Abstract
A simple and reliable method to diagnose cucumber green mottle mosaic virus of watermelon in Korea (CGMMV-WK) was determined by RT-PCR, and coat protein gene for CGMMV-WK was cloned. Comparing to a method reported by Lee et al. (1996), the method developed here showed a better RT-PCR reaction. RT-PCR was possible by one step in the PCR reaction mixture that contains 20 pmol of primer, reverse transcriptase (30 unit), RNasin (5 unit) using the crude RNA solution. RT-PCR condition for specifically diagnosing CGMMV-WK was that cDNA was synthesized at 42$^{\circ}C$ for 45 min followed by pre-denaturation at 95$^{\circ}C$ for 2 min, and then PCR reaction was carried out with a programmed condition that consisted of 36 sequential cycles at 96$^{\circ}C$ for 30 sec, 6$0^{\circ}C$ for 30 sec, and 72$^{\circ}C$ for 1 min. A gene encoding the coat protein of CGMMV-WK was cloned and characterized. Nucleotide sequence of coat protein gene of CGMMV-WK shared 98.77% and 99.38% of sequence identity with those of CGMMV-W and CGMMV-SH, respecitvely, however, all of amino acid sequences were same.
한국산 수박 녹반 모자이크 바이러스(CGMMV-WK)를 TR-PCR 기술에 의해서 간편하고 확실한 진단방법을 구명하고 아울러 CGMMV의 외피단백질 유전자를 클로닝 하였다. 바이러스의 추출은 Lee등 (1996)의 간이 조즙액추출법을 변형하여 정제된 핵산 추출액을 사용하여도 RT-PCR이 양호하였으며, 20 pmol의 primer, reverse transcriptase (30 unit), Rnasin (5unit)이 첨가된 PCR 반응액에서 one step reaction으로 RT-PCR이 가능하였다. CGMMV의 진단을 위한 RT-PCR 조건으로 42$^{\circ}C$에서 45분간 cDNA를 합성하고 있어서 95$^{\circ}C$ 에서 2분간 per-denaturation하고 96$^{\circ}C$ 에서 30초, 6$0^{\circ}C$ 에서 30초 그리고 72$^{\circ}C$에서 1분간으로 36 cycle을 반응을 수행함으로서 간편하고 확실하게 CGMMV를 진단할 수 있었다. 또한 추출된 CGMMV의 외피단백질의 염기서열분석 한 결과 CGMMV-W와는 98.77%, CGMMV-SH와는 99.38%의 상동성을 가지고 있었으며 아미노산 서열은 모두 100%의 상동성을 가지고 있었다.
