Construction of infectious clones of Papaya ringspot virus (PRSV) and comparative studies of genomic analyses among different strains of PRSV
Date Issued
2008
Date
2008
Author(s)
Liu, Ting-Chung
Abstract
Abstract apaya ringspot is one of the most destructive diseases of papaya. This disease invaded Taiwan in 1975, and immediately spread all over the papaya-cultivated areas in Taiwan. This disease is the greatest impact for the papaya industry. It is caused by Papaya ringspot virus（PRSV）,which is the member of the genus Potyvirus. Based on the differences of foliar symptoms, PRSV can be categorized into three main strains such as the severe mottling（SM）, deformation（DF）and severe mottling with necrosis（SMN）strains. The SM strain was ever absolutely predominant in Taiwan, but the DF and SMN have recently become newly rising dominant strains. The alignment of nucleotide sequences reported that three strains are very close with around 97% homology. Their individual genes within the genome also show high homology (95 ~100％) except of the P1 gene (94.5~94.8％) and 5’ UTR（91.8％ ~92.9％）. To facilitate the study of the relation between the symptoms and the nucleotide sequences, this thesis was dedicated to construct the full-length infectious clones to synthesize of PRSV genomic RNA through in vitro transcription for artificial inoculation tests. The DF strain was first chosen as the template for amplification and cloning of several genomic fragments with reverse-transcription polymerase chain reaction (RT-PCR) and the TA-cloning kit (pGEM-T vector). These fragments were further amplified with polymerase chain reaction (PCR), digested with restriction enzymes and assembled into a complete cDNA clone (pT3-NP) of PRSV genome with T4-DNA ligase, The cDNA clone was cut with the XbaI restriction enzyme into a linear form and in vitro transcription was performed. The RNA products were collected (2500~3000 ng/μl) and used in the inoculation tests to confirm their infectivity. PRSV was monitored in several infected papaya plants with RT-PCR assays and the infectious percentage was 25～33％. Sequencing of the RT-PCR products demonstrated these viruses derived from the pT3-NP clone. The leaf-deformation symptoms were observed in the infected papayas 3 weeks after inoculation. The continuous monitoring of PRSV with RT-PCR showed that PRSV was replicating in the hosts, which confirm that the transcripts from pT3-NP are infectious. However, the apparent and typical symptoms of PRSV-DF train such as mottling, green islands and fern-leaf have not been observed so far.
Subjects
papaya
Papaya ringspot virus
infectious clone
in vitro transcript
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