【作者】 李林；

【导师】 李阜棣； 喻子牛；

【作者基本信息】 华中农业大学 ， 微生物学， 1999， 博士

【摘要】 本研究通过筛选苏云金芽胞杆菌受体菌和对受体菌电脉冲转化条件的研究，建立了一种苏云金芽胞杆菌的高频转化系统。分别用溴化乙锭诱变处理、逐级升温培养以及用SDS处理等三种方法对苏云金芽胞杆菌无晶体（Cry^-）和无质粒突变株进行了筛选研究。用限量培养基和42℃培养9株野生菌株，结果筛选到9株Cry^-突变株；继续升温至44℃来培养其中的三个Cry^-突变株，得到内生质粒被进一步消除的突变株。然后进一步升温至46℃来培养突变株BMB170，并用SDS处理，最终筛选到1株无内生质粒的突变株BMB171。对出发菌株YBT-1463和其无质粒突变株BMB171的部分形态、生理生化和遗传学特性进行的比较研究的结果表明，突变株BMB171不形成伴胞晶体，但在个体形态与菌落特征、对红霉素等10种抗生素的敏感性、对葡萄糖等19种碳源和谷氨酸等12种氮源的利用能力及生长性能与出发菌株YBT-1463无明显差异。用pHT3101等5种供体质粒电转化BMB171的效率比电转化YBT-1463的效率显著提高，而转化导入的3种外源质粒在BMB171中的稳定性也明显高于出发菌株YBT-1463。 对用电脉冲法转化苏云金芽胞杆菌受体菌BMB171优化条件的研究结果表明，采用SG溶液作电脉冲缓冲液，用10.0 kV／cm的脉冲场强和1次电脉冲（4.6ms），以及采用对数前期(OD₆₅₀约0.2～0.3)收获的受体菌，可以达到最高转化频率。转化频率随质粒pHT3101浓度的增加，在54.69pg／mL至3.50μg／mL范围内呈线性增加，随后达到饱和。 通过将苏云金芽胞杆菌杀虫晶体蛋白基因cry1Aal、cry1Ac10和cry1Ca转移至不同质粒载体上，分别构建了重组质粒pBMB121L、pBMB9821L和pBMB986L，并分别导入无质粒突变株BMB171，筛选得到携带相应ICPs基因的重组菌株。 研究了苏云金芽胞杆菌Cry^-突变株和无质粒突变株BMB171的转化性能和表达性能。用pHT3101、pBMB3305、pBMB1736、pBMB671、pBTL-1和pHV1249等6种外源质粒电转化无质粒突变株BMB171的转化频率，分别是Bt-4Q7、Bt-4D10和Bti．IPS．78／11等3种常用受体菌相应最高转化频率的2³500倍。无质粒突变株BMB171表达cry1Ac基因的表达量高于Bt-407，略低于Bt-4D10和Bti．IPS．78／11，而表达cry1Ca和cry3Aa的表达量高于这3种对照受体菌。对小菜蛾3龄幼虫和甜菜夜蛾初孵幼虫的毒力测定结果表明，BMB171表达cry1Ac基因的杀虫毒力高于Bt-4Q7和Bt-4D10，略低于Bti．IPS．78／11；而BMB171表达cry1Ca基因的毒力高于这3种受体菌。 对转座子mini-Yn10在苏云金芽胞杆菌野生菌株YBT-1463及其无质粒突变株BMB171中的转座作用进行了初步研究。更多还原

【Abstract】 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更多还原