The Plant Pathology Journal 2002;18(4):187-191.
Published online August 31, 2002.
Cloning of the 5`-end and Amplification of Full-Length Cdna of Genomic RNA of Lily symptomless virus
Seon Ah Choi, Ki Hyun Ryu
Abstract
This paper describes the cloning and sequence analysis of the 5`-terminal region and full-length cDNA production of genomic RNA of Lily symptomless virus (LSV), a species of the genus Carlavirus. A single DNA band about 600 bp harboring the 5`-end of genomic RNA of the virus was successfully amplified by reverse transcription- polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE), and was cloned for nucleotide sequence determination. Sequence analysis of selected RACE cDNA clones revealed that the LSV 5` non-translated region consists of 67 nucleotides long of AT rich stretch followed GC rich from the 5`-end. To produce full-length cDNA products for the viral genomic RNA, a set of LSV-specific primers could be designed based on the obtained sequence in this study and the known sequences of 3`-terminal region for the virus. Full-length cDNA copies of LSV, an 8.4 kb long, were directly amplified by the long-template RT-PCR technique from the purified viral genomic RNA samples. This full-length cDNA copies were analyzed by restriction mapping. The molecules produced in this study can be useful for the production of in vitro infectious cDNA clone, as well as, for the completion of genomic RNA sequence and genome structure for the virus.
Key Words: Carlavirus, 5`-end sequence, full-Length cDNA, Lily symptomless virus, RACE
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