Saf kültür olarak stoklanmış bazı mikrofunsgusların Its, B-Tubulin ve aktin gen dizilerine göre moleküler tanısı
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Dosyalar
Tarih
2016
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Trakya Üniversitesi Fen Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Bu çalışmada, laboratuvar ortamında stok olarak saf kültür halinde saklanan bununla beraber; cins veya tür düzeyinde morfolojik olarak teşhis edilemeyen bazı mikrofunguslar moleküler olarak tanımlanmıştır. Çalışmada kullanılan mikrofunguslar daha önce tür düzeyinde morfolojik-koloniyal olarak tanımlanamamış izolatlardan oluşmuştur. Örnekler, MEA besiyerinde inoküle edilip 25 ºC’de 7 gün inkübe edilmiştir. Besiyerinden alınan numuneler analiz yapılana kadar -20ºC’de saklanmıştır. DNA izolasyonu için funguslara özel, kimyasal (SDS ve CTAB), biyokimyasal (proteinazK vb.) ve fiziksel (0.1 mm çaplı boncuklar) parçalama yöntemlerini bir arada kullanan DNA izolasyon kitleri kullanılmıştır. Çeşitlilik çalışmaları, PCR tabanlı fungal çeşitlilik çalışma kitleri ile yapılmıştır. Tüm izolatlar için ITS gen dizisine bakılmış, cinse bağlı olarak ß-tubulin ve Actin gen dizilimlerini hedeflenmiştir. PCR ile çoğaltılan DNA dizilimleri “Sanger Sequencing Yöntemi” ile dizilenmiştir. Elde edilen dizilerin hangi organizmalara ait olduğu NCBI ve EBI gibi uluslararası nükleik asit data bankalarında mevcut dizilimlerle, elde edilen filotiplerin dizilimleri karşılaştırılarak belirlenmiştir. Toplam 61 mikrofungus örneği, cins düzeyinde 3 gruba (A,B,C) ayrılmış olup, gruplara göre ilgili gen bölgeleri moleküler teşhis için analiz edilmiştir. 3 grup için hem ilgili gen bölgeleri hem de ITS bölgeleri baz alınarak yapılan moleküler analizler sonucunda, 6 örneğin tür teşhisi yapılamamış, 56 tür moleküler düzeyde teşhis edilmiştir.
abstract
Some microfungi deposited in laboratuary as pure culture and unidentified morphologically as genus or species level and these fungi are identified by molecular methods in study. The used microfungi are comprised unidentifed species and can not be identify as colonially and morphologically previously. We used ITS gene for all isolates, also according to the genus we used ß-tubulin and actin genes targeting PCR based fungal biodiversity working kits. DNA sequences were sequenced by Sanger Method, and obtained sequences were analysed as bioinformatics and completed filogenetically analysis. Then samples inoculated to MEA at 25 °C for 7 days. All samples obtained from MEA media are preserved at -20 °C until analysis. For DNA isolation were used the spesific kits, using together digestion method such as chemical (SDS and CTAB), biochemical (proteinaseK etc.) and physical (beat with 0.1 mm diameter) were used for DNA isolation. DNA’s maintained in silica colones. In the last stage of isolation, nucleic acides were dissolved in the water DNase/Pyrogen free. After that, sequence series analysised on spectrophotometer and so determined, purity of DNA. Studies of diversity is made with PCR based fungal biodiversity working kits. After the PCR application, DNA series sequenced using by “Sanger-Sequencing Protocol”. Sequence series found that belong to which organisms on NCBI and EBI. After that, philotypes were compared with similar organisms. A total of 61 Microfungi samples , the genus level 3 groups (A, B, C) is divided , according to group related gene regions were analyzed for molecular diagnostics. Both gene regions related to 3 groups based on the results of molecular analyzes of ITS, 6 species diagnosis has not been made , for example , 56 species have been identified at the molecular level .
abstract
Some microfungi deposited in laboratuary as pure culture and unidentified morphologically as genus or species level and these fungi are identified by molecular methods in study. The used microfungi are comprised unidentifed species and can not be identify as colonially and morphologically previously. We used ITS gene for all isolates, also according to the genus we used ß-tubulin and actin genes targeting PCR based fungal biodiversity working kits. DNA sequences were sequenced by Sanger Method, and obtained sequences were analysed as bioinformatics and completed filogenetically analysis. Then samples inoculated to MEA at 25 °C for 7 days. All samples obtained from MEA media are preserved at -20 °C until analysis. For DNA isolation were used the spesific kits, using together digestion method such as chemical (SDS and CTAB), biochemical (proteinaseK etc.) and physical (beat with 0.1 mm diameter) were used for DNA isolation. DNA’s maintained in silica colones. In the last stage of isolation, nucleic acides were dissolved in the water DNase/Pyrogen free. After that, sequence series analysised on spectrophotometer and so determined, purity of DNA. Studies of diversity is made with PCR based fungal biodiversity working kits. After the PCR application, DNA series sequenced using by “Sanger-Sequencing Protocol”. Sequence series found that belong to which organisms on NCBI and EBI. After that, philotypes were compared with similar organisms. A total of 61 Microfungi samples , the genus level 3 groups (A, B, C) is divided , according to group related gene regions were analyzed for molecular diagnostics. Both gene regions related to 3 groups based on the results of molecular analyzes of ITS, 6 species diagnosis has not been made , for example , 56 species have been identified at the molecular level .
Açıklama
Anahtar Kelimeler
ITS Gen Dizisi, Mikrofungusların Moleküler Teşhisi, Actin Gen Dizisi, Sekans Yöntemleri, ß-Tubulin Gen Dizisi, PCR, PCR, Identification of Microfungi, ß-Tubulin Region, Actin Region, ITS Region, Sequencing Methods
