Synthesis of in vivo Full-Length Transcripts cDNA of Papaya Ringspot Virus (PRSV-P) Thai Strain

Abstract

A whole genome of Papaya ringspot virus (PRSV–P) Thai strain was cloned. Seven complementary DNAs (cDNAs) of subgenomic fragments encompassed the whole genome of PRSV–SMK5 isolate. The PRSV-SMK5 genome was synthesized by reverse transcriptionpolymerase chain reaction (RT–PCR) using primers designed from unique sites of restriction enzymes in the genome. The sizes of the PCR products ranged from 493 to 2,706 nucleotides (nts). Each fragment was cloned into the pGEM^®–T easy plasmid vector to verify the sequence. The confirmed plasmids were used as templates for over lapping PCR and sequentially cloned into pCass2 plant expression vector. The transcription of PRSV genome was driven by Cauliflower mosaic virus (CaMV) 35S promoter and terminated by CaMV terminator. Two fulllength cDNA clones of PRSV-SMK5 isolate, which were designated as pCF17_7 and pCF17_8, were obtained. Each clone showed 10,358 nts in length with 35 nts of an artificial poly(A) tail and containing both 5´ and 3´ non-coding regions consisting of 85 and 206 nts, respectively. A polyprotein of 3,343 deduced amino acids was encoded from 10,032 nts. Both plasmids, pCF17_7 and pCF17_8, were infectious and showed the PRSV typical symptoms at 21 days after mechanical inoculation on papaya seedlings. The in vivo transcribed RNAs in the plant sap were confirmed by RT–PCR, serological detection of PRSV coat protein and PRSV particles were also observed under immuno electron microscopy.