环状芽孢杆菌C-2几丁酶基因在荧光假单胞菌中的表达

Expression in pseudomonas fluorescens of chitinase gene cloned from bacillus circulans C-2

将已克隆到的环状芽孢杆菌(Bacillus circulans)C-2的几丁酶基因chi1片段克隆到质粒载体pUXBF5中，得到重组质粒pUXCH1，该重组质粒在大肠杆菌中可以表达产生具有生物活性的几丁酶并分泌到胞外．将pUXCH1分别利用感受态法和三亲本杂交法转化荧光假单胞菌(Psendomonas fluorescens)，在含有卡拉霉素的金氏B平板上筛得转化子．但将转化子点种于几丁质平板上却不能产生水解圈，提取细胞内容物后用比色法也没有检测到有效的酶活性．该研究表明环状芽孢杆菌的几丁酶基因chi1已经被成功的整合到荧光假单胞菌的染色体上，但却没有表达或表达效率极低，这可能是由于荧光假单胞菌的表达系统不能正确有效的识别存在于该chi1几丁酶基因片段中的环状芽孢杆菌基因启动子而造成的．

A cloned DNA fragment from Bacillus circulans C-2 containing the chitinase gene chi1 was inserted into the carrier plasmid pUXBF5, and got a recombinated plasmid named pUXCH1. This recombinated plasmid showed high efficient expression in E. coli XL1-Blue. The pUXCH1 was transformed into Pseudomonas fluorescens by method of high efficiency transformation and three parents mating. Transconjugants were selected on King's B plates containing Kanamycin. But the transconjuants could not product the clearzone on the chitin overlay plate, and no active chitinase could be found in the cytosol when determine by method of colorimetry. This research evinced that the chitinase gene chi1 was integrated into the chromosome of Pseudomonas fluorescens successfully, but no expression could be measured. Maybe the expression system of Pseudomonas fluorescens can't recognise the gene promoter in the fragment of chitinase gene chi1.