Molecular Biology

, 45:751

Cloning and characterization of trichome-specific promoter of cpr71av1 gene involved in artemisinin biosynthesis in Artemisia annua L.

  • Yueyue Wang
  • Ke Yang
  • Fuyuan Jing
  • Meiya Li
  • Ting Deng
  • Runze Huang
  • Boshi Wang
  • Guofeng Wang
  • Xiaofen Sun
  • Ke-Xuan Tang
Genomics. Transcriptomics

DOI: 10.1134/S0026893311040145

Cite this article as:
Wang, Y., Yang, K., Jing, F. et al. Mol Biol (2011) 45: 751. doi:10.1134/S0026893311040145

Abstract

Artemisinin, a sesquiterpene lactone endoperoxide derived from Artemisia annua L.(Asteraceae), is the most effective antimalarial drug. We used two methods: genome walking and thermal asymmetric interlaced polymerase chain reaction, to isolate the unknown 5′-flanking sequence of the cyp71av1 gene. The subsequent sequence analysis using bio informatics software revealed that there are several cis-acting elements inside the cyp71av1 promoter. The 5′-rapid amplification of the cDNA ends method was used to determine the transcription start site of the cyp71av1 gene. We then mapped it at the 18 base upstream of the ATG initiation codon. For simple functional characterization, we built fusion vectors between the 5′-deletion promoter and the gus reporter gene. The expression levels of the transferred vectors into A. annua L. were analyzed by the transient expression way. The β-glucuronidase assay results indicated that deletion of the region to −1551 bp did not lead to much damage in the GUS activity, whereas further deletion, to −1155 bp, resulted in a 5.5-fold reduction of GUS activity. In stabilized transgenic A. annua L. seedlings we observed that GUS expression was restricted to trichomes, which means that the promoter of the cyp71av1 gene is trichome-specific. Compared with the constitutive CaMV 35S promoter, which can express genes throughout the plant, influence on the trichome system through the trichome-specific expression promoter merely imperils plant growth. In addition, the promoter of the cyp71av1 gene contains several binding sites for transcription factors, which implies that the cyp71av1 promoter responds to more than one form of stimulation.

Keywords

promotergenome walkingthermal asymmetric interlaced polymerase chain reaction5′-rapid amplification of the cDNA endscyp71av1 genecis-acting elementsArtemisia annua L.

Abbreviations

6-BA

6-benzylaminopurine

NAA

naphthaleneacetic acid

SA

salicylic acid

MU

methyl umbelliferone

AS

acetasyringone

Copyright information

© Pleiades Publishing, Ltd. 2011

Authors and Affiliations

  • Yueyue Wang
    • 1
  • Ke Yang
    • 1
  • Fuyuan Jing
    • 1
  • Meiya Li
    • 1
  • Ting Deng
    • 1
  • Runze Huang
    • 1
  • Boshi Wang
    • 1
  • Guofeng Wang
    • 1
  • Xiaofen Sun
    • 2
  • Ke-Xuan Tang
    • 1
  1. 1.Plant Biotechnology Research Center, School of Agriculture and Biology, Fudan-SJTU-Nottingham Plant Biotechnology R&D CenterShanghai Jiao Tong UniversityShanghaiChina
  2. 2.State Key Laboratory of Genetic Engineering, School of Life Sciences, Morgan-Tan International Center for Life Sciences, Fudan-SJTU-Nottingham Plant Biotechnology R&D CenterFudan UniversityShanghaiChina