Study on cloning of diterpene synthase genes from plants

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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.