• BMB Reports
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• v.29 no.2
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• pp.137-141
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• 1996

A partial cDNA encoding a Korean radish isoperoxidase was obtained from a cDNA library prepared from 9 day old radish root. In order to obtain Korean radish isoperoxidase cDNA, 5' RACE (rapid amplification cDNA end) PCR was performed and a cDNA (prxK1) encoding a complete structural protein was obtained by RT (reverse transcription)-PCR. Sequence analysis revealed that the length of the cDNA was 945 base pairs, and that of the mRNA transcript was ca. 1.6 kb. The deduced amino acid of the protein were composed of 315 amino acid residues and the protein was 92% homologous to turnip peroxidase, and 46% to 50% homologous to other known peroxidases. The 945 bp cDNA encoding Korean radish isoperoxidase was overexpressed in Escherichia coli up to approximately 9% of total cellular protein. The recombinant fusion protein exhibited 43 kDa on SDS-PAGE analysis and the activity level of the recombinant nonglycosylated protein was two fold higher in IPTG induced cell extracts than that of uninduced ones.

• The Plant Pathology Journal
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• v.18 no.4
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• pp.187-191
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• 2002

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 sing1e 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.