Tuz Stresi Altındaki Kişniş Bitkisinin (Coriandrum sativum) DNA Metilasyon Modlarının RAPD Markırları ile Belirlenmesi

Abstract

Epigenetik, gen ifadesi ve fonksiyonundaki kalıtsal değişiklikleri tanımlayan bir alanı ifade eder, ancak DNA baz değişimi olmaksızın gerçekleşir. Bitkilerde en çok incelenen epigenetik mekanizmalar, DNA metilasyonu ve histon proteinlerindeki kimyasal modifikasyonlardır. Epigenetik araştırmalarda, düşük ve yüksek işlem hacimli DNA metilasyon tespiti için çeşitli teknikler kullanılmaktadır. Düşük işlem hacimli tekniklerden biri, enzim tabanlı DNA sitozin metilasyonunu belirleme yaklaşımıdır. Bu yöntem, biyolojik aktiviteleri ve terapötik potansiyeli olan tuz uygulaması yapılan kişniş (Coriandrum sativum) fidelerinde rastgele genlerdeki DNA sitozin metilasyonunu incelemek amacıyla kullanılmaktadır. Bu çalışma, bitki genotiplerindeki epigenetik değişiklikleri anlamamıza ve bu değişikliklerin biyolojik etkilerini değerlendirmemize olanak tanır. Bu çalışmada tuz stresine maruz kalan kişniş fidelerinden 12. ve24. Saatlerde alınan doku örnekleri arasındaki sitozin metilasyon polimorfizmini ortaya koymak için touch-down polimeraz zincir reaksiyonları metilasyon duyarlı-rastgele arttırılmış polimorfik DNA (TD-MS RAPD) tekniği uygulanmıştır. Kişniş fidelerinden (Cemre) genomik DNA örnekleri izole edilmiş ve DNA izolatı touch- metilasyona duyarsız olan MspI restriksiyon enzimi ve metilasyona duyarlı HpaII restriksiyon enzimi ile kesilmiştir. Kullanılan 9 oligonükleotid primerinden üç primer (OPA 05-OPD 01-OPH 10) uygulamalar ve saatlik alınan örnekler arasında metilasyon polimorfizmleri belirlenmiştir. TD-MS-RAPD-PZR metodu, basit ve temel cihazlar gerektiren ekonomik bir yaklaşımdır. Bu yöntem, standart bir DNA termal döngü cihazı ve DNA jel elektroforezi sistemi kullanılarak kolayca uygulanabilir. Bu metotla belirlenen sitozin metilasyon polimorfizmi uygulamalar arsında oldukça düşüktür. İki farklı tuz uygulaması arasında (100mM-200mM) düşük düzeyde polimorfizm olduğu sonucuna varılmıştır. Saatlik alınan kişniş fidelerinden elde edilen doku örneklerindeki metilasyona bakıldığında ise 12’nci ve 24’üncü saatlerde alınan örnekler arasında bir metilasyon farkı tespit edilmemiştir.

Keywords

• Kişniş
• DNA metilasyonu
• MspI-HpaII

Determination of DNA Methylation Modes of Coriander Plant (Coriandrum sativum) Under Salt Stress Using RAPD Markers

Abstract

Epigenetics pertains to heritable alterations in gene expression and function that occur without modifications to the DNA base sequence. Among the extensively researched epigenetic mechanisms in plants are DNA methylation and chemical modifications in histone proteins. In the realm of epigenetic exploration, a variety of techniques are employed for both low- and high-throughput DNA methylation detection. One low-throughput approach involves the utilization of an enzyme-based method for detecting DNA cytosine methylation. This technique was applied to investigate DNA cytosine methylation in randomly selected genes within salt-treated coriander (Coriandrum sativum) seedlings, renowned for their biological activities and therapeutic potential. The study facilitates comprehension of epigenetic modifications in plant genotypes and an assessment of the biological implications of these alterations. The research employed the touch-down polymerase chain reaction methylation-sensitive-randomly amplified polymorphic DNA (TD-MS RAPD) technique to unveil cytosine methylation polymorphism between tissue samples collected at 12 and 24 hours from coriander seedlings subjected to salt stress. Genomic DNA samples extracted from coriander seedlings (Cemre) underwent treatment with the methylation-insensitive MspI restriction enzyme and the methylation-sensitive HpaII restriction enzyme prior to touch-down polymerase chain reactions. Methylation polymorphisms were identified using three primers (OPA 05, OPD 01, OPH 10) out of the 9 oligonucleotide primers employed, with samples taken at hourly intervals. The TD-MS-RAPD-PCR method presents an economical approach requiring basic equipment. Implementation involves a standard DNA thermal cycler and DNA gel electrophoresis system. Notably, cytosine methylation polymorphism determined by this method exhibited minimal variance across applications. The findings indicate a low level of polymorphism between two distinct salt treatments (100mM-200mM). Moreover, when scrutinizing methylation in tissue samples obtained from coriander seedlings at hourly intervals, no discernible methylation differences were observed between the samples collected at the 12th and 24th hours.

Keywords

• Coriander
• DNA methylation
• MspI-HpaII

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