香蕉束顶病毒DNA组分2的克隆与序列分析

 香蕉束顶病毒(Banana bunchy top virus,BBTV)基因组中至少含有6个DNA组分,栽们实验室已经对BBTV广东2个株系(NS和NSP)基因组中的DNA分组1、3、6进行了测序和报道。现又对NS和NSP的DNA组分2分别进行了克隆和序列分析,测序结果报道如下。     

香蕉束顶病毒NSP株系DNA组分5的克隆和序列分析

 本文利用PCR技术扩增得到香蕉束顶病毒(BBTV) NSP株系DNA组分5的全基因,该基因全长为1013 nt,具有一个开放阅读框,编码161个氨基酸。NSP株系与南太平洋组澳大利亚、夏威夷、埃及分离物DNA组分5核苷酸和编码的氨基酸序列相比较,核苷酸序列同源率介于88%~89%之间,氨基酸序列同源率介于76%~79%之间,借助进化树分析和生物信息学研究方法,发现NSP株系组分5同样含有保守的与植物体内Rb蛋白结合的、能够调节寄主体内DNA复制相关蛋白积累的功能基序——LYCDE;并预测了蛋白质二级结构和性质。     

贝宁首次报道香蕉束顶病毒

香蕉束顶病毒(Banana bunchy top virus,BBTV)是香蕉(AAA基因型)和大蕉(AAB基因型)上最重要的病原物之一,在撒哈拉以南非洲的中部和南部地区广泛分布。香蕉交脉蚜(Pentalonia nigronervosa)是其重要的传播媒介。     

水稻瘤矮病毒广东分离物基因组第10组分的序列分析及原核表达

 应用RT-PCR技术克隆了水稻瘤矮病毒(Rice gall dwarf virus,RGDV)广东分离物基因组的第10片段,并测定了全序列。结果表明,RGDV广东分离物S10(登录号EF532325)全长1198bp,含有一个ORF,编码一条由320氨基酸组成、推测分子量约36kDa的多肽。与泰国分离物相应组分相比,基因结构基本一致,核苷酸和氨基酸序列同源性分别为96.2%和98.8%;S10编码多肽与水稻矮缩病毒(Rice dwarf virus,RDV)s9编码蛋白及伤瘤病毒(Wound tumor virus,WTV)S11编码蛋白也分别具有29%和33%的相似性。本研究还将S10cDNA克隆至原核表达载体pET28b(+)上,通过IPTG诱导在大肠杆菌BL21(DE3)中得到了高效表达,并利用His,Bind树脂纯化得到电泳纯级制品。本工作为进一步研究S10编码蛋白的结构与功能奠定了一定的基础。     

香蕉分化芽BBTV DAS-ELISA检测方法的建立和应用

为了解海南海口香蕉分化芽携带香蕉束顶病毒(Banana bunchy top virus, BBTV)的情况, 本研究建立了双抗夹心酶联免疫反应(DAS-ELISA)检测法, 对来自海南地区组培厂的香蕉分化芽进行检测。检测结果表明, 6个品种1 852个香蕉分化芽平均带毒率为2.1%, 其中‘皇帝蕉’为1.98%, ‘粤科1号’为2.07%, ‘广粉蕉’为1.98%, ‘大蕉’为6.25%, ‘218’为1.89%, ‘巴西蕉’为2.25%。为了进一步验证ELISA结果的可靠性, 随机选取12个阳性样品提取总DNA进行PCR鉴定。鉴定结果显示, 12个样品中11个检测出有BBTV, 表明DAS-ELISA方法的准确率较高, 与分子检测结果基本一致, 可以胜任日常香蕉组培苗BBTV的检测。     

刺茄中分离的中国番茄黄化曲叶病毒及伴随的卫星DNA分子的全基因组结构

 从云南砚山刺茄(Solanum aculeatissimum)上分离到病毒分离物Y322,全序列测定表明,Y322 DNA-A全长2 730个核苷酸,共编码6个ORF。基因组比较发现,Y322 DNA-A与中国番茄黄化曲叶病毒(TYLCCNV)各分离物的同源性最高(88.3%~99.2%),而与其它双生病毒的同源性均在79.6%以下,表明Y322是TYLCCNV的一个分离物。利用DNAβ的特异性引物在Y322中扩增到DNAβ分子(Y322 β),序列分析表明,其全长1 331个核苷酸,与TYLCCNV伴随的DNAβ的同源性最高,达75.1%~93.1%,而与已报道的其它种类的DNAβ的同源性均低于55.4%。这是首次在刺茄中检测到中国番茄黄化曲叶病毒。     

苹果茎沟病毒梨分离物外壳蛋白基因的克隆和序列分析

 选取生物学和血清学特性存在较大差异的苹果茎沟病毒(Apple stem grooving virus,ASGV)梨分离物P-6-1-17、P-4-1-69、P-L2和P-D-21,采用RT-PCR法对3'端进行扩增,所获扩增片段经序列测定,全长分别为1089nt(P-6-1-17、P-L2)和1069nt(P-4-1-69、P-D-21)。4个分离物的CP基因均由714nt组成,其核苷酸和推导编码蛋白氨基酸序列同源性分别为89.8%~96.4%和94.9%~97.9%。将我国4个分离物的CP基因与已报道ASGV分离物及分子变种进行序列同源性比较和系统进化树分析,结果表明我国ASGV分离物至少可分为2个组群,其中生物学表现明显不同于其它分离物的P-L2,其CP基因核苷酸序列显示较大的变异。     

香蕉束顶病毒海口分离物Rep基因的克隆、原核表达、抗血清制备及检测

［目的］ 对香蕉束顶病毒(Banana bunchy top virus,BBTV)海口分离物起始蛋白(replication initiation proteins, Rep)基因进行克隆、原核表达、抗血清制备,为BBTV有效的检测及分子流行病学等研究奠定基础。［方法］ 以海口地区染病香蕉幼嫩假茎和叶片的总DNA为模板,通过PCR技术克隆BBTV海口分离物的起始蛋白基因,连接到表达载体并进行大肠杆菌原核表达,制备高效价特异性抗血清。［结果］ 应用PCR方法从染毒香蕉幼嫩假茎和叶片总DNA中扩增复制rep基因,回收目的片段后经酶切,获得了含BBTV Rep基因的重组质粒pET32b Rep。将重组质粒转化E.coli BL21(DE3),经不同的时间、温度及IPTG浓度优化,12% SDS PAGE电泳分析,在20 ℃、0.1 mmol/L IPTG条件诱导4h,最终获得大量的可溶性融合蛋白。将可溶性蛋白上清液经Ni2+ NTA亲和层析柱纯化,得到高纯度的融合蛋白。用纯化后的融合蛋白免疫家兔,获得了BBTV Rep蛋白抗血清。以融合蛋白做抗原,间接ELISA法测定抗血清效价大于125 000。以田间样品做抗原时,结果表明抗血清最佳工作浓度为1∶1 000。Western blot鉴定结果表明抗血清能与融合蛋白特异性结合。［结论］ 利用Rep基因制备的特异性抗血清在BBTV病毒粒体的组装机制研究及病毒病诊断上具有重要的应用价值。     

来源于桃和苹果的苹果褪绿叶斑病毒的部分分子生物学特性和cp基因的原核表达

 从桃和苹果上分离得到苹果褪绿叶斑病毒ACLSV-HBP和ACLSV-C2个分离物,采用RT-PCR法进行扩增,所获扩增片段经序列测定,其全长分别为1768nt(ACLSV-HBP)和1751nt(ACLSV-C)。这2个分离物扩增片段全长的同源性为83%,mp基因片段核苷酸和推导编码氨基酸序列同源性分别为82.6%和87.1%;cp基因均由582nt组成,其核苷酸和推导编码氨基酸序列同源性分别为87.8%和95.9%。将2个分离物的cp基因与已报道ACLSV分离物进行序列同源性比较,结果显示ACLSV-HBP与SX/2的cp基因核苷酸序列及推导编码氨基酸序列同源性最高,分别为94.0%和96.4%。将ACLSV-HBP分离物的cp基因克隆到原核表达载体pGEX-KG,在大肠杆菌BL21(DE3)中诱导表达,SDS-PAGE分析表明,融合蛋白大小约为46kDa。Western-blot分析表明,该基因在大肠杆菌内得到高效表达,融合蛋白具有抗原性。     

油桃茎痘相关病毒中国分离物基因组的分子特征分析

为明确我国油桃茎痘相关病毒(nectarine stem-pitting-associated virus,NSPaV)基因组的分子特征,利用RT-PCR和RACE技术对NSPaV中国分离物NSPaV-T04基因组进行克隆,采用最大似然法对得到的NSPaV基因组序列和GenBank中的5条NSPaV基因组序列构建系统发育树,应用RDP软件对NSPaV基因组序列进行重组分析。结果表明:中国分离物NSPaV-T04基因组序列全长为4 991 nt,包括4个开放阅读框(open reading frame,ORF),其中ORF1与ORF2共同编码1个RdRp P1-P2融合蛋白,ORF3编码1个CP,ORF5与ORF3共同编码CP通读蛋白。系统发育树和序列比较分析结果显示,中国分离物NSPaV-T04(MN095353)与美国分离物NSPaV/12P42(KT273410)的亲缘关系最近,核苷酸序列同源性最高,为96.4%;NSPaV-T04的RdRp P1变异较大,与GenBank中5条NSPaV基因组核苷酸序列的同源性为90.5%~96.1%,CP较为保守,核苷酸序列的同源性为96.6%~98.7%。重组分析结果显示,中国分离物NSPaV-T04为鉴定的一个重组体(韩国分离物SK)的亲本序列,表明中国分离物NSPaV-T04可能是一个实际的重组体。     

香蕉束顶病毒株系生物学特性的研究

 在广东香蕉束顶病株症状基本相同的情况下,在8个产区分别随机采集11~15个标样共111个分离物,接种在香蕉苗上,其后又从8个产区的分离物中各选取1个代表分离物用于进行各项研究。寄主范围试验结果,这8个代表分离物可分为能侵染粉蕉的NSP株系(以广州天河分离物为代表的7个分离物)和不能侵染粉蕉的NS株系(高州代表分离物)。用Eco RI对8个毒源地的111个分离物DNA组分1进行酶切分析,结果表明:所有高州分离物共15个和信宜分离物14个中的9个都可以被酶切,应属NS株系;而其余6个毒源地的所有分离物及信宜分离物中的另外5个都不能被酶切,应属NSP株系。在病害潜育期方面,2个株系在大蕉上的差异达到显著水平;在香蕉4个品种上,2株系也存在较明显的差异,但其中有些差异未达到显著水平。在病毒增殖、运转速度及病毒达到高浓度所需的时间上,2个株系也存在显著的差异。     

Characterization and genetic status of Banana bunchy top virus isolated from Okinawa, Japan

Isolates of Banana bunchy top virus (BBTV), which causes bunchy top disease in bananas, were collected in field surveys on seven islands in Okinawa Prefecture, Japan. From 44 banana samples, one isolate from each island was selected, and the DNA-1 and DNA-3 components were sequenced. Analysis of the major common region of DNA-1 showed that BBTV in Okinawa belongs to the Asian group of BBTV. DNA-1 and DNA-3 analysis revealed that Okinawan BBTV had a closer relationship with isolates from Taiwan and the Philippines than with some isolates from China and Vietnam. All the Okinawan BBTV isolates had high homology in the nucleotide sequences of DNA-1 and DNA-3 (%) because of a single, recent BBTV invasion of this area.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB108449 – AB108458     

应用单克隆抗体检测香蕉束顶病

应用香蕉束顶病毒（ＢＢＴＶ）的单克隆抗体（ＭｃＡｂ）建立了检测香蕉束顶病的间接ＥＬＩＳＡ方法，其检测香蕉病组织汁液的最大稀释度为１：１２８０，健康香蕉组织汁液无非特异性反应。用间接ＥＬＩＳＡ方法检测了采自广东、广西９个市（县）的香蕉产区的８０个大田香蕉束顶病样本和２５个荫棚香蕉试管苗样本，其中大田样本ＢＢＴＶ的带毒率为７６．５％，试管苗样本的带毒率为８．０％。香蕉束顶病株不同部位ＢＢＴＶ含量检测表     

香蕉束顶病毒(BBTV)侵染对寄主内源激素的影响

 本试验用ELISA方法测定了香蕉感染束顶病毒(BBTV)后植株体内的3种内源激素,赤霉素(GA₃)、玉米素类(iPAs)和脱落酸(ABA)的变化。结果表明,感株的GA₃水平在侵染过程中虽有微弱增加,但含量和增长速度显著低于健株对照;iPAs在接种BBTV第14天后明显下降,并维持较低水平;ABA在BBTV侵染后被大量诱导增加并不断积累,在接种后第35天测定含量最高,为对照的3.34倍。试验还同时检测了BBTV在侵染过程中的运转。用间接ELISA测定的接种叶和顶叶的BBTV含量显示:接种21天后BBTV在接种叶和顶叶中还大量增殖,呈系统性分布。但寄主的症状在接种35天后才逐渐在顶叶表现。以上结果表明:香蕉束顶病的症状表现似乎主要与病株中的内源激素的失调有关,而与BBTV在体内的运转并不直接相关     

Regeneration of healthy banana plantlets from banana bunchy top virus-infected tissues cultured at high temperature

Exposure of banana bunchy top virus (BBTV)-infected plantlets of Cavendish banana to 40°C for 16 h daily for periods of up to 5 weeks did not free them from BBTV. However, BBTV was less readily transmitted by aphids from treated plants than from untreated controls. The same heat treatment applied for up to 12 weeks also failed to eliminate the virus from BBTV-infected tissue. When similar BBTV-infected tissue was cultured for 3 months at 35°C some of the buds started to produce healthy looking plantlets. Five out of 11 cultures produced healthy-looking plantlets in 6 months. Crude extracts from the leaves of these plantlets did not react with a monoclonal antibody prepared against BBTV. When heat-treated, healthy-looking plantlets were inoculated with viruliferous aphids all plants developed bunchy top symptoms indicating that they were not resistant to BBTV. The results suggest that the uneven distribution and low concentration of virus after treatment at high temperature leads to BBTV-free primordial cells which in turn may develop into healthy plantlets.     

Induction of systemic resistance in banana (<Emphasis Type="Italic">Musa</Emphasis> spp.) against <Emphasis Type="Italic">Banana bunchy top virus</Emphasis> (BBTV) by combining chitin with root-colonizing <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> strain CHA0

Pseudomonas fluorescens strains CHA0 and Pf1 were investigated for their biocontrol efficacy against Banana bunchy top virus (BBTV) in banana (Musa spp.) alone and in combination with chitin under glasshouse and field conditions. Bioformulation of P. fluorescens strain CHA0 with chitin was effective in reducing the banana bunchy top disease (BBTD) incidence in banana under glasshouse and field conditions. In addition to disease control, the bioformulation increased the economic yield significantly compared to the untreated control. Increased accumulation of oxidative enzymes, peroxidase (PO), polyphenol oxidase (PPO), phenylalanine ammonia lyase (PAL), pathogenesis-related (PR) proteins, chitinase, β-1,3-glucanase and phenolics were observed in CHA0 bioformulation amended with chitin-treated plants challenged with BBTV under glasshouse conditions. Indirect ELISA indicated the reduction in viral antigen concentration in P. fluorescens strain CHA0 with chitin-treated banana plants corresponding to reduced disease ratings. The present study revealed that induction of defence enzymes by P. fluorescens with chitin amendment reduced the BBTD incidence and increased bunch yield in banana.     

First report of cucumber mosaic cucumovirus in banana from Pakistan

During regular surveys of banana plantations in the Sindh province of Pakistan for banana bunchy top nanavirus (BBTV), sporadic plants showing mosaic, chlorotic spots intermingled with a dark green area were observed at a few places in the districts of Thatta, Hyderabad and Nawabshah. The infected young plants were uprooted and grown in pots for 2 years to ascertain the cause of the disease. Apart from studying the development of symptoms, the infected plants were tested for the presence of BBTV, cucumber mosaic cucumovirus (CMV), chilli veinal mottle potyvirus (CVMV), tomato mosaic tobamovirus (TMV), potato X potexvirus (PVX) and potato Y potyvirus (PVY) by DAS-ELISA. Virions were also partially purified. The infected plants developed typical symptoms of CMV. All tested plants were ELISA-positive only for CMV, and a partially purified preparation revealed virus particles measuring 25–28 nm in diameter. Based on the characteristic field symptoms, serology and particle morphology, the presence of CMV in banana in Pakistan was established.     

香蕉束顶病毒的寄主及其在病害流行中的作用

 接种测定结果表明:香蕉、粉芭蕉和大蕉等是香蕉束顶病毒的寄主,但美人蕉、芋头、艳山姜、芭蕉芋、姜黄、长春花、甜瓜等其它24种供试植物不是该病毒的寄主。研究结果还认为:在福建省香蕉产区,香蕉束顶病流行的主要侵染源为香蕉病株,而粉芭蕉和大蕉等病株则可在病区中病害的长期定殖和流行起重要的作用。本文还讨论了这些研究结果对该病害的流行学和防治的重要性。     

香蕉束顶病毒株系的研究

 采自福建各地蕉区的不同香蕉束顶病毒株类型之间致病性和介体蚜虫的传病率有明显的差异,进一步依其在香蕉品种台湾蕉和Williams上的反应、香蕉交脉蚜的传病率、毒株类型之间的交互保护和血清学测定结果等,把香蕉束顶病毒区分为BBTV-S(重型)和BBTV-M(轻型)两个株系。
BBTV-M和BBTV-S均能引致香蕉叶片上的青筋症状,两者在血清学上有密切关系,但BBTV-M仅引致植株产生少量青筋,而BBTV-S除引致香蕉植株上有大量青筋之外,还引致严重矮化、束顶以及轻度黄化,BBTV-M的潜育期较BBTV-S明显长,香蕉交脉蚜对BBTV-S的传病率大大高于对BBTV-M的传病率,BBTV-M对BBTV-S有强的保护作用。