Designing Of Degenerate Primers-Based Polymerase Chain Reaction (PCR) For Amplification Of WD40 Repeat-Containing Proteins Using Local Allignment Search Method

Degenerate primers-based polymerase chain reaction (PCR) are commonly used for isolation of unidentified gene sequences in related organisms. For designing the degenerate primers, we propose the use of local alignment search method for searching the conserved regions long enough to design an acceptable primer pair. To test this method, a WD40 repeat-containing domain protein from Beauveria bassiana was used as reference sequence to find out a group of homologous gene sequences similar to some closely related to Verticillium dahliae. The retrieved sequences from Genbank amino acid sequence databases were analyzed for designing degenerate primers. The Multiple allignments of the selected sequences revealed two highly conserve motif regions of amino acids that enabled us to design a degenerate primers. Forward and reverse degenerate primers for amplification of the related WD40 repeat-containing domain gene were designed from an alignment of the translated sequences from some other fungi. By using two rounds of Touchdown Semi-Nested RT- PCR, a partial 249 bp PCR products were isolated and sequenced. The results showed that the amplified fragment between two motifs from V. dahliae with the reference protein has 66.3% similarities at the amino acid level. However, this value when the entire of both sequences were compared is 81.9% . These data suggested that homologues transcripts encoded to WD40 repeat-contaning protein can be isolated from V. dahliae using degenerate primers from the common motif regions through a local allignment search method.