Identification of Class 2 1-Deoxy-D-xylulose 5-Phosphate Synthase and 1-Deoxy-D-xylulose 5-Phosphate Reductoisomerase Genes from Ginkgo biloba and Their Transcription in Embryo Culture with Respect to Ginkgolide Biosynthesis

Abstract

Diterpenoid ginkgolides having potent platelet-activating factor antagonist activity are major active ingredients of ginkgo extract. Class 2-type 1-deoxy-D-xylulose 5-phosphate synthase (GbDXS2) and 1-deoxy-D-xylulose 5-phosphate reductoisomerase (GbDXR), the first two enzymes in 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway, operating in the earlier step of ginkgolide biosynthesis, were cloned from embryonic roots of Ginkgo biloba through a homology-based polymerase chain reaction for role assessment of the enzymes. Plasmids harboring each gene rescued the respective knockout E. coli mutants. The levopimaradiene synthase gene (LPS), responsible for the first committed step in ginkgolide biosynthesis, and GbDXS2 were transcribed exclusively in embryonic root, suggesting a specific role of GbDXS2 in ginkgolide biosynthesis. GbDXR retained a higher transcription level in roots than in leaves, whereas class 1 DXS (GbDXS1) showed 30 to 50 % higher level in leaves. Ginkgolides and bilobalide were found both in leaves and roots from an earlier stage of the embryo culture. Exclusive transcription of ginkgolide biosynthesis-specific LPS and GbDXS2 in roots and the appearance of ginkgolides in leaves was consistent with translocation of the compounds from roots to leaves.

Abbreviations

dd:double distilled

DXR:1-deoxy-D-xylulose 5-phosphate reductoisomerase

DXS:1-deoxy-D-xylulose 5-phosphate synthase

LB:Luria-Bertani

LPS:levopimaradiene synthase

ME:2-C-methyl-D-erythritol

MEP:2-C-methyl-D-erythritol 4-phosphate

ORF:open reading frame

PCR:polymerase chain reaction

RACE:rapid amplication of cDNA end

RT:real time

Ginkgo biloba - 1-deoxy-D-xylulose 5-phosphate synthase - 1-deoxy-D-xylulose 5-phosphate reductoisomerase - ginkgolide

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