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Applied Biochemistry and Biotechnology

, Volume 158, Issue 3, pp 571–581 | Cite as

Transfer of Ephedra Genomic DNA to Yeasts by Ion Implantation

  • Jie Lü
  • Xiang Jin
  • Pei-Hong Mao
  • Xiang-Dong Ma
  • Hai-Qiu Ling
  • Yong-Hong Fan
  • Long Yu
  • Bao-Shan Wu
  • Ping-Kai Ouyang
Article

Abstract

The genomic DNA from Ephedra glauca was randomly transferred to Saccharomyces cerevisiae and Hansenula anomala by argon and nitrogen ion implantation. Through repeated subculturing and using reversed phase high-performance liquid chromatography analysis to quantify the concentrations of the secondary metabolites, l-ephedrine and d-pseudoephedrine, 12 recombinant strains of genetically stable yeast were obtained, each using glucose as a carbon source, NaNO3 as a nitrogen source and producing l-ephedrine and/or d-pseudoephedrine. After culturing in liquid medium for 72 h, extracellular l-ephedrine and d-pseudoephedrine concentrations of 18.85 and 4.11 mg/L, respectively, were detected. Using l-ephedrine and d-pseudoephedrine as the target products, the transformation efficiencies of the genomic DNA from E. glauca transferred to S. cerevisiae and H. anomala were 1.15% (1/87) and 2.13% (8/376), respectively. The addition of the amino acid, L-Phe, to culture media substantially changed the amount of l-ephedrine and/or d-pseudoephedrine produced by the recombined yeasts. However, the change in metabolite production was not consistent among strains, rising in some, while dropping to nondetectable levels in others. After random amplification of polymorphic DNA (RAPD) analysis, four RAPD primers were obtained from the initial 100 RAPD primers, each amplifying different fragments with the recombined yeast Ar_Han0458 genome. Using one primer as polymerase chain reaction primer, the result showed that the recombined yeast Ar_Han0458 genome matched E. glauca genomic DNA at 150 bp, indicating a successful transfer of genetic information, facilitated by ion implantation.

Keywords

Ion implantation Ephedra genomic DNA Transformation Recombined yeast l-Ephedrine d-Pseudoephedrine 

Notes

Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China (no. 10365001 and no. 30760009). We thank Kun-Yun Qiao, Jun Zhou, and Ting Feng for their assistances in sample testing and the Division of Ephedrine, Xinjiang International Industry Co. Ltd. for providing standard samples of l-ephedrine and d-pseudoephedrine for RP-HPLC analysis.

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Copyright information

© Humana Press 2009

Authors and Affiliations

  • Jie Lü
    • 1
    • 2
  • Xiang Jin
    • 2
  • Pei-Hong Mao
    • 2
  • Xiang-Dong Ma
    • 3
  • Hai-Qiu Ling
    • 2
  • Yong-Hong Fan
    • 2
  • Long Yu
    • 1
  • Bao-Shan Wu
    • 2
  • Ping-Kai Ouyang
    • 1
  1. 1.College of Life Science and PharmacyNanjing University of TechnologyNanjingPeople’s Republic of China
  2. 2.Institute of Ion Beam Biotechnology, College of Physics Science and TechnologyXinjiang UniversityUrumqiPeople’s Republic of China
  3. 3.College of Life ScienceHubei UniversityWuhanPeople’s Republic of China

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