Lucilia sericata’nın Lucimycin Geninin Moleküler Karakterizasyonu
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Tarih
2020
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info:eu-repo/semantics/openAccess
Özet
Calliphoridae ailesinin bir üyesi olan Lucilia sericata, Lucilia cinsindeki en yaygın türlerden birisidir.L.sericata’nın tıp açısından önemi, maggot debridman tedavisi (MDT)’nde kullanılmasından kaynaklanmaktadır. MDT, L.sericata larvalarının steril hale getirilerek iyileşmeyen yaraların tedavisinde kullanılmasınaverilen isimdir. Kronik, iyileşmeyen yaraların (dekübit ülseri, venöz bacak ülseri, diyabetik ayak ülseri vb.)tedavisinde kullanılan L.sericata kurtçukları, salgıladıkları proteolitik tripsin ve lucimycin benzeri enzimleryardımıyla yaraları temizler. Bu çalışma, MDT’de görev alan L.sericata larvalarından moleküler yöntemlerkullanılarak elde edilen lucimycin geninin moleküler karakterizasyonunu belirlemek ve literatüre katkıdabulunmak amacıyla yapılmıştır. Çalışma, L.sericata türüne ait erişkin kolonilerinin sürekli üretimi, ışık, nemve sıcaklık gibi koşulların oluşturulduğu insektaryum ünitesinde yapılmıştır. L.sericata yaşam döngüsütakip edilerek, türüne ait yumurta, larva, pupa, erişkin sineklerin üretimi ve sinek kolonileri oluşturulmuştur. İnsektaryum ünitesinde ergin sineklerden elde ettiğimiz üçüncü dönem larvalardan RNA izolasyonuyapılmış ve takiben revers transkripsiyon ile cDNA sentezi gerçekleştirilmiştir. Sentezlenen cDNA’ların,L.sericata’nın lucimycin geni için tasarlanan özgül primerler ile polimeraz zincir reaksiyonu (PCR) analiziyapılmış ve elde edilen amplikonlar pJET1.2/blunt vektörüne klonlanarak plazmit saflaştırılmıştır. Eldeedilen rekombinant plazmitlerin, vektöre özgül primerlerle dizi analizi yapılmış ve hedef gen bölgesidizileri elde edilmiştir. Nükleotit dizileri saptanan izolatın moleküler karakterizasyonu belirlendikten sonra GenBank veritabanına MF964229 aksesyon numarası ile kaydı sağlanmıştır. İnsektaryum ünitesindeüretilen L.sericata larvalarından elde edilen cDNA’dan lucimycin özgül primerleri kullanılarak yapılan PCRsonucunda 288 baz çifti (bp) büyüklüğünde PCR ürünü elde edilmiştir. Agaroz jelde görüntülenen PCRürünü, saflaştırılarak transformasyon işlemi yapılmış ve sonrasında oluşan kolonilerin rekombinant plazmitiçerip içermedikleri PCR ile gösterilmiştir. Elde edilen kolonilerin üç tanesinin PCR ile L.sericata lucimycingeni içeren rekombinant plazmit olduğu tespit edilmiştir. PCR ile pozitifliği doğrulanan üç kolonidenminiprep yapılarak L.sericata lucimycin geni içeren rekombinant plazmitler saflaştırılmıştır. Toplam 20 ?lrekombinant plazmit miniprep ürününe PCR uygulanarak rekombinant plazmit ürünün L.sericata lucimycin geni içerdiği doğrulanmıştır. Klonlama sonrası plazmitin doğrulanması amacıyla DNA dizi analizi yapılmıştır. DNA dizi analizi yapılarak 288 bp uzunluğundaki L.sericata lucimycin gen dizisi doğrulanmıştır. İzole ettiğimiz lucimycin genine, özgül pJET1.2 ileri ve revers primerler kullanılmak suretiyle çift yönlü dizianalizi uygulanarak sonuçlar Blastn algoritması ile tür ve/veya alt tür düzeyinde doğrulanmıştır. İzolat,MF964229 aksesyon numarası ile GenBank veri tabanına kaydedilmiştir. İzole ettiğimiz örneğimize aitDNA dizisi, Pubmed/Blast programı ile GenBank’ta bulunan diğer izolatlarla karşılaştırılmış ve KJ413251.1GenBank nolu izolatla %99 benzer bulunmuştur. İzolatımıza ait dizide 113. nükleotitin C (sitozin) olduğugörülürken, KJ413251.1 GenBank nolu izolattaki dizide ise G (guanin) olması iki dizi arasındaki farklılığıortaya koymuştur. Bu çalışma ile Türkiye’de ilk kez L.sericata larvalarından elde edilen lucimycin genininmoleküler karakterizasyonu yapılmış, elde edilen ve antifungal özelliğe sahip bu molekülün ileride gerçekleştirilecek olan özellikle kutanöz enfeksiyonlara neden olan mikroorganizmaları kapsayan çalışmalardakullanılabileceği düşünülmüştür. Bu çalışma, Türkiye’nin biyolojik varlığı olarak GenBank’a kaydı yapılanizolat olması ve dünyada ikinci çalışma özelliği taşıması açısından önemlidir
Lucilia sericata, a member of the Calliphoridae family, is one of the most common species in the genus Lucilia. Medical importance of L.sericata stems from its use in maggot debridement therapy (MDT). MDT is the name of L.sericata larvae being sterilized and used in the treatment of non-healing wounds. L.sericata maggots used in the treatment of chronic and non-healing wounds (decubitus ulcer, venous leg ulcer, diabetic foot ulcer, etc.) clean the wounds with the help of secreted proteolytic trypsin and lucimycin -like enzymes. The aim of the study was to determine the molecular characterization of lucimycin gene obtained from L.sericata larvae in MDT by using molecular methods and to contribute to the literature. In this study, continuous production of adult colonies of L.sericata species was carried out in insectarium unit where conditions such as light, humidity and temperature were formed. The life cycle of L.sericata was followed and the production of eggs, larvae, pupae, adult flies and fly colonies of the species were formed. In the third stage larvae obtained from adult flies in the insectarium unit, RNA was isolated and subsequently cDNA synthesis was performed by reverse transcription. Polymerase chain reaction (PCR) analysis of the synthesized cDNAs with the specific primers designed for the lucimycin gene of L.sericata was performed and the obtained amplicons were cloned into pJET1.2/blunt vector and the plasmid was purified. The recombinant plasmids were sequenced with vector-specific primers and target gene region sequences were obtained. After the molecular characterization of the isolate with nucleotide sequences was determined, it was registered to GenBank database with the accession number MF964229. The PCR product of 288 bp was obtained from the cDNA obtained from the larvae of L.sericata produced in the insectarium unit by PCR using lucimycin specific primers. The PCR product imaged on the gel was purified by transformation and subsequent colonies were screened to see whether they contained recombinant plasmids. Three of the colonies were identified as recombinant plasmids containing L.sericata lucimycin gene by PCR screening. From three colonies confirmed by PCR screening, recombinant plasmids containing L.sericata lucimycin gene were purified by miniprep. The recombinant plasmid product was confirmed to contain the L.sericata lucimycin gene by PCR from a total of 20 ?l of the recombinant plasmid miniprep product. DNA sequencing analysis was performed to confirm the plasmid after cloning. The 288 bp L.sericata lucimycin sequence was confirmed by DNA sequence analysis. The lucimycin gene isolated was confirmed by specific and pJET1.2 forward and reverse primers using Blastn algorithm as a result of species and/or subspecies using the Blastn algorithm and the related isolate was recorded in GenBank database with the MF964229 accessory number. The DNA sequence of the isolated sample was compared with other isolates found in GenBank by Pubmed/Blast program. KJ413251.1 was found to be 99% similar to the GenBank isolate. The 113th nucleotide was C (cytosine) in the sequence of our isolate, while the existence of G (guanine) in the sequence numbered KJ413251.1 GenBank revealed the difference between the two sequences. In this study the molecular characterization of lucimycin gene derived from L.sericata larvae were determined for the first time in Turkey, it is assumed that this molecule which has an antifungal property, can be used in the studies that will be carried out in the future, especially in microorganisms causing cutaneous infections. The study is important since the isolate is registered as a biological asset of Turkey in GenBank and also being the second study in the world.
Lucilia sericata, a member of the Calliphoridae family, is one of the most common species in the genus Lucilia. Medical importance of L.sericata stems from its use in maggot debridement therapy (MDT). MDT is the name of L.sericata larvae being sterilized and used in the treatment of non-healing wounds. L.sericata maggots used in the treatment of chronic and non-healing wounds (decubitus ulcer, venous leg ulcer, diabetic foot ulcer, etc.) clean the wounds with the help of secreted proteolytic trypsin and lucimycin -like enzymes. The aim of the study was to determine the molecular characterization of lucimycin gene obtained from L.sericata larvae in MDT by using molecular methods and to contribute to the literature. In this study, continuous production of adult colonies of L.sericata species was carried out in insectarium unit where conditions such as light, humidity and temperature were formed. The life cycle of L.sericata was followed and the production of eggs, larvae, pupae, adult flies and fly colonies of the species were formed. In the third stage larvae obtained from adult flies in the insectarium unit, RNA was isolated and subsequently cDNA synthesis was performed by reverse transcription. Polymerase chain reaction (PCR) analysis of the synthesized cDNAs with the specific primers designed for the lucimycin gene of L.sericata was performed and the obtained amplicons were cloned into pJET1.2/blunt vector and the plasmid was purified. The recombinant plasmids were sequenced with vector-specific primers and target gene region sequences were obtained. After the molecular characterization of the isolate with nucleotide sequences was determined, it was registered to GenBank database with the accession number MF964229. The PCR product of 288 bp was obtained from the cDNA obtained from the larvae of L.sericata produced in the insectarium unit by PCR using lucimycin specific primers. The PCR product imaged on the gel was purified by transformation and subsequent colonies were screened to see whether they contained recombinant plasmids. Three of the colonies were identified as recombinant plasmids containing L.sericata lucimycin gene by PCR screening. From three colonies confirmed by PCR screening, recombinant plasmids containing L.sericata lucimycin gene were purified by miniprep. The recombinant plasmid product was confirmed to contain the L.sericata lucimycin gene by PCR from a total of 20 ?l of the recombinant plasmid miniprep product. DNA sequencing analysis was performed to confirm the plasmid after cloning. The 288 bp L.sericata lucimycin sequence was confirmed by DNA sequence analysis. The lucimycin gene isolated was confirmed by specific and pJET1.2 forward and reverse primers using Blastn algorithm as a result of species and/or subspecies using the Blastn algorithm and the related isolate was recorded in GenBank database with the MF964229 accessory number. The DNA sequence of the isolated sample was compared with other isolates found in GenBank by Pubmed/Blast program. KJ413251.1 was found to be 99% similar to the GenBank isolate. The 113th nucleotide was C (cytosine) in the sequence of our isolate, while the existence of G (guanine) in the sequence numbered KJ413251.1 GenBank revealed the difference between the two sequences. In this study the molecular characterization of lucimycin gene derived from L.sericata larvae were determined for the first time in Turkey, it is assumed that this molecule which has an antifungal property, can be used in the studies that will be carried out in the future, especially in microorganisms causing cutaneous infections. The study is important since the isolate is registered as a biological asset of Turkey in GenBank and also being the second study in the world.
Açıklama
Anahtar Kelimeler
Kaynak
Mikrobiyoloji Bülteni
WoS Q Değeri
Scopus Q Değeri
Cilt
54
Sayı
3
Künye
ÇETİN S, AKSOY T (2020). Lucilia sericata’nın Lucimycin Geninin Moleküler Karakterizasyonu. Mikrobiyoloji Bülteni, 54(3), 392 - 403.
