Journal of Plant Biotechnology

The Korean Society of Plant Biotechnology (한국식물생명공학회)
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Molecular Characterization of Cinnamate 4-Hydroxylase gene in Red Hot Pepper (Capsicum annuum L.)

고추에서 분리한 Cinnamate 4-Hydroxylase 유전자의 분자생물학적 특성

  • Kim Kye-Won (National Institute of Agricultural Biotechnology, Institute of Biological Chemistry, Washington State University) ;
  • Ha Sun-Hwa (National Institute of Agricultural Biotechnology) ;
  • Cho Kang-Jin (National Institute of Agricultural Biotechnology) ;
  • Kim Eun-Ju (Department of Crop Science & Biotechnology, Jinju National University) ;
  • Lee Min-Kyung (Department of Crop Science & Biotechnology, Jinju National University) ;
  • Yu Jae-Ju (Department of Crop Science & Biotechnology, Jinju National University) ;
  • Kim Jong-Guk (Department of Microbiology, Kyungpook National University) ;
  • Lee Shin-Woo (National Institute of Agricultural Biotechnology, Department of Crop Science & Biotechnology, Jinju National University)
  • 김계원 (농업생명공학연구원) ;
  • 하선화 (농업생명공학연구원) ;
  • 조강진 (농업생명공학연구원) ;
  • 김은주 (진주산업대학교 작물생명과학과) ;
  • 이민경 (진주산업대학교 작물생명과학과) ;
  • 유재주 (진주산업대학교 작물생명과학과) ;
  • 김종국 (경북대학교 미생물학과) ;
  • 이신우 (농업생명공학연구원, 진주산업대학교 작물생명과학과)
  • Published : 2005.09.01
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Abstract

Three different cDNAS for cinnamate 4-hydroxylase (C4H) which are involved in the second step of the general phenylpropanoid pathway were isolated and designated as pc4h1 (1,755 bp), pc4h2 (1,655 bp), and pc4h3 (1,316 bp), respectively. The nucleotide sequence analysis revealed that both pc4h1 and pc4h2 clones encode polypeptides of 505 amino acids frame but pc4h3 clone was truncated at the 5'-end of coding region. The alignment of the deduced amino acid sequences showed that PC4H1 and PC4H2 are highly homologous (95.8% identical) with each other and contain three conserved domains which are typical in cytochrome P450 monooxygenase: proline-rich region, threonine-containing binding pocket for the oxygen molecule, and heme binding region. In addition, result of the phylogenic tree analysis revealed that both pepper C4Hs belong to Class 1. pc4h2 transcription was strongly induced in wounded fruit (400%) and root (200%) relative to its very low basal level but not in leaf or stem tissue. In case of pc4h1, the basal level of transcription was higher than pc4h2 but induction by wounding was lower in fruit and root while leaf and stem tissues did not respond to wounding. The basal level of pc4h3 transcripts was not, if any, detectable and response to wounding was not observed.

본 연구는 고추열매의 capsaicinoid 생합성 조절 기작을 연구하고자 general phenylpropanoid pathway의 2번째 단계에 작용하는 것으로 알려진 3종류의 c4h cDNA clone을 확보하여 염기서열을 분석한 결과, pc4h1와 pc4h2의 크기는 각각 1,775 bp와 1,655 bp으로 505개의 아미노산으로 구성된 펩티드를 암호하는 full length의 ORF를 갖추고 있었으나 pc4h3는 5'-말단의 coding 영역 일부가 소실 되었다. 이들은 공히 모든 cytochrome P450 효소에서 진화학적으로 보존되어 있는 3종류의 conserved region 즉 domain 1(proline-rich region), domain 2 (threonine-containing binding pocket for the oxygen molecule), 그리고domain 3(heme binding region)을 포함하고 있었으며 evolutionary tree분석을 통하여 pc4h1와 pc4h2는 모두 Class I에 속하는 것으로 서로 간에는 95.8%의 유사성을 나타내어 거의 동일한 유전자인 것으로 조사되었다. 특히 Class II로 분류된 Citrus sinensis C4H1과 Phaseolus vulgaris C4H와는 단지 64.9에서 64.5%의 homology를 나타내었다. 또한 pc4h2는 상처를 주지 않은 조직에서는 비교적 적은 양의 mRNA 발현수준을 보였으나 상처를 가한 후 6시간의 열매에서는 400%, 뿌리에서는 200%까지 그 발현 양이 증가하였다. 이와 유사하게 pc4h1의 전사량도 상처에 의하여 유도는 되나 basal level이 pc4h2보다 높아서 발현증가 비율은 그다지 높지 않았다. 반면에 pc4h3 유전자는 상처를 주지 않은 모든 조직에서 거의 발현되지 않았으며 상처에 의하여서도 전혀 반응을 하지 않았다.

Keywords

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