African Journal of
Biotechnology

  • Abbreviation: Afr. J. Biotechnol.
  • Language: English
  • ISSN: 1684-5315
  • DOI: 10.5897/AJB
  • Start Year: 2002
  • Published Articles: 12278

Full Length Research Paper

Development of stable marker-free nuclear transformation strategy in the green microalga Chlorella vulgaris

Ahmed E. Gomma#
  • Ahmed E. Gomma#
  • Department of Biotechnology, Chonnam National University, Chonnam 550-749, Republic of Korea.
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Sung-Kwon Lee#
  • Sung-Kwon Lee#
  • Center for Nutraceutical and Pharmaceutical Materials, Myongji University, Gyeonggi 449-728, Republic of Korea.
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Sang Mi Sun
  • Sang Mi Sun
  • Department of Biotechnology, Chonnam National University, Chonnam 550-749, Republic of Korea.
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Seung Hwan Yang*
  • Seung Hwan Yang*
  • Center for Nutraceutical and Pharmaceutical Materials, Myongji University, Gyeonggi 449-728, Republic of Korea; Interdisciplinary Program of Biomodulation, Myongji University, Yongin, Gyeonggi-Do, 449-728, Republic of Korea.
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Gyuhwa Chung*
  • Gyuhwa Chung*
  • Department of Biotechnology, Chonnam National University, Chonnam 550-749, Republic of Korea.
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  •  Received: 22 April 2015
  •  Accepted: 14 September 2015
  •  Published: 16 September 2015

Abstract

Although microalgae have valuable resources with great necessity in many biotechnological applications, no tools have been developed yet for a stable genetic transformation without antibiotic marker genes in these organisms. Chlorella is one of the most useful genus for biotechnological applications. The transfer of foreign DNA (vector or linear DNA cassette) into Chlorella by electroporation has very low stability and it is hard to screen the transformants without antibiotic marker genes. However, the marker genes have some disadvantages to host cells. To avoid the negative effects caused by the marker genes, we tried to develop a stable marker-free nuclear transformation system in Chlorella. For this, linear gene expression cassettes (LGEC) were constructed with functional domains, which are responsible for transformation, of SV40 large T antigen. The LGECs were transferred into Chlorella via electroporation and durability of the LGECs were confirmed in transgenic Chlorella. Transcription levels of the LGECs were also determined at different cell cycle sates. The LGECs integrated into the chromosomal DNA of Chlorella were stably replicated and were expressed successfully at G0-, G1-, and G2-phases. This study presents a stable marker-free nuclear transformation system with potential for biotechnological applications.

Key words: Chlorella vulgaris, marker-free nuclear transformation, SV40 large T antigen, microalga.

Abbreviation

LGECs, Linear gene expression cassettes; SV40, Simian virus 40; NLS, nulear localization signal; DNA-BD, DNA binding domain.

#These authors contributed equally to this work.