甘蔗花叶病毒福建分离物外壳蛋白基因的克隆及序列分析

 A fujian isolate of Sugarcane mosaic virus named SCMV-FJ was isolated from infected sugarcane. Cloning and sequence analysis of the coat protein gene of this isolate was carried out. A pair of primers was designed and synthesized based on the nucleotide sequences of coat protein genes of sugarcane mosaic viruses reported. The coat protein gene of SCMV-FJ was amplified from the extracted total RNA of the infected sugarcane by using RT-PCR, and cloned into the pMD18-T vector. The sequencing result indicated that the cloned segment included a 1137 bp open reading frame(ORF) and a 228 bp 3' untranslated region, in which the ORF comprised the whole coat protein and part of the nuclear inclusion b. The nucleotide and the deduced amino acid sequences of the coat protein gene were compared with those of the other isolates or strains of SCMV subgroup reported in GenBank. The result showed that it shares 56.8%-97.1% and 55.3%-99.4% homology in nucleotide and the putative amino acid sequences, respectively, with the highest amino acid homology of 99.4% with SCMV-D. Thus it was identified as a SCMV-D. This experiment provided a rapid, sensitive and relatively inexpensive method for RT-PCR detection of SCMV. At the same time, the cloning of SCMV-FJ coat protein gene provided the foundation for plant gene engineering against SCMV.     

Field resistance against potato virus Y infection using natural and genetically engineered resistance genes

Commercial tobacco cultivars BB16 (burley) and PBD6 (dark air cured) were transformed with the coat protein gene of lettuce mosaic potyvirus (LMV). Transgenic BB16 plants showed resistance to potato virus Y (PVY) infection, against the necrotic strain PVY-N Versailles, as well as the resistance breaking necrotic strain PVY-N 107. Transgenic PBD6, which carries the recessive va gene conferring resistance to PVY, was also protected against PVY-N 107. In the progenies of most BB16 and PBD6 transformants, 45% to 100% of the inoculated plants were immune to PVY infection. The rest of the plants were tolerant, with atypical attenuated non necrotic symptoms and reduced virus accumulation. No recovery was observed in the tolerant plants, which stably expressed detectable level of LMV coat protein. This suggested a protein-mediated mechanism of heterologous protection.     

甘薯羽状斑驳病毒外壳蛋白基因的分子变异

应用单链构象多态性(single-strand conformation polymorphism,SSCP)技术结合核苷酸序列测定的方法,对我国甘薯主产区11个省份的甘薯羽状斑驳病毒(Sweet potato feathery mottle virus,SPFMV)外壳蛋白(CP)基因的分子变异情况进行了研究.结果表明,SPFMV CP基因的RT-PCR产物表现了较丰富的图谱类型,50个分离物共产生9种主要的SSCP带型;对显示不同带型的20个样品的CP基因进行了序列测定和进化树分析,CP基因核苷酸序列一致性为77.2％～99.9％.说明这些样品的SPFMV的CP基因存在较大的分子变异,可划分为EA、RC、O和C4个株系.     

一个马铃薯X病毒分离物的外壳蛋白基因序列分析与株系鉴定

对山东省侵染马铃薯的一个马铃薯X病毒(PVX)分离物PVX—SD1的外壳蛋白(CP)基因进行了克隆和序列分析。以提纯的病毒RNA为模板，应用RT-PCR扩增目的基因，通过常规的基因克隆法将扩增的CP基因导入pUC19载体，测序。结果表明，PVX—SD1的CP基因长719bp，可编码248个氨基酸；与Gen—Bank中报道的15个有代表性的株系或分离物相比较，核苷酸同源性在80．1％-99．7％，氨基酸同源性在89．8％-100％；与欧洲株系UK3仅1个核苷酸不同，同源性为99．7％，氨基酸同源性达100％，表明它们可能为同一株系，属于X^3组．     

Transgenic potato plants expressing the potato virus X (PVX) coat protein gene developed resistance to the viral infection

The gene coding for potato virus X (PVX) coat protein (CP) was expressed in transgenic potato plants obtained byAgrobacterium tumefaciens transformation. One hundred independent clones were analyzed in challenge experiments for resistance to PVX infection under greenhouse conditions as a preliminary test. From this test, 16 clones with the best resistance results were selected for a small-scale field trial. Clones 54, 60, 73 and 91 demonstrated the best values of resistance to PVX in the field. Statistical analysis of the field trial showed significant differences between means of optical density obtained in ELISA from transgenic clones and non-transformed plants (P<0.05). There was correspondence between resistance to virus infection and expression of the CP gene of PVX virus in the analyzed clones. http://www.phytoparasitica.org posting Jan. 21, 2002. Corresponding author [e-mail: vivian.doreste@cigb.edu.cu].     

马铃薯Y病毒衣壳蛋白的表达、纯化与结晶条件筛选

为研究马铃薯Y病毒（potato virus Y,PVY）衣壳蛋白（coat protein,CP）在体外表达及CP重组蛋白的晶体生长条件,通过Eco R Ⅰ/Hind Ⅲ酶切及连接技术构建pET-32a-PVY CP原核表达载体,对PVY CP的诱导剂浓度进行优化,利用蛋白质纯化及脱盐技术对PVY CP进行纯化和脱盐,并分析PVY CP重组蛋白的晶体生长条件。结果表明,成功构建了pET-32a-PVY CP原核表达载体;PVY CP在大肠杆菌Escherichia coli感受态细胞BL21(DE3)原核表达系统中,于16℃下利用0.8 mmol/L异丙基-β-D-硫代吡喃半乳糖苷（isopropyl-beta-D-thiogalactopyranoside,IPTG）诱导表达可获得高浓度的PVY CP可溶性蛋白;PVY CP重组蛋白在二水甲酸镁处理下可生长出棒状结构晶体。     

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。     

芜菁花叶病毒对油菜致病力差异及壳蛋白基因序列分析

 2004年春季参试的湖北、安徽2省11个芜菁花叶病毒(Turnip mosaic virus,TuMV)分离物感染4个油菜品种,病情指数幅度为26.2~76.0;秋季参试5个TuMV分离物感染14个油菜品种,病情指数幅度为38.3~55.9,均值方差分析表明,致病力差异分别达到极显著和显著水平。壳蛋白(CP)基因序列分析表明,来源于2省油菜、白菜、红菜薹、芝麻和萝卜的17个TuMV分离物与浙江分离物ZJB3序列同源性在97%以上,同属于MB类群;而另一个萝卜分离物WRS1与ZJR1、CH1和CH2分离物序列同源性在95.4%~98.7%之间,属于MR类群。类群间分离物序列同源性仅为88.0%~92.2%。遗传进化树分析表明,萝卜分离物WRS2在MB类群中单独构成一个分支,可能是MR类群和MB类群发生重组的后代。     

Characteristics of a resistance-breaking isolate of potato virus Y causing potato tuber necrotic ringspot disease

An Austrian isolate of potato virus Y^NTN, the causal agent of potato tuber necrotic ringspot disease (PTNRD), was serologically compared with seven Dutch PVY^N isolates. Using polyclonal and monoclonal antibodies, it was found indistinguishable from PVY^N. Determination of the nucleotide sequence of the coat protein cistron and comparison of the deduced amino acid sequence with coat protein sequences of other potyviruses revealed a high level of homology with PVY^N coat protein sequences. This confirmed the close taxonomic relationship of PVY^NTN with the PVY^N subgroup of potato virus Y. PVY^NTN is able to overcome all resistance genes known so far in commercial potato cultivars. Remarkably, transgenic PVY-protected tobacco plants are also resistant to PVY^NTN infection upon mechanical and aphid-mediated inoculation. These experiments indicate that genetically engineered resistance offers great potential in protection of potato to new aggressive strains of PVY^N.     

小西葫芦黄花叶病毒外壳蛋白基因的克隆及序列分析

 以小西葫芦黄花叶病毒(Zucchini yellow mosaic virus,ZYMV)的中国分离物(CH-87)接种发病的叶片中提取的总RNA为模板,经RT-PCR扩增获得ZYMV CP基因,将其克隆到pUCm-T质粒上进行序列分析。结果表明该CP基因由837个核苷酸组成,编码279个氨基酸。与已发表序列相比较,该CP基因与国际上已报道的4个基因型不同,应属于新的基因型,暂命名为基因型Ⅴ。     

甘薯潜隐病毒外壳蛋白基因的克隆、表达及其抗血清的制备

 根据已报道的甘薯潜隐病毒(Sweet potato latent virus,SPLV)外壳蛋白(CP)基因的核苷酸序列合成引物,利用RT-PCR方法克隆了SPLV河南分离物(SPLV-HN)的CP基因及部分3'端非编码区序列,序列分析表明,SPLV-HN CP基因由879个核苷酸组成(GenBank登录号为DQ399862),编码293个氨基酸残基。与GenBank中SPLV-CH(X84011)和SPLV-T(X84012)分离物的核苷酸序列相似性分别为96.8%和93.0%;与日本分离物(E15420)的核苷酸序列相似性为83.6%。将CP基因克隆到原核表达载体pET-30a(+)上,SDS-PAGE分析表明,经IPTG诱导,CP基因在大肠杆菌BL21(DE3)pLysS中得到了高效表达。以表达的蛋白为抗原,免疫家兔,制备了SPLV外壳蛋白的特异性抗血清。ACP-ELISA检测结果表明,制备的抗血清可用于田间甘薯样品的检测。     

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.     

甘薯褪绿斑病毒外壳蛋白的分子变异及其特异抗血清制备

 甘薯褪绿斑病毒(Sweet potato chlorotic fleck virus,SPCFV)是侵染甘薯的主要病毒之一。本研究利用RT-PCR方法克隆了SPCFV中国4个分离物的外壳蛋白(CP)基因。序列分析表明,cp基因全长900 bp,编码299个氨基酸残基。4个分离物cp基因的核苷酸序列一致性为78.3%～89.9%,推导的氨基酸序列一致性为91.3%～95.7%,存在较大的分子变异。不同分离物CP氨基酸序列N末端的第3-32位氨基酸为多变区。将四川分离物的cp基因克隆到原核表达载体pET-28a(+)上,SDS-PAGE分析表明,经IPTG诱导,cp基因在大肠杆菌BL21(DE3)中得到了高效表达。以表达的蛋白为抗原免疫家兔,制备了SPCFV CP的特异性抗血清。ACP-ELISA检测结果表明,制备的抗血清效价达1∶128 000,可用于田间甘薯样品的检测。     

Some coat protein constituents from strawberry latent ringspot virus expressed in transgenic tobacco protect plants against systematic invasion following root inoculation by nematode vectors

The coding sequences in RNA2 for the coat proteins (CP) of strawberry latent ringspot virus (SLRSV) were modified and amplified using polymerase chain amplification reactions (PCR) to facilitate their expression inAgrobacterium tumefaciens-transformedNicotiana tabacum Xanthi-nc. The coding sequences for the smaller capsid protein (S, 29kDa) and that for the theoretical precursor of L and S (P, 73kDa) had ATG initiation codon sequences added at the 5-proximal Ser/Gly (S/G) cleavage site in the unmodified sequence. The sequence coding for the larger of the two proteins of mature SLRSV capsids (L, 44kDa) had an ATG codon added at its 5 S/G site and a TAG stop codon sequence added at the 3-proximal S/G site. The P, L and S proteins were expressedin planta to a maximum concentration of 0.01 % of total extractable proteins but did not assemble into virus-like particles. When challenged by mechanical inoculation with virus particles or viral RNA, and compared with control plants, tobacco plants (primary transgenic clones or S1 and S2, kanamycin-resistant seedlings) expressing the virus capsid subunits separately, or their precursor, decreased the accumulation of SLRSV particles in inoculated leaves and fewer plants became invaded systemically. In experiments in which the roots of seedlings were exposed to SLRSV-carrying vector nematodes (Xiphinema diversicaudatum), SLRSV was detected in the roots of non-transformed control tobacco plants (6/20) and in transgenic tobacco expressing the L protein (7/40), but not in any of 25 tobacco plants expressing the S protein or in 35 expressing the P protein. This is the second example of CP-mediated resistance to virus inoculation by nematode vectors.     

四个李属坏死环斑病毒分离物的检测鉴定及CP基因序列分析

采用RT-PCR方法,从北京昌平、云南昆明、陕西西安和浙江海宁的樱桃和月季罹病叶片中检测到李属坏死环斑病毒,为分析我国李属坏死环斑病毒分子生态学特性,克隆了病毒分离物的CP基因并进行序列分析。结果显示,北京分离物的CP基因长675nt、编码224aa,而西安、昆明和海宁分离物的CP基因均为681nt、编码226aa。序列相似性分析表明,4个分离物的CP基因之间具 有很高的同源性, 各个分离物间的核苷酸同源性在94.8%~99.3%之间, 氨基酸同源性在94.2%~98.7%之间。序列比对与系统发生树的分析结果显示,北京分离物属于GroupⅡ组,昆明、西安和海宁分离物属于Group Ⅰ组。     

甘薯脉花叶病毒外壳蛋白基因在大肠杆菌中的表达及其抗血清的制备

 根据已报道的甘薯脉花叶病毒(Sweet potato vein mosaic virus,SPVMV)外壳蛋白（CP）基因的核苷酸序列合成引物,利用RT-PCR方法克隆了SPVMV河南分离物（SPVMV-HN）基因组3′端1.8 kb的基因片段,包括部分NIb 基因序列和完整的CP基因及3′端非编码区序列(3′UTR)。序列分析表明,SPVMV-HN的CP基因由996个核苷酸组成（GenBank登录号为FJ687211）,编码332个氨基酸残基。与已发表的SPVMV其他分离物相比,其推导的氨基酸序列一致性为95.2%～98.5%,与 SPVMV广东分离物的氨基酸序列一致性为97.9%。将CP基因克隆到原核表达载体pET-28a（+）上,SDS-PAGE分析表明,经IPTG诱导,CP基因在大肠杆菌BL21(DE3) pLysS中得到了高效表达。以表达的蛋白为抗原,免疫家兔,制备了SPVMV外壳蛋白的特异性抗血清。ACP-ELISA检测结果表明,制备的抗血清可用于田间甘薯样品的检测。利用SPVMV的抗血清,对采自全国14个省（市）的田间甘薯样品以及嫁接的巴西牵牛样品进行了检测,结果表明,SPVMV在我国甘薯上普遍存在。     

南瓜果实中黄瓜绿斑驳花叶病毒的RT-PCR检测及cp基因序列分析

以来自甘肃的南瓜果实样品中提取的总RNA为模板进行RT-PCR扩增，序列测定和分析结果表明：扩增片段包含CGMMV的外壳蛋白基因和3’非编码区，证明南瓜果实中存在黄瓜绿斑驳花叶病毒，将其印基因序列与来自国内不同地区的其他CGMMV分离物进行序列比对，发现国内各分离物间的同源率高，有很近的亲缘关系。