S-Space College of Agriculture and Life Sciences (농업생명과학대학) Dept. of Food and Animal Biotechnology (식품·동물생명공학부) Theses (Master's Degree_식품·동물생명공학부)
cDNA isolation and characterization of guaiadiene synthase from lxeris dentata form. albiflora hara
흰씀바귀(Ixeris dentata form. Albiflora hara)에서 guaiadiene synthase 유전자의 분리 및 발현
- Issue Date
- 서울대학교 대학원
- Auaiadiene synthase; Sesquiterpene; Rapid amplification of cDNA ends; Lxeris dentata form. albiflora Hara.
- Thesis (master`s)--서울대학교 대학원 :농생명공학부,2003.
- various sesquiterpenes, whose first committed step in biosynthesis is
cyclization of farnesyl diphosphate by terpene synthase. To isolate the synthase
gene, we amplified 184-bp DNA fragment of a sesquiterpene synthase from I.
dentata genomic DNA using homology-based PCR technique. Cloning and sequencing
of the PCR product showed two groups of partial sequences of sesquiterpene
synthases, which suggest the presence of at least two enzymes. The sequence
information derived from rapid amplification of cDNA ends was used to produce
1956 bp of a full-length cDNA sequence including 1755 bp of an open reading
frame for 584 amino acids with a deduced size of 67.1 kDa and a pI of 5.16. The
deduced amino acid sequence displayed 38% identity with 5-epi-aristolochene
synthase of Nicotiana tabacum. The partially purified recombinant synthase has
temperature and pH optimum at 37 ¡É and pH 7.5 - 8.0, and Km of 4.3 and 14.9 M
at 25 and 37 ¡É, respectively. Expressed protein was inactive with geranyl
diphosphate, but catalyzed the cyclization of farnesyl diphosphate to produce a
sesquiterpene, which was identified through GC-MS and NMR analyses as guaiadiene.
This is the first report on cDNA isolation of the sesquiterpene synthase
producing a guaiane-type skeleton.
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