刺山柑(Capparis spinosa)总RNA不同提取方法的比较

以野生刺山柑叶片为材料,采用70%丙酮抽提研磨材料,结合TRIZOL,CTAB,SDS 3种常见的RNA提取方法提取总RNA,通过RNA获得率、纯度、电泳图谱以及RT-PCR反应等分析,初步确立了一种有效的提取刺山柑叶片总RNA的改良SDS法.该方法提取的RNA A<,260>/A<,280>值在1.88～1.94之间,电泳图谱完整,具有产量高、时间短、成本低等特点,所得的RNA可以用于RT-PCR和cDNA文库构建以及Northern杂交等后续实验.     

啤酒花潜隐类病毒RNA提取方法及检测技术的研究

本研究采用纳米磁珠提取法(Magnetic Nanoparticles,MNP)、改良的Li Cl沉淀法、CTAB法、TRIzol法和RNeasy Plant M ini Kit 5种方法提取感染啤酒花潜隐类病毒(Hop latent viroid,HLVd)的啤酒花叶片总RNA,结果显示Li Cl沉淀法、CTAB法和M NP法提取的RNA的质量较好,而M NP法具有提取时间短、操作简便,环境友好和批量提取的优势,适用于啤酒花潜隐类病毒RNA的快速提取。体外转录制备HLVd RNA标准品,利用实时荧光定量RT-PCR绘制标准曲线,并对其特异性、灵敏度进行评估。应用纳米磁珠提取啤酒花总RNA,结合实时荧光RT-PCR技术,建立了HLVd的快速、高效的M NP-RT-q PCR定量检测方法。     

一种提取荒漠拟步甲昆虫基因组DNA的新方法

昆虫基因组DNA的提取是研究昆虫功能基因的关键环节。一些荒漠昆虫个体较小,不易除去的外壳会造成多糖污染,用普通动物组织DNA提取方法效果不佳。通过紫外分光光度测定、凝胶电泳检测、PCR反应及限制性酶切分析表明,CTAB-NaCl法是一种适用于拟步甲科小个体昆虫提取基因组DNA的好方法,蛋白质、多糖及RNA影响很低,完全能够满足后续DNA研究的需求。与传统方法相比简便快速,且成本低,尤其适用于拟步甲科昆虫DNA的分离。     

利用RT-PCR检测库尔勒香梨苹果茎痘病毒的研究

 以库尔勒香梨新鲜、冷藏、冷冻叶片和皮层为材料,对提取双链RNA (dsRNA)的2种方法和提取总RNA的3种方法进行了分析比较,并对总RNA的提取方法进行了改进,获得了纯度较高、完整性较好的dsRNA和总RNA,在此基础上进行了反转录(RT)和PCR扩增。在国内首次完成了对苹果茎痘病毒(ASPV)的RT-PCR检测,建立了ASPV有效RT-PCR反应体系。用此体系扩增到ASPV一个长约316 bp的片段。实验表明以dsRNA和总RNA为模板均能成功进行RT-PCR检测,且dsRNA优于总RNA。     

检疫性真菌DNA提取方法的比较

为获得一种简便、快速、较低费用的DNA提取方法,用于口岸检疫性真菌后续识别检测工作。本文系统比较了8种不同提取方法对DNA浓度、纯度、DNA中蛋白质含量、以及后续PCR扩增、测序的影响,同时对不同方法的提取时间、价格、原理等进行了比较。结果表明,基于磁珠法提取的DNA得率较高,耗时较少,但较易蛋白质污染;CTAB法得率与磁珠法相似,但耗时最长,人力成本高;膜吸附法得率较低,但纯度较高。     

蚜虫内共生菌基因组DNA提取方法的比较和优化

为探究蚜虫共生菌基因组DNA的提取方案,以桃蚜为试验材料,比较了目前较为常用的4种蚜虫基因组DNA提取方法,从DNA纯度、完整性、PCR扩增效率及稳定性等方面进行了比较和评价,并通过调整蛋白酶K用量和水浴温度及时间对STE法进行了优化。结果表明,4种方法提取的蚜虫共生菌基因组DNA均可用于共生菌的PCR扩增检测;CTAB法和SDS法提取的DNA纯度较高,条带较完整,稳定性相对较高,不易降解,而STE法和PCR缓冲液法操作简便,适于快速提取单头蚜虫共生菌的基因组DNA,但纯度相对较低;可根据试验条件和要求进行选择。STE法优化条件为:用30 μL STE缓冲液将蚜虫匀浆,加入1.5 μL 20 mg/mL蛋白酶K,于56 ℃水浴1.5 h;再加入0.1 μL 10 mg/L RNA酶,于37 ℃培养1 h,95 ℃下处理5 min,5 000 r/min离心3 min,将提取到的DNA于-20 ℃保存或直接用于PCR扩增。优化后的STE法可作为提取蚜虫共生菌基因组DNA经济而快捷有效的方法。     

一种改进的水稻总DNA的快速提取方法

植物总DNA的提取质量是植物分子生物学操作成败的关键,一个优秀的总DNA提取方法往往可以达到事半功倍的效果.本文以水稻叶片为材料,提出了一种改进的植物叶片总DNA快速提取方法,该方法能在35min之内提取到水稻总DNA.电泳结果表明此改进方法提取的总DNA产量与传统CTAB法相当,PCR扩增结果表明其质量可靠,完全可以满足分子生物学操作的要求.     

大豆干种子中大豆花叶病毒的RT-PCR检测

 应用改进的SDS-酚氯仿法,在沉淀RNA前先加入1/4体积无水乙醇、1/10体积5mol/L乙酸钾沉淀多糖,然后再用异丙醇沉淀RNA,成功地从大豆干种子中提取了大豆花叶病毒(SMV)总RNA;应用RT-PCR技术对大豆种子中携带的SMV进行了检测,同时以DAS-ELISA方法作比较,建立了能直接以大豆干种子为检测对象的SMV快速、灵敏、特异的RT-PCR检测技术。     

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.     

一种测定斯氏线虫对亚洲玉米螟侵染力的新方法

目前国内外常用测定斯氏线虫对Sleinernema spp.寄主侵染力的方法是沙埋法(Bedding 1983),把目标昆虫埋入一装有含水量为7％的80克细沙的标准盒底部,沙上滴加线虫悬浮液。对非土栖昆虫,是不适宜的。York(1957)在测定DD-136线虫对欧洲玉米螟(ECB)的侵染力时,采用培养皿底部放滤纸,再加入线虫液和接入ECB,但由于ECB的逃逸而使试验失败。后来他在培养皿里加鲜玉米组织,才使试验成功。作者在研究斯氏线虫对亚洲玉米螟(ACB)侵染力的过程中,设计了一种兼有密封性、透气性,并能满足昆虫生存与习性要求的测试方法,现介绍如下。     

Extraction and electrophoretic analysis of large dsRNAs from desiccated plant tissues infected with plant viruses and biotrophic fungi

Extraction and electrophoretic analysis of large dsRNAs from plant and fungal tissues has been used successfully to detect RNA viruses infecting plants, fungi, and oomycetes. We modified a previously reported dsRNA extraction protocol and used it to detect a wide variety of plant and putative fungal viruses from infected plant tissues. The modified protocol was used successfully to extract large dsRNAs from 50 to 70 mg of desiccated plant tissues infected with acute and persistent RNA viruses and from plant tissues infected with biotrophic fungi causing rusts and powdery mildew diseases. The protocol proved to be efficient, fast, economic and versatile and requires relatively small amounts of desiccated tissue.     

Characterization and Detection of Several Filamentous Viruses of Cherry: Adaptation of an Alternative Cloning Method (DOP-PCR), and Modification of an RNA Extraction Protocol

For the identification and analysis of new RNA plant viruses infecting fruit trees, an initial step often involves the laborious procedure of isolation and cDNA synthesis and cloning from purified viral dsRNA. For subsequent RT-PCR detection of these and other viruses from tissue with high phenolic and polysaccharide concentrations, a simple and efficient extraction protocol for viral nucleic acid is also important. A method for rapid cDNA cloning from small amounts of purified dsRNA using a modification of degenerate oligo primed polymerase chain reaction mbox(DOP-PCR), and a modification of a protocol for effective extraction of viral RNA for use in RT-PCR are presented. Both methods were used to analyze a number of mottling diseases described in cherry. The causal agents for two of these diseases have been previously described, Cherry green ring mottle virus, a tentative member of the foveaviruses, and Cherry mottle leaf virus, a member of the trichoviruses. For the diseases cherry rusty mottle and cherry necrotic rusty mottle, data are presented identifying viruses associated with each disease. Viruses associated with cherry rusty mottle, cherry necrotic rusty mottle and European isolates of cherry mottle leaf diseases, are closely related to Cherry green ring mottle virus and can be tentatively included in the foveavirus genus. An additional virus, related to cherry green ring mottle virus, was discovered by RT-PCR cloning and appears to be a common latent virus of cherry. Finally, isolates of cherry necrotic mottle disease could be assayed positive by RT-PCR for a virus     

利用内标为基础的RT-PCR技术检测草莓斑驳病毒

 利用改进的CTAB法提取出优质的草莓总RNA,可以稳定地进行RT PCR。针对草莓斑驳病毒(SMoV)基因组序列设计筛选引物,对病毒基因组的不同区域进行扩增,扩增产物经克隆、测序证明为SMoV的特异片段,与国外序列的同源性为91%~97%。为了监测RNA的质量和RT PCR反应的正常进行,在检测体系中引入线粒体NADH脱氢酶基因ND2亚基作为内标,内标引物跨越内含子区域,只对剪接后的mRNA进行特异扩增,可以较好地监测整个检测过程。在国内首次建立了SMoV的RT PCR检测体系,以优质的草莓总RNA为模板,结合扩增内标的检测体系,对草莓病毒指示植物和草莓栽培品种都可以进行快速稳定的检测。     

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     

南方水稻黑条矮缩病毒一步双重RT-PCR 检测技术及其应用

 An one-step dual RT-PCR method was developed for Southern rice black-streaked dwarf virus (SRBSDV) detection from host plants and insect vector white-backed planthopper (Sogatella furcifera Horvath,Hemiptera: Delphacidae), from which two cDNA fragments of the viral genome S5 and S10 were amplified
simultaneously. Two primer pairs, S5-F1 / S5-R2 (5′-ttacaactggagaagcattaacacg-3′ / 5′-atgaggtattgcgtaactgagcc -3′) and S10-oF / S10-oR(5cgcgtcatctcaaactacag -3′ / 5′-tttgtcagcatctaaagcgc -3′), were selectedfrom 40 primer pairs based on SRBSDV genome sequences, and amplified viral S5 ORF1 fragment (819 bp) and cp gene fragment ( 682 bp), respectively. Using total RNA extracts from infected plant leaf tissue or individual planthopper adult as templates, one step RT-PCR reaction in the conditions of annealing temperature of 53℃ with the final concentrations of 240 nmol / L S5-F1 / S5-R2 and 120 nmol / L S10-oF / S10-oR generated a similar yield of two amplicons in argrose electrophoresis analysis. Sequence analysis confirmed the correct
result of the amplification. Additionally, several commercal RNA extraction kits was proven to be fit for the template preparation, and the protocol for total RNA extraction from plant leaf tissue and individual planthopper was simplified by sample grinding direct in eppendorf tube. This method can be used for SRBSDV detection of dried or 70% alcochol soaked planthopper samples stored over one month in room temprature. Key words: Southern rice black-streaked dwarf virus; Sogatella furcifera Horvath     

Abrasive und hydrophil/lipophile Effekte unterschiedlicher inerter Stäube im Einsatz gegen Schadinsekten am Beispiel des Kornkäfers Sitophilus granarius L.

Modern research on the insecticidal effects of inert dusts as a stored-grain protectant and for plant protection purposes began in the 1920s. The main advantage of inert dusts is their low-mammalian toxicity. One group of inert dusts used for pest control is diatomaceous earth (DE). DE has been tested as a whole and evaluated as a Group 3 carcinogen by the International Agency for Research on Cancer (IARC). A Group 3 listing indicates that DE is not classifiable as to its carcinogenicity to humans, since definitive conclusions cannot be drawn from the research conducted to date. DE is a form of naturally occurring amorphous silica that can kill insects by ab/adsorbing the lipids such as waxes and triglycerides of the outer cuticle layer by direct contact. When the thin, waterproof layer is lost, the insect looses water and dies. Desiccation follows Ficks law of diffusion into the surrounding atmosphere. In addition to its desiccant action the theory that DE works abrasively to rupture insect cuticles has been proposed. To evaluate the abrasive effects of DE we have tested 27 inert dusts with different surface properties. Materials were compared based on the time needed to kill 50% (LT₅₀) of Sitophilus granarius weevils in laboratory experiments. Substances with hydrophilic and/or hydrophobe properties were significantly more effective against S.?granarius than substances which are mainly abrasive. There is a strong correlation between LT₅₀ and weight loss (=?water loss) of insects (p?=?0.014). Particle size played only a secondary role (p?=?0.077). Commercial DE formulations and activated charcoal (Carbopal MB4) were the most effective hydrophilic substances tested. The most abrasive substances were corundum and silica sand with small particle sizes. It is concluded, that those materials were effective because of a good coverage of the insects outer cuticle and existing shear stress. All results presented are discussed in context of the insecticidal mode of action of inert dusts and possible registration of silica based formulations as biocide.     

温度胁迫下昆虫表观遗传机制的研究进展

温度是限制物种适应性分布的重要因子，决定着物种的分布和扩散区域。昆虫作为小型变温动物，对温度变化十分敏感，温度胁迫反应是其抵御极端环境的最保守的机制之一。其中表观遗传学为昆虫提供了更快的温度胁迫响应机制，与经典遗传学不同，表观遗传学在不改变DNA序列的前提下响应环境变化对基因表达产生快速而持久的影响。表观遗传调控包括DNA甲基化、组蛋白修饰、染色质重塑、非编码RNA调控等，本文主要阐述了以上四个方面在昆虫响应温度胁迫中的应用和研究进展，以探究昆虫在温度变化中的适应能力。