烟草花叶病毒外壳蛋白嵌合基团的重组

普通烟草花叶病毒外壳蛋白基因已和花椰菜花叶病毒35S启动子及3′未端重组成嵌合基因。在连接了用于筛选在土壤杆菌中导入外源基因的新霉素磷酸转移酶Ⅰ(NPT Ⅰ)基因和用于筛选在植物细胞中导入外源基因的嵌合的新霉素磷酸转移酶Ⅱ(NPT Ⅱ)基因后,已导入一个去致瘤基因的Ti载体T-DNA区中。这种在Ti载体T-DNA区带嵌合的烟草花叶病毒外壳蛋白基因的土壤杆菌菌株,可以用来转化植物。观察在转化植物中表达的这种外壳蛋白能否延缓或减轻烟草花叶病毒对它们的危害。     

Identification of a peptide of the membrane protein of tobacco mosaic virus, cross-linked with intraviral RNA under the effect of UV-radiation

As reported previously, UV-irradiation induces crosslinking between tobacco mosaic virus (TMV) coat protein molecules and intraviral RNA nucleotides. We have irradiated [3H]-uridine labeled TMV and isolated TMV coat protein subunits with the attached nucleotide label. These TMV protein subunits were hydrolyzed with trypsin. The tryptic peptides were separated by high-performance liquid chromatography and [3H]-labeled peptides were identified. The UV-irradiation of TMV was found to result in crosslinking to intraviral RNA of the T8 tryptic peptide (residues 93-112) of TMV coat protein.     

Coat protein-mediated protection: analysis of transgenic plants for resistance in a variety of crops

Since 1986, research has shown that plants expressing the coat protein gene of a plant virus exhibit degrees of resistance or protection when challenge inoculated with that virus or closely related isolates. This phenomenon, called coat protein-mediated protection, sparked research efforts to develop transgenic plants that resist infection to a range of plant viruses. This report summarizes the research efforts that deal with viral coat protein gene-crop combinations of commercial potential. The viruses include tobacco mosaic, potato virus X and Y, cucumber mosaic and papaya ringspot; the crops include tomato, cucumber, tobacco and papaya.     

Early cell death caused by TMV mutants with defective coat proteins

Summary Four closely related strains of TMV, which differ in the ability to kill infected turkish tobacco leaves, were compared cytologically and with respect to the amounts of soluble and insoluble coat proteins synthesized. The results suggest that the cytopathic effects might be caused by coat proteins with unusual solubility properties.     

Expression of alfalfa mosaic virus coat protein gene confers cross-protection in transgenic tobacco and tomato plants

A chimeric gene encoding the alfalfa mosaic virus (AlMV) coat protein was constructed and introduced into tobacco and tomato plants using Ti plasmid-derived plant transformation vectors. The progeny of the self-fertilized transgenic plants were significantly delayed in symptom development and in some cases completely escaped infection after inoculated with AlMV. The inoculated leaves of the transgenic plants had significantly reduced numbers of lesions and accumulated substantially lower amounts of coat protein due to virus replication than the control plants. These results show that high level expression of the chimeric viral coat protein gene confers protection against AlMV, which differs from other plant viruses in morphology, genome structure, gene expression strategy and early steps in viral replication. Based on our results with AlMV and those reported earlier for tobacco mosaic virus, it appears that genetically engineered cross-protection may be a general method for preventing viral disease in plants.     

Calcium ion binding by isolated tobacco mosaic virus coat protein

Calcium ion titrations were performed on solutions of tobacco mosaic virus coat protein using a calcium-specific ion-exchange electrode. Isolated coat protein was found incapable of binding calcium ions under equilibrium conditions at pH values above its iso-ionic point (pH 4.3 to 4.6). However, calcium ions were found to bind to coat protein under non-equilibrium conditions, which suggests that the isolated coat protein has the proper conformation to bind calcium ions at the iso-ionic point.     

Oleosin-like proteins are not present on the surface of tobacco pollen

Pollen-stigma interactions on wet- and dry-type stigmas involve similar processes: the hydration of the pollen, followed by pollen tube growth and penetration of the stigma. Furthermore, in some species, identical molecules, namely lipids, are used to achieve this. In addition to lipids, oleosin-like proteins of the pollen coat of dry-type stigma plants have been shown to be involved in pollen-stigma interactions. However, little information is present about the proteins on the surface of pollen of wet-type stigma plants, in particular that of the Solanaceae. To analyze proteins from the surface of pollen of Nicotiana tabacum (tobacco), a solanaceous plant, we used an antiserum raised against Brassica pollen coat, a dry-type stigma plant of the Brassicaceae. In addition we used a molecular approach to identify tobacco homologues of oleosin-like genes. Our results show that no proteins similar to Brassica oleracea pollen coat proteins are present on the surface of tobacco pollen, and that oleosin-like genes are not expressed in tobacco anthers or stigmas.     

Subcellular localization of the coat protein in tobacco cells infected by cucumber mosaic virus isolated from Catharanthus roseus

Electron microscopy and immunolabelling with antiserum specific to cucumber mosaic virus coat protein were used to examine tobacco leaf cells infected by cucumber mosaic virus isolated from Catharanthus roseus (CMV-Cr). Crystalline and amorphous inclusions in the vacuoles were the most obvious cytological modifications seen. Immunogold labelling indicated that the crystalline inclusion was made up of virus particles and amorphous inclusions contained coat protein. Rows of CMV-Cr particles were found between membranes of dictyosomes, but membranous bodies and tonoplast-associated vesicles were not evident. Virus particles and/or free coat protein were easily detected in the cytoplasm by immunolabelling. No gold labelling was found within nuclei, chloroplasts and mitochondria.     

A non-coat protein synthesized in tobacco mesophyll protoplasts infected by tobacco mosaic virus

Summary A high molecular weight protein unrelated to the viral coat was detected in tobacco mesophyll protoplasts infected by tobacco mosaic virus.     

两个缺陷型TMV粒子在烟草中的互补表达

根据对ＴＭＶ高效复制和基因表达的顺式作用元件的分析,在体外重组包装了２个缺失型ＴＭＶ粒子：ＴＭＶＲＰ和ＴＭＶＣＰ。前者缺失了ＴＭＶ外壳蛋白ＣＰ基因的３′端及后序区域,后者缺失了大部分复制酶基因。把两者分别或共同电击感染烟草原生质体：１．用ＣＰ抗体进行免疫印渍检测,单独感染的原生质体内的ＣＰ在１６小时内无增加,而在共同感染的原生质体内,ＣＰ在感染２小时后就开始明显增加。２．用ＲＴ一两次ＰＣＲ法专一地检测新生负链ＲＮＡ的合成情况,在单独感染的原生质体内没有检测到,但在混合感染的原生质体内在感染１小时后就检测到ＣＰ基因特异的负链ＲＮＡ的形成,并用Ｓｏｕｔｈｅｒｎ杂交得到进一步验证。这些结果表明,复制酶缺失型ＴＭＶＣＰ内的ＣＰ基因不能表达,但可以在ＴＭＶＲＰ存在时,通过其所表达的复制酶互补作用得到复制从而有效表达．     

A proteinless mutant of tobacco mosaic virus: Evidence against the role of a viral coat protein for interference

Summary A coat-protein-free mutant of tobacco mosaic virus as well as mutants with a non-functional coat protein were found to interfere with the establishment and spread of challenging strains of TMV. The results do not support an earlier concept, according to which the genome of a related challenging virus could be captured by the coat protein of the virus introduced in advance. The presence of a viral coat protein is obviously not essential and a competition among the viral genomes for some specific site seems to be a more likely mechanism of cross protection.A part of the data was presented at the 5th International Congress for Virology, Strassburg, August 2–7, 1981     

Expression of Multiple Virus-derived Resistance Determinants in Transgenic Plants Does Not Lead to Additive Resistance Properties

Plants can be protected against infection by potyviruses by expressing different portions of potyviral genomes as transgenes. This strategy has proven effective with several potyvirus genes, including the Nla, Nlb, and coat protein coding regions. Given the effectiveness of separate potyvirus coding regions as determinants of resistance, we tested the hypothesis that combinations of potyvirus coding regions would provide additively greater protection of plants against potyviruses. For this, we compared transgenic plant lines that expressed either the coat protein (CP) or the Nla+Nlb+coat protein (NNC) coding regions from tobacco vein mottling virus (TVMV). We found that plants that carry the NNC gene combination were invariably less resistant to TVMV than were lines that contain a CP gene alone. Additionally, we found that NNC lines displayed virtually no resistance to tobacco etch virus (TEV), in contrast to the CP lines. We conclude that combining more than one virus-derived resistance determinant in a single construct is detrimental to the production of virus-resistant plants.     

Protein Synthesis Directed by the RNA from a Plant Virus in a Normal Animal Cell

RNA from tobacco mosaic virus can be translated inside oocytes of the frog Xenopus laevis. The main product is a polypeptide with a molecular weight of 140,000. There is no evidence for coat protein synthesis, and it is unlikely that the polypeptide that is made contains either a whole or a partial coat protein sequence.The picture of translation of tobacco mosaic virus RNA obtained using oocytes is very much simpler than that found using cell-free protein-synthesizing systems, in which a great many polypeptides are made under the direction of tobacco mosaic virus RNA. The reasons for this difference are discussed, and the relative merits of in vivo and in vitro protein-synthesizing systems are compared.     

Infectivity suppressing and virus-binding activities of a membrane material isolated from tobacco leaves.

TMV binding substance (R) was isolated from a tobacco leaf membrane fraction and was purified by extraction with organic solvents and by column chromatography. Experimental results suggest that the binding of R with TMV results in inactivation of TMV. When tobacco leaves were inoculated with the R-TMV complex, it was found that the formation of polysome containing infecting viral RNA was inhibited. Model experiments showed that the mode of R-TMV adsorption to the membrane is different from that of TMV adsorption and that stripping of coat protein from TMV by SDS was inhibited by R. A possible explanation for the mechanism of this inhibition by R is that the R-TMV complex follows a pathway which does not lead to establishment of infection. Although less efficient, R was still active when it was applied after virus inoculation. Due to its affinity to coat protein, R might also interfere with a later process of viral multiplication.     

Synthesis,Biological Activity and Action Mechanism Study of Novel Chalcone Derivatives Containing Malonate

A series of novel chalcone malonate derivatives were synthesized and their antibacterial and antiviral activities were evaluated. All target compounds were characterized by spectral data. The results of antimicrobial bioassay showed that one compound (diethyl [3‐(naphthalen‐2‐yl)‐1‐(3‐nitrophenyl)‐3‐oxopropyl]propanedioate) showed excellent antibacterial activity against Xanthomonas oryzae pv. oryzae (Xoo), with an EC₅₀ value of 10.2 μg/mL, which is significantly superior to bismerthiazol (71.7 μg/mL) and thiodiazole copper (97.8 μg/mL). At the same time, the mechanism of two compounds was confirmed by scanning electron microscopy. In addition, another compound (diethyl [3‐(naphthalen‐2‐yl)‐1‐(4‐nitrophenyl)‐3‐oxopropyl]propanedioate) showed significant curative activity to tobacco mosaic virus, with a value of 74.3 %, which was superior to 53.3 % of ningnanmycin. The results of microscale thermophoresis also showed that the Kd value of the combination of two compounds with the coat protein of tobacco mosaic virus was 0.211 and 0.166 μmol/L, which was better than 0.596 μmol/L of ningnanmycin. At the same time, the molecular docking of two compounds with tobacco mosaic virus‐coat protein shows that the compound is well embedded in the pocket between the two subunits of tobacco mosaic virus‐coat protein. These results show that chalcone derivatives containing malonate group can be considered as activators in the design of antibacterial and antiviral agents.