Home > 2006, (6) > Genetic transformation of marine Actinomycete sp. isolate M048 and expression of a recombinant plasmid carrying the apc gene

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

2006, (6): 145-152.

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

1.  Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China
2.  Graduate School, Chinese Academy of Sciences, Beijing 100039, China
3.  School of Ocean, Harbin Institute of Technology, Weihai 264209, China,Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China,Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China and School of Ocean, Harbin Institute of Technology, Weihai 264209, China

Received Date: 2006-02-13
Accepted 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).

Key words: allophycocyanin , protoplast , intergeneric conjugantion , exconjugant , marine Actinomycetes

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

HOU Yanhua, LI Fuchao, QIN Song and WANG Quanfu,