IDENTIFICATION, ISOLATION, CLONING AND SEQUENCING A PARTIAL ANNEXIN GENE FROM AUREOBASIDIUM PULLULANS

Annexin is the common name for genes and proteins that identified as calcium-dependent phospholipid-binding proteins but recently has been recognized more complex set of functions for these superfamily of proteins in vesicle trafficking, cell division, apoptosis, calcium signalling, mineralization, crystal nucleation inside the extracellular organelles-matrix vesicles (MVs) and growth regulation. In the present work Aureobasidium pullulans strain PRAFS8 genomic DNA was extracted and using designed primers from highly conserve region of anexxin genes of Aspergillus fumigatus and Aspergillus niger a 800 bp PCR product obtained from degenerated PCR. The 800 bp PCR product was gel purified and cloned into E. coli Top-10F´ (Stratagene) using the pGEM®-T easy vector system (Promega) and standard cloning procedures. From grown transformed E. coli Top-10F´ cells, pGEM®-T easy vector was extracted and the presence of expected insert into plasmid, was confirmed by digestion of plasmid by Eco R1 restriction enzyme. 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 understanding the evolutionary relationship of annexin genes between some microrganisim. Also southern analysis of 800 bp PCR product using digoxigenin labeled probe with DIG-High Prime DNA Labeling kit (Roche) demonstrated the probability of two copies of annexin genes existence in the A. pullulans genome.

کلیدواژه ها:

Annexin ، Gene ، Sequencing ، Aureobasidium pullulans