Establishment of ELISA method for testing tobacco mosaic virus (TMV) infecting Zingiber officinale Rosc / 中国免疫学杂志

Objective:In order to establish ELISA method for testing tobacco mosaic virus (TMV) infecting Zingiber officinale Rosc.Methods: Tobacco mosaic virus(TMV) infecting Zingiber officinale Rosc was tested by RT-PCR.Zingiber officinale Rosc leaves which only contained TMV were choiced.TMV particle was purified by centrifugation method.TMV CP was purified through preparation electrophoresis including 12％ SDS-PAGE first and then 5％-20％ gradient SDS-PAGE.Polyacrylamide gel contained TMV CP was ground into suspension.Mice were immuned with the suspension and antiserum was obtained.Antiserum quality was tested by Western blot and ELISA test.IgG was purified through affinity chromatography method.IgG solution was concentrated and dialyzed to a suitable concentration.The IgG then mixed with glycerol.Results: IgG in antiserum only combined with TMV CP protein and it could combine with nature TMV particle CP protein.Its quality was up to standard.Conclusion: Establishment of ELISA method for testing TMV infecting Zingiber offcinale Rosc is successfull by using this IgG.

A new phenylpropanoid from Dai Medicine Arundina graminifolia and its anti-tobaco mosaic virus activities / 中草药

Objective: For the purpose of finding new bioactive agents from ethnic medicines, the chemical study on Dai Medicine Arundina graminifolia was carried out. Methods: The chemical constituents in the whole plant of A. graminifolia were isolated by silica gel, MCI-GEL resin, Sephadex LH-20 column chromatography, and high performance liquid chromatography (HPLC) method. Their structures were elucidated by spectroscopic methods, including extensive 1D and 2D NMR techniques. The inhibitory activities of compound 1 against tobaco mosaic virus (TMV) replication were tested using the half-leaf method. Results: Four phenylpropanoids, 6-(3-hydroxypropanoyl)-5-hydroxymethyl-isobenzofuran-1(3H)-one (1), coumaric acid (2), ω-hydroxypropioguaiacone (3), and 3-methoxy-4-hydroxy-propyl alcohol (4) were isolated from the whole plant of A. graminifolia. Conclusion: Compound 1 is isolated from the whole plant of A. graminifolia for the first time. Compound 1 is a new compound named Arundina phenylpropanoid. It is tested for its anti-TMV activities. The results show that compound 1 exhibits the potential anti-TMV activities with the inhibitory rate of 22.6%.

Isolation and molecular characterization of an ethylene response factor NtERF1-1 in Nicotiana tabacum cv. Xanthi.

Apetala2/Ethylene Response Factors (AP2/ERF) play important roles in regulating gene expression under abiotic and biotic stress in the plant kingdom. Here, we isolated a member of the AP2/ERF transcription factors, NtERF1-1, from Nicotiana tabcum cv. Xanthi NN carrying the N gene, which is resistant to Tobacco mosaic virus (TMV). NtERF1-1 encoded a putative protein of 229 amino acids with a predicted molecular mass of 24.58 kDa. Nucleotide sequence analysis showed that NtERF1-1 contained a conserved DNA-binding domain at the N-terminal. Comparison of amino acid sequences revealed that NtERF1-1 possessed high similarities to ERFs from diverse plants. Semi-quantitative and real-time quantitative RT-PCR analyses indicated that NtERF1-1 was up-regulated following TMV infection. In addition, we speculated that NtERF1-1 might participate in the signal transduction pathway of defence response inducted by the interaction between the N gene and TMV.

Detection and identification of TMV infecting tomato under protected cultivation in Paraná State

During an inspection in plastic houses in Sapopema, Paraná, 90 percent of tomato plants showed leaf abnormalities, probably associated with herbicide toxity. However, virus like symptoms developed in selected hosts after mechanical inoculatation. RT-PCR reactions using primers for an internal region within the movement protein gene of TMV and ToMV resulted in the amplification of a 409 bp cDNA fragment only by TMV primers. Deduced amino acids showed 100 percent identity when compared to TMV movement protein and 94 percent with ToMV. The RT-PCR protocol was efficient for quick and conclusive determination of virus species. The virus was purified and a polyclonal antiserum was raised for future surveys in tomato crops of Paraná. The partial genomic sequence obtained for TMV-Sapopema has been deposited under the accession number DQ173945, which is the first partial genomic sequence of an isolate of TMV from Brazil in the GenBank, and the first tomato virus isolate from Paraná to have some of its biological and molecular properties determined.

Durante uma inspeção em cultivos protegidos de tomate em Sapopema, Paraná, foram observadas anormalidades foliares em 90 por cento das plantas, indicando possivelmente a existência de um problema de fitotoxidade causada por herbicidas. Todavia, os sintomas manifestados nas hospedeiras após os ensaios de inoculação mecânica revelaram que os sintomas estariam relacionados a uma infecção por Tobamovirus. As reações de RT-PCR com oligonucleotídeos específicos para uma região interna da proteína de movimento de dois vírus comuns em tomate, TMV e ToMV, resultaram na amplificação de um fragmento de 409 pares de bases, apenas com os oligonucleotídeos específicos para o TMV. Após o sequenciamento, os aminoácidos deduzidos apresentaram identidade de 100 por cento quando comparados com as seqüências das proteínas de movimento de outros isolados do TMV, e 94 por cento de identidade com seqüências do ToMV. A RT-PCR demonstrou ser um método eficiente para a rápida e conclusiva determinação da espécie viral envolvida na infecção do tomateiro. A seqüência parcial do genoma do isolado de TMV de Sapopema, está depositada no GenBank sob o número de acesso DQ173945, sendo esta a primeira seqüência genômica parcial de um isolado de TMV do Brasil, e conforme o nosso conhecimento, o primeiro isolado de TMV do Paraná a ter algumas de suas propriedades biológicas e moleculares determinadas. Um anti-soro policlonal foi produzido, o que permitirá futuros levantamentos da ocorrência de Tobamovirus nas principais áreas de cultivo de tomate do Paraná.

Expression of a Kind of Curcin Induced from Jatropha curcas (curicin 2) in Tobacco / 中国生物工程杂志

A new kind of curcin (curcin 2), induced by several kinds of stresses from Jatropha curcas leaves, under the control of the 35S CaMV (cauliflower mosaic virus) promoter, was introduced into tobacco genome by Agrobacterium tumefaciens-mediated transformation method. Curcin 2 protein was only detected in the transgenic tobacco plants transformed with the cur2p fragment (coding premature curcin 2 protein), but not in the plants transformed with cur2m fragment (coding mature curcin 2 protein). The transgenic lines expressing curcin 2 showed increased tolerance to tobacco mosaic virus (TMV).