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Molecular Biology and Genetics |
Electronic Journal of Biotechnology ISSN: 0717-3458 |
Vol. 10 No. 4, Issue of October 15, 2007 |
© 2007 by Pontificia Universidad Católica de Valparaíso -- Chile |
Received July 24, 2006 / Accepted December 26, 2006 |
DOI: 10.2225/vol10-issue4-fulltext-1 |
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Amplification of genome-integrated BeYDV replicons by transient expression of Rep in Arabidopsis thaliana
Yong-Sun Moon
College of Natural Resources
Department of Horticulture
Yeungnam University
214-1 Dae-dong, Gyeongsan-si
Gyeongsangbuk-do, 712-749
Republic of Korea
Kathleen L. Hefferon*
Cornell Research Foundation
Cornell University
20 Thornwood Dr., Ithaca
NY 14850 USA
Tel: 607 257 1081
Fax: 607 254 1015
E-mail: klh22@cornell.edu
*Corresponding author
Financial support: This work comprises a portion of Dr. Moon’s doctoral thesis at Cornell University, and was partially supported by DARPA grant N65236-98-1-5411, the Department of Horticulture and an Albert S. Roe fellowship at Cornell University.
Keywords: Agrobacterium-mediated expression, BeYDV-based vector, gene amplification, GUS expression, Rep expression, transgenic Arabidopsis.
Abbreviations: |
BeYDV: |
bean yellow dwarf geminivirus |
CaMV: |
cauliflower mosaic virus |
ChsA: |
chalcone synthase A gene |
CP: |
coat protein |
DAI: |
days after infiltration |
DIG: |
Digoxygenin |
dsRNA: |
double-stranded RNA |
MSV |
maize streak virus |
ORFS: |
open reading frames |
PCR: |
polymerase chain reaction |
PTGS: |
post-transcriptional gene silencing |
RDR: |
RNA-dependent RNA polymerase |
TGMV: |
tomato golden mosaic virus |
TMV: |
tobacco mosaic virus |
TYDV: |
tobacco yellow dwarf virus |
WDV: |
wheat dwarf virus |
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Bean yellow dwarf geminivirus (BeYDV) has been used as a potential vector to improve foreign gene expression, specifically, to achieve higher yields of vaccine proteins in plants. Previously, we have shown that when the BeYDV replication initiator protein Rep was provided in trans, replication and gene expression of GUS were enhanced enormously from a BeYDV expression vector in a transient assay system. In this paper, transgenic lines of Arabidopsis (cv. Columbia) were generated harboring the BeYDV cis-acting elements required for replication. Constructs encoding BeYDV Rep or intronless Rep open reading frames (ORFs) were transiently introduced into transgenic plants via Agrobacterium-mediated infiltration in order to examine the relative levels of replication and expression of the genome-integrated GUS reporter gene. This study shows that expression of Rep protein was regulated in trans from a separate cassette which enabled the rescue, replication and enhancement of the genome-integrated GUS gene in transgenic Arabidopsis. We conclude that Rep expression can be effectively controlled in Arabidopsis plants, and that regulation of Rep expression can result in the amplification of a genome-integrated foreign gene by circumventing the negative effects of gene silencing.
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