كلونينگ و تعيين توالي بخشي از ژن تانناز در آسپرژيلوس نيجر

Isolation and Cloning of Partial Tannase Gene from Aspergillus niger

Background and Objective: Tannase (tannin acyl liydrolase) is an industrially important enzyme produced by a large number of fungi, which hydrolyzes the ester and depside bonds of gallotannins and gallic acid esters. In the present work- a partial gene of tannase from .tfv rgilltts /tiger as an important bioreaci.or has been cloned and sequenced. Materials and Methods: Genomic DNA was extracted from Aspergilius /tiger using a modified version of the method described by Menniuiu (1977). Degenerated primers were designed against highly conserved amino acid sequences of tannase from f1.spergillu.s awwnori. rlspergillus uculeatus and A.spergillus ory.-ae and used for PCR after determining optimum annealing temperature and magnesium concentration. Southern analysis was carried out with a number of restriction enzymes using DIG labelled tannase probe and a single band between I .5-6 kb were observed depending on the restricting enzymes. Results: A PCR product of the predicted size (about 990 bp) were obtained and ligated into pGEMT®- Easy vector and cloned into E, coil Top-10Fʹ (Stratigen) using the standard cloning procedures. Following transformation of competent cells, blue/white selection performed on ampicillin agar plates containing x-gal and IPTG. Plasmid DNA was extracted from tra.nsformant cells restricted with EcoR I and digested DNA run out by gel electrophoresis. Three colonies containing the expected insert size for tannase gene(s) were sent for sequencing. Conclusion: All three nucleotide sequences were identical and shared a high degree of homology to Aspergll/us awamori. Aspergilhts uculeaius and A.spergillus orvzae tannase sequences (64-52% identity at the nucleotide level). Result of southern blotting indicating probability of single copy oftannase gene in the genome of A. niger.