高通量测序技术在植物及昆虫病毒检测中的应用

在过去的十几年中,测序技术的发展为分子生物学领域带来了革命性的变化。第二代测序(next-generation sequencing,NGS)技术以快速、高灵敏性、高通量、非序列依赖性等特点极大地促进了病毒诊断学研究领域的发展。NGS技术可以在不了解病毒的生物学特性、血清学特点及基因组信息情况下快速检测未知病毒。通过对总核酸样本进行NGS可以获得某个特定生态环境或种植系统中的所有病毒序列,即病毒组。通过大量的NGS数据可以分析寄主中某一病毒的基因组变化、构建病毒的准种以及研究病毒的进化和起源。本文介绍了高通量测序的方法在植物和昆虫病毒检测中的应用。     

双生病毒——一类值得重视的植物病毒

 双生病毒是世界范围内广泛发生的一类植物单链DNA病毒,已在多种作物上引起严重危害。重组是导致双生病毒变异和病害流行的重要原因。近年来,在单组份双生病毒中发现有新型卫星DNA分子——DNAβ,DNAβ是病毒致病所必需的。本文就双生病毒的危害性、病毒的重组、DNAβ的发现及与致病性的关系、双生病毒与伴随的小分子DNA形成病害复合体、双生病毒DNA-A和小分子DNA的进化关系等问题进行了简要评述。     

山茶褪绿矮缩伴随病毒杭州分离物的序列和进化分析

正双生病毒(Geminivirus)是一类基因组为单链环状DNA的植物病毒,具有孪生的病毒粒子。国际病毒分类委员会(International Committee on Taxonomy of Viruses,ICTV)最新的分类报告将双生病毒分为9个属[1]。随着测序技术的发展,利用小RNA或转录组测序可鉴定植物中的已知和未知病毒,新的双生病毒也不断被发现和鉴定[2]。山茶褪绿矮缩伴随病毒(Camellia chlorotic dwar     

高通量测序技术在植物病毒检疫中的应用与发展

高通量测序技术是当前主流的测序技术,具有快速、灵敏、高通量以及非序列依赖性等特点。本文详细阐述了高通量测序技术的发展以及检测植物病毒的流程,并探讨了该技术应用于植物病毒检测的优势和不足。同时,介绍了利用该技术发现的新的病毒或类病毒,尤其是一些已被欧洲和地中海国家植物保护组织列入检疫性有害生物名录的病毒。目前,有些国家已应用该技术筛查进口植物材料携带的病毒,这对我国的口岸检疫工作提供了新思路。     

湖南省番茄和牵牛花上双生病毒的分子鉴定

双生病毒是一类在全世界范围内广泛发生的单链环状DNA病毒。本文对从湖南采集到的6例(洋姜、番茄、萝卜、赛葵、甘薯、牵牛花)疑似双生病毒侵染的植物叶片进行了分子鉴定。利用滚环扩增技术(RCA)对样品DNA进行扩增,分别对其RCA产物进行酶切,并将酶切得到的片段测序后进行BLAST比对,结果显示番茄样品中的病毒分离物与番茄黄化曲叶病毒相似性最高(99%),牵牛花样品中的病毒分离物与甘薯卷叶病毒相似性最高(99%),证明这两个分离物是单组分DNA-A双生病毒。这是在湖南省首次发现并报道双生病毒的全核酸序列。     

广西粉虱传染类植物双生病毒调查

广西粉虱传染类植物双生病毒调查植物双生病毒（Ｇｅｍｉｎｉｖｉｒｕｓ）具有独特的球状对生病毒形态和单链ＤＮＡ，由于其经济和理论研究上的重要性，近年成为病毒学研究的热点之一，但是，国内有关此组病毒的报道极少。笔者自１９９１年以来在广西对粉虱传染类植物双生...     

河北省番茄黄化曲叶病毒病的分子鉴定初报

双生病毒是一类具有孪生颗粒形态的植物单链病毒,目前双生病毒病害已在多个国家和地区的作物上造成严重危害。近年来,我国多省报道作物上有这类病毒的发生,且有逐年加重和扩散的趋势。根据危害番茄的双生病毒DNA A序列保守区设计简并引物,对2009年4月采自河北省魏县表现叶片黄化、曲叶症状的4个番茄样品进行PCR检测,均为双生病毒阳性,对样品的扩增片断进行了克隆测序,经序列比对分析,与番茄黄化曲叶病毒（Tomato leaf curl virus,TYLCV）山东分离物（FJ646611.1）序列相似性为99.25%~99.55%,说明河北番茄黄化曲叶病由TYLCV引起。     

安徽省太和县中国南瓜曲叶病毒自然侵染南瓜的检测分析

中国南瓜曲叶病毒(squash leaf curl China virus, SLCCNV)是一种植物单链DNA病毒, 属于双生病毒科Geminiviridae菜豆金色花叶病毒属Begomovirus?在自然条件下该病毒可以侵染多种葫芦科作物, 如南瓜?甜瓜等?该病毒由烟粉虱进行持久性传播, 是影响瓜类产量和品质的重要病害?2021年4月-5月, 安徽省太和县温室大棚中的南瓜叶片出现斑驳?皱缩?卷曲以及植株矮化等现象, 我们采集了具有典型病毒病症状的南瓜病叶样品以及烟粉虱虫体进行RT-PCR鉴定, 结合测序结果鉴定危害南瓜的病毒为SLCCNV?为了进一步明确安徽太和地区SLCCNV的系统进化特征, 我们测定了SLCCNV的外壳蛋白基因(coat protein, CP)序列并进行系统发育分析, 结果表明安徽太和县所检测到的SLCCNV分离物与中国广东和海南地区的分离物亲缘关系较近, 并且与越南?菲律宾?柬埔寨?泰国等一些国家的分离物处于同一个大分支, 存在较小的地域差异性, 而与印度和孟加拉国地区的分离物亲缘关系相对较远, 处于不同的大分支?本研究是安徽省SLCCNV侵染南瓜的首次报道, 期望为该病害的预警和防控提供理论依据?     

双生病毒复制增强蛋白C3/AC3的研究进展

双生病毒作为全球范围内引起作物严重病害的单链DNA病毒，编码多种多功能蛋白，其帮助自身完成复制、转录、组装、移动和致病等生命过程，其中双生病毒的复制增强蛋白C3/AC3在病毒侵染初期可促进病毒复制，提高病毒积累水平以帮助其快速建立侵染。为深入解析C3/AC3蛋白的功能和双生病毒致病机制，该文概述双生病毒C3/AC3蛋白的基本生物学特性及其在病毒侵染过程中的功能，重点阐述C3/AC3蛋白与其他蛋白及寄主因子互作以促进病毒复制的分子机制，同时针对双生病毒复制机制和C3/AC3蛋白的功能解析进行展望。     

利用小RNA测序技术鉴定吉林省甘薯病毒

在吉林省7个主要甘薯种植区共采集85份甘薯叶片样品,利用小RNA深度测序技术对混合样品进行检测,经RT-PCR和测序验证,鉴定出样品中存在10种病毒,包括6种RNA病毒和4种DNA病毒。分别是马铃薯Y病毒科马铃薯Y病毒属的甘薯羽状斑驳病毒Sweet potato feathery mottle virus (SPFMV)、甘薯潜隐病毒Sweet potato latent virus (SPLV)、甘薯G病毒Sweet potato virus G (SPVG)、甘薯C病毒Sweet potato virus C (SPVC)、甘薯2号病毒Sweet potato virus 2 (SPV2);长线形病毒科毛形病毒属的甘薯褪绿矮化病毒Sweet potato chlorotic stunt virus (SPCSV);双生病毒科菜豆金色花叶病毒属的甘薯曲叶病毒Sweet potato leaf curl virus(SPLCV);玉米线条病毒属的甘薯无症状1号病毒Sweet potato symptomless virus 1 (SPSMV1);花椰菜花叶病毒科杆状DNA病毒属的甘薯杆状DNA病毒B Sweet potato badnavirus B (SPBV-B)和甘薯隐症病毒Sweet potato pakakuy virus (SPPV)。     

Diagnosis of plant diseases using the Nanopore sequencing platform

Reliable detection and identification of plant pathogens are essential for disease control strategies. Diagnostic methods commonly used to detect plant pathogens have limitations such as requirement of prior knowledge of the genome sequence, low sensitivity and a restricted ability to detect several pathogens simultaneously. The development of advanced DNA sequencing technologies has enabled determination of total nucleic acid content in biological samples. The possibility of using the single-molecule sequencing platform of Oxford Nanopore as a general method for diagnosis of plant diseases was examined. It was tested by sequencing DNA or RNA isolated from tissues with symptoms from plants of several families inoculated with known pathogens (e.g. bacteria, viruses, fungi, phytoplasma). Additionally, samples of groups of 200 seeds containing one infected seed of each of two or three pathogens, as well as samples with symptoms but unidentified pathogens were tested. Sequencing results were analysed with Nanopore data analysis tools. In all the inoculated plants, pathogens were identified in real time within 1–2 h of running the Nanopore sequencer and were classified to the species or genus level. DNA sequencing or direct RNA sequencing of samples with unidentified disease agents were validated by conventional diagnostic procedures (e.g. PCR, ELISA, Koch test), which supported the results obtained by Nanopore sequencing. The advantages of this technology include: long read lengths, fast run times, portability, low cost and the possibility of use in every laboratory. This study indicates that adoption of the Nanopore platform will be greatly advantageous for routine laboratory diagnosis.     

The transmission of geminiviruses by Bemisia tabaci

The cosmopolitan whitefly species, Bemisia tabaci (Gennadius) and Trialeurodes taporariorum (Westwood) have always been regarded as pests to a large range of worldwide crops. Both species are capable of transmitting plant viruses, with T. vaporariorum being the vector of only a few ‘clostero’-like viruses and B. tabaci the vector of viruses in several groups. The largest group of viruses transmitted by B. tabaci are the geminiviruses and B. tabaci is known to transmit around 60 members. Until recently, B. tabaci had been associated with only a limited range of host plants within any one region, although its total potential host range was large. Virus transmission was confined within the plant host range of each regional population of B. tabaci. The emergence of the polyphagous ‘B’ biotype of B. tabaci with its increased host range of more than 600 plant species, has resulted in geminiviruses infecting previously unaffected crops. As the ‘B’ biotype spreads further into Europe, European field and glasshouse crops have been shown to be susceptible to whitefly-transmitted viruses already endemic to other parts of the world. More than 20 colonies of B. tabaci, including both ‘B’ and non-‘B’ biotypes from disparate global locations have been compared for their ability to transmit more than 20 geminiviruses. All but two highly host-specific colonies were capable of transmitting most geminiviruses tested. However, some viruses were transmitted more efficiently than others. The virus coat protein or capsid is essential for vector recognition and transmission. By comparing transmissible viruses at the molecular level to viruses that are no longer whitefly-transmissible, the active epitope on the virus coat protein could be identified for designing future virus control strategies.     

山茶褪绿矮缩伴随病毒杭州分离物的序列和进化分析

Through sequencing and assembly of small RNAs, a Camellia chlorotic dwarf-associated virus (CaCDaV) was identified on a camellia plant showing chlorosis and distorting leaves from Hangzhou (CaCDaV-HZ1). The full genome of CaCDaV-HZ1 was determined and compared with that of a CaCDaV isolate from Chongqing (CaCDaV-CA1). Results showed that they shared 85.7% overall nucleotide sequence identity. Insert and delete mutations were mainly distributed in the non-coding region, whereas the coding regions mainly contained synonymous substitution. All genes except V3 have identical length among the two isolates. Additionally, the small RNA profile of CaCDaV-HZ1 was also analysed and results showed that 24 nt is the major class of viral-derived siRNA indicating that methylation is a major resistance mechanism of camellia plants against geminiviruses.     

10种植物病毒的基因芯片检测技术研究

 本研究设计了10种植物病毒和各种对照的探针, 将探针固定在醛基化玻璃片基上制备基因芯片。用常规的Trizol法提取植物总RNA, 根据病毒的外壳蛋白基因或复制酶基因设计特异性引物, 经RT-PCR扩增相应区段并用Cy3-dCTP进行标记。将标记的PCR产物与芯片杂交, 扫描仪对杂交结果扫描, GenePix Pro 4.0软件对杂交图像进行分析。结果表明, 该芯片可以从病毒感染样本中检测到特异性识别信号, 检测灵敏度比RT-PCR高10~100倍, 所以, 基因芯片能对植物病毒作出快速、准确的检测。     

用滚环扩增技术检测双生病毒

 本文报道了利用滚环扩增技术(rolling circle amplification,RCA)放大双生病毒的靶核酸信号,进而利用限制性内切酶片段长度多态性(RFLP)检测不同类型双生病毒的新技术体系。研究结果表明,利用RCA-RFLP对于已知的单组份和双组份以及带有卫星的菜豆金色花叶病毒属病毒都能够有效检测,对采自浙江杭州地区的田间样品进行RCA-RFLP检测的结果与利用简并引物PA和PB进行PCR扩增的结果相吻合。相比传统的PCR检测方法,用RCA-RFLP体系检测双生病毒灵敏性更高,操作更简便。     

RNA沉默转基因技术在植物抗病虫害研究中的作用

RNA沉默技术由于其简便、高效及高特异性在各种模式和非模式生物系统中得到了广泛的应用。植物转基因技术是将植物、动物或微生物中分离到的目的基因,通过各种手段整合到植物基因组上,使其稳定遗传并赋予植物新的优良性状的生物技术。RNA沉默在植物转基因工程领域的研究十分广泛,目前已经成功研究培育出许多抗病毒、抗虫转基因植物,为农业病虫害的防控提供了新的思路和方法。本文重点综述了近年来植物转基因介导RNA沉默在抗虫与抗病毒研究方面的进展,加深人们对转基因植物抗虫与抗病毒研究的认识。