Document Details
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Document Type |
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Thesis |
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Document Title |
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Identification of Genes Related to PCD in Plant via Modern Biotechnology تحديد الجينات المرتبطة بالموت الخلوي المبرمج في النبات باستخدام التقنيات الحيوية الحديثة |
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Subject |
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Faculty of Sciences |
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Document Language |
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Arabic |
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Abstract |
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Programmed cell death (PCD) is a fundamental process that occurs in eukaryotes and prokaryotes. The idea behind this research was the use of a natural exciting phenomenon suggesting that suppression of genes inducing programmed cell death (PCD) might help the plant cells to tolerate abiotic stresses more efficiently. The main objective of the current research study is the detection of PCD-related genes in tobacco (N. benthamiana) to be knocked down by inducing the RNAi machinery via virus induced gene silencing )VIGS) to ensure their role during PCD. Then, gene analogues in Arabidopsis were tested under salt stress.
First, PCD-related genes in tobacco were induced due to oxalic acid (OA) treatment (20 mM) that was proven to trigger PCD after 24 h. Plant cells were, then, characterized at the biochemical and molecular levels to confirm the incidence of PCD. Plant cells undergoing PCD were detected by the staining with Evans blue, then DNAs extracted during PCD were tested for laddering as the basic DNA molecular marker for plant PCD and results were positive after 24 h. Expression of eleven standard genes known for their effects during PCD was detected via VIGS (lines were kindly provided by Professor Dr. Gregory Martin, Boyce Thompson Institute, Cornell University, Ithaca, NY 14853-1801, USA). The results indicated that the standard genes promoted PCD except for two genes, e.g., ALD2H and plastocyanin that showed opposite results. Then, we detected expression rates of these eleven genes during PCD across time of OA exposure via qRT-PCR. The results indicated the occurrence of gene regulation in six of them, e.g., MEK2, SGT1, cathepsinB, BAK1, ALD2H and plastocyanin, where five genes were upregulated and one (e.g., cathepsinB) was down-regulated. On the other hand, the other five genes were found, unexpectedly, non-regulated during PCD. These genes are MAP3Kα, NRC1, WIPK, RAR1 and SIPK. Then, RNAs were extracted from tobacco cells 0, 2, 6, 12 and 24 h after OA treatment for deep sequencing.
Then, RNA-Seq analyses were done at Genomics Unit, Michigan State University, USA with a special emphasis to clusters whose PCD-related genes were upregulated after 2 h of OA treatment. Accordingly, 23 tobacco PCD-related genes were knocked down via virus-induced gene silencing (VIGS), whereas our results indicated the influence of five of them on inducing or suppressing PCD. Knockout T-DNA insertion mutants of these five genes in Arabidopsis (provided by SALK Institute, Genomic Analysis Laboratory, USA) were tested under salt stress (0, 100, 150, and 200 mM NaCl), and the results indicated that a mutant of an antiapoptotic gene, namely Bax Inhibitor-1 (BI-1), whose VIGS induced PCD in tobacco, was salt sensitive, while a mutant of an apoptotic gene, namely mildew resistance locus O (Mlo), whose VIGS suppressed PCD, was salt tolerant as compared to the WT (Col) control. These data support our hypothesis that retarding PCD-inducing genes can result in higher levels of salt tolerance, while retarding PCD-suppressing genes can result in lower levels of salt tolerance in plants. |
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Supervisor |
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Prof. Ahmed Bahieddin Ahmed |
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Thesis Type |
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Doctorate Thesis |
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Publishing Year |
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1438 AH
2017 AD |
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Co-Supervisor |
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Dr. Idris Ahmed Idris |
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Added Date |
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Tuesday, February 7, 2017 |
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Researchers
| ظافر علي القرني | Alqarni, Dhafer Ali | Researcher | Doctorate | |
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