كلونينگ و توالي يابي بخش ازژن فسفوليپاژ B1 در آسپرژيلوس فوميگاتوس

Cloning and Sequencing of Partial Plbl Gene of Aspergillus fumigatus

Background and Objectives: Aspergillus species are primarily opportunistic pathogens and, with a few exceptions, cause invasive disease only in an immunocompromised hosts. During last two decades, the importance of fungal disease has been increased dramatically due to an increased occurrence of tuberculosis, chemotherapy-induced neutropenia, AIDS, open chest surgery and use of antibiotics and immunosuppressive therapy. The production of extracellular phospholipases by pathogenic fungi such as A. fumigatus are involved in the degradation of target cell membrane phospholipids in invaded tissues. Materials and Methods: Using degenerate primers based on homologous amino acid sequences of fungal phospholipase B (plb) gene, PCR products of 545bp were generated. Sequence analysis revealed that the sequence had a high homology to published plb sequences sharing highest homology to Aspergillus oryzae PLB (76% identity). Inverse PCR was used to attempt to clone the entire plbl genes. Initially, genomic DNA was digested with selected restriction enzymes and self-ligated with T4 DNA ligase to give circular DNA for inverse PCR (IPCR) for afplbl. IPCR with Xho I self-ligated digest gave product of 1.8 Kb for afplbl and was found to encode the 3ʹ ends of gene and using CAP assembly software this sequence was assembled with 545 bp sequence. Results: BLST X and GeneFinder analysis revealed that the first 960 bp of assembled total 2197 bp (after deletion of similar bp from both sequences) encoded the 3ʹ end of afplbl gene. Conclusion: The sequence availability of partial afplbl gene and after that full sequence of this gene represents a major breakthrough in the study of this opportunistic pathogen and function of plb gene in pathogenesis of A. fumigatus. Also phylogenic tree analysis of afplbl gene shows that plbs and potential plb analogues are contained in a large cluster of the plb family. A. fumigatus is more closely related to A. oryzae, A.niger, P.notatum,and N. crassa plb than other fungi.