Lactobacillus plantarum'un moleküler yöntemler ile tanılanması ve genomik anti-faj sistemleri
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Dosyalar
Tarih
2019
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Ege Üniversitesi, Fen Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Laktik asit bakterileri (LAB), endüstriyel gıda fermentasyonlarında en sık kullanılan mikoorganizmalardır. LAB'ın probiyotik özelliği, çoklu ilaç dirençliliği, bakteriyosin üretimi, çeşitli enzim aktiviteleri gibi özelliklerinin yanı sıra bakteriyofajlara karşı dirençliliği de endüstriyel kullanım alanlarında önem arz etmektedir. Mikroorganizmalar evrimsel süreçte çeşitli hayatta kalma stratejileri geliştirmişlerdir. Bunlardan biri de genomik anti-faj sistemi olan CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats) savunmasıdır. CRISPR düzenli aralıklarla kümelenmiş kısa palindromik tekrarlar olarak tanımlanmaktadır. Bu tekrar dizileri arasına yerleştirilen yabancı genetik elemanların DNA dizileri ile CRISPR ilişkili (cas) genlerinin iş birliği ile gerçekleşen RNA temelli bir immün sistemdir. Bu çalışmada 40 Lactobacillus sp. izolatından Lactobacillus plantarum türlerinin hızlı tanısı, türe spesifik prob ve primerler ile Real-Time PCR 'da gerçekleştirilmiştir. İzolatlardan 25'i Lactobacillus plantarum olarak tespit edilmiştir. Tespit edilen 25 Lactobacillus plantarum için moleküler ve biyoinformatik yöntemler ile genomik anti-faj sistemi olan CRISPR-Cas lokuslarının varlığı araştırılmıştır. L.plantarum'un crispr dizilerine spesifik primer dizaynı gerçekleştirilmiştir. Klasik pcr denemeleri ile L. plantarum izolatlarından 14 'ünde Crispr lokusu saptanmıştır. Sonuçlar NCBI-Blast, CRISPRFinder ve CrisprTarget veritabanları kullanılarak analiz edilmiştir. Lactobacillus plantarum subsp. plantarum nF1-FD straininde 6 repeat ,5 spacer dizisi bulunmuştur. Diğer izolatlarda ise Crispr ilişkili proteinler (Dead/Deah box helikazlar) tespit edilmiştir.
Lactic acid bacteria (LAB) are the most commonly used microorganisms in industrial food fermentations. The multidrug resistance, bacteriocin production, various enzyme activities and probiotic properties of LAB , as well as resistance against bacteriophages are also important in industrial use areas. Microorganisms have developed various survival strategies in the evolutionary process. One of these is the defense of the genomic anti-phage system CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats). CRISPR is defined as clustered short palindromic repeats at regular intervals. This is an RNA-based immune system that is involved in the collaboration of CRISPR-related (cas) genes with DNA sequences of foreign genetic elements inserted between repeat sequences. In this study, rapid diagnosis of Lactobacillus plantarum species from 40 Lactobacillus sp. isolates was carried out in Real-Time PCR with species-specific probes and primers. 25 isolates were identified as Lactobacillus plantarum. For the determined 25 Lactobacillus plantarum, the presence of CRISPR-Cas loci of genomic anti-phage system was investigated by molecular and bioinformatics methods. Specific primer design for L.plantarum crispr arrays was performed. Crispr loci was found in 14 of L. plantarum isolates with classical polymerase chain reaction experiments. The results were analyzed using NCBI-Blast, CRISPRFinder and CrisprTarget databases. At Lactobacillus plantarum subsp. plantarum nF1-FD, 6 repeat, 5 spacer arrays were found. Crispr-related proteins (Dead / Deah box helicases) were detected in other isolates.
Lactic acid bacteria (LAB) are the most commonly used microorganisms in industrial food fermentations. The multidrug resistance, bacteriocin production, various enzyme activities and probiotic properties of LAB , as well as resistance against bacteriophages are also important in industrial use areas. Microorganisms have developed various survival strategies in the evolutionary process. One of these is the defense of the genomic anti-phage system CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats). CRISPR is defined as clustered short palindromic repeats at regular intervals. This is an RNA-based immune system that is involved in the collaboration of CRISPR-related (cas) genes with DNA sequences of foreign genetic elements inserted between repeat sequences. In this study, rapid diagnosis of Lactobacillus plantarum species from 40 Lactobacillus sp. isolates was carried out in Real-Time PCR with species-specific probes and primers. 25 isolates were identified as Lactobacillus plantarum. For the determined 25 Lactobacillus plantarum, the presence of CRISPR-Cas loci of genomic anti-phage system was investigated by molecular and bioinformatics methods. Specific primer design for L.plantarum crispr arrays was performed. Crispr loci was found in 14 of L. plantarum isolates with classical polymerase chain reaction experiments. The results were analyzed using NCBI-Blast, CRISPRFinder and CrisprTarget databases. At Lactobacillus plantarum subsp. plantarum nF1-FD, 6 repeat, 5 spacer arrays were found. Crispr-related proteins (Dead / Deah box helicases) were detected in other isolates.
Açıklama
Anahtar Kelimeler
L.plantarum, Anti-Faj, CRISPR-Cas, Anti-Phage
