复合RT-PCR方法同步检测百合X病毒、百合无症病毒及百合斑驳病毒

建立了特异性检测百合X病毒(LVX)、百合无症病毒(LSV)及百合斑驳病毒(LMoV)的单一、双重及三重PCR体系并对各体系的灵敏度进行了研究。结果表明,单一PCR体系可检测的LVX最低浓度在1×10^-7μg/mL,LSV最低浓度在1×10^-8μg/mL,LMoV最低浓度在1pg/mL;双重PCR体系可明显检测的LVX、LSV最低浓度在1pg/mL,LSV、LMoV最低浓度在1ng/mL,LVX、LMoV最低浓度在1pg/mL;三重PCR体系可明显检测的LVX、LSV和LMoV的最低浓度为1ng/mL。     

A study of viral coat protein accumulation in lily chloroplasts from mixed virus infections of Lily mottle virus and Cucumber mosaic virus

Two viruses that frequently occur in many Lilium species are Lily mottle virus (LMoV) and Cucumber mosaic virus (CMV), which usually co-infect lilies causing severe disease symptoms. Recent reports have revealed that the viral coat protein (CP) affects chloroplast ultrastructure and symptom development. This study used western blot analysis to confirm that in leaves infected by mixed virus infections of LMoV and CMV, CPs of both viruses were accumulated in lily chloroplasts. Immunogold labelling further demonstrated that both the LMoV CP and CMV CP were localized in the stroma and the thylakoid membranes of the chloroplasts. In addition, it was found that CPs of both viruses were rapidly transported into isolated, intact chloroplasts (in vitro), and their transport efficiencies were positively related to CP concentrations. The lowest transmembrane concentration of CMV CP decreased from 38 μg mL⁻¹ recorded in the single CMV CP import system to 10 μg mL⁻¹ in the mixed import system of LMoV CP and CMV CP. CPs of both viruses exhibited species selection in their transmembrane transport into chloroplasts. This is the first report that the CPs from two viruses (LMoV and CMV) are simultaneously present in lily chloroplasts. Accumulation of high levels of LMoV CP and CMV CP inside the chloroplast appears to contribute to a synergistic interaction inducing the development of mosaic symptoms.     

侵染白附子的芋花叶病毒分子变异研究

为明确侵染白附子的芋花叶病毒(dasheen mosaic virus, DsMV)的分子变异情况,对51个DsMV白附子分离物(DsMV-BF)的外壳蛋白(Coat Protein,CP)基因和3个分离物的近全长基因组序列进行了克隆和测定,DsMV-BF的CP基因大小有855个和942个核苷酸两种类型,51个白附子分离物之间CP基因的核苷酸和氨基酸一致率分别为88.3%～100%和91.9%～100%,BF8、BF30和BF38分离物之间多聚蛋白的核苷酸和氨基酸序列一致率分别为82.9%～95.9%和90.7%～95.9%,与GenBank中其他分离物之间多聚蛋白的核苷酸和氨基酸序列一致率分别为76.9%～99.4%和85.6%～99.0%;P1基因的分子变异较大,P1基因大小有987个和990个核苷酸两种类型;CP基因核苷酸序列系统进化树分析结果表明,侵染白附子的DsMV分离物可分为两个亚组;重组分析结果表明BF8和BF30分离物各检测到1个重组事件,BF38检测到2个重组事件。     

Evolutionary Characterization of Two Lily Isolates of Cucumber mosaic virus Isolated in Japan and Korea

We analyzed the evolutionary histories of two lily strains of Cucumber mosaic virus (CMV) isolated in Japan and Korea (HL- and Ly2-CMVs). They share common biological characteristics in that their host ranges are very restricted perhaps from a unique adaptation to lily plants. Although HL and Ly2 were isolated independently from different lily species in separate countries, their RNA3 sequences had a very high sequence similarity (97%). The evolutionary relationships between the two isolates were characterized by comparing their phylogenetic trees for the 3a and CP genes. The two lily CMVs always formed a distinct cluster within subgroup IB in 3a, but within IA in CP. Together, the phylogenetic tree topology and the sequence identity between the two lily CMVs suggest that they evolved from a common progenitor. Received 5 November 2001/ Accepted in revised form 11 January 2002     

两个马铃薯Y病毒黑龙江马铃薯分离物株系鉴定

为明确分离自黑龙江省克山县马铃薯上的2个病毒分离物KS4和KS7的分类地位,通过RTPCR扩增、克隆获得其基因组序列,利用重组分析程序包和最大似然法分别进行重组分析和系统发育分析。结果显示,分离物KS4和KS7的开放阅读框均有9 186个核苷酸,编码3 061个氨基酸,分离物KS4的核苷酸和氨基酸序列均与马铃薯Y病毒(potato virus Y,PVY)分离物Mb112一致率最高,分别为96.9%和98.4%;分离物KS7的核苷酸序列与PVY分离物12-94一致率最高,为97.4%,其氨基酸序列与PVY分离物SYR-Ⅱ-Be1一致率最高,为97.8%。重组分析表明,分离物KS4和KS7均为分离物N-605和Oz的重组体,其中KS4基因组5′-端的2 392个核苷酸来自分离物N-605,其余核苷酸来自分离物Oz;KS7基因组的第800～2 227个核苷酸和第5 637～8 950个核苷酸来自分离物N-605,其余核苷酸来自分离物Oz。系统发育分析发现,分离物KS4被聚类到N:O株系(PVY~(N:O)),分离物KS7被聚类到NTN株系(PVY~(NTN))b型。     

百合无症病毒对百合光合生理、酶活性及叶绿体超微结构的影响

 以东方百合“西伯利亚”为试验材料,研究百合无症病毒(LSV)侵染百合对其叶片生理生化以及叶绿体超微结构的影响。检测结果表明:叶片中叶绿素a、b以及总叶绿素含量与健康对照相比分别下降了28.6%、33.3%和23.5%,净光合速率、气孔导度及胞间CO₂浓度分别下降33.3%、25%和13.8%;超氧化物歧化酶(SOD)、过氧化物酶(POD)、多酚氧化酶(PPO)和苯丙氨酸解氨酶(PAL)与健康对照相比,分别增加了16.6%、29.4%、16.7%和22.2%。电镜观察发现:感病植株叶绿体膨胀变形,基质片层散乱,叶绿体内淀粉粒肿大且数目增多,从而证明LSV侵染破坏叶绿体结构,影响植株的光合作用。     

白草花叶病毒承德玉米分离物3′-cDNA 片段序列分析

 采自河北承德11 个表现矮花叶症状的玉米样品,用甘蔗花叶病毒(Sugarcane mosaic virus, SCMV)和白草花叶病毒
(Pennisetum mosaic virus, PenMV)简并引物扩增了基因组3′ 端约2. 1 kb 的片段并进行测序。Blast 结果表明其中8 个样
品含有PenMV。扩增到的PenMV 序列均为2 135 nt,包括部分NIb 基因(985 nt)、完整的CP 基因(909 nt)和3′-UTR(241
nt)。这8 个分离物CP 基因和3′-UTR 与GenBank 上其他PenMV 分离物相应序列的核苷酸一致率分别为89. 8% ~ 93． 4%
和95. 9% ~ 97. 9% 。根据扩增的2 135 nt 序列和CP 基因序列构建系统发育树,8 个分离物与GenBank 上其他PenMV 分离
物都分为2 个组:山西组和承德组。重组分析表明CD9 的CP 基因存在重组。     

Genetic variability and molecular evolution of arabis mosaic virus based on the coat protein gene sequence

Arabis mosaic virus (ArMV) is an important pathogen infecting a wide range of plant species worldwide. In the present study, we carried out exhaustive phylogenetic analyses based on the coat protein (CP) sequence of ArMV isolates originating from different host plants and geographic regions. Maximum-likelihood reconstruction revealed the presence of three distinct, well-supported phylogroups. BaTS analyses indicated that the diversity of ArMV isolates may be correlated with both host plant and geographic origin. Moreover, population genetic analyses showed significant differences in the level of genetic diversity among variants within ArMV that originated from rhubarb plants, as expected by quasispecies. Further analysis revealed that both selection and recombination are shaping the population structure of ArMV. In total 14 groups of residues were detected as coevolving for different amino acid properties.     

马铃薯Y病毒N-Wi株系广西分离物的鉴定

 马铃薯Y病毒 (potato virus Y,PVY) 是一种重要的农作物病毒,可造成产量损失和产品质量下降。其宿主范围广泛,包括马铃薯、烟草、番茄和辣椒等经济作物。在广西从叶片表现斑驳褪绿症状的马铃薯上分离到一株PVY分离物DX,其基因组包含一个大的开放阅读框 (open reading frame,ORF),由9 186 nt组成,编码3 061个氨基酸。系统发育进化分析显示,分离物DX与PVY^N-Wi株系分离物IUNG-12、SGS-AG、MAF-VOY聚类成一个分支。重组分析表明分离物DX基因组在496 nt和2 388 nt存在重组位点,分别位于P1和HC-Pro/P3结合区,是分离物Oz和N605的重组体。通过机械摩擦接种,分离物DX可侵染茄科9种作物,引起本生烟叶片花叶、皱缩和泡状突起等症状;引起普通烟草和番茄的轻微花叶症状;引起马铃薯叶片花叶症状,接种辣椒没有发病。序列比对分析显示,分离物DX缺乏引起烟草叶脉坏死相关的氨基酸位点N₂₀₅、K₄₀₀和E₄₁₉。本研究比较了分离物DX对茄科共12种作物的侵染能力和病害症状差异,结果表明分离物DX可用于探索PVY在不同寄主中的致病机理。     

Diversity of the coat protein-coding region among Ilarvirus isolates infecting hop in Australia

Coat protein (CP) sequences of 17 Ilarvirus isolates were obtained from hops at three farms in Tasmania, Australia. Phylogenetic analysis of these sequences and additional database sequences indicated several Apple mosaic virus (ApMV) isolate clusters distinct from Prunus necrotic ringspot virus (PNRSV): one containing isolates from apple; one containing a single isolate from almond; a third containing Australian hop isolates of the 'apple' serotype and a German isolate of unknown origin; and a fourth containing Australian hop isolates of the 'intermediate' serotype. Isolates from hop, pear and prune from the Czech Republic either formed a fifth grouping, or were divergent members of the 'intermediate' serotype group. Deduced amino acid (aa) residue differences between the coat proteins of the two hop isolate serotype groups were highlighted as possible regions of serological differentiation. No evidence for coinfection of plants with both serotypes was found. Tests of ApMV-infected hop buds using the Shirofugen flowering cherry assay revealed a possible differentiation of the two strains based on hypersensitivity. Because of serological similarities to PNRSV, these viruses have commonly been reported as strains of PNRSV. However, this study shows ilarviruses from Australian hops are strains of ApMV, but distinct from those infecting Malus spp.     

小西葫芦黄花叶病毒山东南瓜分离物的分子特性

 Zucchini yellow mosaic virus (ZYMV) was detected by RT-PCR from pumpkin (Cucurbita moschata) plant showing yellowing and mosaic symptom from Liaocheng, Shandong Province. The 3'-termial 1 684 bp genomic sequence covered 633 bp of NIb encoding sequence, 840 bp of cp gene and 211 bp of 3'-untranslated region of the isolate ZYMV-Liaocheng was determined. The cp gene of ZYMV-Liaocheng shared identities of 81.4%-98.8% and 89.4%-99.5% at nucleotide and amino acid levels, respectively, with other ZYMV sequences available in the GenBank. Phylogenetic analysis indicated that ZYMV could be clustered to 6 genotypes. ZYMV-Liaocheng belonged to genotypeⅠ, which contained isolates from Asia, Europe and America. Genotypes Ⅲ and Ⅴ were unique and contained only isolates from East Asia. The isolates from East Asia had the highest variability.     

小麦黄花叶病毒(WYMV)2个山东分离物的全基因组序列及进化分析

利用5'RACE结合一步法RT-PCR分别从山东泰安与临沂冬小麦上克隆了小麦黄花叶病毒(Wheat yellow mosaic virus,WYMV)的全基因组序列。这2个分离物(TADWK和LYJN)基因组间核苷酸一致性分别为97.21%(RNA1)和95.12%(RNA2)。通过对目前已报道的共14个分离物的基因组不同部分的分析,表明5'UTR是WYMV基因组变化幅度最大的区域,而编码区(ORF)及3'UTR的序列一致性较高且变动幅度小。另外,发现LYJN RNA2的5'UTR与已报道的所有分离物RNA2的核苷酸一致率仅为90%左右。综合分析RNA1和RNA2的系统发生树,表明WYMV各基因组片段呈单独进化特征,不同分离物间存在RNA重排。RNA重组分析显示在LYJN RNA1和TADWK RNA2发现了RNA重组。此研究说明WYMV在山东地区存在分离物分化现象,WYMV的5'UTR是基因组中的突变热点。     

马铃薯Y病毒中国分离物的分子株系组成

 马铃薯Y病毒(potato virus Y,PVY)主要侵染马铃薯和烟草等茄科作物,给世界农业造成巨大经济损失。本文对测定的23个及GenBank中注册的52个中国PVY分离物ORF序列进行了系统发育、重组和选择压等分析。系统发育分析表明,根据ORF序列可把我国75个PVY分离物和国外30个参比分离物分成O、C、E、NTN-NW(SYR-I型)、NTN-NW(SYR-II型)、NTN(NTN-a型)、NTN(NTN-b型)、NA-N/NTN、Eu-N、N-Wi(N:O型)和N-Wi(N-Wi型)等11个分子株系,其中中国PVY分离物属于除E和C株系外的9个分子株系。除ME162、guiyang、PVYzu、SD-G、WA-13和CN:JL-1:17等 6个分离物基因组中未检测到重组,其余69个分离物均存在明显重组。根据重组位点的不同,中国PVY可分为11种重组类型,其中5种为新的重组类型。选择压分析表明,中国PVY分离物的11个基因均处于负选择,其中核内含体b基因受到的选择压最大,PIPO受到的选择压最小。基因流分析表明,黑龙江、河南和山东PVY分离物间基因交流频繁,马铃薯与烟草PVY分离物之间基因交流频繁。本研究的结果明确了中国PVY分离物的分子株系组成,对指导PVY的检测和防控具有积极作用。     

来源于猕猴桃的柑橘叶斑驳病毒的RT-PCR检测及外壳蛋白基因序列分析

柑橘叶斑驳病毒(Citrus leaf blotch virus,CLBV)为柑橘病毒属(Citrivirus)代表种。本研究采用RT-PCR技术从我国栽培猕猴桃上首次检测到CLBV,总检出率为13.3%。测定了5个CLBV分离物的外壳蛋白基因(coat protein gene,cp)序列,全长均为1 092核苷酸(nt),分离物间cp基因核苷酸和编码氨基酸序列相似性分别为83.2%~99.7%和91.9%~98.9%,这些分离物与新西兰报道的CLBV猕猴桃分离物M3-A核苷酸序列相似性仅为83%左右,与来源于柑橘及近缘种的CLBV分离物cp基因核苷酸序列相似性为84%~86%。在基于该病毒cp基因核苷酸序列的系统进化树中,本研究所测定的CLBV猕猴桃分离物与新西兰报道的除M3-A外的猕猴桃分离物聚在同一组群。研究结果为进一步明确CLBV在我国猕猴桃上的侵染和危害特点及建立该病毒的分子检测技术提供了重要信息。     

引起玉米粗缩病的水稻黑条矮缩病毒山东分离物的分子特性

 Four isolates of Rice black-streaked dwarf virus (RBSDV) were collected from the maize plants showing rough dwarf symptom in Linyi and Tai'an,Shandong province.The S10 genomic sequences of these isolates were determined and compared with those of 14 other RBSDV isolates.All of the four sequences were 1 801 base pairs (bp) long including the 5'-UTR of 21 bp and the 3'-UTR of 103 bp.They all contained an open reading frame of 1 677 bp (22-1698),encoding the coat protein (CP) of 558 amino acids.The sequences of these four RBSDV isolates and those of the major cp gene of 14 other isolates available in the GenBank were divided into two groups in the phylogenetic tree.Recombination analysis indicated that the isolate Lym2 was likely a recombinant of isolates Lym1 and Zhjs.     

西瓜花叶病毒2号新疆昌吉分离物外壳蛋白基因核苷酸序列分析

 利用RT-PCR从新疆昌吉地区表现花叶、疱斑、扭曲等症状的南瓜病株上检测到西瓜花叶病毒2号新疆昌吉分离物(简称WMV-2-XJ-CJ),并测定了该分离物外壳蛋白(CP)基因序列。序列分析表明,新疆昌吉分离物CP基因全长850个核苷酸,编码197个氨基酸。与国内外报道的12个WMV-2CP基因相比,其核苷酸序列同源性为92.6%~98.3%,由此推导的氨基酸序列同源性为94.7%~99.3%。新疆昌吉分离物在CP N'端可变区明显不同于国内外报道的核苷酸序列。WMV-2新疆昌吉分离物与日本和郑州分离物较其它国家和地区的分离物多出6个核苷酸,但其核苷酸及其推导的氨基酸序列差异较大。新疆昌吉分离物外壳蛋白有2个氨基酸残基明显不同于其它分离物,其中蚜传株系的特征结构域DAG突变为DAE。     

广东省国兰病毒病害调查及CymMV和ORSV基于cp基因的系统进化分析

为调查广东省国兰病毒病发生情况,采用双抗体夹心-酶联免疫吸附测定(double sandwich enzyme-linked immunosorbent assay,DAS-ELISA)法对采集自广州市、汕头市和韶关市兰花主产区的53份具有典型病毒病症状的国兰叶片进行检测,选取35份病叶应用RT-PCR技术对检测结果进行验证,并基于cp基因序列对检测到的病毒进行系统进化分析。DAS-ELISA检测结果显示,53份病叶中有44份检出病毒,总检出率为83.02%;其中,有14份仅检出建兰花叶病毒(Cymbidium mosaic virus,CymMV),有19份仅检出齿兰环斑病毒(Odontolossum ring spot virus,ORSV),有11份检出CymMV和ORSV复合侵染,检出率分别为26.42%、35.85%和20.75%。RT-PCR检测结果显示,35份病叶中有30份检出病毒,总检出率为85.71%;其中,有21份仅检出CymMV,有6份仅检出ORSV,有4份检出CymMV和ORSV复合侵染,检出率分别为60.00%、17.14%和11.43%,但未检测到其它病毒。表明广东省国兰病毒病害主要由CymMV和ORSV侵染引起。序列和系统进化分析显示,来源于不同地区和寄主的CymMV和ORSV分离物的cp基因序列极为保守,CP氨基酸序列相似性分别大于97.31%和93.67%,且CymMV和ORSV多样性种群的分化与寄主种属和地理隔离关系不大。     

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?     

潍坊萝卜红心病病原鉴定

 本文用生物学、血清学和分子生物学证据证明,引起潍坊萝卜红心病的病原为芜菁花叶病毒(Turnip mosaic virus,TuMV).该病毒可系统侵染曼陀罗、油菜、咸阳黄瓜、丝瓜、大白菜和普通烟,局部侵染苋色藜和假酸浆,不侵染豌豆.病毒粒体弯曲线状,长约700 nm,可由蚜虫传播,在红心病组织内形成风轮状和片层凝集状内含体,它在SDS-琼脂糖凝胶免疫双扩散试验中可与TuMV的抗血清形成明显的沉淀线.该病毒的CP基因共867个核苷酸,编码288个氨基酸,分子量为32.98 kD.该序列与国内外20个TuMV分离物的CP氨基酸序列比较结果表明,这些分离物可以分为5组,其中引起萝卜红心病的病毒与日本的H1J、KYD8lJ、意大利的ITA7同属一组.     

侵染猕猴桃的柑橘叶斑驳病毒外壳蛋白多克隆抗体的制备及检测应用

柑橘叶斑驳病毒(Citrus leaf blotch virus,CLBV)是β线形病毒科(Betaflexiviridae)柑橘病毒属(Citrivirus)的代表种,前期研究表明CLBV在陕西栽培猕猴桃中广泛发生.为进一步简化CLBV的检测,并探究其致病机制,通过RT-PCR扩增得到CLBV猕猴桃分离物外壳蛋白(c...