النمو، الفعالية الانزيمية والتعبير الجيني للانزيمات المحللة للبروتين (Protease) في الفطر الجلدي Trichophyton rubrum == Growth, Enzyme Activity And Gene Expression of Proteases In A Dermatophyte Trichophyton Rubrum
Author name:
سارة كريم كاظم الزبيدي
Supervisor name:
جواد كاظم الجنابي | عدنان حمد الحمداني
General topic:
Biology
Specific topic:
Microbiology
Degree:
Doctorate
University:
University of Babylon - College Of Science - Department Of Biology
Language:
English
University location:
Babylon
First pages:
24T2721 - p.pdf
Abstract:
صممت الدراسة الحالية لتوصيف الانماط الجزيئية والمظهرية للنمو والفعالية الانزيمية للانزيم المحلل للبروتين والتنوع الوراثي وعلاقته بالتعبير الجيني بين عزلات الفطر الجلدي.Trichophyto rubrum ولهذا الغرض جمعت 150 عينة سريرية (قشطات جلدية (80), اجزاء شعر (60) | The present study was designed to characterize the morphological and the molecular patterns of growth, enzymic activity of proteases, genetic diversity and their correlation with gene expression among strains of Trichophyton rubrum. For these purpose, A total of 150 clinical specimens (skin scrapings (80), hair fragments (60) and nail clippings (10)) were collected from 100 patients (70 males&80 females) whom clinically diagnosed with dermatophytosis after attendingto the dermatology and venereal diseases centre of Mergan Teaching hospital and private clinic in Babylon city from Feb. 2014 to May 2014. T. rubrum were isolated and identified using cultural, biochemical and physiological tests. Isolates were then subjected for confirmation by PCR, genotyping using RFLP - PCR, sequencing and registration of these sequences in GeneBank for obtaining accession numbers then phylogeny. The growth conditions, were tested, in addition to, the genetic expression of proteases (exocellular protease and endocellular aminopeptidase) were determined using Real - Time PCR. Out of 150 specimens, 24 (16%) isolates were dermatophytic fungi and only 5 (20.8%) of them belong to T. rubrum. The influence of cultural conditions in growth of T. rubrum showed that the 30 ?C, pH 6, SDA medium and 7 days incubation were the optimal conditions for its growth. Proteolytic activity of isolates of T. rubrum against casein as a substrate revealed an ability to produce protease in solid and liquid media after 14 days of incubation. This activity was varies according to the type of isolate where the isolate No.1 (isolated from skin) gave a high proteolytic activity (5.6cm) in solid media and (80.1U/ml) in liquid media in comparison with other tested isolates (No.2 - No.8). on the other hands, the 9 days of incubation, 30 ?C, pH 7 and 0.5% substrate concentration were the optimal condition for proteolytic activity of these isolates. The molecular tests confirmed that all tested isolates belong to T. rubrum with amplicon size (601bp) after amplification of ITS1 primers using PCR technique. While the RFLP - PCR technique showed the presence of two genotypes (I%II) belong to T. rubrum with subgenotypes (Ia - Id) and (IIa - IId) respectively. The relative quantification of proteolytic activity (exocellular protease and endocellular aminopeptidase) produced by T. rubrum genotypes were expressed by using Real - Time PCR after amplification of the target gene of ptotease and aminopeptidase in comparison with housekeeping gene (? - actin) as a reference gene. The results showed the up - regulation of gene encoded to exocellular protease than the down - regulation of endocellular aminopeptidase produced by T. rubrum in the presence of casein as a substrate. The internal transcribed spacer 1, partial sequence, 5.8S ribosomal RNA gene and internal transcribed spacer 2, complete sequence and 28S ribosomal RNA gene, partial sequence of local isolates of T. rubrum were used for sequencing, registration in Genebank - NCBI and phylogeny. Five accession numbers were recorded and available to NCBI, EMBL in Europe and the DNA Bank of Japan. These accession numbers were : KP979787, KP979788, KP979789, KP979790, KP979791. Phylogenetic relation between local strains and world strain showed a high identicasl with T. rubrum (GQ376105.1). This is the first study in Iraq which employed sequencing, registration of sequences in Genebank - NCBI and carrying out phylogeny of local and world strains of T. rubrum.
