基于原生质体法保加利亚乳杆菌电转化条件的研究
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摘要
目的:研究不同因素对保加利亚乳杆菌原生质体法电转化效率的影响,通过研究电转化的最适条件,以提高电转化的效率。方法:以保加利亚乳杆菌为受体菌株,大肠杆菌-乳酸菌穿梭型质粒pMG36e为载体,通过原生质体电转化的方法研究溶菌酶浓度、质粒浓度、电转化参数、复苏培养基组成对电转化效率的影响。结果:用SMM在37℃下制备溶菌酶(30 mg/mL)对细胞进行酶解,直至通过显微镜发现约60%的原生质体形成。用1μL质量浓度为1.2μg/μL的质粒pMG36e,在电场强度7.5 kV/cm,电阻200Ω,电容25μF的条件进行电击转化,以含有0.5 mol/L蔗糖和20 mmol/L的MgCl_2、CaCl_2且无吐温80的MRS再生培养基复苏培养2.5 h,并以含红霉素的MRS平板筛选,获得1.42×10~5CFU/μgDNA的电转化效率。结论:本研究实现了保加利亚乳杆菌的高效遗传转化,并为遗传育种提供了技术支持。
Objective: The effects of different factors on the electroporation efficiency of Lactobacillus bulgaricus protoplasts were studied. The optimum conditions of electroporation were studied to improve the efficiency of electrotransformation. Methods: Lactobacillus delbrueckii subsp. bulgaricus was used as the host, and the E. coli-lactic acid bacteria shuttle plasmid pMG36e were used as the vector. The effects of the concentration of lysozyme, the concentration of plasmid, the parameters of the transformation and the composition of the recovery medium on the efficiency were studied by the method of protoplast transformation. Results: The cells were hydrolyzed by SMM at 37 ℃ with lysozyme(30 mg/mL)until approximately 60% of the protoplasts were found by microscopy. 1 μL of plasmid pMG36e with a concentration of 1.2 μg/μL was used for electric shock at the parameters of 8 kV/cm, 200 Ω and 25 μFu U. The cells were incubated in MRS medium containing 0.5 mol/L sucrose and 20 mmol/L MgCl_2, CaCl_2(without Tween 80) for 2.5 hours, and screened in MRS medium containing erythromycin, the best electroporation efficiency was obtained. As a result, the transformational efficiency reaches 1.66×10~5 cfu/μg DNA. Conclusion: This study achieved efficient genetic transformation of Lactobacillus bulgaricus and provided technical support for genetic breeding.
Objective: The effects of different factors on the electroporation efficiency of Lactobacillus bulgaricus protoplasts were studied. The optimum conditions of electroporation were studied to improve the efficiency of electrotransformation. Methods: Lactobacillus delbrueckii subsp. bulgaricus was used as the host, and the E. coli-lactic acid bacteria shuttle plasmid pMG36e were used as the vector. The effects of the concentration of lysozyme, the concentration of plasmid, the parameters of the transformation and the composition of the recovery medium on the efficiency were studied by the method of protoplast transformation. Results: The cells were hydrolyzed by SMM at 37 ℃ with lysozyme(30 mg/mL)until approximately 60% of the protoplasts were found by microscopy. 1 μL of plasmid pMG36e with a concentration of 1.2 μg/μL was used for electric shock at the parameters of 8 kV/cm, 200 Ω and 25 μFu U. The cells were incubated in MRS medium containing 0.5 mol/L sucrose and 20 mmol/L MgCl_2, CaCl_2(without Tween 80) for 2.5 hours, and screened in MRS medium containing erythromycin, the best electroporation efficiency was obtained. As a result, the transformational efficiency reaches 1.66×10~5 cfu/μg DNA. Conclusion: This study achieved efficient genetic transformation of Lactobacillus bulgaricus and provided technical support for genetic breeding.
引文
[1]韩璞,田洪涛,苑社强,等.罗伊氏乳杆菌原生质体的制备与再生条件的研究[J].中国食品学报,2010, 10(1):10-18.
[2]张莉滟,陈林,张德纯.保加利亚乳杆菌原生质体的制备与回复研究[J].中国微生态学杂志, 2004,16(2):73-74.
[3]罗红霞,陈志刚,黄彦芳,等.原生质体融合技术选育弱后酸化乳酸菌菌株的研究[J].中国乳品工业,2010, 38(1):8-11.
[4]王玉华,张桂荣,刘景圣.原生质体融合提高嗜酸乳杆菌耐酸及耐胆盐能力[J].食品科学, 2006, 27(3):96-99.
[5]贾建波,陈军,时号.乳酸菌融合菌株的构建及其特性研究[J].食品科学, 2009, 30(21):309-311.
[6]曾献春,孟冬丽.乳酸菌原生质体制备与再生研究[J].食品科学, 2006, 27(10):269-272.
[7]凌代文,东秀珠.乳酸细菌分类鉴定及实验方法[M].中国轻工业出版社, 1999:75-86.
[8]韩璞,田洪涛,苑社强,等.罗伊氏乳杆菌原生质体的制备与再生条件的研究[J].中国食品学报,2010, 10(1):10-18.
[9]任婧,李楠,陈臣,等.干酪乳杆菌LC2W最优电转化条件的建立[J].食品安全质量检测学报, 2014,5(4):1028-1032.
[10]PATNAIK R, LOUIE S, GAVRILOVIC V, et al.Genome shuffling of Lactobacillus for improved acid tolerance[J]. Nature Biotechnology, 2002, 20(7):707-712.
[11]韩鹿,李政,王凤寰,等.鼠李糖乳杆菌遗传转化方法的优化[J].生物加工过程, 2010, 8(3):44-47.
[12]张波,李晨,董慧,等. 1种快速高效提取乳酸菌质粒的方法[J].中国食品学报, 2014,(9):7-13.
[13]NEUMANN E, ROSENHECK K. Permeability changes induced by electric impulses in vesicular membranes[J]. The Journal of Membrane Biology, 1972, 10(1):279-290.
[14]MCINTYRE D A, HARLANDER S K. Improved electroporation efficiency of intact Lactococcuslactis subsp. lactis cells grown in defined media[J]. Applied and Environmental Microbiology, 1989, 55(10):2621-2626.
[15]SERROR P, SASAKI T, EHRLICH S D, et al.Electrotransformation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis with various plasmids[J]. Applied and Environmental Microbiology, 2002, 68(1):46-52.
[16]WEI M Q, RUSH C M, NORMAN J M, et al. An improved method for the transformation of Lactobacillus strains using electroporation[J]. Journal of Microbiological Methods, 1995, 21(3):97-109.
[2]张莉滟,陈林,张德纯.保加利亚乳杆菌原生质体的制备与回复研究[J].中国微生态学杂志, 2004,16(2):73-74.
[3]罗红霞,陈志刚,黄彦芳,等.原生质体融合技术选育弱后酸化乳酸菌菌株的研究[J].中国乳品工业,2010, 38(1):8-11.
[4]王玉华,张桂荣,刘景圣.原生质体融合提高嗜酸乳杆菌耐酸及耐胆盐能力[J].食品科学, 2006, 27(3):96-99.
[5]贾建波,陈军,时号.乳酸菌融合菌株的构建及其特性研究[J].食品科学, 2009, 30(21):309-311.
[6]曾献春,孟冬丽.乳酸菌原生质体制备与再生研究[J].食品科学, 2006, 27(10):269-272.
[7]凌代文,东秀珠.乳酸细菌分类鉴定及实验方法[M].中国轻工业出版社, 1999:75-86.
[8]韩璞,田洪涛,苑社强,等.罗伊氏乳杆菌原生质体的制备与再生条件的研究[J].中国食品学报,2010, 10(1):10-18.
[9]任婧,李楠,陈臣,等.干酪乳杆菌LC2W最优电转化条件的建立[J].食品安全质量检测学报, 2014,5(4):1028-1032.
[10]PATNAIK R, LOUIE S, GAVRILOVIC V, et al.Genome shuffling of Lactobacillus for improved acid tolerance[J]. Nature Biotechnology, 2002, 20(7):707-712.
[11]韩鹿,李政,王凤寰,等.鼠李糖乳杆菌遗传转化方法的优化[J].生物加工过程, 2010, 8(3):44-47.
[12]张波,李晨,董慧,等. 1种快速高效提取乳酸菌质粒的方法[J].中国食品学报, 2014,(9):7-13.
[13]NEUMANN E, ROSENHECK K. Permeability changes induced by electric impulses in vesicular membranes[J]. The Journal of Membrane Biology, 1972, 10(1):279-290.
[14]MCINTYRE D A, HARLANDER S K. Improved electroporation efficiency of intact Lactococcuslactis subsp. lactis cells grown in defined media[J]. Applied and Environmental Microbiology, 1989, 55(10):2621-2626.
[15]SERROR P, SASAKI T, EHRLICH S D, et al.Electrotransformation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis with various plasmids[J]. Applied and Environmental Microbiology, 2002, 68(1):46-52.
[16]WEI M Q, RUSH C M, NORMAN J M, et al. An improved method for the transformation of Lactobacillus strains using electroporation[J]. Journal of Microbiological Methods, 1995, 21(3):97-109.