抄録
Transformed adventitious roots, so-called hairy roots, induced in a plant by Ri pia smid of Agrobacteium rhizogenes are expected to be a valuable culture system for production of secondary metabolites due to the high inoculation ratio and different cells. Hairy roots (HAHR) induced in Hyoscyamus albus by inoculation with A rhizogenes showed the production of sesquiterpen-type phytoalexin, lubimin, by treatment with CuSO_4. HAHR also produced solavetivone and its oxygenated and acylated derivatives by treatment with methyl jasmonate (MeJA). Seven compounds including four new compounds were isolated, and their structures were elucidated to be solavetivone, and its hydroxy- and acyl-derivatives. We found that HAHR co-treated with CuSO_4-MeJA gave different products from those derived by treatment with MeJA Thus we tried to isolate the products from the large scale hairy root culture co-treated with CuSO_4-MeJA. Eleven metabolites including four new compounds were isolated and their structures were determined to be further acylated and oxygenated derivatives of solavetivone. To study details of sesquiterpene-type phytoalexins production in HAHR, we tried to identify terpene synthase (TPS), cytochromeP450 (CYP) and transcription factor WRKY. As the results of degenerate PCR and RACE experiments, full length cDNA of these genes named HaTIN, HaCYP and HaWRKY were obtained from HAHR. From the results of promoter analysis using GenomeWalker (Clontech), we confirmed the presences of W-boxs, which is a cis-element for WRKY, in the promoter region of HaTPS and HaCYP.lb analyze the relationship between these genes and phytoalexin production, RT-PCR experiments were carried out with specific primers of these genes and RNA prepared from HAHR treated with CuSO_4, MeJA and CuSO_4-MeJA, respectively. HaTPSrelated to formation of carbon skeleton of the sesquiterpene-type phytoalexin was induced by treatment with the all chemical elicitors. Expressions of HaCYP and HaWRKYwere confirmed in the HAHR at 6 hr after treatment with MeJA and at 24 hr after treatment with CuSO_4-MeJA, but not with CuSO_4. These result indicated that HaTPS was related to the first committed step in the pathway of the phytoalexins, and HaCYP and HaWRKYwere related to the oxidative modification of the phytoalexins.
