CAT为报告基因的铜绿假单胞菌启动子文库的建立及初步鉴定

Construction and Preliminary Identification of Pseudomonas aeruginosa Promoter Library Using CAT as the Reporter Gene

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。

To construct pCAT2 promoter probe vector using chloramphenicol acetyltransferase （CA T） as the reporter gene and screened promoter gene from Pseudomortas aeruginosa library. The genome of P. aeruginosa was digested by Sau3A, then the fragments （500-1 500 bp） were inserted to pCAT2 vector to construct genomic libraries of P. aeruginosa. PCR was employed to identificate the diversity of the library. Sixteen posi- tive strains were isolated using plate containing chloramphenicol （10μg/mL） and kanamycin （50 μg/mL）. In- serted fragments of those candicates were identified by PCR, sequenced and blast at NCBI. The capacity of this genomic library was up to 50 000, covering 3.5 times full length of genome. 90% of these clones contained random DNA fragments of P. aeruginosa. The efficiency of promoters screening using double resistant plate was up to 96%. Nine promoters were homology. Nine promoters were screened and preliminarily identificated and the homology to P. aeruginosa genome in GenBank is 99%, which laid a foundation for further study of P. aeruginosa gene expression and regulation.