RESUMEN
Endogenous pararetroviral sequences are the most commonly found virus sequences integrated into angiosperm genomes. We describe an endogenous pararetrovirus (EPRV) repeat in Fritillaria imperialis, a species that is under study as a result of its exceptionally large genome (1C = 42 096 Mbp, approximately 240 times bigger than Arabidopsis thaliana). The repeat (FriEPRV) was identified from Illumina reads using the RepeatExplorer pipeline, and exists in a complex genomic organization at the centromere of most, or all, chromosomes. The repeat was reconstructed into three consensus sequences that formed three interconnected loops, one of which carries sequence motifs expected of an EPRV (including the gag and pol domains). FriEPRV shows sequence similarity to members of the Caulimoviridae pararetrovirus family, with phylogenetic analysis indicating a close relationship to Petuvirus. It is possible that no complete EPRV sequence exists, although our data suggest an abundance that exceeds the genome size of Arabidopsis. Analysis of single nucleotide polymorphisms revealed elevated levels of CâT and GâA transitions, consistent with deamination of methylated cytosine. Bisulphite sequencing revealed high levels of methylation at CG and CHG motifs (up to 100%), and 15-20% methylation, on average, at CHH motifs. FriEPRV's centromeric location may suggest targeted insertion, perhaps associated with meiotic drive. We observed an abundance of 24 nt small RNAs that specifically target FriEPRV, potentially providing a signature of RNA-dependent DNA methylation. Such signatures of epigenetic regulation suggest that the huge genome of F. imperialis has not arisen as a consequence of a catastrophic breakdown in the regulation of repeat amplification.
Asunto(s)
Citosina/metabolismo , Fritillaria/genética , Fritillaria/virología , Retroviridae/genética , Centrómero , Metilación de ADN , Epigénesis Genética , Regulación de la Expresión Génica de las Plantas , Genoma de Planta , Hibridación Fluorescente in Situ , Filogenia , Polimorfismo de Nucleótido Simple , ARN Pequeño no TraducidoRESUMEN
OBJECTIVE: To prepare antiserum against the CP of Lilg mottle virus (LMoV). METHODS: Specific primer was designed according to Genbank to amplify CP gene of LMoV of Fritillaria thumbergii and its sequence was analyzed. Then the CP gene was inserted into pSBET and expressed in Escherichia coli BL21 (DE3) plys E strain. The objective protein was purified by 12% SDS-PAGE firstly and subsequently 5% - 20% gradient SDS-PAGE. The antiserum against the CP was raised in mouse and their specificity was determined by Western blot. The ability to combine with nature LMoV particles was confirmed by ELISA analysis. RESULTS: LMoV CP gene shared 95% - 99% nucleotide identities and 98% - 100% amino acid identities with the CP genes reported on Genbank. The antiserum was special to LMoV CP and IgG against LMoV could combine LMoV particles. CONCLUSION: The antiserum prepared in this study is suitable for LMoV detection.
Asunto(s)
Proteínas de la Cápside/biosíntesis , Fritillaria/virología , Sueros Inmunes/aislamiento & purificación , Potyvirus/genética , Proteínas Recombinantes/biosíntesis , Secuencia de Aminoácidos , Animales , Anticuerpos/inmunología , Anticuerpos/aislamiento & purificación , Western Blotting , Proteínas de la Cápside/genética , Proteínas de la Cápside/inmunología , Clonación Molecular , Cartilla de ADN , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Fritillaria/genética , Vectores Genéticos , Sueros Inmunes/inmunología , Ratones , Potyvirus/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunologíaRESUMEN
OBJECTIVE: To prepare antiserum against Fritillary virus Y (FVY) CP for detecting FVY and study serological relationships with other viruses. METHODS: Specific primer was designed according to Genbank (accession: AM039800) to amplify CP gene of FVY infecting Thunberg fritillary. Sequence relationship with other potyviruses was made by Blast. The CP gene was inserted into pSBET and expressed in Escherichia coli BL21 (DE3) plys E strain. The object protein was purified by 12% SDS-PAGE firstly and subsequently 5% - 20% gradient SDS-PAGE. The antiserum against the CP was raised in mouse and its specificity was confirmed by Western blot analysis. The reactivity of the antiserum produced to FVY CP was tested by Western blot against the over-expressed coat proteins of 17 potyviruses. The ability to combine with nature FVY particles was confirmed by ELISA analysis. RESULTS: It shared 81.2% nucleotide acids identities with TrVY (Tricyrtis virus Y, AY 864850) CP gene, 68.1% with SMV-P (Soybean mosaic virus Pinellia strain, AJ507388. 2) CP gene and 67.2% with ZYMV (Zucchini yellow mosaic virus Luan isolate) CP gene. The prepared antiserum was special to FVY CP, also reacted moderately to the expressed CP of SMV-P (Soybean mosaic virus Pinellia strain) and weakly to that of ZYMV (Zucchini yellow mosaic virus Luan isolate). CONCLUSION: The antibody could combine to nature FVY particles and the antiserum is suitable for FVY detection by ELISA in large scale.
Asunto(s)
Proteínas de la Cápside/biosíntesis , Fritillaria/virología , Sueros Inmunes/aislamiento & purificación , Potyvirus/genética , Proteínas Recombinantes/biosíntesis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Western Blotting , Proteínas de la Cápside/genética , Proteínas de la Cápside/inmunología , China , Clonación Molecular , Cartilla de ADN , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Sueros Inmunes/inmunología , Ratones , Datos de Secuencia Molecular , Filogenia , Pinellia/virología , Reacción en Cadena de la Polimerasa , Potyvirus/clasificación , Potyvirus/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Glycine max/virologíaRESUMEN
Plants of Thunberg fritillary (Fritillaria thunbergii Miq.) from Zhejiang Province, were found to be co-infected with two distinct potyviruses. One was an isolate of the recently reported Thunberg fritillary mosaic virus (TFMV; Wei et al., (2005) Arch Virol 150: 1271-1280), while the other was a distinct virus that did not react with TFMV antiserum nor with antisera to 17 other potyviruses, except for a weak reaction with antibodies produced to soybean mosaic virus (SMV) Pinellia strain. Both viruses could be transmitted mechanically to their original host but not to any of a range of commonly used indicator plants. No local lesion host was identified that would enable the viruses to be propagated independently. The complete sequences of both viruses were determined; that of the new virus (9656 nt) had the typical genome organisation and recognised sequence motifs of a potyvirus, encoding a putative polyprotein of 351 kDa. Phylogenetic analysis, sequence comparisons, and the pattern of polyprotein cleavage sites all indicated that it was a member of the Bean common mosaic virus subgroup. The most closely related species are Soybean mosaic virus and Wisteria vein mosaic virus, with 68-69% amino acid identity between their polyproteins. This is sufficiently different for the new virus to be regarded as a distinct species, which we have tentatively named Fritillary virus Y.
Asunto(s)
Fritillaria/virología , Potyvirus/clasificación , Potyvirus/aislamiento & purificación , Secuencia de Bases , China , ADN Viral/genética , Genoma Viral , Microscopía Electrónica , Filogenia , Potyvirus/genética , Potyvirus/patogenicidad , ARN Viral/genética , ARN Viral/aislamiento & purificaciónRESUMEN
A potyvirus causing mosaic symptoms in Thunberg fritillary (Fritillaria thunbergii) was found at two sites in Zhejiang province, China. The virus was readily mechanically transmitted to its original host but not to any of 17 other widely used plant virus indicators. A polyclonal antiserum raised to purified virus particles reacted with its homologous virus but not with a range of other viruses (including 16 potyvirus species). In electron microscopy, virus particles and inclusion bodies typical of a potyvirus were seen. The complete nucleotide sequence of an isolate from Ningbo was determined. It was 9723 nt long and sequence analyses predicted the standard potyvirus organisation. The partial sequence (1664 nts at the 3'-terminus) of an isolate from Panan was also determined; the two sequences had 96.9% nt identity. In sequence comparisons and phylogenetic analyses with completely sequenced potyviruses, the new virus was most closely related to Lily mottle virus (53.0% aa identity) and Leek yellow stripe virus. The most closely related incomplete sequence in the international databases was for Lycoris mild mottle virus (72.8% nt identity in their coat proteins). These results suggest that the virus studied is a new species in the genus Potyvirus, which we have tentatively named Thunberg fritillary mosaic virus.