烟草脉扭病毒基因组部分序列的克隆和分析

烟草脉扭病毒(Tobacco vein distorting virus,TVDV)是引起烟草丛顶病的两种病毒之一.TVDV被归为黄症病毒科的暂定成员.应用黄症病毒科的通用引物和根据马铃薯卷叶病毒属成员核酸序列设计的简并引物,通过RT-PCR从烟草丛顶病烟株总RNA中扩增到了TVDV基因组的部分序列.序列分析获得了长度为1654 bp的序列,编码推测的TVDV复制酶基因的部分序列、外壳蛋白基因及运动蛋白基因的全部序列.根据这三个基因编码的氨基酸序列构建的分子进化树分析表明,TVDV为黄症病毒科的确定成员.根据其基因间隔区的长度特征和各ORF编码的氨基酸的分子进化分析,我们推测TVDV应当是马铃薯卷叶病毒属的一个新成员.这是TVDV的分子生物学特征的首次报道.     

马铃薯卷叶病毒56kD蛋白基因及3‘端非编码区克隆和序列分析

根据已报道的马铃薯卷叶病毒基因组序列,设计合成一对特异性引物,以马铃薯卷叶病毒中国分离株的ＲＮＡ为模板,反转录合成了ｃＤＮＡ第一条链,经ＰＣＲ扩增后克隆于ｐＵＣ１９质粒中,进一步用ＰＣＲ鉴定,限制酶切分析用序列分析。结果表明,ＰＬＲＶ－Ｃｈ５６ｋＤ蛋白基因由１５２７个核苷酸组成,编码５０８个氨基酸,３‘端非编码区由１４１个核苷酸组成,与国外报道的苏格兰ＰＬＲＶ－Ｓ,荷兰ＰＬＲＶ－Ｎ,加拿大ＰＬＲＶ     

幽影病毒属病毒的研究现状与展望

幽影病毒是一类较为特殊的植物病毒,其基因组不编码外壳蛋白,因此不形成通常的病毒粒体结构。这类病毒可以通过机械摩擦方式传播,在黄症病毒的帮助下也可以通过蚜虫传播。幽影病毒属病毒由7个确定种和3个暂定成员组成,烟草丛顶病毒是目前国内发现的幽影病毒属唯一成员。幽影病毒的寄主范围较窄,体外抗性也较差。幽影病毒感病组织中含有大量dsRNA。有些病毒还含有卫星RNA。幽影病毒的基因组为单分体的 ssRNA,编码4个非结构蛋白,其中的ORF3蛋白在病毒RNA的稳定性及寄主体内的长距离运输中起到非常重要的作用。文章综述了国内外幽影病毒的研究现状,并对未来的相关研究趋势和研究领域进行了展望。     

中国小反刍兽疫病毒分离株China/Tib/07全基因组序列测定与分析

小反刍兽疫病毒属于副黏病毒科麻疹病毒属成员。本研究对我国首次分离的小反刍兽疫病毒株China/Ti-bet/07进行了全基因组序列测定及分子生物学特征分析。根据GenBank公布的小反刍兽疫病毒基因组序列设计引物通过RT-PCR扩增病毒基因组内部序列,通过3′和5′-RACE获得病毒基因组末端序列。序列测定与分析的结果表明,China/Tib/07株全长15948bp,预测编码6种结构蛋白和2种非结构蛋白,与已发表小反刍兽疫病毒基因组的长度和结构相似;在系统进化上与西南亚流行毒株有很高的同源性(91.6%～98.1%);与麻疹病毒属的其它成员相比,与牛瘟病毒的同源性最高(64.3%)。     

甘蔗黄叶病毒P0蛋白基因细菌双杂交诱饵载体的构建和自激活鉴定

由甘蔗黄叶病毒所引起的甘蔗黄叶病是甘蔗生产中潜在的重要病毒病害之一。该病毒属黄症病毒科(Luteoviridae)中的马铃薯卷叶病毒属(Poleroviruses)。根据甘蔗黄叶病毒海南分离物(SCYLV-CHN-HN1)全基因组序列(Gen Bank No.HQ342888),设计P0蛋白基因的一对特异性引物P0-Bait F/P0-Bait R。以实验室保存的重组质粒p MD18T-P0为DNA模板,PCR扩增P0蛋白基因。然后将P0蛋白基因和细菌双杂交诱饵质粒p BT分别进行双酶切后,连接构建重组诱饵质粒p BT-P0。经PCR扩增、双酶切鉴定和序列分析,表明获得了细菌双杂交重组诱饵载体p BT-P0,且编码框正确。将重组载体p BT-P0转化细菌双杂交系统Bacterio Match RⅡScreening Reporter感受态细胞,进行自激活和毒性验证。结果显示p BT-P0重组载体在细菌双杂交系统中无自激活作用及毒性作用,表明本试验构建的p BT-P0可应用于该系统筛选与之互作的寄主蛋白。     

薯蓣褪绿坏死花叶病毒CP基因的原核表达及抗血清的制备

薯蓣褪绿坏死花叶病毒(yam chlorotic necrotic mosaic virus,YCNMV)是马铃薯Y病毒科柘橙病毒属暂定成员。采用RT-PCR方法,以Sprimer/M4简并引物对扩增获得的YCNMV分离物3'端基因组序列为参考序列,根据该序列设计CP基因特异引物获得YCNMV CP基因并插入p ET-30a表达载体中,在BL21(DE3)菌株中诱导表达。获得的融合蛋白经Ni+-NTA柱纯化后免疫新西兰大白兔,制备抗血清。结果表明,连接在表达载体中的CP基因序列及表达的蛋白氨基酸序列与预期的CP基因核苷酸和氨基酸序列同源性均为99.65%。聚丙烯酰胺凝胶电泳结果表明,获得的融合蛋白大小约为44 k D,与预期蛋白大小相符。制备的抗血清经Western blotting和ID-ELISA检测表明,获得的多克隆抗体效价较高,能可靠、灵敏、特异地与YCNMV病毒颗粒结合,可应用到该病毒的检测中。     

广东番茄曲叶病毒G2分离物基因组DNA-A的分子特征

从采集于广东的番茄曲叶病病株上分离到病毒分离物G2 ,序列分析结果表明 ,其DNA_A为单链环状 ,全长2 74 4nt,共有 6个ORF ,其中病毒链上编码AV1(CP)、AV2 ,互补链上编码AC1、AC2、AC3和AC4。BLAST结果显示 ,与G2基因组有同源关系的病毒均属双生病毒科菜豆金色花叶病毒属。序列比较结果显示 ,G2与菜豆金色花叶病毒属病毒的DNA_A序列同源率均不超过 83% ,其中同源率最高的是PaLCuCNV_[G10 ](82 8% )。进一步比较发现 ,它们的基因间隔区 (IR)变异最大 (同源率为 30 9%～ 81 8% ) ;CP氨基酸序列的同源率较高 (77 6 %～ 99 2 % ) ,AC4蛋白氨基酸序列的同源率较低 (4 3 5 %～ 78 8% )。系统进化关系分析结果也显示 ,G2与已报道的菜豆金色花叶病毒属病毒的亲缘关系均较远。因此 ,G2可能是双生病毒科菜豆金色花叶病毒属中一个未报道的新种 ,命名为广东番茄曲叶病毒 (TomatoleafcurlGuangdongVirus ,ToLCGDV)     

小菜粉蝶颗粒体病毒颗粒体蛋白基因的序列测定及在大肠杆菌中的表达

根据颗粒体病毒颗粒体蛋白(Granulin)基因在其起始密码子上游的12个碱基高度保守序列(TATAAGGAATTT)以及大菜粉蝶颗粒体病毒(PbGV)的颗粒体蛋白基因的序列[1]设计引物,PCR扩增得到850bp左右大小的片段,核苷酸序列测定结果表明该病毒的granulin基因全长为855bp,起始密码位于第38~40位碱基,终止密码位于779~781位碱基,编码框序列全长为744;推测该基因编码一段由247个氨基酸组成的多肽,分子质量约为2.9178×104道尔顿。与其它颗粒体病毒颗粒体蛋白基因进行同源性比较,核苷酸同源性都在70%以上,氨基酸同源性都在75%以上,最高的为大菜粉蝶颗粒体病毒(PbGV),核苷酸同源性为97%,氨基酸同源性为98%。构建了重组表达载体pet-28a-Gran,IPTG诱导后经SDS-PAGE检测,表明获得了颗粒体蛋白基因在大肠杆菌BL21中的特异表达。     

T4病毒研究进展

T4病毒科由一类单股正链RNA病毒组成,分为松天蛾β样病毒属和松天蛾ω样病毒属。这2个属的病毒具有不同的基因组结构,β样病毒含单组分基因组,其结构蛋白由一亚基因组RNA表达; 而ω样病毒含双组分基因组,2个RNA分子分别编码复制酶蛋白和结构蛋白。在T4病毒基因组RNA 3′端有类似tRNA的二级结构。ω样病毒壳蛋白的氨基酸序列一致性高达66%～86%, 而β样病毒壳蛋白的氨基酸同源性则要低得多。在昆虫细胞中表达壳蛋白基因时都能形成病毒类似粒子。该文还介绍了T4病毒复制机理以及T4病毒与其他病毒的进化关系。     

一个侵染山东大葱的胡葱黄条病毒分离物的鉴定

在山东章丘大葱上发现了一种引起畸形黄条症状的线状病毒。该病毒容易摩擦接种侵染大葱和胡葱,但不侵染其它22种测试植物,能经由豌豆蚜传播。病毒粒子形态和细胞病理特征具有马铃薯Y病毒科成员的典型特征。Western blot分析表明,提纯病毒制备的抗血清能与病毒自身外壳蛋白起特异性的强反应,与薤花叶病毒、晚香玉轻斑驳病毒、小西葫芦黄花叶病毒和芋花叶病毒外壳蛋白有较弱的反应。采用马铃薯Y病毒科简并引物PCR技术扩增了该病毒的基因组3′-末端部分序列,序列比较表明它为胡葱黄条病毒(SYSV),系统进化树分析表明世界范围内的SYSV分离物可以区分为4个主要群体,在一定程度上与寄主植物种类及地理分布相关。     

Molecular identification and phylogenetic analysis of a viral RNA associated with the Chinese tobacco bushy top disease complex

Tobacco bushy top disease is caused by a complex of the viruses tobacco bushy top virus (TBTV, a member of the genus Umbravirus) and tobacco vein distorting virus (TVDV, a member of the genus Polerovirus), which acts as a helper virus encapsidating the TBTV genomic RNA. RNA from purified virions is separated as five bands. The two largest (6.0 and 4.2 kb) were shown by Northern blot analysis to be the genomic RNAs of TVDV and TBTV, respectively. A band of about 3 kb was cloned and sequenced and shown to be the RNA of a previously undescribed virus with two open reading frames (ORFs), the second of which is an RNA‐dependent RNA polymerase (RdRp) and is probably expressed by readthrough of the ORF1a stop codon. BLAST and phylogenetic analyses of the RdRp show that it is related to two RNAs previously reported in association with the poleroviruses Beet western yellows virus and Carrot red leaf virus. These three RNAs appear to represent species of a new genus of plant viruses dependent upon a helper polerovirus for their transmission.     

幽影病毒引起的几种主要植物病害

幽影病毒的基因组不编码外壳蛋白,不形成通常的病毒粒体结构。这类病毒往往和黄症病毒复合侵染引起植物病害,蚜虫传播是病害在田间传播流行的主要方式。对幽影病毒引起的胡萝卜杂色矮缩病、花生丛簇病以及烟草丛顶病等几种主要病害的症状、发生与危害、病原物特性以及病害的控制等进行了综述。     

Complete genome sequence of Aminobacterium colombiense type strain (ALA-1)

Aminobacterium colombiense Baena et al. 1999 is the type species of the genus Aminobacterium. This genus is of large interest because of its isolated phylogenetic location in the family Synergistaceae, its strictly anaerobic lifestyle, and its ability to grow by fermentation of a limited range of amino acids but not carbohydrates. Here we describe the features of this organism, together with the complete genome sequence and annotation. This is the second completed genome sequence of a member of the family Synergistaceae and the first genome sequence of a member of the genus Aminobacterium. The 1,980,592 bp long genome with its 1,914 protein-coding and 56 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Non-contiguous finished genome sequence of Aminomonas paucivorans type strain (GLU-3)

Aminomonas paucivorans Baena et al. 1999 is the type species of the genus Aminomonas, which belongs to the family Synergistaceae. The species is of interest because it is an asaccharolytic chemoorganotrophic bacterium which ferments quite a number of amino acids. This is the first finished genome sequence (with one gap in a rDNA region) of a member of the genus Aminomonas and the third sequence from the family Synergistaceae. The 2,630,120 bp long genome with its 2,433 protein-coding and 61 RNA genes is a part of the GenomicEncyclopedia ofBacteria andArchaea project.     

Complete genome sequence of Sanguibacter keddieii type strain (ST-74)

Sanguibacter keddieii is the type species of the genus Sanguibacter, the only genus within the family of Sanguibacteraceae. Phylogenetically, this family is located in the neighborhood of the genus Oerskovia and the family Cellulomonadaceae within the actinobacterial suborder Micrococcineae. The strain described in this report was isolated from blood of apparently healthy cows. Here we describe the features of this organism, together with the complete genome sequence, and annotation. This is the first complete genome sequence of a member of the family Sanguibacteraceae, and the 4,253,413 bp long single replicon genome with its 3735 protein-coding and 70 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Complete genome sequence of Sulfurimonas autotrophica type strain (OK10)

Thermocrinis albus Eder and Huber 2002 is one of three species in the genus Thermocrinis in the family Aquificaceae. Members of this family have become of significant interest because of their involvement in global biogeochemical cycles in high-temperature ecosystems. This interest had already spurred several genome sequencing projects for members of the family. We here report the first completed genome sequence a member of the genus Thermocrinis and the first type strain genome from a member of the family Aquificaceae. The 1,500,577 bp long genome with its 1,603 protein-coding and 47 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.