利用小RNA深度测序技术鉴定半夏病毒病病原

RNA沉默是真核生物体内由病毒来源的干扰小RNA（virus derived small interfering RNA, vsiRNA）沉默复合物介导目标RNA特异降解的一种保守机制,通过对vsiRNA分析可进行植物病毒病原鉴定。本文利用小RNA深度测序技术对感病半夏叶片进行鉴定,结果发现,表现典型花叶症状的半夏叶片受到大豆花叶病毒（Soybean mosaic virus, SMV）、黄瓜花叶病毒（Cucumber mosaic virus, CMV）、芋花叶病毒（Dasheen mosaic virus, DsMV）、魔芋花叶病毒（Konjac mosaic virus, KoMV）、烟草花叶病毒（Tobacco mosaic virus, TMV）等多种病毒的复合侵染。为明确SMV山西半夏分离物（SMV-SXBX）的进化关系,进行SMV-SXBX全基因组克隆与分析,获得SMV-SXBX全长为9 735 nt,编码一个由3 105个氨基酸组成的多聚蛋白质。通过核苷酸与氨基酸序列比对发现,SMV-SXBX与半夏分离物P同源性最高,分别为91.1%和94.1%,且系统发育分析表明,SMV-SXBX与半夏SMV分离物P聚为一簇。同时,也对vsiRNA进行了系统分析,研究结果有望为半夏SMV的有效防治提供一定科学依据。     

利用小RNA深度测序技术检测分析小黄姜病毒

对贵州省六盘水市田间发病小黄姜进行病毒检测,分析小黄姜病毒种类。采用小RNA高通量测序技术结合生物信息学方法对小RNA数据库进行拼接组装,预测小黄姜病毒种类。设计特异性引物,采用反向PCR及分段克隆测序方法验证小黄姜病毒的存在。检测发现有3条拼接序列与DNA杆状病毒属(Badnavirus)中的薯蓣杆状病毒(Dioscorea bacillifrom virus,DBV)相似,氨基酸相似度均高于80%。并在小黄姜样品中扩增得到1 883 bp病毒序列。RT和RNase H基序核苷酸序列与来自古巴的Banana streak virus(Gen Bank登录号:KF386733)相似性最高,为74%。根据目前的国际病毒分类委员会(the international committee on taxonomy of viruses,ICTV)病毒分类方案,Badnavirus病毒中RT和RNase H核苷酸基序相似度低于80%的可划为不同病毒种,该病毒可能是Badnavirus中的一种新病毒,暂命名为小黄姜杆状病毒(Zingiber bacilliform virus)。小黄姜杆状病毒在30个田间小黄姜样品中存在且存在多态性,该病毒可能是一个古老的病毒,在小黄姜进化过程中将病毒基因组整合到小黄姜基因组中来防御杆状病毒属病毒的侵染。     

蚕豆萎蔫病毒2号板蓝根分离株全基因组序列测定与分析

板蓝根(Isatidis Radix)病毒病害的发生已对其产量和品质造成了严重影响。因此,建立一套灵敏、快速、有效的板蓝根病毒病害检测手段十分重要。本研究利用双链RNA(double-stranded RNA,dsRNA)和非序列依赖PCR扩增(sequence-independent amplification,SIA)等技术对感病板蓝根进行鉴定,确定其被蚕豆萎蔫病毒2号(Broad bean wilt virus 2, BBWV2)所侵染。为明确BBWV2板蓝根分离物(BBWV2-IR)的进化关系,对其全基因组进行序列扩增与分析,获得其RNA1序列全长为5 955 bp,RNA2序列全长为3 602 bp,分别编码由1 870和1 064个氨基酸组成的多聚蛋白质。序列比对发现,BBWV2-IR RNA1与BBWV2-Am分离物的同源性最高,RNA2与BBWV2-SN分离物的同源性最高。全基因组变异情况分析表明,BBWV2-IR RNA1和RNA2分别与其同源性最高的株系存在多个氨基酸变异位点。系统进化分析表明,BBWV2-IR RNA1与BBWV2-Am RNA1聚为一簇,RNA2与BBWV2-SN RNA2聚为一簇,亲缘性最近。本研究获得了BBWV2板蓝根分离株的全基因组序列,并明确其在进化过程中的地位和区域变化情况,为进一步研究BBWV2-IR致病性变异提供一定的理论基础。     

山东省玉米矮花叶病毒的生物学特性及基因组全序列测定

对山东省玉米矮花叶病毒原分离物(SD)进行了寄主范围、血清学等普通生物学鉴定,测定了该病毒的基因组核苷酸全序列。该病毒基因组RNA由9596个核苷酸组成(不包括3’—polyA的长度),整个基因组按一个ORF编码一个3063个氨基酸的多聚蛋白。序列比较表明,该病毒分离物(SD)与玉米矮花叶病河南分离物(HN,EMBL登录号：AF94510)核苷酸全序列同源性最高,为98．2％,与已报道的甘蔗花叶病毒(SCMV)7个分离物同源性也高达79．5％-98．2％,但与玉米矮花叶病毒保加利亚分离物(MDMV—B8,AJ001691)和高梁花叶病毒萧山甘蔗分离物(SrMV—XoS,AJ310197)的同源性仅为67．8％和69．3％,与约翰逊草花叶病毒(226920,JGMV)差异最大。系统进化树分析也表明,该病毒与SCMV分离物位于同一进化簇,而与MDMV进化关系很远。     

蚕豆萎蔫病毒2号山西分离株的全基因组序列测定与分析

本试验在生物学接种的基础上,利用非序列依赖性PCR扩增（sequence-independent amplification,SIA）对感病青椒（Capsicum annuum）进行了分子鉴定. 序列测定及分析发现,侵染青椒的病毒为蚕豆萎蔫病毒2号（Broad bean wilt virus 2, BBWV2）. 为明确BBWV2青椒分离物（BBWV2 Ca）的分类地位,对BBWV2 Ca的全基因组序列进行了分段克隆、序列测定和分析. BBWV2-Ca RNA1全长5 929 bp,含有1个ORF. BBWV2 Ca RNA2全长3 559 bp,含有1个ORF,编码3种蛋白：VP53/VP37、外壳蛋白大亚基（large coat protein, LCP）和外壳蛋白小亚基(small coat protein, SCP). 全序列核苷酸同源性分析显示,BBWV2-Ca RNA1与BBWV2其它分离物核苷酸同源性在77.9%～93.7%之间|RNA2与BBWV2其它分离物核苷酸同源性在80.2%～93.8%之间. 全序列系统进化分析显示,BBWV2-Ca RNA1与BBWV2-XJ14-3以及BBWV2-RP3株系聚为一簇,亲缘关系最近|RNA2与BBWV2-Am形成一个独立分支,亲缘关系最近.     

猫尾草花叶病相关的新型马铃薯Y病毒属病毒

猫尾草(Uraria crinita)为豆科中草用药植物,亦称狗尾苔,在中国主要分布于福建、江西、广东、海南、广西、云南等地。江西地区发现叶片呈现花叶、黄化等病毒感染的病株。电子显微镜镜检,可观察到长度约800nm的长丝状病毒颗粒,患病细胞内可观察到风车状病毒内含体。机械接种于藜麦进行病毒单离,9～10 d后可观察叶脉处出现坏疽病斑。单离的病毒分离株回接猫尾草并未引起感染。萃取猫尾草患病叶组织总量RNA,配合Potyvirus属病毒简并式引物进行反转录聚合酶连锁反应(RT-PCR)所扩增出的cDNA片段,其序列与大豆花叶病毒(soybean mosaic virus,SMV)和watermelon mosaic virus(WMV)最为相似。解析病毒的全长度基因序列为9651个核苷酸,对应产生一个聚合蛋白。病毒基因结构与Potyvirus属病毒相同。病毒全长度基因核苷酸序列与WMV和SMV的相似度分别为67.6%和70.5%;外鞘蛋白基因核苷酸序列与WMV和SMV的相似度分别为74.0%和74.7%。本研究表明,猫尾草花叶病相关的病毒,应是Potyvirus属病毒的一个新病毒种。     

甜菜黑色焦枯病毒新疆分离物基因组RNA的序列分析及侵染性cDNA克隆构建

甜菜黑色焦枯病毒(Beet black scorch virus,BBSV)新疆分离物(BBSV-X)和宁夏分离物(BBSV-N)分别来自生态环境不同的新疆和宁夏的甜菜产区,自然条件下,BBSV-X含有卫星RNA,BBSV-N不含有卫星RNA。为明确二者的分子差异,以BBSV-X基因组RNA为模板,通过RT-PCR扩增获得了全长cDNA克隆。序列分析显示,BBSV-X基因组RNA全长为3 644个核苷酸,与以往报道的BBSV-N相同,且核苷酸序列一致性高达99.45%。除5′和3′非翻译区各有一个核苷酸发生变异外,核苷酸序列差异主要存在于ORF1通读区和ORF6,氨基酸序列差异仅存在于ORF1的通读区。构建获得了BBSV-X的侵染性cDNA克隆,用体外转录物定量接种了苋色藜(Chenopodiumamaranticolor)和豇豆(Vigna sinensis),结果显示BBSV-X与BBSV-N之间在致病性上存在一定的差异。     

侵染白术的大豆花叶病毒的全基因序列测定及分析

大豆花叶病毒(soybean mosaic virus,SMV)是影响大豆产量与质量的重要病原体,且自然寄主范围窄。本研究对疑似感病白术叶片进行非序列依赖性扩增(sequence-independent amplification,SIA)检测,结果表明,感病白术为SMV侵染,并命名为SMV-Am。为明确其基因组结构特征及系统进化关系,本研究利用双链RNA(double-stranded RNA,dsRNA)提取、RT-PCR及RACE技术对其进行全基因组扩增,并进行生物信息学分析。结果表明,SMV-Am基因组全长9 587 nt,具有显著的马铃薯Y病毒属(Potyvirus)基因组结构特征;核苷酸与氨基酸序列相似性分析表明,SMV-Am与来自中国的SMV-Liaoning分离物相似性最高,核苷酸与氨基酸序列相似性分别为96.57%和98.86%;系统进化分析也显示与SMV-Liaoning分离物亲缘关系最近;对SMV-Am蛋白序列进一步分析发现,存在与功能位点相关的氨基酸突变,经I-TASSER和PyMOL软件进行蛋白建模分析,发现SMV-Am的P1、HC-Pro、P3、6K2、NIa-Pro、NIb蛋白结构均发生不同程度的变化,且P1蛋白最明显;重组分析结果显示,在SMV-Am基因组的6 560~8 950 nt位置存在重组事件,其主要亲本和次要亲本分别为SMV-XFQ012(GenBank登录号:KP710875.1)和SMV-pCB301-SC15(GenBank登录号:MH919386.1)。这是SMV侵染白术的首次报道,研究结果有望为防范SMV病害在白术上爆发流行以及防治提供科学依据。     

应用深度测序技术鉴定平菇球形病毒

旨在鉴定采集于北京顺义的畸形平菇样品中的病毒,提取总RNA,构建小RNA库,对小RNA库进行深度测序。测序结果表明,与平菇球形病毒(Oyster mushroom isometric virus,OMSV)基因组序列(NC_004560.1)匹配的小RNA共有3 075条,分布在OMSV基因组的不同位置,形成一些热点区域。经拼接,获得了3条长度大于500个核苷酸的序列。根据其中覆盖OMSV外壳蛋白(Coat protein,CP)基因的序列(seq22)中相对保守区域,设计引物,利用RT-PCR扩增获得cp基因部分序列。序列比对表明,其与OMSV相应区域序列相似度为91%,证实了OMSV在畸形平菇样品中的侵染。同时,将深度测序结果与现有4种si RNA预测软件的预测结果进行比较,发现测序结果与软件预测结果之间存在差异,讨论了这一差异出现的原因。     

我国禽脑脊髓炎病毒分离株全基因组的测定

测定了我国禽脑脊髓炎病毒(avian encephalomyelitis virus,AEV)分离株L2Z株的全基因组核苷酸序列.该病毒株的3′和5′非编码区核苷酸序列用3′和5′RACE(cDNA末端快速扩增)法获得.基因组全长为7 059个核苷酸残基,包括494个核苷酸残基的5′非编码区、6 402个核苷酸残基的开放阅读框和136个核苷酸残基的3′非编码区及poly(A)尾巴.与已发表的AEV疫苗株1 143的基因组序列比较发现,它们之间核苷酸和氨基酸的同源性分别为98%和97.6%.结构蛋白(VP1～VP4)中,主要宿主保护性免疫原蛋白VP1氨基酸之间差异较小.与小RNA病毒科其它病毒属相比,在非结构蛋白3D中,预测的8个RNA依赖性RNA聚合酶主要结构域中的4个高度保守.从而进一步确认了AEV的分子特性.     

黄瓜花叶病毒卫星RNA XJs1侵染性cDNA克隆构建及其生物学功能初步研究

利用RT_PCR的方法,获得了黄瓜花叶病毒卫星RNA XJs1的全长侵染性cDNA克隆pMSC20。序列分析显示,XJs1全长384nt(GenBank登录号:DQ070748),比较XJs1与具有代表性的CMV卫星RNA的序列结构表明,在XJs1核苷酸序列的325nt~350nt间,具有典型的坏死型卫星RNA保守序列。通过体外转录,将XJs1与不含卫星RNA的辅助病毒分离物CMV_AH组合接种普通烟和心叶烟并进行检测。初步研究结果表明,XJs1为一致弱卫星RNA。     

Sequence analysis of a soil-borne wheat mosaic virus isolate from Italy shows that it is the same virus as European wheat mosaic virus and Soil-borne rye mosaic virus

The complete sequence of the two RNAs of a furovirus isolate from durum wheat in Italy was determined. Sequence comparisons and phylogenetic analysis were done to compare the Italian virus with Soil-borne wheat mosaic virus (SBWMV) from the USA and with furovirus sequences recently published as European wheat mosaic virus (EWMV), from wheat in France, and Soil-borne rye mosaic virus (SBRMV), from rye and wheat in Germany. Over the entire genome, the Italian isolate RNA1 and RNA2 had respectively 97.5% and 98.6% nucleotide identity with EWMV, 95.5% and 85.8% with SBRMV-G and 70.6% and 64.5% with SBWMV. The Italian isolate was therefore clearly distinct from SBWMV. The European isolates all appear to belong to the same virus and the name Soil-borne cereal mosaic virus may resolve earlier ambiguities.     

Antiviral RNA silencing is restricted to the marginal region of the dark green tissue in the mosaic leaves of tomato mosaic virus-infected tobacco plants

Mosaic is a common disease symptom caused by virus infection in plants. Mosaic leaves of Tomato mosaic virus (ToMV)-infected tobacco plants consist of yellow-green and dark green tissues that contain large and small numbers of virions, respectively. Although the involvement of RNA silencing in mosaic development has been suggested, its role in the process that results in an uneven distribution of the virus is unknown. Here, we investigated whether and where ToMV-directed RNA silencing was established in tobacco mosaic leaves. When transgenic tobaccos defective in RNA silencing were infected with ToMV, little or no dark green tissue appeared, implying the involvement of RNA silencing in mosaic development. ToMV-related small interfering RNAs were rarely detected in the dark green areas of the first mosaic leaves, and their interior portions were susceptible to infection. Thus, ToMV-directed RNA silencing was not effective there. By visualizing the cells where ToMV-directed RNA silencing was active, it was found that the effective silencing occurs only in the marginal regions of the dark green tissue ( approximately 0.5 mm in width) and along the major veins. Further, the cells in the margins were resistant against recombinant potato virus X carrying a ToMV-derived sequence. These findings demonstrate that RNA silencing against ToMV is established in the cells located at the margins of the dark green areas, restricting the expansion of yellow-green areas, and consequently defines the mosaic pattern. The mechanism of mosaic symptom development is discussed in relation to the systemic spread of the virus and RNA silencing.     

Characterisation of cucumber leaf spot virus isolated from recycled irrigation water of soil-less cucumber cultures

Greenhouse‐grown cucumber plants showed mosaic‐type symptoms and irregular yellow spots on their leaves. The disease did not affect plant growth and the fruits remained symptom free. A virus having isometric particles, 30 nm in size, was isolated from the infected tissues and from recycled drainage water collected from tuff (volcanic rock) raised beds on which plants were grown. The virus was identified as a variant of cucumber leaf spot virus (CLSV) that has a host range similar but not identical to that of a previously described CLSV isolate. The overall nucleotide sequence identity between the RNAs of the Israeli isolate and the type isolate virus (accession numbers: DQ227315 and AY571334, respectively) amounts to 96%.     

An Isolate of Brome Mosaic Virus Specifically Supports the Replication of a Defective Form of its RNA 3

In one German isolate of brome mosaic virus, a defective form of RNA 3 was detected in addition to intact RNA 3. It was propagated only in barley, but was lost in Chenopodium quinoa and depended for its replication on the RNAs of the virus isolate from which it was derived. It did not induce any change of the symptoms produced in barley.     

甜菜坏死黄脉病毒内蒙分离物自然缺失突变体缺失区域的定位分析

以甜菜坏死黄脉病毒内蒙分离物（ＢＮＹＶＶ ＮＭ）总ＲＮＡ为模板,经ＲＴ－ＰＣＲ扩增,分别获得ＲＮＡ２、ＲＮＡ３和ＲＮＡ４自然缺失突变体ｃＤＮＡ克隆。序列分析结果表明,ＲＮＡ２自然缺失突变体在７５ｋＤ通读蛋白编码区Ｃ端缺失３４８个核苷酸（缺失位置ｎｔ１４８８￣ｎｔ１８３５）。ＲＮＡ３在其２５ｋＤ蛋白编码区内缺失３６０个核苷酸（缺失位置ｎｔ７２９￣ｎｔ１０８８）。ＲＮＡ４的自然缺失区域位于３１ｋＤ蛋白     

Symptom-modulating properties of peanut stunt virus satellite RNA sequence variants.

The symptom-modulating properties of three peanut stunt virus (PSV) satellite RNA (satRNA) sequence variants were studied. The (V)-satRNA did not affect symptom development in tobacco plants infected with PSV. The (G)- or (WC)-satRNA, on the other hand, attenuated the symptoms. In these plants, the symptoms of PSV were restricted primarily to the inoculated leaves, and in some cases, a few leaves above the inoculated leaf showed small chlorotic areas. Northern blot analysis of total nucleic acids from PSV-infected plants containing the (V)-satRNA revealed the presence of both satellite and viral RNAs in inoculated leaves as well as in systemically infected leaves. On the other hand, satellite and viral RNAs were detected in the inoculated but not in the noninoculated leaves from infected plants containing either (G)- or (WC)-satRNA. Although a decrease in the quantities of genomic RNAs 1, 2, and 3 was characteristic of all satRNA-containing plants, this effect was more evident in the case of (G)- and (WC)-satRNAs. The complete nucleotide sequences of the three satRNAs were determined and compared to the published sequence of PSV satRNA. The (V)-satRNA differed from the published sequence at two positions, whereas the (G)- and (WC)-satRNAs differed at six and eight positions, respectively. Comparison of the nucleotide sequence of the satRNA having no effect on PSV-induced symptoms with those reducing virus symptoms suggests that a single nucleotide change or as many as five nucleotide changes may distinguish between attenuating and nonattenuating satRNAs.     

Sequence analysis of a soilborne wheat mosaic virus isolate from Italy shows that it is the same virus asEuropean wheat mosaic virus andSoilborne rye mosaic virus

The complete sequence of the two RNAs of a furovirus isolate from durum wheat in Italy was determined. Sequence comparisons and phylogenetic analysis were done to compare the Italian virus withSoilborne wheat mosaic virus (SBWMV) from the USA and with furovirus sequences recently published asEuropean wheat mosaic virus (EWMV), from wheat in France, andSoilborne rye mosaic virus (SBRMV), from rye and wheat in Germany. Over the entire genome, the Italian isolate RNA1 and RNA2 had respectively 97.5% and 98.6% nucleotide identity with EWMV, 95.5% and 85.8% with SBRMV-G and 70.6% and 64.5% with SBWMV. The Italian isolate was therefore clearly distinct from SBWMV The European isolates all appear to belong to the same virus and the nameSoilborne cereal mosaic virus may resolve earlier ambiguities.