单亲灭活柠檬酸杆菌与奇球菌原生质体融合

Protoplast Fusion Between Single Inactivated Citrobacter freundii and Deinococcus radiodurans

摘要:

利用单亲灭活原生质体技术对柠檬酸杆菌和奇球菌原生质体进行了融合, 考察了原生质体制备条件与融合中的影响因素。试验表明, 随着溶菌酶浓度, 酶解时间的增加和温度的上升, 原生质体的形成率呈上升趋势, 而再生率则逐渐下降。另外, 正交试验结果表明, 在PEG (6000)浓度40%、融合时间10 min、融合温度42°C、pH值为8的条件下促融, 最大融合频率可达2.74 × 10-7。筛选出来的融合子传代10 次, 性状稳定。进一步研究发现融合子在铀溶度为85 mg/L时, 比柠檬酸杆菌耐受性好；融合子和亲本吸附比较试验中, 融合子总体吸附性能比柠檬酸杆菌略高。研究结果为耐辐射基因工程菌的构建提供了基础。

Abstract:

Single inactivated protoplast fusion technique was adopted in the fusion of Citrobacter freundii and Deinococcus radiodurans, the condition of protoplast preparation and influence factor of fusion process were examined in this paper. With the lysozyme concentration, enzymolysis time increased and temperature rose, an upward trend of protoplast formation rate was showed, while regeneration rate was found gradually decreased in the test. In addition, the results of orthogonal experiment on fusion factors indicated that the optimal fusion condition was 40% PEG (6000) which induced protoplast for 10 minutes at 42°C, pH 8, and the highest fusion rate reached to 2.74 × 10-7. The screened fusants had been stably transferred for ten generations. Furthermore, the tolerance to uranium for fusant was greater than that of Citrobacter freundii under the concentration of 85 mg/L, and biosorption capability of fusant was better than that of parental strains appreciably in the comparison experiment. This works lay foundation for constructing radioresistant engineering strains.