identification of the diterpene synthase in salvia divinorum used to synthesize salvinorin a adrian...
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Identification of the diterpene synthase in Salvia divinorum used to synthesize salvinorin A
Adrian LaurenziTucson High Magnet SchoolResearch MethodsMarch 26, 2008
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What is Salvia divinorum?
Cultigen found in Sierra Mazatec region of Oaxaca, Mexico
Traditionally used by the Mazatec Indians for its hallucinogenic and therapeutic effects.
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Why are we interested in it?
Produces salvinorin A; a potent k-opioid receptor agonist
Medicinal potential:
Diseases w/ hallucinations: schizophrenia, dementia, Alzheimer's, depression (Roth et al., 2002)
Drug addiction (Roth et al., 2002)
HIV and AIDS (Peterson et al., 2001)
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Localization of salvinorin A
Salvinorin A is a diterpenoid found in glandular trichomes
Salvinorin A is likely produced within glandular trichomes
Possible anti-feeding properties (insect or mammal herbivores)
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Purpose
Identify the protein that acts as the first step in the biosynthesis of salvinorin A
Future applications:● Synthesis of salvinorin A
● Further understanding of chemical mechanisms of k-opioid receptor in the brain
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Salvinorin A quantification
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Biosynthesis of salvinorin A
IPP and DMAPP are synthesized via the non-mevalonate (MEP) pathway (Kutrzeba et al., 2007)
GGPP is assembled by joining 4 units of IPP
A diterpene synthase converts GGPP into a precursor of salvinorin A
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cDNA library & sequencing
The cDNA library was sequenced via 454 sequencing
RNA was isolated from S. divinorum trichomes and used to construct a cDNA library
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BLAST searching & assemblyBLAST diterpene synthases against S. divinorum library
Results assembled into a small contig, which is then Netblasted
Contig1 assembled from 112 fragments and Netblasted
BLAST results from Netblast against S. divinorum library
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BLAST searching & assembly
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Conclusion
Enzyme likely to be an ent-copalyl diphosphate synthase
Future methods:
Express protein and assay with GGPP
Test for enzyme activity in S. divinorum
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Acknowledgments
Margaret Wilch
Kevin Vogel
Dr. David Gang, Ph.DDepartments of Plant Sciences,Biochemistry, and Molecular BiophysicsBIO5 InstituteUniversity of Arizona