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

Yıl 2023, Cilt: 54 Sayı: 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

Öz

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.

Anahtar Kelimeler

β-aktin, reaktif oksijen radikalleri, Triticum aestivum

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

Öz

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.

Anahtar Kelimeler

β-Actin, reactive oxygen radicals, Triticum aestivum

Kaynakça

• Bowler, C., Montagu, M. V., & Inze, D. (1992). Superoxide dismutase and stress tolerance. Annual Review of Plant Physiology and Plant Molecular Biology, 43(1), 83–116. [CrossRef]
• Bustin, S. A., Benes, V., Garson, J. A., Hellemans, J., Huggett, J., Kubista, M., Mueller, R., Nolan, T., Pfaffl, M. W., Shipley, G. L., Vandesompele, J., & Wittwer, C. T. (2009). The MIQE guidelines: Minimum information for publication of quantitative real-time PCR experiments. Clinical Chemistry, 55(4), 611–622. [CrossRef]
• Dudley, L., & Shani, U. (2003). Modeling plant response to drought and salt stress. Vadose Zone Journal, 2, 751–758.
• Gachon, C., Mingam, A., & Charrier, B. (2004). Real-time PCR: What relevance to plant studies? Journal of Experimental Botany, 55(402), 1445–1454. [CrossRef]
• Gutierrez, L., Mauriat, M., Guénin, S., Pelloux, J., Lefebvre, J. F., Louvet, R., Rusterucci, C., Moritz, T., Guerineau, F., Bellini, C., & Van Wuytswinkel, O. (2008). The lack of a systematic validation of reference genes: A serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants. Plant Biotechnology Journal, 6(6), 609–618. [CrossRef]
• Huggett, J., Dheda, K., Bustin, S., & Zumla, A. (2005). Real-time RT-PCR normalisation; strategies and considerations. Genes and Immunity, 6(4), 279–284. [CrossRef]
• Hunter, T., & Garrels, J. I. (1977). Characterization of the mRNs for alpha-, beta- and gamma-actin. Cell, 12(3), 767–781. [CrossRef]
• Jain, M., Nijhawan, A., Tyagi, A. K., & Khurana, J. P. (2006). Validation of housekeeping genes as internal control for studying gene expression in rice by quantitative real-time PCR. Biochemical and Biophysical Research Communications, 345(2), 646–651. [CrossRef]
• Jiang, W., Yang, L., He, Y., Zhang, H., Li, W., Chen, H., Ma, D., & Yin, J. (2019). Genome-wide identification and transcriptional expression analysis of superoxide dismutase (SOD) family in wheat (Triticum aestivum). PeerJ, 7, e8062. [CrossRef]
• Kahya, S., Büyükcangaz, E., & Çarlı, K. F. (2013). Polimeraz Zincir Reaksiyonu (PCR) optimizasyonu. Uludag University Journal of the Faculty of Veterinary Medicine, 32(1), 31–38.
• Kavousi, H. R., Marashi, H., Mozafari, J., & Bagheri, A. R. (2009). Expression of phenylpropanoid pathway genes in chickpea defense against race 3 of Ascochyta rabiei. Plant Pathology Journal, 8(3), 127–132. [CrossRef]
• Knight, H., & Knight, M. R. (2001). Abiotic stress signaling pathways: Specificity and cross-talk. Trends in Plant Science, 6(6), 262–267. [CrossRef]
• Manoli, A., Sturaro, A., Trevisan, S., Quaggiotti, S., & Nonis, A. (2012). Evaluation of candidate reference genes for qPCR in maize. Journal of Plant Physiology, 169(8), 807–815. [CrossRef]
• McCord, J. M., & Fridovich, I. (1968). The reduction of cytochrome c by milk xanthine oxidase. Journal of Biological Chemistry, 243(21), 5753–5760. [CrossRef]
• McCord, J. M., & Fridovich, I. (1969). Superoxide dismutase an enzymic function for erythrocuprein (hemocuprein). Journal of Biological Chemistry, 244(22), 6049–6055. [CrossRef]
• Mittler, R. (2006). Abiotic stress, the field environment and stress combination. Trends in Plant Science, 11(1), 15–19. [CrossRef]
• Novo, E., & Parola, M. (2008). Redox mechanisms in hepatic chronic wound healing and fibrogenesis. Fibrogenesis and Tissue Repair, 1(1), 5. [CrossRef]
• Paolacci, A. R., Tanzarella, O. A., Porceddu, E., & Ciaffi, M. (2009). Identification and validation of reference genes for quantitative RT-PCR normalization in wheat. BMC Molecular Biology, 10, 11. [CrossRef]
• Perl-Treves, R., & Galun, E. (1991). The tomato Cu, Zn superoxide dismutase genes are developmentally regulated and respond to light and stress. Plant Molecular Biology, 17(4), 745–760. [CrossRef]
• Scandalios, J. G. (1993). Oxygen stress and superoxide dismutases. Plant Physiology, 101(1), 7–12. [CrossRef]
• Tsang, E. W. T., Bowler, C., Hérouart, D., Van Camp, W., Villarroel, R., Genetello, C., Van Montagu, M., & Inzé, D. (1991). Differential regulation of superoxide dismutase in plants exposed to environmental stress. Plant Cell, 3(8), 783–792. [CrossRef]
• Tseng, M. J., Liu, C. W., & Yiu, J. C. (2007). Enhanced tolerance to sülfür dioxide and slat stress of transgenic Chinese cabbage plants expressing both superoxide dismutase and catalase in chloroplasts. Plant Physiology and Biochemistry, 45(10–11), 822–833. [CrossRef]
• Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A., & Speleman, F. (2002). Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biology, 3(7), RESEARCH0034. [CrossRef]
• Wang, W., Xia, M. X., Chen, J., Yuan, R., Deng, F. N., & Shen, F. F. (2016). Gene expression characteristics and regulation mechanisms of superoxide dismutase and its physiological roles in plants under stress. Biochemistry. Biokhimiia, 81(5), 465–480. [CrossRef]
• Wei, L., Wang, L., Yang, Y., Wang, P., Guo, T., & Kang, G. (2015). Abscisic acid enhances tolerance of wheat seedlings to drought and regulates transcript levels of genes encoding ascorbate-glutathione biosynthesis. Frontiers in Plant Science, 6, 458. [CrossRef]
• Zhang, S., Zeng, Y., Yi, X., & Zhang, Y. (2016). Selection of suitable reference genes for quantitative RT-PCR normalization in the haplotype Halostachys caspica under salt and drought stress. Scientific Reports, 6, 30363. [CrossRef]