IDENTIFICATION, ISOLATION, CLONING AND SEQUENCING APARTIALANNEXIN GENE FROM AUREOBASIDIUM PULLULANS

Background and Objectives: Annexin is the common name for genes and proteins that were identified as calcium-dependent phospholipid-binding proteins. Recently a more complex set of functions has been recognized for this superfamily of proteins including in vesicle trafficking, cell division, apoptosis, calcium signalling, mineralization, crystal nucleation inside the extracellular organelles-matrix vesicles (MY s) and growth regulation. Methods: In the present work Aureobasidium pullulans strain PRAFS8 genomic DNA was extracted. Using designed primers from a highly conserved region of annexin genes of Aspergillus Jumigatus and Aspergillus niger a 800 bp PCR product was obtained from degenerated PCR. The 800 bp PCR product was gel purified and cloned into E. coli using the suitable plasmid and standard cloning procedures. From grown transformed E. coli, plasmid was extracted and the presence of expected insert in the plasmid, was confirmed by digestion of plasmid by Eco RI restriction enzyme. Results: Gel purified 800 bp band was sequenced and submitted at NCBI gene bank with accession No.: AY848856. A phylogenetic tree for obtained partial gene of annexin was drawn using bioinformatic software in order to understand the evolutionary relationship of annexin genes between some microrganisms. Also southern analysis of800 bp PCR product using digoxigenin (DIG) labeled probe demonstrated the probability of two copies of annexin genes existence in the A. pullulans genome. Conclusion: This study for the first time presented the presence of annexin gene in yeast-like fungi and this result is important due to the existence of this superfamily of genes in moulds but not in yeasts. We emphasize for future additional work to clone and sequence the full length of annexin gene(s) from A. pullulans and also additional studies for this gene expression and annexin mRNA transcription to understand the effective factors for expression of annexin.