| After screening the relevant Bacillus thuringiensis recipients and studied the optimized parameters of high-frequency transformation by electroporation, this Ph. D dissertation established a high-frequency transformation system for Bacillus thuringiensis. The acrystalliferous and plasmid-free derivatives of Bacillus thuringiensis were screened with ethidium bromide treatment, elevating growth temperature from 42 to 44 then to 46 by degrees, and treating it with 0.05% Sodium Docecyl Sulphate (SDS) as the plasmid-curing agent at 46 . There is no acrystalliferous derivative was screened after treating 4.5 g/mL ethidium bromide, but after elevating growth temperature of YBT-1463 and other 8 Bacillus thuringiensis parental strains to 42 , 9 acrystalliferous derivative were obtained. A series of partially plasmid-cured derivatives were further obtained from 3 acrystalliferous derivatives originated from wild-type strain YBT-1463 by elevating temperature to 44 . One of which, BMB170, was further treated by elevating temperature to 46C and adding 0.05% SDS as the plasmid-curing agent, a plasmid-free derivative which designated BMB171, was finally obtained.The morphological, physiological, biochemical and genetic diversities between Bacillus thuringiensis wild-type strain YBT-1463 and its plasmid-free mutant BMB171 was comparatively studied. It showed that the plasmid-free mutant strain BMB171 lost the ability to form the parasporal crystal, but there was on obvious diversities were observed on the sensitivity to 10 antibiotics, the utilization of 19 carbon sources and 12 nitrogen sources, as well as the growth properties between YBT-1463 and BMB171, whereas the electro-transformation frequencies of BMB171 were much higher than those of YBT-1463, respectively with 5 exogenous plasmids as the donor DNAs. The transformation frequencies of exogenous plasmids in BMB171 were 7.5 103-8.1 108 folds as much as those in YBT-1463, and stability of exogenous introduced in 8MB 171 was obviously higher than in YBT-1463.The optimized parameters of high-frequency in Bacillus thuringiensis plasmid-free mutant BMB171 by electroporation was studied. It showed that a highest electro-transformation frequency could be obtained, when SG solution was used as the buffer, and a 10.0 kV/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 transformation frequencies increased at linear velocity with the concentration increase of pHT3101 from 54.69 pg/mL to 3.50 g /mL, then reached saturation afterwards.The recombinant plasmids pBMB121L, pBMB9821L and pBMB986L were constructed after transferring Bacillus thuringiensis ICPs gene crylAal, crylAclO and crylCa from their original plasmid vectors to different plasmid vector. The relevant recombinant strains were obtained after introducing the 3 recombinant plasmids into Bacillus thuringiensis plasmid-free mutant strain 8MB 171 by electroporation.The transformation and expression properties of 8MB 171 were studied. A high transformation efficiency in BMB171 was found, whose transformation frequencies were 1000-, 6.7-, 12.5-, 66.7-, 3500-, and 2-fold as much as the highest values of 3 recipients Bt-4Q7, Bt-4D10 and Bti.IPS.78/11, respectively using 6 exogenous plasmids (pHT3101, pBMB3305, etc.) as the donor DNA. The highest of transformants could reach 107 with per ng DNA in BMB171. The stability of exogenous plasmids introduced in BMB171 was correlated with the patterns of replicons, and sizes of plasmids. All plasmids bearing ICPs genes introduced in BMB171 could produce characteristic insecticidal crystal proteins through expression of relevant ICPs genes. Meanwhile, these insecticidal crystal proteins could form parasporal crystals, which have characteristic geometric shapes. The properties of expressing cry1Ac and crylCa in BMB171 was also studied by quantifying the expression level and determining the toxicity of the relevant transformants against two Lepidopteran larvae. I...
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