状态下的基因表达模式及分子机理。象甲科昆虫拥有动物界中最多的物种种类,该科许多昆虫也是我国粮食仓储和农林产业上的重大害虫。通过研究象甲科昆虫转录组,从分子水平揭示其生命过程中相关的基因及其功能,对寻找害虫防治的新方法具有重要意义。本文对象甲科昆虫转录组研究现状进行了浅析,涉及象甲科不同发育阶段基因差异、昆虫触角、与植物互作、防治、免疫机制、进化、基因沉默7个方面的分析,可在理论上丰富对象甲科昆虫遗传发育、免疫、潜在RNA干扰靶点筛选等方面的理解,并在此基础上提出了象甲科转录组今后的研究热点与应用前景,为害虫防控提供理论指导。