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以CAT(chloramphenicol acetyltransferase,CAT)为报告基因,构建p CAT2启动子探针载体从绿脓杆菌基因文库中筛选启动子。Sau3AⅠ酶切铜绿假单胞菌的基因组,回收(500~1 500 bp)片段并与p CAT2载体连接构建铜绿假单胞菌的基因组文库,PCR鉴定文库的多样性,利用氯霉素(10μg/m L)和卡那霉素(50μg/m L)双抗平板筛选了16株阳性菌株,并用PCR、测序、NCBI比对进行鉴定。构建的基因文库的库容量可达50 000个,覆盖基因全长的3.5倍,插入率达90%,具有较强的随机性。采用双抗平板进行启动子的筛选,筛选效率高达96%。获得了绿脓杆菌基因组中的9个启动子,同源性达99%以上,并对其活性进行初步鉴定,为铜绿假单胞菌基因表达调控的进一步研究奠定基础。
Using CAT (chloramphenicol acetyltransferase, CAT) as a reporter gene, a p CAT2 promoter probe vector was constructed to screen for promoters from the Pseudomonas aeruginosa genetic library. The genome of Pseudomonas aeruginosa was digested with Sau3A Ⅰ, the fragment of 500 ~ 1 500 bp was recovered and ligated with p CAT2 vector to construct the genomic library of Pseudomonas aeruginosa. The diversity of the library was identified by PCR. Chloramphenicol (10 μg / 16 positive strains were screened by double anti-kanamycin (50 μg / mL) and PCR, sequencing and NCBI. The constructed gene library has a library capacity of 50,000, covering 3.5 times of the full-length of the gene and an insertion rate of 90%, with strong randomness. The double anti-plate promoter screening, screening efficiency up to 96%. Nine promoters were obtained from the genome of Pseudomonas aeruginosa, and the homology was over 99%. The preliminary identification of the activity of Pseudomonas aeruginosa in the genome of Pseudomonas aeruginosa laid the foundation for the further study of gene regulation of Pseudomonas aeruginosa.