A cryptic promoter in potato virus X vector interrupted plasmid construction
1 Department of Pathological Sciences, School of Veterinary Medicine, 2015 Linden Dr. Madison, WI53706, USA
2 Department of Entomology, College of Agricultural and Life Sciences, University of Wisconsin-Madison, 1630 Linden Dr. Madison, WI53706, USA
BMC Molecular Biology 2007, 8:17 doi:10.1186/1471-2199-8-17Published: 5 March 2007
Potato virus X has been developed into an expression vector for plants. It is widely used to express foreign genes. In molecular manipulation, the foreign genes need to be sub-cloned into the vector. The constructed plasmid needs to be amplified. Usually, during amplification stage, the foreign genes are not expressed. However, if the foreign gene is expressed, the construction work could be interrupted. Two different viral genes were sub-cloned into the vector, but only one foreign gene was successfully sub-cloned. The other foreign gene, canine parvovirus type 2 (CPV-2) VP1 could not be sub-cloned into the vector and amplified without mutation (frame shift mutation).
A cryptic promoter in the PVX vector was discovered with RT-PCR. The promoter activity was studied with Northern blots and Real-time RT-PCR.
It is important to recognize the homologous promoter sequences in the vector when a virus is developed as an expression vector. During the plasmid amplification stage, an unexpected expression of the CPV-2 VP1 gene (not in the target plants, but in E. coli) can interrupt the downstream work.