arabidopsis thaliana bitkisinden izole edilen na+/h+ antiporter (atnhx1) geninin nicotiana tabacum bitkisine aktarılarak tuz direncinin değerlendirilmesi

Large, membrane-bound vacuoles of plant cells are suitable organelles for the compartmentation of ions. These vacuoles contain Na+/H+ antiporters for movement of Na+ within the organelle in exchange for H+. They provide an efficient mechanism to prevent the occurance of detrimental outcomes of Na+ accumulation in the cytosol. Identification of AtNHX1 gene that confers resistance to salinity by expressing a Na+/H+ antiport pump facilitates the understanding of the salt stress tolerance mechanisms of plants. The aim of the present study was to isolate and clone the Arabidopsis thaliana AtNHX1 coding sequence for transformation of Nicotiana tabacum plants via Agrobacterium tumefaciens mediated gene transfer. For this purpose, total RNA was isolated from Arabidopsis thaliana plants and cDNA synthesis was performed. AtNHX1 (1614bp) was amplified by using cDNA of Arabidopsis via specific primers. The amplified PCR product was verified by sequencing. AtNHX1 coding sequence was cloned into the plant transformation vector pCVB1 and 10 independent putative transgenic tobacco plants were obtained via Agrobacterium tumefaciens mediated gene transfer sysytem. Transfer of selected 8 putative transgenic plants to soil provided the regeneration of T1 seeds. Germination of the seeds under different salt treatments (0, 50, 100, 150, 200, 250 mM NaCl) was observed for evaluating the salt tolerance of transformed plants. The 82% and 60% of the transgenic T1 seeds were germinated on 150 mM NaCl and 200 mM NaCl containing media, respectively. In contrast the germination percentage of wild type tobacco seeds under 150 mM NaCl and 200 mM NaCl concentrations were 39% and 21%, respectively. The germination rate of the transgenic T1 seeds were significantly higher (p=0,001) when compared to the control seeds especially under high salt stress conditions (150 and 200 mM NaCl). Taken all together, our results demonstrated that the germination efficiencies and growth of the plants transformed with AtNHX1 were higher than the wild type tobacco plants under high salt concentrations.M.S. - Master of Scienc

Thellungiella halophila ekotiplerinde abiyotik stresle ilişkili MiRNA'ların belirlenmesi.

Environmental stress with climate change currently contributes to approximately 50% of crop loss occurring world-wide. Thellungiella halophila is a plant that is native to highly saline and semiarid environments and exhibits an exceptional ability to tolerate abiotic stress. Primarily, the determination of relatively high GSH/GSSG ratios (glutathione assay), elevated proline accumulation, less MDA (lipid peroxidation) production and increased anthocyanin levels and less reduced RWC of the T. halophila (Shandong and Yukon ecotypes) after exposure to increasing concentrations of NaCl allowed us to support that Thellungiella tolerate environmental stress conditions. Genomic studies have shown that differences in stress tolerance arise not only from the presence or absence of stress-associated genes but also from their regulation. In plants, miRNAs are the key gene regulators that have been shown to control various developmental processes and responses to environmental stresses. In order to assess the expression profiles of miRNAs in abiotic stress tolerance of T. halophila ecotypes, salt, cold and drought treatments were carried out. As a result, we identified the changes in the expression profiles of seven conserved miRNAs under 250 mM NaCl, 4°C and 200 mM mannitol using Northern-Blot analysis and TaqMan qRT-PCR assays. Four of the stress related miRNAs (miR156, miR169, miR319 and miR393) were found to be downregulated and three (miR159, miR398 and miR169) were found to be upregulated during exposure to salt, cold and drought stress. Based on the conservation of mature miRNA sequences, potential target genes for miRNAs were also predicted by using conservation of miRNA target sites among species. The target mRNA expression analysis were done by RT-qPCR after exposure to salt stress. RT-qPCR results showed decrease in the expression of miR156, miR169, miR319 and miR393 with a corresponding upregulation of their putative target mRNA transcripts (SPL10, NF-YA5, ITD1 and WRKY33, respectively). In addition, the induction of miR159 was accompanied by the down regulation of its probable target mRNA (HEC1). Taken together, these findings suggest that a number of conserved miRNAs may function in Thellungiella halophila ecotypes tolerance to environmental stresses.Ph.D. - Doctoral Progra

Generating salt-tolerant Nicotiana tabacum and identification of stress-responsive miRNAs in transgenics

Identification of vacuolar Na+/H+ antiporters facilitates the basis of salt stress tolerance mechanisms. Na+ accumulation into the vacuole is crucial for the avoidance of cytoplasmic Na+ toxicity. In this study, we show that the introduction of AtNHX1 into tobacco generated more tolerant plants when compared to wild-type plants. Transgenic tobacco plants exhibited higher germination rates in the presence of increasing salt concentrations compared to wild-type plants. In addition, proline levels were higher under salt stress conditions in both the shoots and the roots of transgenics compared to wild-type plants. Increase in malondialdehyde production during lipid peroxidation by salinity was lower in transgenic plants compared to the controls. Being important and newly discovered determiners of plant stress responses, microRNAs (miRNAs) are noncoding small RNAs and essential indicators of plant stress response mechanisms. For further identification of stress responses, the expression levels of growth and abiotic stress-related miRNAs (miR319a, miR319b, miR159b, miR398a, and miR398b) were quantified. miR319a, miR319b, and miR159b expression levels were higher in wildtype plants, and miR319a and miR159b expression was restored in transgenic plants. There was a slight decrease in the expression levels of miR398a and miR398b of wild-type plants. However, the recovery of the miR398a and miR398b expressions was especially significant in transgenic plants. Furthermore, an investigation of miRNAs in transgenic plants can help to understand the stress tolerance mechanisms of the plants

Hepatoprotective Role of Thymol in Drug-Induced Gastric Ulcer Model

Introduction and aim. Indo is widely one of the non-steroidal anti-inflammatory drugs and one of the common toxic effects of this drug is hepatic failure. Thymol is a monoterpene phenol with many different pharmacological activities. However, up to now its hepatoprotective effects on Indo-induced gastric ulcer model in rats have not been explored yet.Material and methods. Thirty five Sprague-Dawley rats were divided into seven groups: control, ulcer control (30 mg/kg Indo), Indo + reference standard (50 mg/ kg Rantidine), Indo + Thymol (75, 100, 250 and 500 mg/kg) groups. 10 minutes after the induction of ulcer with Indo; Thymol was orally administered to the rats. Liver function enzymes (AST, ALT and LDH) were measured from serum samples. TOS/TAC, TNF-α and PGE2 levels, eNOS and Caspase-3 activity were assessed from tissue homogenate samples. In addition, histopathologic analysis on liver sections was performed.Results. Indo significantly increased the levels of hepatic enzymes, TNF-α and eNOS, and caspase-3 activation, while decreased PGE2 levels. Furthermore, it induced oxidative stress as evidenced by elevated TOS and decreased TAC levels. However, Thymol treatment induced a significant improvement in these parameters, especially in 250 mg/ kg dose. On the other hand, treatment with Thymol 500 mg/kg dramatically affected the parameters much worse than the Indo treated group.Conclusion. The findings of the current study demonstrated that Thymol administration significantly ameliorated liver injury due to Indo toxicity. This effect of Thymol (250 mg/kg) may be mediated by its anti-oxidative or anti-inflammatory effect, and up-regulation the synthesis of PGE2

Propolis and Its Combination with Boric Acid Protect Against Ischemia/Reperfusion-Induced Acute Kidney Injury by Inhibiting Oxidative Stress, Inflammation, DNA Damage, and Apoptosis in Rats

Ischemia reperfusion (I/R) injury which causes kidney dysfunction is one of the most studied diseases directly linked to oxidative stress. In this regard, it is important to protect cells against damage by inducing antioxidant response. Herein, we aimed to evaluate the therapeutic roles and possible mechanisms of propolis and boric acid in kidney I/R injury based on relevant basic research and clinical studies. Sprague-Dawley rats were subjected to 50 min of ischemia followed by 3 h of reperfusion. Animals were randomly divided into a control group (the abdominal wall was just opened and closed), an I/R injury group, the propolis intervention group (200 mg/kg, intragastric administration, 1 h before ischemia), boric acid intervention group (14 mg/kg, intragastric administration 1 h before ischemia), and the propolis + boric acid intervention group (intragastric administration 1 h before ischemia). Kidney function, the antioxidant defensive system, and renal damage were assessed. In addition, the oxidative stress and inflammatory status were estimated in renal tissue. Furthermore, DNA damageand apoptosis were detected by immunohistochemistry. When compared with I/R group, propolis alone and especially propolis + boric acid groups significantly improved functional parameters. While the antioxidant response was increased, renal injury size and apoptosis were significantly decreased in both groups. Also, the MDA and TNF-alpha levels besides the 8-OHdG formation were downregulated. According to these outcomes, it can be said that especially propolis together with boric acid ameliorates kidney injury caused by I/R through acting as an antioxidant, anti-inflammatory, and antiapoptotic agent. In conclusion, propolis alone and its combination with boric acid could be developed as therapeutic agents against serious renal I/R injuries