短小芽孢杆菌碱性蛋白酶基因启动子的克隆、鉴定及其应用.pdfVIP

HEREDITAS (Beijing) 2007 7 , 29(7): 874880 ISSN 0253-9772 DOI: 10.1360/yc-007-0874 , , , 610064 : TAIL-PCR(Thermal asymmetric interlaced PCR) , 797 bp, 390 bp , , , 160 bp DNA WApQ3 − pSUGV4 , pSUBpWApQ3 , , 466.5 U/mL 3060 U/mL : ; ; ; ; Cloning, characterization and application of the promoter of alkaline protease gene in Bacillus pumilus YANG Chun-Hui, WANG Hai-Yan College of Life Science, Sichuan University, Sichuan Key Laboratory of Molecular Biology and Biotechnology, Chengdu 610064, China Abstract: A 797 bp promoter fragment of alkaline protease gene was cloned from the Bacillus pumilus genome by employing TAIL-PCR strategy. The sequence analysis of this promoter fragment showed that the sequence accounting for gene expression was approximately 390bp. Deletion analysis of the fragment defined the minimal required se- quence of promoter for initiating transcription lies on a 160 bp region upstream of the start codon. The alkaline pro- tease gene WApQ3 containing the cloned promoter fragment was inserted into the shuttle vector pSUGV4 and the constructed expression plasmid pSUBpWApQ3 was transformed into Bacillus subtilis and B. pumilus . Active alkaline protease was successfully expressed in both host strains. The peak value of extracellular alkaline protease activities of B. subtilis and B. pumilus recombinants reached to 465.5 U/mL and 3060 U/mL, respectively. Keywords: Bacillus pumilus ; Bacillus subtilis ; alkaline protease; promoter; gene expression , , - , : 2006− 10−26;