Buğdayda Gerçek Zamanlı Polimeraz Zincir Reaksiyonu Analizi için Bazı Süperoksit Dismutaz Genlerinin Optimizasyonu

Year 2023, Volume: 54 Issue: 3 - Research in Agricultural Sciences, 153 - 158, 19.10.2023

Aslıhan Esra Bildirici , Muhammed Tatar Fatih Ölmez , Zemran Mustafa , Mortaza Hajyzadeh

https://doi.org/10.5152/AUAF.2023.23074

Abstract

Çevresel stress her yıl mahsul kalitesinde önemli düşüşlere ve verimlilik kayıplarına neden olmaktadır. Olumsuz çevre koşullarında bitkilerin zarar görmesine neden olan mekanizmalardan biri de süperoksit, hidrojen peroksit ve hidroksil radikalleri gibi reaktif oksijen türlerinin aşırı üretimidir. Ayrıca, bu tür oksidatif streslerin, yüksek veya düşük sıcaklıklara, özellikle yüksek ışık yoğunluklarına, kuraklığa, ultraviyole ışığa, herbisitlere ve ozon veya kükürt dioksit gibi hava kirleticilerin varlığına maruz kalan bitkilerde meydana geldiği gösterilmiştir. Hidroksil radikalleri, proteinler, lipitler ve DNA ile anında reaksiyona girerek hızla hücre hasarına neden olur. Bu nedenle bitkiler, oksijen radikallerini verimli bir şekilde temizleyen enzimatik ve enzimatik olmayan mekanizmalar geliştirmiştir. Bununla birlikte, hidroksil radikalleri enzimatik olarak elimine edilemeyecek kadar reaktiftir, bu nedenle radikallerin oluşumu O2 ve H2O2 salınımı ile sınırlandırılır. Bu çalışmanın da konusu olan süperoksit dismutazlar (SOD), O2 radikallerini ortadan kaldıran (2O2 + 2H+ → H2O2 +O2), metal içeren ve antioksidan savunmanın ilk hattı olarak görev yapan anahtar enzimlerdir. Bu çalışmada, real-time PCR optimizasyonu için sekiz SOD geni seçilmiştir. Spesifik primerler dizayn edilmiş ve buğday örneğinden elde edilen DNA kullanılarak, real-time PCR analizi için annealing (bağlanma) sıcaklığı optimizasyonu yapılmıştır. Ayrıca, aynı DNA örnekleri kullanılarak β-aktin spesifik primerleri için de bağlanma sıcaklığı optimizasyonu yapılmıştır. β-aktin, ekspresyon profili çalışmalarında normalizasyon faktörü olarak yaygın bir şekilde kullanılan, sabit ekspresyon profiline sahip bir housekeeping gendir. Mevcut real-time PCR protokolleri kullanılarak; SOD gen ekspresyonunun bağıl ve mutlak değerlerinin değerlendirilmesi ve SOD gen ekspresyonlarının zaman içinde ve farklı koşullar altında değişimlerinin incelenmesi kolayca gerçekleştirilebilir. Bu tür çalışmalar, buğdayın farklı stres koşulları ile başa çıkma mekanizmaları hakkında önemli bilgiler sağlayabilir.

Keywords

β-aktin, reaktif oksijen radikalleri, Triticum aestivum

Optimization of Real-Time Polymerase Chain Reaction Conditions of Some Superoxide Dismutase Genes for Analysis in Wheat

Abstract

Environmental stress causes a significant decrease in crop quality and losses of productivity every year. One of the important mechanisms by which plants are damaged in adverse environmental conditions is the overproduction of reactive oxygen species such as superoxide, hydrogen peroxide, and hydroxyl radicals. In addition, such oxidative stresses have been shown to occur in plants exposed to high and low temperatures, particularly to high light intensities, drought, the presence of air pollutants such as ozone or sulfur dioxide, ultraviolet light, and herbicides. Hydroxyl radicals instantly react with proteins, lipids, and deoxyribonucleic acid, causing rapid cell damage. Therefore, plants have developed enzymatic and nonenzymatic mechanisms that efficiently scavenge oxygen radicals. However, hydroxyl radicals are too reactive to be eliminated enzymatically, so the formation of radicals is limited by the release of O2 and H2O2. Superoxide dismutases, which are also the subject of this study, are key enzymes that scavenges superoxide radicals (2O2 + 2H+ → H2O2 +O2), contain metals, and act as the first line of antioxidant defense. In this study, eight superoxide dismutase genes were chosen for real-time polymerase chain reaction optimization. Specific primers were designed, and annealing temperature optimization was performed for realtime polymerase chain reaction analysis using deoxyribonucleic acid from wheat sample. In addition, annealing temperature optimization for β-actin specific primers were performed using the same deoxyribonucleic acid sample. β-actin is a housekeeping gene with a constant expression profile that is commonly used as a normalizing factor in expression profile studies. Evaluation of the relative and absolute values of superoxide dismutase gene expressions and the changes of superoxide dismutase gene expressions over time and under different conditions can be easily studied using the established real-time polymerase chain reaction protocols. These studies can provide important information on wheat coping mechanisms under different stress conditions.

Keywords

β-Actin, reactive oxygen radicals, Triticum aestivum

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