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The Minor Outer Capsid Protein P9 Of Rice Dwarf Virus Showed Transcriptional Activation In Vivo

Posted on:2008-06-19Degree:MasterType:Thesis
Country:ChinaCandidate:Z YinFull Text:PDF
GTID:2143360215494526Subject:Plant pathology
Abstract/Summary:PDF Full Text Request
Rice dwarf virus (RDV) is a member of the genus, Phytoreovirus, the family of Reoviridae. The genome of RDV is composed of 12 segmented double-stranded RNAs encapsided within an icosahedral double-shelled particle having a diameter of approximately 700?. RDV can propagate in cells of host plants and insect vectors and is transmitted by leafhoppers (Nephotettix cincticeps or Resilia dorsalis) in nature.RDV encoded seven structural proteins and five nonstructural proteins. The seven structural proteins, namely P1, P2, P3, P5, P7, P8, P9, are the products of segments S1, S2, S3, S5, S7, S8, and S9. The five nonstructural proteins, namely Pns4, Pns6, Pns10, Pns11, and Pns12, are encoded by the S4, S6, S10, S11, and S12 segments, respectively.The RDV S6, S7, S9 and S12 were cloned from infected rice leaves by using RT-PCR. gene The ORF of each segment was fused with HA-tag or Flag-tag, and were cloned into plant expression vector pCAMBIA1301. Western blotting anaysis revealed all fusion protein were expressed.The RDV gene segment S9 was cloned into yeast expression vector pGBKT7 for growth test assay. pGBK-S9 plasmid was transformed into host strain AH109, and could grow on the plates synthetic medium lacking tryptophan, histidine, and adenine (SD/-Trp/-His/-Ade).The results showed that RDV P9 could activate transcription of the reporter genes in yeast. To further examine transcriptional activity of P9 in yeast, we used o-nitrophenylβ-D-galactopyanoside (ONPG) as a substrate for quantitative assay. Based on the growth ability test, the quantitative analysis strongly supported that RDV P9 showed transcriptional activation in yeast. To verify the transcriptional activation of P9 in planta, the transiently expressed system were conducted by usingβ-glucuronidase(GUS) as a reporter. The results indicated that strong GUS activities were visible in tissues.Quantitative analysis of relative GUS activities in leaves indeed showed P9 performed the transcriptional activation in planta by Fluorometry spectromonitor. Western blotting analysis confirmed the expression of P9 in yeast and N. benthamiana plants.Transient expression of a P9: green fluorescent protein (GFP) fusion in tobacco bright yellow 2 (BY2) cells and fluorescence microscopy analyses suggested P9 was located in the nuclear. These results suggested that RDV P9 might play an important role in regulation of host and gene transcription for the benefit of virus infection.
Keywords/Search Tags:Rice dwarf virus, Minor outer capsid P9, Transcriptional activation, GUS, Subcellular localization
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