Cloning And Functional Analysis Of 5'flanking Region Of Genomic DNA Encoding Alkaline Lipase From Penicillium Expansum PF898

A 5' flanking region DNA encoding alkaline lipase from Penicillium expansumPF898 with about 2000 bp was specially amplified by LA-PCR, then sequenced andsubmitted into the GenBank data (GenBank accession NO. DQ677520). The sequencecontained a novel promoter region with TATA box,CAAT box and GC box-like elementsin the identical positions shared by the eucaryote promoter sequence regions incomparison with the sequences in the GenBank.The amplified sub-cloned products of 5' flanking region were ligated into the vectorwhich contained the green fluorescence protein (GFP) gene as the reporter gene toconstruct the expression plasmids. The recombined plasmids were transfered toEscherichia.coil TOP10. Many Escherichia.coil cells with the recombined plasmid couldemit fluorescence. The result indicated that the 5' flanking region had the promoter genesegment, which could promote GFP gene expression in Escherichia.coil cells.The sub-cloned products of 5' flanking region were ligated into the promoter-probevector pSUPV4 with kanamycin-resistant gene (Kan~r) as the reporter gene to construct therecombined plasmids, which were transfered to Escherichia.coil DH5a to determine thepromoter function of the sub-cloned PCR produces. Escherichia.coils with therecombinant Kan~r gene promoted by the above fragments showed the different resistancesto Kanamycin in the range of 1000-3500μg/mL. The result was similar to the analysisresult of promoter function using GFP as the reporter gene, showing that the promoterfunction was sured.