Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/68304
標題: Construction and evaluation of transgenic tobacco plants expressing the coat protein gene of papaya ringspot virus with different translation leaders
作者: Cheng, Y.H.
Yeh, S.D.
關鍵字: coat protein gene
papaya ringspot virus
potyvirus
translation leader
phosphorothioate-modified dna
nucleotide-sequence
mosaic-virus
potyvirus
protection
resistant
infection
rna
phenotypes
serology
期刊/報告no:: Botanical Bulletin of Academia Sinica, Volume 41, Issue 1, Page(s) 1-10.
摘要: Papaya ringspot virus (PRSV) YK isolate used in this study is a local mosaic strain isolated from Yung-Kang, Tainan, and its genome has been cloned and completely sequenced. A NcoI site before the coat protein (CP) reading frame of PRSV YK was generated by oligonucleotide-directed mutagenesis, and then the CP reading frame with the 3' noncoding region of PRSV YK was ligated with the gus leader sequence from the pGEM vector to create the construct pGGCP. To express the CP with a homologous viral translation sequence, the gus leader was replaced by the cDNA sequence corresponding to the 5' region (nt 1-347) of PRSV genome to generate a protein containing 9 kDa polypeptide of PRSV P1 protein fused with the CP, and the construct was designated as pG5'CP. In vitro translation from the transcripts derived from pGGCP and pG5'CP generated protein products of 36 kDa and 45 kDa, respectively. Both proteins reacted with the antiserum to PRSV CP, and the level of 36 kDa protein was higher than that of 45 kDa protein. The CP reading frame with the gus or PRSV 5' leaders was individually subcloned into a Ti binary vector. Transgenic tobacco plants (Nicotiana tabacum L. Havana 423) expressing the PRSV CP gene with the gus leader (GCP lines) or with the viral leader (5'CP lines) were obtained by Agrobacterium-mediated transformation. When the transgenic lines were analyzed by western blotting, the protein products of 36 kDa and 45 kDa reacting to PRSV CP antiserum were detected in the GCP lines and 5'CP lines, respectively. The presence of the CP gene in the transgenic tobacco was also confirmed by polymerase chain reaction (PCR) using primers specific to the CP gene. Analysis of segregation ratios in the R-1 plants of four GCP lines and four 5'CP lines indicated that the CP gene in all of them was nuclearly inherited as a single dominant trait. R-0 and R-1 plants of the four GCP lines and four 5'CP lines were inoculated with tobacco etch virus (TEV), potato virus Y (PVY), or pepper mottle virus (PepMoV). The transgenic lines showed significant delay in symptom development and the severity of symptoms was attenuated. The GCP lines expressing the PRSV CP gene by the gus leader accumulated higher levels of CP and showed higher degrees of resistance than the 5'CP lines with the PRSV 5' leader. Our results indicate that the homologous viral leader does not enhance CP expression either in vitro or in vivo, nor does it provide better resistance in transgenic tobacco.
URI: http://hdl.handle.net/11455/68304
ISSN: 0006-8063
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