利用RNA干扰介导抗病性获得兼抗四种病毒的转基因马铃薯

为获得兼抗马铃薯X病毒(Potato virus X,PVX)、马铃薯Y病毒(Potato virus Y,PVY)、马铃薯卷叶病毒(Potato leaf roll virus,PLRV)和马铃薯潜隐花叶病毒(Potato virus S,PVS)4种病毒的转基因马铃薯新材料,分别以这4种病毒全长CP基因为模板,通过设计PCR引物和亚克隆获得4种病毒CP基因相对保守区段的基因片段,并将其拼接成融合基因,以载体pHANNIBAL和pBI121为基础,构建RNA干扰(RNA interference,RNAi)载体,利用农杆菌介导的转基因体系进行马铃薯遗传转化,并对获得的转基因马铃薯进行病毒抗性检测。结果表明,所获得的融合基因片段RH1和RH2,酶切鉴定分别得到长度为1 200 bp的条带,与预期片段相符;构建了含pdk内含子和RH1、RH2融合基因的RNAi植物表达载体,经Bam H I/Sac I双酶切,获得长度约3 200 bp的片段,表明RNAi植物表达载体pBI121-pRH构建成功;转化易感病毒马铃薯品种陇薯11号,PCR检测和PCRSouthern杂交分析表明融合基因已整合到陇薯11号马铃薯基因组中;抗病性检测显示4株转基因马铃薯植株对4种病毒均免疫。表明利用RNAi可筛选出抗多种病毒的转基因马铃薯新种质。     

基于小RNA深度测序和RT-PCR检测侵染广东省冬种马铃薯的病毒

为明确侵染广东省冬种马铃薯的病毒种类及优势病毒,结合小RNA深度测序技术及RTPCR检测方法,对采集于广东省冬种马铃薯7个主产区的189份疑似病样进行检测分析。结果表明,经小RNA深度测序技术检测马铃薯病毒病混合样,发现存在马铃薯Y病毒(Potato virus Y,PVY)、马铃薯S病毒(Potato virus S,PVS)和马铃薯卷叶病毒(Potato leaf-roll virus,PLRV)3种病毒。进一步设计3种病毒的特异性引物并利用国内已报道的其它5种马铃薯病毒的特异性引物进行RT-PCR检测,发现189份马铃薯病毒病疑似病样中仅检测到PVY、PVS和PLRV这3种病毒,检出率依次为75.13%、10.05%和4.76%,且3种病毒在马铃薯上还存在复合侵染,复合侵染率为14.19%,其中PVY在各马铃薯产区均可检测到。表明侵染广东省冬种马铃薯的病毒为PVY、PVS和PLRV,其中PVY是优势病毒。     

马铃薯4种病毒多重PCR检测体系的建立

我国马铃薯病毒主要有马铃薯Y病毒(PVY)、马铃薯X病毒(PVX)、马铃薯S病毒(PVS)、马铃薯卷叶病毒(PLRV),常发生复合侵染。根据GenBank中4种马铃薯病毒的外壳蛋白(coat protein,CP)基因全长设计引物,通过RT-PCR扩增得到4种病毒CP基因全长片段,测序结果显示序列同源性96%以上;针对4种病毒CP基因的保守序列分别设计引物,在一个PCR体系中同步对4种病毒进行扩增,得到421、202、516、330bp的特异性条带,优化建立了能同步检测PVY、PVX、PVS和PLRV的多重RT-PCR检测体系。检测结果证明优化后的多重RT-PCR体系能在田间样品中快速、高效地检测出4种病毒。     

湖南省马铃薯病毒病发生情况调查

为了解湖南省马铃薯种薯质量和主要病毒病发生情况,2019年-2020年马铃薯秋作和冬作期间,对长沙、益阳、湘潭、澧临等马铃薯生产区的155个马铃薯样品,运用反转录-聚合酶链式反应(RT-PCR)和双抗体夹心酶联免疫吸附检测(DAS-ELISA)技术,筛查6种主要马铃薯病毒,包括马铃薯X病毒Potato virus X(PVX)、马铃薯Y病毒Potato virus Y(PVY)、马铃薯M病毒Potato virus M(PVM)、马铃薯S病毒Potato virus S(PVS)、马铃薯A病毒Potato virus A(PVA)、马铃薯卷叶病毒Potato leaf roll virus(PLRV)。检测结果表明:6种马铃薯病毒病在湖南均有不同程度的发生,单一和两种病毒复合感染植株占比最高,其次是3种病毒复合感染,存在极少数植株复合感染4～5种病毒病情况。在秋作马铃薯中,PVY检出率达到29.41%;PVS和PVA检出率均为27.94%;PVM、PVX、PLRV的检出率分别为20.59%、19.12%、17.65%。在冬作马铃薯中,PVX检出率最高,达到31.03%;其次是PLRV,...     

应用蛋白宏阵列快速检测西瓜细菌性果斑病菌

蛋白阵列(Protein array)也叫蛋白芯片,是生物芯片(Biochip)的一种,主要利用抗原/抗体可特异性结合原理制备而成。按阵列样点大小,蛋白阵列又可分为微阵列(Microarray)和宏阵列(Mac-roarray)两类。微阵列集成度极高,可进行高通量     

Detection of tobacco rattle virus in nematodes by reverse transcription and polymerase chain reaction

An assay, based on amplification of cDNA synthesized from genomic viral RNA, has been developed to detect tobacco rattle virus in infected plant material and viruliferous nematodes. A range of different TRV strains could be detected using the procedure developed. The presence of one to three viruliferous nematodes in a nematode suspension was sufficient for the detection of TRV. The minimum amount of purified virus detectable in the assay was 15 fg, indicating an increased sensitivity of the PCR-based assay as compared to serological detection methods, like ELISA. A dot-blot hybridization procedure was developed for the detection of the PCR products, making agarose gel electrophoresis dispensable.     

梨褪绿叶斑伴随病毒的RT-PCR和巢式RT-PCR检测技术建立

 梨褪绿叶斑伴随病毒(Pear chlorotic leaf spot-associated virus,PCLSaV)是新近发现的为害梨树的欧洲花楸环斑病毒属(Emaravirus)病毒,该病毒基因组由5条负义单链RNA组成。本研究比较分析了反转录引物pd(N)6、3C和5H及基于该病毒基因组RNA3和RNA5链序列设计的4对引物用于RT-PCR检测梨样品中PCLSaV的效果,结果显示,采用与该病毒基因组RNA链3′末端互补的引物3C用于cDNA合成及基于该病毒RNA5链序列的引物5-F/R用于PCR扩增时,检测PCLSaV的灵敏度相较采用引物pd(N)6和5H合成cDNA为模板时高10~100倍;不同部位和不同发病状况的梨树组织中PCLSaV检测结果差异明显。进一步建立了具有高灵敏度的巢式RT-PCR技术,采用外侧引物5-F/R和内巢引物5-IF/IR结合可用于梨不同组织样品中PCLSaV的检测。本研究为系统分析PCLSaV在我国栽培梨树上的危害状况及无病毒梨种质培育奠定了技术基础。     

二重RT-PCR快速检测马铃薯病毒的方法

本研究采用传统的蛋白酶K法和病毒RNA简易浸提法,从马铃薯块茎、茎干、叶梗、叶片中提取马铃薯X病毒,马铃薯Y病毒,马铃薯A病毒及马铃薯卷叶病毒RNA,并设计了4种马铃薯病毒引物,优化了二重RT-PCR反应条件,可以同步扩增出上述4种病毒,扩增产生的靶带分别为562bp(PVX)、480bp(PVY)、336bp(PLRV)、255bp(PVA).应用病毒RNA简易制样技术和优化的二重RT-PCR反应条件,可以同步快速检测田间自然感染的马铃薯病毒,此研究还可适合于检测马铃薯脱毒种薯及试管苗,对马铃薯病毒病早期监测有一定的作用.     

Aphid Acquisition and Cellular Transport of Potato leafroll virus-like Particles Lacking P5 Readthrough Protein

ABSTRACT Lepidopteran cells (Spodoptera frugiperda) produced isometric virus-like particles (VLP) when infected with a recombinant baculovirus Ac61 that contained the Potato leafroll virus (PLRV) coat protein gene modified with an N-terminal histidine tag (P3-6H). Cells infected with AcFL, a recombinant baculovirus that expressed cDNA copies of the PLRV genome RNA, did not produce virus-like particles (VLP). In cell lines doubly infected with Ac61 and AcFL, VLP were formed that contained PLRV-RNA packaged in P3-6H coat protein (FL). Both the P3-6H and the FL particles were morphologically indistinguishable from particles of PLRV despite the fact that they lacked the P5 readthrough protein present in wild-type PLRV. When aphids (Myzus persicae) were fed on, or injected with, purified PLRV, or VLP of either type (FL or P3-6H) and examined by electron microscopy, no differences were observed among treatments for particle endocytosis, transcellular transport, or exocytosis at the aphid midgut or accessory salivary glands. Particles were observed in the salivary canals and in the salivary duct leading out of the aphid. These results suggest that P5 readthrough protein of PLRV may not be essential for cellular transport of virus through aphid vectors.     

Distribution of Potato virus Y in potato plant organs, tissues, and cells

The distribution of Potato virus Y (PVY) in the systemically infected potato (Solanum tuberosum) plants of the highly susceptible cultivar Igor was investigated. Virus presence and accumulation was analyzed in different plant organs and tissues using real-time polymerase chain reaction and transmission electron microscopy (TEM) negative staining methods. To get a complete insight into the location of viral RNA within the tissue, in situ hybridization was developed and optimized for the detection of PVY RNA at the cellular level. PVY was shown to accumulate in all studied leaf and stem tissues, in shoot tips, roots, and tubers; however, the level of virus accumulation was specific for each organ or tissue. The highest amounts of viral RNA and viral particles were found in symptomatic leaves and stem. By observing cell ultrastructure with TEM, viral cytoplasmic inclusion bodies were localized in close vicinity to the epidermis and in trichomes. Our results show that viral RNA, viral particles, and cytoplasmic inclusion bodies colocalize within the same type of cells or in close vicinity.     

Seasonal dynamics and virus translocation of Grapevine leafroll‐associated virus 3 in grapevine cultivars

Grapevine leafroll‐associated virus 3 (GLRaV‐3) is associated with grapevine leafroll disease, one of the most economically important viral diseases of grapevines. This disease impacts on both vine health and grape quality; reduction in yield, brix and wine colour are among its detrimental effects. Many methods, including serological and molecular procedures, have been developed for the detection of GLRaV‐3; however, there is no PCR‐based assay available to quantify virus populations within plant tissues. A real‐time RT‐PCR assay with TaqMan probe was developed for specific and reliable quantitative detection of GLRaV‐3 in infected tissues. The designed primers and probes target the conserved sequence in the RNA‐dependent RNA polymerase (RdRp) domain of the viral genome to prevent amplification of most subgenomic and defective RNAs. This protocol was used to examine the seasonal dynamics and translocation of GLRaV‐3 in field‐grown grapevines. The results showed that the virus spread quickly from trunks to new growing shoots and leaves early in the growing season, and most samples still harboured detectable virus during late summer and autumn. The seasonal progress of one GLRaV‐3 isolate was compared in four grapevine cultivars (Chardonnay, Cabernet Sauvignon, Italia and Thompson Seedless). Within cultivars there was little variability in the distribution and translocation of GLRaV‐3, except for in Thompson Seedless. This quantitative detection assay will be a valuable tool for GLRaV‐3 diagnosis, disease monitoring and population ecology studies.     

Possible correlations between the characteristics of Potato leafroll virus isolates occurring in different geographical regions in Tunisia

Biological and molecular characterization supported by transmission efficiency, symptom expression and Open Reading Frame 0 (ORF0) nucleotide sequence analysis were carried out to assess nine isolates of Potato leafroll virus (PLRV) collected from three Tunisian geographic and bioclimatic zones. Plant-to-plant transmission by Tunisian Myzus persicae aphid clones showed high transmission efficiency for all isolates tested. Symptom expression analysis on a Physalis floridana plant test distinguished viral isolates as very severe, severe and mild. The ORF0 sequences of the Tunisian PLRV isolates showed an assignment to two aggregates when compared with GenBank PLRV sequences. A significant correlation between symptom severity and ORF0 nucleotide sequence or between symptom severity and geographic origins of the PLRV isolates was established. However, the transmission efficiency and the ORF0 sequence were not affected by the bioclimatic origin. No significant correlation between transmission and symptom or between transmission and the ORF0 sequence was detected.     

深度测序发现贵阳发生的辣椒病毒病由多种病毒复合侵染所致

2016年9月贵州省贵阳市发生严重的辣椒病毒病,症状复杂,主要表现为植株矮化,叶片黄化、花叶、皱缩、畸形以及枯死斑,果实有坏死斑等,根据症状难以判断病毒种类。本文采用小RNA深度测序技术对田间自然发病的2株辣椒标样进行了毒源鉴定,发现样品1由蚕豆萎蔫病毒2号(Broad bean wilt virus2,BBWV2)、辣椒脉斑驳病毒(Chilli veinal mottle virus,ChiVMV)、黄瓜花叶病毒(Cucumber mosaic virus,CMV)和辣椒内源RNA病毒(Bell pepper endornavirus,BPEV)4种病原复合侵染;样品2中除鉴定到上述4种病毒外,还检测到马铃薯Y病毒(Potato virusY,PVY)。进一步通过反转录PCR(RT-PCR)对深度测序结果进行了验证,证明其准确可靠。其中4个辣椒标样中均有的辣椒内源RNA病毒(BPEV)为贵州省首次报道。多种病毒复合侵染是辣椒产量和品质的重要限制因素之一,是辣椒生产的主要威胁。     

Incidence of potato viruses and characterisation of <Emphasis Type="Italic">Potato virus Y</Emphasis> variability in late season planted potato crops in Northern Tunisia

Surveys were conducted of symptomatic potato plants in late season crops, from the major potato production regions in Northern Tunisia, for infection with six common potato viruses. The presence of Potato leafroll virus (PLRV), Potato virus Y (PVY), Potato virus X (PVX), Potato virus A (PVA), Potato virus S (PVS) and Potato virus M (PVM) was confirmed serologically with virus infection levels up to 5.4, 90.2, 4.3, 3.8, 7.1 and 4.8%, respectively. As PVY was prevalent in all seven surveyed regions, further biological, serological and molecular typing of 32 PVY isolates was undertaken. Only one isolate was shown to induce PVY^O-type symptoms following transmission to tobacco and to react only against anti-PVY^O-C antibodies. Typical vein necrosis symptoms were obtained from 31 samples, six of which reacted against both anti-PVY^N and anti-PVY^O-C antibodies showing they contained mixed isolates, while 25 of them reacted only with anti-PVY^N antibodies. An immunocapture RT-PCR molecular test using a PVY^NTN specific primer pair set in the 5’NTR/P1 genomic region and examination of recombinant points in three genomic regions (HC-Pro/P3, CI/NIa and CP/3’NTR) showed that all 25 serotype-N PVY isolates were PVY^NTN variants with similar recombinations to the standard PVYNTN-H isolate. This is the first report of the occurrence of the PVY^NTN variant and its high incidence in late season potatoes in Tunisia.     

Diagnosis and molecular analysis of Potato leafroll virus isolates in Tunisia

Potato leafroll virus (PLRV) is a major constraint to potato production in North Africa. Serological (sandwich and cocktail ELISA) and molecular (RT-PCR) tests were used to detect PLRV in 131 potato samples collected in different areas of Tunisia. RT-PCR proved to be usable as a routine diagnostic test for epidemiological purposes, being more sensitive and reliable, and less time-consuming, than serological tests. One RT-PCR-amplified portion of ORF3 (336 nt) was cloned and sequenced, and used for molecular characterization of Tunisian PLRV isolates. These showed high sequence identity with PLRV retrieved from GenBank.     

Localization of potato leafroll virus in leaves of secondarily-infected potato plants

Potato leafroll virus (PLRV) antigen was localized by immunogold labelling in semi-thin leaf sections of secondarily-infected potato plants cv. Bintje. Viral antigen was present in all cell types of the phloem tissue. but occurred most abundantly in the companion cells. Detectable amounts of PLRV antigen were found only in the sieve elements in veins with a large number of infected companion cells. Occasionally, parenchyma cells were also found to be infected. PLRV was not exclusively limited to the phloem tissue in the infected potato plants, but was also found in mesophyll cells neighbouring minor phloem vessles. Spread of virus from cell to cell in the mesophyll was not observed. The distribution of PLRV in the potato leaf tissue has implication on its availability, for acquisition by aphids.     

Molecular Detection of Phytophthora ramorum, the Causal Agent of Sudden Oak Death in California, and Two Additional Species Commonly Recovered from Diseased Plant Material

ABSTRACT Sudden oak death is a disease currently devastating forest ecosystems in several coastal areas of California. The pathogen causing this is Phy-tophthora ramorum, although species such as P. nemorosa and P. pseudo-syringae often are recovered from symptomatic plants as well. A molecular marker system was developed based on mitochondrial sequences of the cox I and II genes for detection of Phytophthora spp. in general, and P. ramorum, P. nemorosa, and P. pseudosyringae in particular. The first-round multiplex amplification contained two primer pairs, one for amplification of plant sequences to serve as an internal control to ensure that extracted DNA was of sufficient quality to allow for polymerase chain reaction (PCR) amplification and the other specific for amplification of sequences from Phytophthora spp. The plant primers amplified the desired amplicon size in the 29 plant species tested and did not interfere with amplification by the Phytophthora genus-specific primer pair. Using DNA from purified cultures, the Phytophthora genus-specific primer pair amplified a fragment diagnostic for the genus from all 45 Phytophthora spp. evaluated, although the efficiency of amplification was lower for P. lateralis and P. sojae than for the other species. The genus-specific primer pair did not amplify sequences from the 30 Pythium spp. tested or from 29 plant species, although occasional faint bands were observed for several additional plant species. With the exception of one plant species, the resulting amplicons were smaller than the Phytophthora genus-specific amplicon. The products of the first-round amplification were diluted and amplified with primer pairs nested within the genus-specific amplicon that were specific for either P. ramorum, P. nemorosa, or P. pseudo-syringae. These species-specific primers amplified the target sequence from all isolates of the pathogens under evaluation; for P. ramorum, this included 24 isolates from California, Germany, and the Netherlands. Using purified pathogen DNA, the limit of detection for P. ramorum using this marker system was approximately 2.0 fg of total DNA. However, when this DNA was spiked with DNA from healthy plant tissue extracted with a commercial miniprep procedure, the sensitivity of detection was reduced by 100- to 1,000-fold, depending on the plant species. This marker system was validated with DNA extracted from naturally infected plant samples collected from the field by comparing the sequence of the Phytophthora genus-specific amplicon, morphological identification of cultures recovered from the same lesions and, for P. ramorum, amplification with a previously published rDNA internal transcribed spacer species-specific primer pair. Results were compared and validated with three different brands of thermal cyclers in two different laboratories to provide information about how the described PCR assay performs under different laboratory conditions. The specificity of the Phytophthora genus-specific primers suggests that they will have utility for pathogen detection in other Phytophthora pathosystems.