烟草花叶病毒蚕豆株系基因组全序列分析及结构特征

根据已报道的TMV-U1株系核苷酸序列合成引物 ,利用RT-PCR技术获得了覆盖整个烟草花叶病毒蚕豆株系 (TMV-B)基因组的cDNA重组克隆 .结合末端测序技术 ,完成了TMV-B全基因组序列测定 .TMV-B全基因组共有 6 395个核苷酸组成 ,包括 4个开读框 (ORF) ,分别编码 1 2 6ku(含 1 1 1 6个氨基酸 )、1 83ku(含 1 6 1 6个氨基酸 )、30ku(含 2 6 8个氨基酸 )和 1 7.5ku蛋白 (含 1 5 9个氨基酸 ) .TMV-B与TMV-U1相比全基因组同源率达 99.4% ,两病毒基因组 5′ ,3′非编码区和CP基因完全相同 .TMV-B与TMV-U1之间在 1 2 6ku蛋白中有 6个氨基酸差异 ,5 4ku蛋白中有 2个差异 ,30ku蛋白中有 3个差异 .对导致TMV-B侵染蚕豆的可能致病机理进行了分析 .     

烟草花叶病毒蚕豆株系基因组全序列 分析及结构特征*1)

根据已报道的TMV-U1株系核苷酸序列合成引物,利用RT-PCR技术获得了覆盖整个烟草花叶病毒蚕豆株系（TMV-B）基因组的cDNA重组克隆.结合末端测序技术,完成了TMV-B全基因组序列测定.TMV-B全基因组共有6 395个核苷酸组成,包括4个开读框（ORF）,分别编码126 ku（含1 116个氨基酸）、183 ku（含1 616个氨基酸）、30 ku（含268个氨基酸）和17.5 ku蛋白（含159个氨基酸）.TMV-B与TMV-U1相比全基因组同源率达99.4%,两病毒基因组5′,3′非编码区和CP基因完全相同.TMV-B与TMV-U1之间在126 ku蛋白中有6个氨基酸差异,54 ku蛋白中有2个差异,30 ku蛋白中有3个差异.对导致TMV-B侵染蚕豆的可能致病机理进行了分析.     

黄瓜绿斑驳花叶病毒辽宁分离物全基因组序列测定

以感病组织总RNA为模板,采用RT-PCR方法扩增并测定黄瓜绿斑驳花叶病毒(Cucumber green mottle mosaic virus,CGMMV)辽宁分离物(CGMMV-LN)的基因组全序列。CGMMV-LN基因组全长6 422 nt,5'非编码区(noncoding region,NCR)和3'NCR分别为59 nt和175 nt。CGMMV-LN编码的4个蛋白依次是186 kD和129kD的复制酶,29 kD的移动蛋白和17.4 kD的外壳蛋白。CGMMV-LN与其他4个CGMMV分离物基因组核苷酸序列同源性为97.6%～99.3%,与同属其他3种病毒基因组核苷酸序列同源性仅为61.7%～62.8%。基于186kD复制酶和外壳蛋白氨基酸序列的同源树显示:侵染葫芦科作物的烟草花叶病毒属病毒可分为2个亚组,亚组I包括所有CGMMV分离物,亚组II包括Kyuri绿斑驳花叶病毒(Kyuri green mottle mosaic virus,KGMMV)、黄瓜果实斑驳花叶病毒(Cucumber fruit mottle mosaic virus,CFMMV)和小西葫芦绿斑驳花叶病毒(Zucchini ...     

烟草花叶病毒蚕豆株系外壳蛋白基因及3‘端非编码区的克隆与序列分析

根据烟草花叶病毒Ｕ１株系序列,人工合成引物,用ＲＴ法合成了ｃＤＮＡ后,通过ＰＣＲ技术扩增并克隆了烟草花叶病毒蚕豆株系的外壳蛋白的基因和３‘端非编码区。ＤＮＡ序列测定结果表明,外壳蛋白基因全长４８０个碱基,编码１５８个氨基酸,３’端非编码区全长２０４个碱基,与ＴＭＶ－Ｕ１株系的同源率为１００％。     

黄瓜绿斑驳花叶病毒辽宁分离物外壳蛋白基因与3''''非编码区的序列分析

黄瓜绿斑驳花叶病毒(Cucumber green mottle mosaic virus, CGMMV) 为烟草花叶病毒属(Tobamovirus)成员,Tobamovirus属病毒基因组至少编码4个蛋白,靠近5'端的126 kDa和183 kDa两个蛋白与病毒的复制有关,其中183 kDa是由126 kDa 蛋白终止子超读产生的;另外两个蛋白分别为约30 kDa的移动蛋白(Movement protein, MP)和约17.5 kDa 外壳蛋白(Coat protein, CP),这两个蛋白分别由不同的亚基因组RNA表达产生;病毒基因组5'和3'端均含有一段非编码区(Noncoding region, NCR),5'端含帽子结构,3'端有一个可接受组氨酸的类似tRNA状结构[1].     

汉滩病毒S基因的分段表达及其线性和构象型抗原表位分析

构建汉滩病毒76—118N蛋白及其分别从N-端和C-端缺失的共6个突变体,在大肠杆菌BL-21中进行表达,并对其中一些蛋白进行了纯化。通过Western blot、酶联免疫吸附试验(ELISA)进行汉滩病毒N蛋白的抗原表位分析,N蛋白及6个缺失突变体都与组特异性抗体L13F3呈阳性反应,而缺失突变体与型特异性抗体AH30呈阴性反应。构建汉滩病毒76—118N蛋白及其6个缺失突变体的真核表达载体,并在COS-7细胞中进行表达。通过间接免疫荧光试验(IFA)进行汉滩病毒N蛋白的抗原表位分析,病人血清与真核表达的N蛋白及6个缺失突变体呈阳性反应。而仅有N蛋白及缺失N端1～30位氨基酸序列的NPN30与型特异性抗体AH30呈阳性反应。证实组特异性抗体L13F3结合的抗原表位位于N端1～30位氨基酸;而C端抗原表位对于型特异性抗体AH30与N蛋白的识别和结合具有重要意义,缺失N端100位氨基酸序列可能破坏羧基端构象型表位,也可以影响N蛋白与AH30的结合。     

番茄条斑坏死病毒的研究

从上海郊区番茄条斑坏死病果分离的棒状病毒(ToSNV)的外壳蛋白亚基的分子量小于烟草花叶病毒普通株(TMVc)和长叶车前花叶病毒(HRV),ToSNV的外壳蛋白比TMVc少10个氨基酸残基,比HRV少8个氨基酸残基,氨基酸组成中含甲硫氨酸及组氨酸。ToSNV与长叶车前花叶病毒上海分离株(HRVsh)及油菜花叶病毒(YMV_(15))有免疫交叉反应,但与TMVc却无血清学亲缘关系,而且ToSNV外壳蛋白的酶解图谱不同于TMVc。因此推测ToSNV是烟草花叶病毒组中既不同于TMVc又有别于HRVsh和YMV_(15)的一个分离株。     

番茄条斑坏死病毒的研究

从上海郊区番茄条斑坏死病果分离的棒状病毒(ToSNV)的外亮蛋白亚基的分子量小于烟草花叶病毒普通株(TMVc)和长叶车前花叶病毒(HRV),ToSNV 的外壳蛋白比TMVc少10个氨基酸残基,比HRV 少8个氨基酸残基,氨基酸组成中含甲硫氨酸及组氨酸。ToSNV 与长叶车前花叶病毒上海分离株(HRVsh)及油菜花叶病毒(YMV_(15))有免疫交叉反应,但与TMVc 却无血清学亲缘关系,而且ToSNV 外壳蛋白的酶解图谱不同于TMVc。因此推测ToSNV 是烟草花叶病毒组中既不同于TMVc 又有别于HRVsh 和YMV_(15)的一个分离株。     

植物生物技术的现状及展望

1　大田转基因植物研究现状1.1　病原体诱导的病毒抗性自从Beachy等发现转基因植物表达烟草花叶病毒外壳蛋白基因可以抑制或延缓病毒病的发生之后 ,又发现许多病毒序列可以产生一定水平的抗病性。病原体诱导的病毒抗性 (PDR)基因包括一些非编码蛋白序列 (如缺陷干扰型RNAs和DNA     

毛头鬼伞(Coprinus comatus)中一种抗病毒蛋白y3特性和氨基酸序列分析

食用茵中含有多种抗病毒蛋白,可用于植物保护,利用离子交换层析技术和凝胶层析技术,从食用茵毛头鬼伞中提取到抗植物病毒蛋白y3,实验结果表明,y3是一种糖蛋白,利用Western杂交方法可以在发酵茵丝体和子实体中同时检测到,说明可能是组成型表达,根据其N端氨基酸序列,使用RACE-PCR克隆技术,获得了蛋白的氨基酸序列和部分cDNA序列,浓度为2.0 μg/ml时,蛋白y3对烟草花叶病毒(TMV,20 μg/m1)侵染心叶烟的抑制率为50%,实验同时表明,y3还可抑制病毒在寄主普通烟Nicotirma tabacum Var.k326中的复制.     

Expression and purification of a neuropeptide nocistatin using two related plant viral vectors

Both odontoglossum ringspot virus (ORSV) and tobacco mosaic virus (TMV) were investigated as expression viral vectors for the expression of a neuropeptide nocistatin. Chimeras of ORSV and TMV were constructed by fusion of 17 amino acids of mouse nocistatin (mNST) to the C-terminal of the coat protein (CP) gene via a Factor Xa cleavage linker to yield ORSV-mNST and TMV-mNST. Expression of the mNST peptide was demonstrated by immuno-transmission electron microscopy, western blot, mass spectrometry and radioimmunoassay. Serial passaging of the chimeric viruses revealed loss of mNST from TMV-mNST by the fifth passage. The mNST was maintained in ORSV-mNST throughout six passages. The mNST peptide could be effectively cleaved and purified from chimeric ORSV CP. To our knowledge, this is the first successful attempt in obtaining a complete peptide with no additional amino acid sequence after expression and purification through the use of either ORSV or TMV as vectors.     

Crystal Structure of a Four-Layer Aggregate of Engineered TMV CP Implies the Importance of Terminal Residues for Oligomer Assembly

Background

Crystal structures of the tobacco mosaic virus (TMV) coat protein (CP) in its helical and disk conformations have previously been determined at the atomic level. For the helical structure, interactions of proteins and nucleic acids in the main chains were clearly observed; however, the conformation of residues at the C-terminus was flexible and disordered. For the four-layer aggregate disk structure, interactions of the main chain residues could only be observed through water–mediated hydrogen bonding with protein residues. In this study, the effects of the C-terminal peptides on the interactions of TMV CP were investigated by crystal structure determination.

Methodology/Principal Findings

The crystal structure of a genetically engineered TMV CP was resolved at 3.06 Å. For the genetically engineered TMV CP, a six-histidine (His) tag was introduced at the N-terminus, and the C-terminal residues 155 to 158 were truncated (N-His-TMV CP¹⁹). Overall, N-His-TMV CP¹⁹ protein self-assembled into the four-layer aggregate form. The conformations of residues Gln36, Thr59, Asp115 and Arg134 were carefully analyzed in the high radius and low radius regions of N-His-TMV CP¹⁹, which were found to be significantly different from those observed previously for the helical and four-layer aggregate forms. In addition, the aggregation of the N-His-TMV CP¹⁹ layers was found to primarily be mediated through direct hydrogen-bonding. Notably, this engineered protein also can package RNA effectively and assemble into an infectious virus particle.

Conclusion

The terminal sequence of amino acids influences the conformation and interactions of the four-layer aggregate. Direct protein–protein interactions are observed in the major overlap region when residues Gly155 to Thr158 at the C-terminus are truncated. This engineered TMV CP is reassembled by direct protein–protein interaction and maintains the normal function of the four-layer aggregate of TMV CP in the presence of RNA.     

Misfolded plant virus proteins: elicitors and targets of ubiquitylation

Mutant tobacco mosaic virus (TMV) coat proteins (CPs) with known amino acid replacements provide well defined examples of destabilized tertiary structures. Here we show that misfolded TMV CPs, but not functional wild-type CPs, induce massive ubiquitylation in tobacco cells and that denatured, insoluble CP subunits are the main substrates of ubiquitin conjugation. As TMV CPs can be easily manipulated they are unique tools to study the molecular basis of the plant cell's response to aberrant protein structures and the associated intracellular stress reactions.     

Nucleotide sequence analysis of a cDNA clone encoding the 34K movement protein gene of odontoglossum ringspot virus,ORSV-Cy,the Korean isolate

The partial nucleotide sequence of the 3-terminal region of the Korean isolate of odontoglossum ringspot tobamovirus (ORSV-Cy) from cool-growing Cymbidium was determined. The sequence contained a full length open reading frame (ORF) coding for the viral cell-to-cell movement protein (MP). The ORF was located upstream of the coat protein gene and 105 nucleotides longer than that of tobacco mosaic virus (TMV). The ORF predicts a polypeptide chain of 303 amino acids with a molecular weight of 33573. The ORF contained a similar region of conserved sequence motif of tobamoviruses and putative assembly origin of the viral RNA was located at about 1,100 nucleotides away from the 3 end. The predicted amino acid sequence for the MP gene of ORSV-Cy is more closely related to pepper mild mottle virus (PMMV), TMV-vulgare and TMV-Rakkyo than to tobacco mild green mosaic virus (TMGMV), TMV-L, cowpea strain of TMV (SHMV), and cucumber green mottle mosaic virus (CGMMV).     

Location of the cistron of the tobacco mosaic virus coat protein.

Treatment of tobacco mosaic virus (TMV) RNA with T1 RNase under mild conditions cuts the RNA molecule into a large number of fragments, only a few of which may be specifically recognized by disks of TMV protein. It has been shown elsewhere that these specifically recognized RNA fragments are a part of the coat protein cistron, the portion coding for amino acids 95 to 129 of the coat protein. It is reported that different size classes of partially uncoated virus particles were prepared by limited reconstitution between TMV RNA and protein or by partial stripping of intact virus with DMSO. Both procedures produce nucleoprotein rods in which the 5'-terminal portion of the RNA is encapsidated and the 3'-terminal region is free. The free and the encapsidated portions of the RNA were each tested for the ability to give rise to the aforesaid specifically recognized fragments of the coat protein cistron upon partial T1 RNase digestion. It was found that only the 3'-terminal third of the virus particle need to be uncoated in order to expose the portion of the RNA molecule from which these fragments are derived. We conclude, therefore, that the coat protein cistron is situated upon the 3'-terminal third of the RNA chain, i.e. within 2000 nucleotides of the 3'-end.     

Local and systemic spread of tobacco mosaic virus in transgenic tobacco.

Expression of a chimeric gene encoding the coat protein (CP) of tobacco mosaic virus (TMV) in transgenic tobacco plants confers resistance to infection by TMV. We investigated the spread of TMV within the inoculated leaf and throughout the plant following inoculation. Plants that expressed the CP gene [CP(+)] and those that did not [CP(-)] accumulated equivalent amounts of virus in the inoculated leaves after inoculation with TMV-RNA, but the CP(+) plants showed a delay in the development of systemic symptoms and reduced virus accumulation in the upper leaves. Tissue printing experiments demonstrated that if TMV infection became systemic, spread of virus occurred in the CP(+) plants essentially as it occurred in the CP(-) plants although at a reduced rate. Through a series of grafting experiments, we showed that stem tissue with a leaf attached taken from CP(+) plants prevented the systemic spread of virus. Stem tissue without a leaf had no effect on TMV spread. All of these findings indicate that protection against systemic spread in CP(+) plants is caused by one or more mechanisms that, in correlation with the protection against initial infection upon inoculation, result in a phenotype of resistance to TMV.