Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Cloning of Cytochrome P450 Gene involved in the Pathway of Capsidiol Biosynthesis in Red Pepper Cells

고추세포에서 Capsidiol 생합성을 유도하는 Cytochrome P450 유전자의 탐색

  • Kwon, Soon-Tae (School of Bioresources Science, Andong National University) ;
  • Kim, Jae-Sung (Korean Atomic Energy Research Institute) ;
  • Jung, Do-Cheul (Department of Plant Resources, Sangju National University) ;
  • Jeong, Jeong-Hag (School of Bioresources Science, Andong National University) ;
  • Hwang, Jae-Moon (School of Bioresources Science, Andong National University) ;
  • Oh, Sei-Myoung (School of Bioresources Science, Andong National University)
  • Published : 2003.12.01
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Abstract

In order to measure the enzyme activity of 5-epi-aristolochene hydroxylase, one of cytochrome P450 (P450) enzymes in eicitor-treated pepper cell, we used in vivo assay method and demonstrated a dramatic suppression of the activity by P450-inhibitors, ancymidol and ketocornazole. Using RT-PCR method with degenerate primer of the well conserved domains found within most P450-enzymes, and using cDNA library screening method, one distinct cDNA, being designated P450Hy01, was successfully isolated from elicitor-treated pepper cells. P450Hy01 mRNA was all induced in elicitor-treated cells whereas never induced in control cells. Moreover, levels of P450Hy01 expression were highly correlated with the levels of extracellular capsidiol production by different elicitors in cell cultures. P450Hy01 transcript was also induced by several other elicitors such as, cellulase, arachidonic acid, jasmonic acid, yeast extract as well as UV stress. P450Hy01 sequence contained high probability amino acid matches to known Plant P450 genes and ORF with a conserved FxxGxRxCxG heme-binding domain. P450Hy01 cDNA showed 98% of homology in sequence of nucleotide as well as amino acid to 5-epi-aristolochene-1, 3-hydroxylase (5EAl, 3H) which has been isolated in tobacco cells, suggesting that P450Hy01 is prominent candidate gene for P450-enzyme encoding 5EAl, 3H in pepper cell.

추에서 항균 phytoalexin으로 알려진 capsidiol의 생합성을 촉매하는 5-epi-aristolochene hydroxylase는 cytochrome P450 (P450) 억제제인 ancymidol과 ketocornazol에 의해 그 활성이 특이적으로 억제되어, 이 효소가 P450계 효소임을 알 수 있었다. P450 효소가 공통으로 보유하는 염기서열을 지닌 primer를 이용하여 RT-PCR과 cDNA screening을 실시한 결과 고추배양세포에서 elicitor 처리에 의해 강하게 유도되는 cDNA (P450Hy01)를 cloning하였다. 배양세포에 cellulase, arachidonic acid, jasmonic acid, 자외선 등을 처리하여 capsidiol을 생합성하는 량과 P450Hy01 mRNA의 발현정도는 밀접한 유사성이 있었다. P450Hy01의 염기서열은 담배에서 밝혀진 5-epi-aristolochene-1,3-hydroxylase와 98%의 유사성이 있었으며, P450 효소가 공통으로 지니는 heme-binding domain인 FxxGxRxCxG를 보유하고 있었다 이러한 결과는 본 연구에서 cloning된 P450Hy01이 고추의 세포에서 capsidiol의 생합성을 촉매하는 5-epi-aristolochene hydroxylase 효소를 coding 하고 있음을 시사한다.

Keywords

References

  1. Proc. Natl. Acad. Sci. USA v.87 Sequence analysis of ripening-related cytochrome P450 cDNA from avocado fruit Bozak,K.R.;H.Yu;R.Sirebak;R.E.Christoffersen https://doi.org/10.1073/pnas.87.10.3904
  2. Annu. Rev. Plant Physiol. Plant Mol. Biol. v.46 Biochemistry and molecular biology of the isoprenoid biosynthetic pathway in plants Chappell,J. https://doi.org/10.1146/annurev.pp.46.060195.002513
  3. Phytochemistry v.26 Accumulation of capsidiol in tobacco cell cultures treated with fungal elicitor Chappell,J.;R.Nable;P.Fleming;R.A.Andersen;H.R.Burton https://doi.org/10.1016/S0031-9422(00)84695-6
  4. Plant Physiol. v.96 Multiple forms of plant cytochrome P450 Donaldson,R.P.;D.G.Luster https://doi.org/10.1104/pp.96.3.669
  5. Plant Physiol. v.110 Cloning of wound-induced cytochrome P450 monooxygenases expressed in pea Frank,M.R.;J.M.Deyneka;M.A.Schuler https://doi.org/10.1104/pp.110.3.1035
  6. Phamacol. Rev. v.40 The molecular biology of cytochrome P450s Gonzalez,F.J.
  7. Phytochemistry v.38 5-epi-Aristolochene 3-hydroxylase from green pepper Hoshino,T.;T.Yamaura;H.Imaishi;M.Chida;Y.Yoshizawa;K.Higashi;H.Ohkawa;J.Mizutani https://doi.org/10.1016/0031-9422(94)00695-P
  8. Kor. J. Plant Tissue Culture v.28 Elicitation of seedlings and cultured cells for the production of capsidiol in Capsicum annuum Kwon,S.T.;E.A.Jung;H.Y.Park;K.H.Son
  9. Korean J. Life Science v.8 Cloning of elicitor-inducible 5-epi-aristolochene hydroxylase in tobacco cell suspension cultures Kwon,S.T.;I.J.Lee;J.Chappell
  10. Physiol. Plant v.15 Revised medium for rapid growth and bioassay with tobacco tissue culture Murashige,T.;F.Skoog https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
  11. Plant Physiol. v.87 Cytochrome P450 from the mesocarp of avocado (Persea americana) Okeefe,D.P.;K.J.Leto
  12. Arch. Biochem. Biophys. v.393 Cloning, heterologous expression, and functional characterization of 5-epi-aristolochene-1,3-dihydroxylase from tobacco (Nicotiana tabacum) Ralston,L.;S.T.Kwon;M.Schoenbeck;J.Ralston;D.J.Schnek;R.M.Coates;J.Chappell https://doi.org/10.1006/abbi.2001.2483
  13. Molecular Cloning: A Laboratory Manual(2nd Ed.) Sambrook,J.;E.F.Fritsch;T.Maniatis
  14. Critical Rev. in Plant Sci. v.15 Plant cytochrome P450 mono-oxygenases Schuler,M.A. https://doi.org/10.1080/713608134
  15. Proc. Natl. Acad. Sci. USA v.90 Isolation and sequence of a cDNA encoding the Jerusalem artichoke, cinnamic acid 4-hydroxylase, a major plant cytochrome P450 involved in the general phenylpropanoid pathway Teutsch,H.G.;M.P.Hasenfrantz;A.Lesot;C.Stoltz;J.M.Garnier;F.Durst;D.Werck-Reichhart
  16. Phytochemistry v.29 Synthesis of (+)-5-epi-aristolochene and (+)-1-deoxycapsidiol from capsidiol Whitehead,I.M.;D.F.Ewing;D.R.Threlfall;D.E.Cane;P.C.Prabhakaran https://doi.org/10.1016/0031-9422(90)85100-T
  17. Phytochemistry v.28 5-epi-aristolochene is a common precursor of the sesquiterpenoid phytoalexins capsidiol and debneyol Whitehead,I.M.;D.R.Threlfall;D.F.Ewing https://doi.org/10.1016/0031-9422(89)80114-1

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