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

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 。

SCREENING OF ACRYSTALLIFEROUS MUTANTS FROM BACILLUS THURINGIENSIS AND THEIR TRANSFORMATION PROPERTIES

After elevating growth temperature of YBT-1463 and other 8 Bacillus thuringiensis parental strains to 42 degrees C, 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 degrees C. One of which, BMB170, was further treated by elevating temperature to 46 degrees C 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 efficiency 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/microgram DNA, and the stability of those exogenous plasmids in BMB171 were also higher than in other Cry- mutants and YBT-1463 strain.