Tissue Specificity and Developmental Pattern of Amorpha-4,11-diene Synthase (ADS) Proved by ADS Promoter-Driven GUS Expression in the Heterologous Plant, Arabidopsis thaliana

Abstract

Amorpha-4,11-diene synthase (ADS) of Artemisia annua L. is a sesquiterpene cyclase that catalyzes the conversion of farnesyl diphosphate into amorpha-4,11-diene in the biosynthesis of the antimalarial artemisinin. To explore the mechanisms regulating the tissue-specific and developmental distributions of ADS, a full ADS promoter was generated using PCR, and fused to GUS for introduction into Arabidopsis thaliana. ADSpro::GUS fusion transcripts were organ-specific, mainly present in the anthers and trichomes of the green tissues of the juvenile leaves. This result was consistent with the ADS transcription pattern observed in A. annua as examined by RT-PCR. To determine the subcellular localization of ADS, an open reading frame (ORF) of ADS was fused to the green fluorescent protein (smGFP) gene and introduced into the A. thaliana protoplasts. GFP fluorescence was located exclusively in the cytosol, an indication that ADS is a cytosol-localized protein.

Abbreviations

ADS:amorpha-4,11-diene synthase

ADSpro:amorpha-4,11-diene synthase promoter

MS medium:Murashige and Skoog medium

ORF:open reading frame

PCR:polymerase chain reaction

RACE:rapid amplification of cDNA ends

PLM-RACE:RNA ligase mediated RACE

RT-PCR:reverse transcriptase-PCR

TAIL-PCR:thermal asymmetric interlaced-PCR

Key words

Artemisia annua - Arabidopsis thaliana - Asteraceae - amorpha-4,11-diene synthase (ADS) - amorpha-4,11-diene synthase promoter (ADSpro)

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