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cDNA Isolation and Characterization of (+)-Germacrene A Synthase from Ixeris dentata form. albiflora Hara

Kim Min-Young    (School of Agricultural Biotechnology, Seoul National University   ); Chang Yung-Jin    (School of Agricultural Biotechnology, Seoul National University   ); Bang Myun-Ho    (Graduate School of Biotechnology and Plant Metabolism Research Center, Kyung Hee University   ); Baek Nam-In    (Graduate School of Biotechnology and Plant Metabolism Research Center, Kyung Hee University   ); Jin Jianming    (School of Agricultural Biotechnology, Seoul National University   ); Lee Cheol-Ho    (School of Agricultural Biotechnology, Seoul National University   ); Kim Soo-Un    (School of Agricultural Biotechnology, Seoul National University, Graduate School of Biotechnology and Plant Metabolism Research Center, Kyung Hee University  );
  • 초록

    Ixeris dentata form. albiflora Hara, a perennial medicinal herb, accumulates various sesquiterpenes whose first committed step in biosynthesis is the cyclization of farnesyl diphosphate by terpene synthase. To isolate the synthase gene IdGAS, we amplified a 184-bp DNA fragment of sesquiterpene synthase gene from I. dentata genomic DNA, using a homology-based PCR technique. Sequence information derived from the rapid amplification of cDNA ends was used to produce a 1956-bp full-length cDNA sequence. This included a 1755-b open reading frame for 584 amino acids, with a deduced size of 67.1 kDa and a pi of 5.16. The partially purified recombinant synthase had an optimum temperature and pH at $37^{\circ}C$ and 7.5 to 8.0, respectively, as well as a $K_m$ of 11.0 and 14.9mM at 25 and $37^{\circ}C$ , respectively. The expressed protein was inactive with geranyl diphosphate, but did catalyze the cyclization of farnesyl diphosphate to produce a sesquiterpene that was then identified through GC-MS and NMR analyses as (+)-germacrene A. When 44 residues were deleted from its N-terminal, the mutant lost $90\%$ of its activity, suggesting that additional residues are necessary for full enzymatic activity. Transcript levels were comparable between roots and leaves, but began to decline in leaves near the onset of flowering.


  • 주제어

    Compositae .   (+)-germacrene A synthase .   Ixeris dentata .   rapid amplification of cDNA ends .   sesquiterpene.  

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