2004 Fiscal Year Final Research Report Summary
Study on cloning of diterpene synthase genes from plants
| Project/Area Number |
14360061
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Bioproduction chemistry/Bioorganic chemistry
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| Research Institution | Hokkaido University |
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Principal Investigator |
OIKAWA Hideaki Hokkaido Univ., Grad.School of Science, Prof., 大学院・理学研究科, 教授 (00185175)
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| Co-Investigator(Kenkyū-buntansha) |
ITO Hiroyuki Hokkaido Univ., Grad.School of Agric., Associate Prof., 大学院・農学研究科, 助教授 (10241366)
TOSHIMA Hiroaki Ibaraki Univ., Fac.of Agric., Associate Prof., 農学部, 助教授 (50237088)
TOYOMASU Tomonobu Yamagata Univ., Fac.of Agric., Associate Prof., 農学部, 助教授 (60272085)
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| Project Period (FY) |
2002 – 2004
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| Keywords | diterpenes / terpene synthases / phorbol / biosynthesis / phytoalexins |
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| Research Abstract |
Extensive GC-MS analysis of hydrocarbon fractions in Aleurites fordii Hemsl showed the presence of ent-kaurene, abietadiene and pimaradiene unidentified constituent whose spectrum resembled to that of macrocyclic diterpene cembrane. Similar analysis of those of Euphobia lachyris was also investigated to identify more than 11 novel hydrocarbons. Since limited amounts of samples were obtained, synthesis of an expected biosynthetic precursor from major constituent L3 in E.lachyris was employed to obtain the 5,10-dideoxy compound which has correct stereochemistry and location of C-C double bonds. Several PCR products were obtained when we performed RT-PCR with cDNA from A.fordii and with degenerated primers designed for plant diterene synthase genes. Currently, expression and characterization of the resultant genes are investigated.
Diterpene syntheses responsible for production of phytotoxins in rice plant were investigated. Genes induced by the rice plant treated with UV-irradiation were identified by RT-PCR with degenerated primers designed for diterene synthase genes. The identified genes were cloned and expressed in Esherichia coli. GC-MS analysis of enzymatic reaction products with GGDP showed that OsCyc1 and OsCyc2 were responsible for production of syn-CDP and ent-CDP, respectively. On the other hand, similar enzymatic reactions with syn-CDP and ent-CDP established that OsDTC1, OsDTC2, OsKS4, OsKS10 produced ent-cassa-12,15-diene, stemarene, ent-sandaracopimarradiene and ent-pimara-8(14),15-diene, respectively.
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