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/cm^3, 37 ℃, 40 min...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/cm^3, 37 ℃, 40 min）, and the concentration of sucrose in protoplast buffer was 0.4 mol/dm^3 for keeping the balance of osmotic pressure. Using PEG-mediated pmtoplasts transformarion, 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 （pUZS002） using shuttle vectors pPM801, pPM803 and a φC31-derived integration vector pIJ8600 containing onT and attP 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）.展开更多
文摘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/cm^3, 37 ℃, 40 min）, and the concentration of sucrose in protoplast buffer was 0.4 mol/dm^3 for keeping the balance of osmotic pressure. Using PEG-mediated pmtoplasts transformarion, 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 （pUZS002） using shuttle vectors pPM801, pPM803 and a φC31-derived integration vector pIJ8600 containing onT and attP 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）.
