59 イネ培養細胞におけるファイトアレキシン生合成に関与するジテルペン環化酵素遺伝子 OsDTC1 の cDNA クローニングと機能解析

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In suspension-cultured rice cells, diterpenoid phytoalexins such as momilactones and ent-phytocassanes are produced in response to exogenously applied elicitors such as chitin oligomers and cerebrosides. We have attempted to isolate cDNAs encoding diterpene cyclases involved in the phytoalexin production by RT-PCR with the degenerate primers designed on the basis of conserved motifs of plant diterpene cyclases, indicating the presence of two cDNAs encoding putative diterpene cyclases, Os DTC1 and OsDTC2. The full-length OsDTC1 cDNA was isolated by RACE-PCR and overexpressed in Escherichia coli as a fusion protein with glutathion S-transferase. The recombinant OsDTC1 converted ent-copalyl diphosphate (ent-CPP) into a diterpene hydrocarbon-like compound X and (-)-pimara-8,15-diene in ca. 10% and less than 1% yields, respectively. We also found that compound X occurs in elicited rice cells with known diterpene hydrocarbons, 9β-pimara-7,15-diene, stemer-13-ene, pimara-8,15-diene, aphidicol-15-ene, and aphidicol-16-ene. Considering the possibility that compound X is ent-cassa-12,15-diene, a hypothetical biosynthetic intermediate leading to ent-phytocassanes, we synthesized ent-cassa-12,15-diene from (R)-Wieland-Mischer ketone. The retention time and full-scan mass spectrum of compound X in capillary GC-MS corresponded completely to those of the synthetic ent-cassa-12,15-diene. On the other hand, when syn-CPP was used as a substrate, the recombinant OsDTC1 converted the substrate into (+)-pimara-8,15-diene and aphidicol-15-ene in less than 1% yield. Both pimara-8,15-diene and aphidicol-15-ene are minor components in the elicited rice cells, and their metabolites have not been identified as rice phytoalexins. These results indicate that OsDTC1 functions as ent-cassa-12,15-diene synthase. Isolation of a full-length OsDTC2 cDNA is now in progress.
植物化学調節学会の論文
2002-10-31

59 イネ培養細胞におけるファイトアレキシン生合成に関与するジテルペン環化酵素遺伝子 OsDTC1 の cDNA クローニングと機能解析

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