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

作　　者：HOU Yanhua LI Fuchao QIN Song WANG Quanfu 

机构地区：[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

出　　处：《Acta Oceanologica Sinica》2006年第6期145-152,共8页海洋学报（英文版）

摘　　要：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）.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）.

关 键 词：ALLOPHYCOCYANIN PROTOPLAST intergeneric conjugantion exconjugant marine Actinomycetes 

分 类 号：Q178[生物学—水生生物学]