德氏乳杆菌保加利亚亚种电转化平台的构建和优化

德氏乳杆菌保加利亚亚种(Lactobacillus delbrueckiisub sp.bulgaricus)是最具经济价值的乳酸菌之一,在世界上广泛应用于酸奶和其它发酵乳生产。当前对该菌的代谢机制研究甚少。外源基因的转化效率是制约其分子代谢机制研究的重要因素。本研究以pMG36c为材料,对L.delbrueckiisub sp.bulgaricus CH3进行电转化条件研究。结果表明,在电转化过程中,电场强度、质粒的浓度、细胞生长状态均对转化效率有明显影响,得到了该菌株的最适电转化条件为:对数初期的细胞,质粒浓度为100 ng/50μl菌液,在10 kV/cm电场强度下电转化,转化后细胞在复壮培养液中培养3 h后涂布选择性培养基,转化率可达2.6×103CFU/μg DNA。以甘氨酸、醋酸锂、二硫苏糖醇处理细胞壁,发现醋酸锂和二硫苏糖醇共同处理对转化效率有明显改善,可提高转化效率。

Construction and optimization of electrotransformation for Lactobacillus delbrueckii subsp. bulgaricus

Lactobacillus delbrueckii subsp.bulgaricus has been widely used in yogurt and other fermentation milk in the world,is recognized as the higher economic value of lactic acid bacteria.The genetic transformation of L.delbrueckii subsp.bulgaricus is a key factor which restricts its genetic manipulation.The method of electrotransformation of L.delbrueckii subsp.bulgaricus CH3 was constructed by means of plasmid pMG36c.The factors were evaluated,including electric field strength,concentration of plasmid and cell growth phage.The optimum factors are the cell harvested at the early exponential growth phase,electrotransformation at the 10kV/cm,with 100 ng plasmid per 50 μl sample,and 3h cell incubation time.The transformation efficiency is 2.6×103CFU/μg DNA at the optimum conditions.The cell wall-weakening agents were studied,including glycine,lithium acetate（LiAc） and dithiothreitol（DTT）.The transformation efficiency was improved under the treatment with LiAc and DTT.