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Potato Virus X-Based microRNA Silencing (VbMS) In Potato.
Published on: May 11, 2020
A cryptic promoter in potato virus X vector interrupted plasmid construction
Yuyuan Guo1, Thomas L German, Ronald D Schultz
1Department of Pathological Sciences, School of Veterinary Medicine, Madison, WI 53706, USA. yuyuanguo@wisc.edu <yuyuanguo@wisc.edu>
BMC Molecular Biology
|March 7, 2007
Summary
A cryptic promoter in the Potato virus X (PVX) vector caused unexpected gene expression during plasmid amplification. This discovery is crucial for developing plant expression vectors and preventing downstream experimental interruptions.
Area of Science:
- Molecular Biology
- Virology
- Biotechnology
Background:
- Potato virus X (PVX) is a widely used plant expression vector for foreign gene expression.
- Sub-cloning foreign genes into PVX vectors requires plasmid amplification, typically without gene expression.
Purpose of the Study:
- To investigate the cause of failed sub-cloning and amplification of the canine parvovirus type 2 (CPV-2) VP1 gene into the PVX vector.
- To identify and characterize a cryptic promoter within the PVX vector.
Main Methods:
- Reverse transcription polymerase chain reaction (RT-PCR) to discover the cryptic promoter.
- Northern blot analysis and Real-time RT-PCR to study promoter activity.
Main Results:
- A cryptic promoter was identified within the PVX vector using RT-PCR.
- The activity of this cryptic promoter was confirmed through Northern blot and Real-time RT-PCR analyses.
- Unexpected expression of the CPV-2 VP1 gene occurred in E. coli during plasmid amplification due to the cryptic promoter.
Conclusions:
- Homologous promoter sequences within viral expression vectors must be recognized to avoid experimental issues.
- The identified cryptic promoter in the PVX vector can lead to premature foreign gene expression in E. coli, interrupting downstream molecular cloning and manipulation.
