【摘要】 以蓝藻高效降解菌云芝F21a(Trametes versicolor)为出发菌株,对其原生质体的制备、再生条件及限制性内切酶介导的遗传转化进行研究。结果表明:在PDA培养基上,28℃培养4 d的菌丝最适于原生质体制备;0.6 mol/L KCl为最适等渗条件;30℃混合酶液(2%cellulase+2%snailase+2%lywallzyme)酶解4 h,原生质体产量达到2.375×107个/m L,此时再生率达0.74%。采用限制性内切酶介导技术可将质粒p AN7-1导入云芝F21a的原生质体,在含有150μg/m L潮霉素的RM培养基中能够获得再生菌株,PCR验证初步表明该质粒已经转入云芝F21a原生质体。连续传代转接显示,阳性转基因菌株外源基因可以稳定表达,本结果为研究真菌高效消除蓝藻的分子机制奠定基础。更多还原

【Abstract】 Cyanobacteria high-degrading fungus Trametes versicolor F21 a as a starting strain was studied for its protoplasts preparation,regeneration conditions,and restriction endonuclease mediated genetic transformation. The results showed that the fungal mycelia cultured on the PDA medium at 28 ℃ for 4 days is the most suitable for protoplast preparation; 0. 6 mol / L KCl was the optimal isotonic conditions for the protoplasts; 30 ℃ mixed enzyme solution( 2%cellulase + 2% helicase + 2% cell-wall-lysozyme) zymolyzed for 4 hours. The protoplast production reached as high as 2. 375 × 107/ m L with the regeneration rate as high as 0. 74%. The plasmid p AN7-1 could be introduced into T. versicolor F21 a protoplasts using restriction endonuclease-mediated technology,could be verified by PCR. Continuous transfer of the mycelia indicated that the exogenous gene could be stable expression in the positive strains. The results of this study supplied a good foundation for elucidation the molecular mechanism of the algicidal fungi.更多还原