벼의 arginine decarboxylase DNA clone의 재조합 및 염기서열 분석

Molecular Cloning and Nucleotide Sequencing of a DNA Clone Encoding Arginine Decarboxylase in Rice (Oryza sativa L.)

ADC는 diamine인 putrescine 생합성의 두가지 경로중에서 식물계에서 특히 중요한 효소이며, ADC 유전자는 E. coli, 귀리, 토마토 genome에서 이미 cloning된 바 있다. 벼 (Oryza sativa L.) 게놈 DNA의 PCR 증폭을 위해서 토마토와 E. coli의 ABC cDNA의 보존된 부분과 일치하는 두개의 degenerate oligonucleotides (17mer)를 인위 합성하였으며, 증폭의 결과 약 1 kbp 크기의 DNA가 관찰되었다. 증폭된 DNA 절편은 1,022bp 염기서열을 포함하고 있는 ORE (open reading frame)으로 확인되었다. 이 PCR product는 POEM-originated T vector에 재조합하였으며 PstI 제한효소로 약 500bp 크기로 절단하여 pGEM-3Zf(+/-) vector에 subcloning하였다. 벼 ADC clone의 염기서열은 귀리와 토마토 ADC cDNA 서열의 같은 부분과 각각 74%와 70%의 동질성을 갖는 것으로 나타났으며, 예상되는 아미노산 서열은 귀리와 토마토 ADC 단백질과 각각 45%와 62%의 동질성이 관찰되었다. 귀리와 E. coli, 토마토와 귀리 그리고 토마토와 E. coli ADC 아미노산 서열에서 각각 34%, 47%, 그리고 38%의 유사성 정도가 보고된 것을 비교하여 볼 때, 벼와 귀리 및 토마토 사이의 유사성 정도는 다른 비교 보다도 월등히 높았다. 벼 유묘기 잎조직에서 추출한 RNA를 이용한 Northern blot 분석에서 ADC는 약 2.5kbp의 전사체로 발현됨이 확인되었다.

Arginine decarboxylase (ADC) is the first enzyme in one of the two pathways of diamine putrescine biosynthesis in plants. The genes encoding ADC have previously been cloned from Escherichia coli, oat and tomato genome. Two degenerate oligonucleotides (17-mer) corresponding to two conserved regions of ADC were used as primers in polymerase chain reaction of rice (Oryza sativa L.) genomic DNA, and an approximately 1.0 kbp fragment was obtained. This amplified PCR product showed an open reading frame which contains 1,022 bp of nucleotide sequences. This PCR product was cloned into pGEM-originated T vector and the short 500 bp PstI digested fragment was subcloned into pGEM-3zf(+/-) vectors to facilitate sequencing. The nucleotide sequence of this PCR product showed about 74% and 70% identity with the same regions of the oat and tomato ADC cDNA sequences, respectively. The predicted amino acid sequence exhibited 45% and 62% identity with oat and tomato ADC polypeptide fragments, respectively. The sequence similarities of 34%, 47% and 38% were previously reported in oat and E. coli, tomato and oat, and tomato and E. coli ADC amino acids, respectively. Therefore, similarities and identities between rice and oat or tomato are remarkably higher than those others of the previous reports. In the highly conserved regions in both the amino acid sequence and spacing regions among the sequences of these three, rice ADC open reading frame also has the exactly same regions with the striking similarity. RNA blot analysis showed that hnc is expressed as a transcript of approximately 2.5 kbP in the rice seedling leaf tissues.