HOU Yanhua, LI Fuchao, QIN Song, WANG Quanfu. Genetic transformation of marine Actinomycete sp. isolate M048 and expression of a recombinant plasmid carrying the apc gene[J]. Acta Oceanologica Sinica, 2006, (6): 145-152.
Citation: HOU Yanhua, LI Fuchao, QIN Song, WANG Quanfu. Genetic transformation of marine Actinomycete sp. isolate M048 and expression of a recombinant plasmid carrying the apc gene[J]. Acta Oceanologica Sinica, 2006, (6): 145-152.

Genetic transformation of marine Actinomycete sp. isolate M048 and expression of a recombinant plasmid carrying the apc gene

  • Received Date: 2006-02-13
  • Rev Recd Date: 2006-06-20
  • Optimal conditions for protoplasts formation of marine Actinomycete sp.isolate M048 were described, dense and disperse mycelia were cultured in SGGP medium, 0.5% glycine, lysozyme exposure (2 mg/cm3, 37℃, 40 min), and the concentration of sucrose in protoplast buffer was 0.4 mol/dm3 for keeping the balance of osmotic pressure.Using PEG-mediated protoplasts transformation, the transformation frequency was 89 transformants per microgramme of pIJ702.Meanwhile, an effective transformation procedure was established based on intergeneric conjugation from E.coli ET12567 (pUZ8002) using shuttle vectors pPM801, pPM803 and a (ψ)C31-derived integration vector pIJ8600 containing ori T and att P fragments.Transformation frequencies were 5.30×10-4±0.26×10-4, 8.92×10-4±0.19×10-4 and 6.38×10-5±0.41×10-5, respectively.Further, the heterologous expression of the allophycocyanin gene (apc) in the strain M048 was used to demonstrate this transformation system.SDS-PAGE and Western blot analysis confirmed the expression of recombinant APC (rAPC).
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    沈阳化工大学材料科学与工程学院 沈阳 110142

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Genetic transformation of marine Actinomycete sp. isolate M048 and expression of a recombinant plasmid carrying the apc gene

Abstract: Optimal conditions for protoplasts formation of marine Actinomycete sp.isolate M048 were described, dense and disperse mycelia were cultured in SGGP medium, 0.5% glycine, lysozyme exposure (2 mg/cm3, 37℃, 40 min), and the concentration of sucrose in protoplast buffer was 0.4 mol/dm3 for keeping the balance of osmotic pressure.Using PEG-mediated protoplasts transformation, the transformation frequency was 89 transformants per microgramme of pIJ702.Meanwhile, an effective transformation procedure was established based on intergeneric conjugation from E.coli ET12567 (pUZ8002) using shuttle vectors pPM801, pPM803 and a (ψ)C31-derived integration vector pIJ8600 containing ori T and att P fragments.Transformation frequencies were 5.30×10-4±0.26×10-4, 8.92×10-4±0.19×10-4 and 6.38×10-5±0.41×10-5, respectively.Further, the heterologous expression of the allophycocyanin gene (apc) in the strain M048 was used to demonstrate this transformation system.SDS-PAGE and Western blot analysis confirmed the expression of recombinant APC (rAPC).

HOU Yanhua, LI Fuchao, QIN Song, WANG Quanfu. Genetic transformation of marine Actinomycete sp. isolate M048 and expression of a recombinant plasmid carrying the apc gene[J]. Acta Oceanologica Sinica, 2006, (6): 145-152.
Citation: HOU Yanhua, LI Fuchao, QIN Song, WANG Quanfu. Genetic transformation of marine Actinomycete sp. isolate M048 and expression of a recombinant plasmid carrying the apc gene[J]. Acta Oceanologica Sinica, 2006, (6): 145-152.

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