人参尖镰孢菌原生质体制备条件优化及绿色荧光蛋白转化

Protoplast Preparation Optimization and Green Fluorescent Protein Transformation of Fusarium oxysporum in Ginseng

为人参根腐病菌的侵染及防治提供可视化的检测和分析手段,通过摸索分生孢子最适萌发时间、酶解液组成、酶解温度、摇床转数及渗透压稳定剂种类,研究人参根腐病菌-尖镰孢菌(Fusarium oxysporum)原生质体制备技术。结果表明:将人参尖镰孢菌野生型菌株0083分生孢子在YEPD培养基中培养11 h,酶解温度37℃、摇床转数180 r/min,渗透压稳定剂为0.7 mol/L NaCl的条件下,酶解液(崩溃酶、溶菌酶、纤维素酶和蜗牛酶质量浓度分别为0.01,0.03,0.02,0.005 g/mL)酶解0.25 g菌丝时,收获的菌丝体最有利于原生质体的形成和释放,共获得2.8×10^(7)/mL原生质体。用聚乙二醇PEG介导外源DNA随机插入的方法,成功获得具有绿色荧光信号和潮霉素B筛选标记的转化子,转化效率(以单位质量的DNA的转化子数计)为1 400/mg。选取转化子FOG1菌株经过6代继代培养后,菌落形态、产孢量和绿色荧光蛋白的表达与菌株0083无明显差异。因此,利用聚乙二醇(PEG)为媒介的遗传转化体系是稳定高效的遗传转化体系。

To provide a visible material for the study of infection and control of fusarium root rot of panax, through observing the optimal spores germination time, the composition of lyases and the type of osmotic pressure stabilizer, the preparation and transformation technologies of ginseng root rot pathogen-Fusarium oxysporum′s protoplast were studied in the experiment. The results revealed that the optimal conditions were young hyphae obtained in YEPD cultured for 11 h, hydrolysis temperature 37 ℃, speed of the shaker 180 r/min, and osmotic stabilizer 0.7 mol/L NaCl. When hydrolysates containing 0.01 g/mL driselase, 0.03 g/mL lysozyme, 0.02 g/mL cellulose and 0.005 g/mL snailase were used to hydrolyze 0.25 g young hyphae, the hyphae harvested were the most favorable for the formation and release of protoplasts, with a total yield of 2.8×10^(7) protoplasts/mL. The exogenous DNA was successfully transferred to F. oxysporum by the PEG-mediated transformation and the transformation frequency of 0083 was 1 400 transformants per mg DNA, which showed hygromycin resistance and fluorescence signal. After six generations of subculture, the representative transformant FOG1 showed stable morphology, sporulation and fluorescence. In conclusion, PEG-mediated protoplast transformation is quite stable and efficient.