电脉冲法转化苏云金芽孢杆菌BMB171的研究  

TRANSFORMATION OF BACILLUS THURINGIENSIS RECIPIENT BMB171 BY ELECTROPORATION

作　　者：李林[1] 邵宗泽[1] 喻子牛[1] 

机构地区：[1]华中农业大学微生物科学技术系,武汉430070

出　　处：《微生物学通报》2000年第5期331-334,共4页Microbiology China

摘　　要：研究了用电脉冲法转化苏云金芽孢杆菌受体菌ＢＭＢ１７１的优化条件以及由转化导入的几类ｃｒｙ基因在ＢＭＢ１７１中的表达效果。结果表明，采用ＳＧ溶液作电脉冲缓冲液，用１０．ｏｋＶ／ｃｍ的脉冲场强和１次电脉冲（４．６ｍｓ）以及采用对数前期（ＯＤ６５０ｎｍ为０．２～０．３）收获的受体菌，可以达到最高转化频率，其中用ｐＨＴ３１０１电转化 ＢＭＢ１７１的最高频率达 ８×１０７转化子／μｇ ＤＮＡ。转化频率随质粒ｐＨＴ３１０１浓度的增加，在５４．６９ｐｇ／ｍＬ至３．５０μｇ／ｍＬ范围内直线性增加，随后达到饱和。转入的几种外源质粒在ＢＭＢ１７１中可分别表达其携带的ｃｒｙｌＡｃ１０，ｃｒｙｌＡｂ，ｃｒｙｌＣａ和ｃｒｙ３Ａａ基因，产生特征性的杀虫晶体蛋白，并形成典型的伴孢晶体。This paper reports the optimized electro-transformation parameters of Bacillus thuringiensis plasmid-free mutant strain BMB171 by electroporation, and expressing effect of several cry genes introduced in this recipient. It showed that a highest electro-transformation frequency could be obtained, when SG solution was used as the buffer, and a 10.0V/cm of field strength, one time of pulse as well as a growth phase of recipient cells at the exponential phase (OD650nm value was 0.2~0.3) were selected. The highest of electro-transformation frequency with pHT3101 could reach at 8 ×107 hansformants/μg DNA. The transformation frequencies increased at linear velocity as the concentration increase of pHT3101 from 54.69pg/ml to 3.50μg/mL, then reached saturation afterwards. All plasmids introduced in BMB171 could produce characteristic insecticidal crystal proteins through expression of relevent cry genes carried by them. Meanwhile, these insecticidal crystal proteins could form parasporal crystals, which have characteristic geometric shapes.

关 键 词：苏云金芽孢杆菌 电脉冲转化法 CRY基因 基因表达 

分 类 号：Q933[生物学—微生物学] Q78