摘要
乳杆菌的遗传转化是限制对其遗传操作的关键因素之一。本实验用广宿主质粒pHY300PLK电转化植物乳杆菌(Lactobacillus plantarum)B0080,并优化了转化条件。系统地考察了细胞的生长时期、生长培养基组分、质粒浓度、电击时电场强度、复苏培养基组分等因素对转化效率的影响。选择含5.13%甘氨酸和0.54mol/L蔗糖的MRS培养基制备乳杆菌受体细胞,加入500ng质粒DNA,在电场强度7.41kV/cm电击转化,以含有0.3mol/L蔗糖的MRS培养基复苏培养,获得最佳转化率为3.6×104CFU/μg DNA,比使用初始方法得到的转化率提高了40倍。采用优化后条件,同样实现了pHY300PLK、pBBR1MCS-5对L.amylovorus B0112和L.acidophilus B0068的高效转化,为进一步对这些菌株进行基因工程改造奠定了有利的方法学基础。
One of the genetic manipulation resection factors in Lactobacillus strains is the efficient and reproducible transformation method. To optimize the conditions for electroporation of Lactobacillus plantarum B0080 cells, a shuttle vector for Escherichia coli and Bacillus subtilis, pHY300PLK, were used. Factors including growth stage, plasmid concentration and composition of the recovery medium were investigated. Response surface methodology was employed to optimize the growth medium and pulse strength. Results showed that the optimal values for concentration of glycine and of sucrose and the pulse strength are 5.13% （W/V）, 0.54 mol/L and 7.41 kV/cm, respectively. Best electroporation efficiency is 3.6× 10^4 CFU/μg DNA obtained after incubation in 0.3 mol/L sucrose containing MRS broth for another 2 h. Several strains of Lactobacillus, such as L.amylovorus B0112 and hacidophilus B0068, are transformed with different kinds of plasmids with the optimized processing.
出处
《食品科学》
EI
CAS
CSCD
北大核心
2008年第2期205-209,共5页
Food Science
基金
新世纪优秀人才支持计划资助项目(NCET04-9704)
