Mercimek (Lens culinaris M.) DEHYDRATION RESPONSIVE ELEMENTBINDING2A (DREB2A) Geninin Kuraklık Stresi Koşullarındaki İfadesinin Belirlenmesi

Amaç: Dehydration responsive element binding (DREB) proteinleri, bitkilerde strese yanıt ve sinyal iletiminde önemli rol oynarlar. Bu çalışmada, DREB gen ailesinin üyesi olan ve LcDREB2A olarak isimlendirilen mercimek (Lens culinaris M.) DREB2A geninin kısmi cDNA’sı izole edilmiş ve kuraklık stresi ile ilişkisi belirlenmiştir.Materyal ve Metot: Kuraklık stresi ve LcDREB2A gen ifadesi arasındaki ilişkiyi anlamak için, 2 hafta süre ile yetiştirilen mercimek fidelerine 6, 13 ve 20 gün süre ile sulamama şeklinde kuraklık stresi uygulanmıştır. Kök ve yapraklarda meydana gelen gen ifadesi değişimleri eş zamanlı kantitatif PCR (Real-time qPCR) ile belirlenmiştir.Bulgular: LcDREB2A gen ifadesi, Fırat87 ve Özbek çeşidinin yapraklarında artan kuraklık stresi ile artmış ve kuraklığın 20. gününde en yüksek seviyeye ulaşmıştır. Diğer taraftan Özbek çeşidinin köklerinde gen ifadesi seviyesi 6. günde en yüksek seviyeye ulaşırken 13. günde azalmış ve 20. günde tekrar artış göstermiştir. Fırat87 çeşidinde ise, artan kuraklık stresi ile birlikte düzenli bir artış görülmüş ve 20. günde en yüksek seviyeye ulaşmıştır.Sonuç: LcDREB2A geninde gözlenen doku spesifik gen ifadesi profili, bu transkripsiyon faktörünün mercimek bitkisinde kuraklık stresindeki rolünü ve kompleks regülasyonunu göstermektedir.

Anahtar Kelimeler:

Mercimek, DREB2A, kuraklık, gen ifadesi, eş zamanlı kantitatif PCR

Determination of Lentil (Lens culinaris M.) DEHYDRATION RESPONSIVE ELEMENT-BINDING2A (DREB2A) Gene Expression under Drought Stress Conditions

Objective: Dehydration responsive element binding (DREB) proteins are important transcription factors in plant stress response and signal transduction. In this study, partial cDNA of lentil (Lens culinaris M.) DREB2A, namely LcDREB2A, which belongs to the DREB gene family was isolated and identified its relationship with drought stress.Material and Methods: Two-week-old plants were subjected to drought stress through irrigations for 6, 13 and 20 days to elucidate the relationship between the expression profile of LcDREB2A gene and drought stress. Changes of gene expression in lentil roots and leaves were assayed by quantitative real time PCR.Results: The expression of LcDREB2A in both Fırat87 and Özbek lentil leaves increased with decreasing water contents and reached to a maximum on the 20th day of dehydration. On the other hand, in Özbek roots, the highest level of expression was observed on the 6th day of dehydration. The levels decreased on 13th day and increased on 20th day. Level of expression in Fırat87 roots decreased with increasing water contents and the greatest level of expression was observed on the 20th day of dehydration.Conclusion: Observed tissue-specific expression profile of LcDREB2A suggested a complex regulation and indicated the role of this transcription factor in lentil drought response.

Keywords:

Lentil, DREB2A, drought, gene expression, real-time quantitative PCR,

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Agarwal P, Agarwal PK, Joshi AJ, Sopory SK, Reddy MK (2010) Overexpression of PgDREB2A transcription factor enhances abiotic stress tolerance and activates downstream stress-responsive genes. Molecular Biology Replication 37:1125-1135.
Bahl PN, Lal S, Sharma BM (1993). An overview of the production and problems in southeast Asia”. In: Erskine W, Saxena MC. (Ed) Lentil in South Asia. Proceedings of the seminar on lentils in South Asia, ICARDA. Aleppo, Syria. pp. 1-10.
Bartels D, Souer E (2004) Molecular responses of higher plants to dehydration. In: Hirt H, Shinozaki K (Ed). Plant Responses to Abiotic Stress. Berlin: Springer-Verlag, pp. 9–38.
Bartels D, Sunkar R (2005) Drought and salt tolerance in plants. Critical Reviews in Plant Sciences 24:23–58.
Bhargava S, Sawant K (2013) Drought stress adaptation: metabolic adjustment and regulation of gene expression. Plant Breeding 2013;132: 21–32.
Çakır B (2015). Asmada (Vitis vinifera cv. Sultani Çekirdeksiz) Bax İnhibitor-1 Geninin İzolasyonu ve Moleküler Karakterizasyonu. Ege Üniversitesi Ziraat Fakültesi Dergisi, 49: 249-254. DOI: 10.20289/zfdergi.69696
Chen H, Liu L, Wang L, Wang S, Cheng X (2016). VrDREB2A, a DREB-binding transcription factor from Vigna radiata, increased drought and high-salt tolerance in transgenic Arabidopsis thaliana. Journal Plant Resources. 129:263-273.
FAOSTAT (2016). http://faostat3.fao.org/faostat-gateway/go/to/download/Q/QC/E. 4 Temmuz 2018
Güneş A, Adak S, İnal A, Alpaslan M, Eraslan F, Çiçek N, Kayan N, Soylu B (2006): Mercimek ve Nohut Bitkilerinde Kuraklığa Bağlı Oksidatif Stres ve Fizyolojik Tolerans Mekanizmalarının Belirlenmesi. Ankara Üniversitesi, Bilimsel Araştirma Projesi Kesin Raporu, Ankara.
Huang BL, Zhang XK, Li YY, Li DY, Ma MY, Cai DT, Wu WH, Huang BQ. (2016) Cloning and characterization of the dehydration-responsive element-binding protein 2A gene in Eruca vesicaria subsp sativa Huang. Genetics and Molecular Research 15 (3): gmr. 15038540
Ito Y, Katsura K, Maruyama K, Taji T, Kobayashi M, Seki M, Shinozaki K. Yamaguchi-Shinozaki K. (2006) Functional analysis of rice DREB1/CBF-type transcription factors involved in cold-responsive gene expression in transgenic rice. Plant Cell Physiology 47:141–153.
Joshi R, Karan R (2013). Physiological, biochemical and molecular mechanisms of drought tolerance in plants, In: Gaur RK, Sharma P, (Ed). Molecular Approaches in Plant Abiotic Stress. Boca Raton, FL: CRC Press;pp. 209–231.
Jovanović Z, Stanisavljević N, Mikić A, Radović S, Maksimović V (2013) The expression of drought responsive element binding protein (DREB2A) related gene frompea (Pisum sativum L.) as affected by water stres. Australian Journal of Crop Science 7:590-1596.
Kidokoro S, Watanabe K, Ohori T, Moriwaki T, Maruyama K, Mizoi J, Myint Phyu Sin Htwe N. Fujita Y, Sekita S, Shinozaki K, Yamaguchi-Shinozaki K (2015) Soybean DREB1/CBF‐type transcription factors function in heat and drought as well as cold stress‐responsive gene expression. Plant Journal 81:505–518.
Lata C, Prasad M (2011) Role of DREBs in regulation of abiotic stress response in plants. Journal of Experimental Botany 62:4731-4748.
Liu Q, Kasuga M, Sakuma Y, Abe H, Miura S, Yamaguchi-Shino-zaki K, Shinozaki K (1998) Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought- and low-temperature-respon-sive gene expression, respectively, in Arabidopsis. Plant Cell 10:1391–1406.
Liu L, Zhu K, Yang Y, Wu J, Chen F, Yu D (2008) Molecular cloning, expression profiling and transactivation property studies of a DREB2-like gene from chrysanthemum (Dendranthema vestitum). Journal of Plant Resources 121:215-226.
Livak JK, Schmittgen DT (2001) Analysis of Relative Gene Expression Data Using RealTime Quantitative PCR and the 2-ΔΔCT Method. Methods 25:402–408.Maruyama K, Sakuma Y, Kasuga M, Ito Y, Seki M, Goda H, Shimada Y, Yoshida S, Shinozaki K, Yamaguchi-Shinozaki K (2004) Identification of cold-inducible downstream genes of the Arabidopsis DREB1A/ CBF3 transcriptional factor using two microarray systems. Plant Journal 38:982–993.
Nakashima K, Ito Y, Yamaguchi-Shinozaki K (2009) Transcriptional regulatory Networks in response to abiotic stresses in Arabidopsis and grasses. Plant Physiology 149:88–95.
Nayak S, Balayi J, Upadhyaya H, Hash C, Kishor P,Chattopadhyay D, Rodrigez L, Blair M, Baum M, McNallyK, This D, Hoisington D, Varshney R (2009) Isolation andsequence analysis of DREB2A homologues in three cerealand two legume species. Plant Science 177:460-467.
Qin F, Sakuma, Y, Li J, Liu Q, Li YQ, Shinozaki K, Yamaguchi-Shinozaki K (2004) Cloning and functional analysis of a novel DREB1/CBF transcription factor involved in cold-responsive gene expression in Zea mays L. Plant Cell Physiology 45:1042–1052.
Qin F, Kakimoto M, Sakuma Y, Maruyama K, Osakabe Y, Tran LamSon P, Shinozaki K, Yamaguchi-Shinozaki K (2007) Regulation and functional analysis of ZmDREB2A in response to drought and heat stress in Zea mays L. Plant Journal 50:54–69.
Saha GC, Vandemark GJ. (2013) Stability of expression of reference genes among different lentil (Lens culinaris) genotypes subjected to cold stress, white mold disease, and aphanomyces root rot. Plant Molecular Biology Reporter 31:1109–1115.
Sakuma Y, Maruyama K, Osakabe Y, Qin F, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2006a) Functional analysis of an Arabidopsis transcription factor, DREB2A, involved in drought-responsive gene expression. Plant Cell 18:1292–1309.
Sakuma Y, Maruyama K, Qin F, Osakabe Y, Shinozaki K, Yamaguchi-Shinozaki K (2006b) Dual function of an Arabidopsis transcription factor DREB2A in water-stress-responsive and heat-stress-responsive gene expression. PNAS 103:18822–18827.
Tavakol E, Sardaro ML, Shariati JV, Rossini L, Porceddu E (2014) Isolation, promoter analysis and expression profile of DREB2 in response to drought stres in wheat ancestors. Gene 549:24-32.