Stability of Infectious Full–Length cDNA Clones of Papaya Ringspot Virus Type P Genome

Abstract

Two recombinant plasmids of the infectious clones of Papaya ringspot virus type P (PRSV–P) containing the full–length cDNA of PRSV–P Thai strain, pCF17_7 and pCF17_8, were investigated for plasmid stability. The in vivo transcript cDNA clones derived from a full–length of PRSV–SMK5 RNA with 10,323 nucleotides of PRSV–P RNA with ten non–viral nucleotides at the 5´ end and fourteen non–viral nucleotides at the 3´end following with poly(A)35 tail. The full–length cDNA clones of PRSV–SMK5 were constructed by sequential cloning and overlap extension PCR and under the control of a partially duplicated Cauliflower mosaic virus (CaMV) 35S promoter and terminated with a CaMV 35S terminator on pCass2 plasmid vector. Stability of pCF17_7, pCF17_8 and their backbone plasmid, pCass2, during their amplifications in a bacterial host, Escherichia coli DH5–α strain, ten randomly selected colonies were separately grown in LB liquid medium containing 100 ppm of ampicillin for 24 hours with constant shaking. Then, each culture was refreshed by diluting and growth was continued to a total of ten passages. At the end of each passage, the plasmid was rapidly extracted by using alkaline lysis and visualized by agarose gel electrophoresis. The results showed that pCF17_7, pCF17_8 and pCass2 were stable, intact and infection on tested papaya seedlings after ten passages of plasmid propagation in E. coli.