RESUMO
Vernonia cinerea plants with yellow vein symptoms were collected around crop fields in Madurai. A portion (550 bp) of the AV1 gene amplified using degenerate primers from the total DNA purified from diseased leaf sample was cloned and sequenced. Specific primers derived from the above sequence were used to amplify 2,745 nucleotides with the typical genome organization of begomoviral DNA A (EMBL Accession No. AM182232). Sequence comparison with other begomoviruses revealed the greatest identity (82.4%) with Emilia yellow vein virus (EmYVV-[Fz1]) from China and less than 80% with all other known begomoviruses. The International Committee on Taxonomy of Viruses (ICTV) has therefore recognized Vernonia yellow vein virus (VeYVV) as a distinct begomovirus species. Conventional PCR could not amplify the DNA B or DNA beta from the diseased tissue. However, the beta DNA (1364 bp) associated with the disease was obtained (Accession No. FN435836) by the rolling circle amplification-restriction fragment length polymorphism method (RCA-RFLP) using Phi 29 DNA polymerase. Sequence analysis shows that DNA beta of VeYVV has the highest identity (56.8%) with DNA beta of Sigesbeckia yellow vein Guangxi betasatellite (SibYVGxB-[CN: Gx111:05]) and 56-53% with DNA beta associated with other begomoviruses. This is the first report of the molecular characterization of VeYVV from V. cinerea in India. The complete molecular characterization, phylogenetic analysis, and putative recombination events in VeYVV are reported.
Assuntos
Fagos Bacilares/genética , Begomovirus/genética , DNA Polimerase Dirigida por DNA/genética , Doenças das Plantas/virologia , Vernonia/virologia , Fagos Bacilares/enzimologia , Sequência de Bases , DNA Polimerase Dirigida por DNA/metabolismo , Genoma Viral , Dados de Sequência Molecular , Filogenia , Recombinação Genética , Alinhamento de SequênciaRESUMO
The sequence motifs present in the replication initiator protein (Rep) of geminiviruses have been compared with those present in all known rolling circle replication initiators. The predicted secondary structures of Rep representing each group of organisms have been compared and found to be conserved. Regions of recombination in the Rep gene and the adjoining 5' intergenic region (IR)of representative species of Geminiviridae have been identified using Recombination Detection Programs. The possible implications of such recombinations on the increasing host range of geminivirus infections are discussed.
Assuntos
DNA Helicases/química , DNA Helicases/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Geminiviridae/química , Recombinação Genética , Transativadores/química , Transativadores/genética , Proteínas Virais/química , Proteínas Virais/genética , Sequência de Aminoácidos , Geminiviridae/classificação , Geminiviridae/genética , Dados de Sequência Molecular , Filogenia , Estrutura Secundária de Proteína , Alinhamento de Sequência , Replicação ViralRESUMO
The replication-initiator protein (Rep) from a soybean-infecting geminivirus was overexpressed in E. coli as a fusion protein with maltose binding protein (MBP). In spite of the presence of the highly soluble MBP as the fusion partner, the overexpressed MBP-Rep fusion protein formed insoluble inclusion bodies. The protein was solubilized from the inclusion bodies and refolded. The refolded MBP-Rep protein was purified using ion exchange and amylose affinity chromatography. The activity of the purified MBP-Rep was assessed using an in vitro cleavage assay. Soluble and stable MBP-Rep protein was obtained in high abundance, providing the feasibility of large-scale production of active Rep protein for functional characterization and X-ray crystallographic structure determination.
Assuntos
DNA Helicases/isolamento & purificação , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Geminiviridae/genética , Glycine max/virologia , Dobramento de Proteína , Transativadores/isolamento & purificação , Transativadores/metabolismo , Proteínas Virais/isolamento & purificação , Proteínas Virais/metabolismo , Cromatografia de Afinidade , Cromatografia por Troca Iônica , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Corpos de Inclusão , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Transativadores/genética , Proteínas Virais/genéticaRESUMO
The complete nucleotide sequences of two soybean-infecting begomoviruses have been determined from central and southern parts of India. Sequence analyses show that the isolate from central India is a strain of Mungbean yellow mosaic India virus (MYMIV) and the southern Indian isolate is a strain of Mungbean yellow mosaic virus (MYMV). Multiple DNA B components could be detected with the soybean strain of Mungbean yellow mosaic virus species. The nucleotide sequence similarity between the DNA A components of the two isolates is higher (82%) than that between the corresponding DNA B components (71%). Analyses of the common region of the genomic components of these two virus isolates indicate considerable divergence in the origin of replication (ori), which did not impair their infectivity as demonstrated for the central Indian isolate by agroinfection with partial tandem repeats (PTRs) of the genomic components. Detailed sequence and phylogenetic analyses reveal the distribution and possible recombination events among legume-infecting begomoviruses from South-East Asia.
