几丁质酶ChiAC不同时期表达对苏云金杆菌的影响

Effect of the Expression of Heterologous ChiAC in Bacillus thuringiensis at Different Stage

目的:研究几丁质酶在苏云金杆菌不同生长时期的异源表达对菌体生长、芽胞形成及杀虫活性的影响。方法:构建了几丁质酶基因chiAC分别在其自身营养期启动子和芽胞特异性强启动子-二元毒素启动子调控下的两个重组质粒pBC和pB-bC,将其转化进苏云金杆菌以色列亚种IPS78菌株(得到的重组菌株分别命名为Bti-pBC和Bti-pBbC)。结果:菌株Bti-pBC在营养期表达(0～40h)ChiAC,ChiAC的表达显著影响其芽胞形成,进而导致对致倦库蚊毒力的显著下降(LC50>1×105 ng/ml,原始菌株为5.61 ng/ml);菌株Bti-pBbC则在其整个生长阶段(0～80 h)均能大量稳定的表达ChiAC,且ChiAC的表达不影响其芽胞形成及对蚊幼的毒力(LC50为7.16 ng/ml)。结论:几丁质酶基因chiAC在二元毒素启动子调控下能在苏云金杆菌中强效稳定的表达且不影响其芽胞形成和杀虫活性。

Objective： To investigate the effects of the expression of a vegetative stage-specific chitinase gene chiAC in Bacillus thuringiensis at different stage on bacterial growth and larvicidal activities.Method： Two plasmids named pBC and pBbC,containing the chitinase gene chiAC under the control of its native vegetative promoter and a sporulation-dependant binary toxin promoter respectively,were transfomed into B.thuringiensis subsp.israelensis IPS78 strain（resulted recombinant strains named Bti-pBC and Bti-pBbC respectively）.Result： The recombinant strain Bti-pBC,which contained the chiAC gene under its native vegetative promoter,could express ChiAC during its vegetative stage（0-40 h） and that the expression of heterologous ChiAC resulted in the significant reduction of sporulation and the loss of toxicity against Culex quinquefasciatus of recombinant（LC501×105 ng/ml,compare to the native strain＇s 5.61 ng/ml）.However,the recombinant Bti-pBbC,containing chiAC gene under the control of a sporulation-dependant binary toxin promoter,expressed ChiAC throughout the bacterial growth stage（0-80h）,and no deleterious effect on sporulation and toxicity of the recombinant was observed in comparison with the parental strain（LC50 is 7.16ng/ml）.Conclusion： By replacing its native vegetative promoter with the sporulation-dependant binary toxin promoter,ChiAC got a higher efficiency and more stable expression in B.thuringiensis strain without deleterious effect on bacterial growth and larvicidal activities.