苏云金芽孢杆菌无晶体突变株的逐级升温筛选及其转化性能

SCREENING OF ACRYSTALLIFEROUS MUTANTS FROM BACILLUS THURINGIENSIS AND THEIR TRANSFORMATION PROPERTIES

逐级从４２ ℃到４４ ℃和４６ ℃升温培养、并用０－０５ ％ ＳＤＳ 处理苏云金芽孢杆菌ＹＢＴ１４６３ ，获得了一系列内生质粒被部分或完全消除的无晶体（Ｃｒｙ －） 突变株，对４ 种Ｃｒｙ － 突变株的转化性能及导入的外源质粒的稳定性进行了研究。用限量培养基和４２ ℃培养筛选到Ｃｒｙ － 突变株后，升温至４４ ℃，从Ｃｒｙ － 突变株得到内生质粒被进一步消除的突变株；然后升温至４６ ℃来培养其中突变株ＢＭＢ１７０ ，并用０－０５ ％ 的ＳＤＳ进行处理，最终筛选到１ 株无质粒突变株ＢＭＢ１７１ 。用ｐＨＴ３１０１ 、ｐＢＭＢ１７３６ 、ｐＢＴＬ１ 和ｐＨＶ１２４９ 等４ 种外源质粒进行的转化及稳定性研究表明，转化频率的大小及导入质粒的稳定性与用作受体菌的Ｃｒｙ － 突变株携有的内生质粒数之间呈现一定的相关性，Ｃｒｙ－ 突变株的转化频率显著高于出发菌株，其中ＢＭＢ１７１ 的转化频率最高达１０７ 转化子／μｇ ＤＮＡ，且所导入的外源质粒的稳定性也高于其它Ｃｒｙ － 突变株及出发菌株ＹＢＴ１４６３ 。

After elevating growth temperature of YBT\|1463 and other 8 Bacillus thuringiensis parental strains to 42℃,9 acrystalliferous(Cry -)mutants were obtained,and then a series of partial plasmid\|cured mutants were further obtained from 3 Cry - mutants BMB151,BMB154 and BMB160 by elevating temperature to 44℃.One of which,BMB170,was further treated by elevating temperature to 46℃ and adding 0.05% SDS as the plasmid\|curing agent.The resulting mutant,which was designated BMB171 and had no resident plasmid, was finally obtained.The studies on the transformation effciency and stability of Cry - mutants showed that the transformation frequencies,as well as stability of several exogenous plasmids(pHT3101,pBMB1736,etc.)introduced into Cry - mutants by electroporation,were correlated with the numbers of resident plasmids bearing by the relevant recipients,the patterns of plasmid replicons and the sizes of those exogenous plasmids.Meanwhile,a much higher electro\|transformation frequencies of Cry - mutants than the parental strain YBT\|1463 was found,and the highest of the transformation frequency,in BMB 171,could reach up to 10 7 transformants/μg DNA, and the stability of those exogenous plasmids in BMB171 were also higher than in other Cry - mutants and YBT\|1463 strain.