枯草芽孢杆菌感受态细胞的制备及质粒转化方法研究  

Competent Preparation and Plasmid Transformation of Bacillus subtilis

作　　者：李瑞芳[1] 薛雯雯[1] 黄亮[1] 熊前程[1] 王彬[1] 

机构地区：[1]河南工业大学生物工程学院,郑州450001

出　　处：《生物技术通报》2011年第5期227-230,共4页Biotechnology Bulletin

基　　金：国家自然科学基金项目(31071922);河南省重点科技攻关项目(0623030300)

摘　　要：为便于枯草芽孢杆菌工业化生产应用,对Spizizen创立的枯草芽孢杆菌DNA转化方法进行改进。用GMI和GMII溶液处理枯草芽孢杆菌野生型菌株BS501a、营养缺陷型突变株DB1342和非营养缺陷型突变株WB800,用改进的方法制备感受态细胞,用7.5 kb质粒pSBPTQ进行转化,并研究RNA、酵母粉、水解酪蛋白、培养方法对枯草芽孢杆菌质粒转化的影响。结果表明,该方法适用于不同基因型枯草芽孢杆菌的质粒转化,营养缺陷型突变株DB1342的转化率为750 CFU/μgDNA,非营养缺陷型突变株WB800转化率为1 070 CFU/μg DNA,野生型菌株BS501a转化率为270 CFU/μg DNA。根据影响转化效率的因素,推测在该方法中,枯草芽孢杆菌质粒转化原理:一定生物量的枯草芽孢杆菌在外界营养条件和钙、镁离子作用下,细胞壁和细胞膜形成缺陷,使外源DNA转入枯草芽孢杆菌细胞内。In order to facilitate the usage of B.subtilis in industrial production,the DNA transformation method founded by Spizizen was modified.Wild-type strain BS501a,nutrient deficiency mutant DB1342 and nutrient non-deficiency mutant WB800 were induced to be competent by GMI and GMII solutions using modified transformation method,which a 7.5 kb plasmid pSBPTQ was transformed into.The effects of RNA,yeast extract,hydrolyzed casein and culture method on the transformation efficiency were studied.The results showed that the modified transformation method was suitable for B.subtilis strains of all kinds of gene type,the transformation efficiency of nutrient deficiency mutant DB1342 was 750 CFU/μg DNA,the transformation efficiency of nutrient non-deficiency mutant WB800 was 1 070 CFU/μg DNA,and the transformation efficiency of wild-type strain BS501a was 270 CFU/μg DNA.According to the results of the affecting factors on the transformation efficiency,the transformation mechanism were speculated：the forming-defects of the cell wall and cell membrane of B.subtilis gave chance of heterogeneous DNA to enter the cells of B.subtilis under the effects of external nutritional conditions,Calcium ions and Magnesium ions.

关 键 词：枯草芽孢杆菌 感受态细胞 质粒转化方法 

分 类 号：Q782[生物学—分子生物学]