الفهرس | Only 14 pages are availabe for public view |
Abstract This Study was conducted in the Molecular and Biochemical Genetic Laboratory of the Department of Genetics, Faculty of Agriculture, Ain Shams University, Shoubra El-Kheima, Egypt and Plant Pathogen Interaction Laboratory at Agricultural Genetic Engineering Research Institution (AGERI) of Agricultural Research Center (ARC), Giza, Egypt during the period from 2012 to 2016. The aim of study was to detect of up-regulation and downregulation of the drought tolerance of ESTs fragments of the two wheat cultivars; Sahel 1 (drought tolerance) and Gemmeiza 7 (drought sensitive) using DD-PCR technique. The seedlings of the two cultivars were germinated in pots for three weeks and taken carefully from potmoss, then placed on towels under the same conditions (drought treatments) and it was collected at definite time points (control, six hours, nine hours and twelve hours), after that it was kept at -80 refrigerator for study physiological evaluation and molecular genetics analysis. a-Physiological evaluation 1-Osmotc potential measurement (OP) The effect of osmotic potential on shoots and roots were detected using Osmomate machine. The result showed that the drought sensitive wheat cultivar (Gemmeiza 7) tended to have a lower osmotic potential than did the drought tolerant wheat cultivar (Sahel 1) because droughttolerant cultivar might be better able to survive under dry condition than the drought-sensitive cultivar. 2-Determination of Ca2+ and K+ ions The samples were digested and treated by specific chemicals for determination Ca2+ and K+ ions by photometry.Potassium and calcium levels were higher in Gemmeiza 7 (sensitive) than in sahel 1 (tolerant) which could be due to that the sensitive cultivar always needs to maintaine the osmotic potential by increasing (K+ and Ca+2%) as osmoregulators. The physiological evaluation data were analyzed by using SPSS analysis program showed that: 1- The calcium, potassium percentage and osmotic potential were markedly significantly affected by drought treatment in the two cultivars. 2- There was a significant difference in Ca2+ and K+ between the two cultivars, whereas OP wasn’t significantly affected. 3- The drought time points factor wasn’t significantly affected between cultivars for Ca2+, K+ and OP. b-Molecular genetic analysis Isolation of RNA from shoots and roots, then measurement of the concentration of product by nanoDROP machine to unique the concentration of RNA followed by conversion to cDNA with reverse transcriptase were carried out. Amplification of cDNA was applied with anchor in addition to three arbitrary primers. A Differential Display Polymerase Chain Reaction (DD-PCR) technique was selected for gene expression profiling to screen the up and down regulation of gene expression in shoots and roots of the two cultivars; Sahel 1 (tolerant-drought) and Gemmeiza 7 (sensitive-drought) in response to drought in wheat bread (Triticum aestivum L.). The expression profiles of the resulted fragments were divided into four clusters: (1) up-expression patterns, which either were expressed due to stress, or gradually increased as time of treatment increased,2) up & down-expression patterns, in which the expression showed increase in trancript accumulation in comparison to control, then decreased after 6 hrs of drought treatment, (3) down & up- expression patterns, in which the expression showed decrease in transcript accumulation in comparison to control, then increased after 6 hrs, (4) down-expression patterns, which were either un-expressed or gradually showed decreased expression as the time of treatment increased. The ESTs were eluted and purified from polyacrylamide gel. The purified ESTs were sent to abroad for reading the sequences. Twenty differentially expressed ESTs were obtained from shoots and roots. Seven of which were from shoots and 13 were from roots. The sequences of ESTs were identified by using bioinformatics analysis, then NCBI was used and BLASTn option was selected to identify the name and function of homologous proteins. Some of the sequence fragments showed significant homology to know predicted proteins were as follows: 1- Metabolic (Fructan 1-exohydrolase and RNA polymerase II gene). 2- Osmoregulation (TaAP2, NRC-1 and Nelumbo nucifera protein NEN- 4). 3- Signal transduction pathways (Glycosyltransferases (GTs), Ageratina adenophora ICE1, Prolamine genes and serine/threonine-protein kinase). 4- Unnamed proteins (proteins don’t identify or characterize till now). Finally, these results could help to elucidate the molecular basis of drought tolerance. This calls for the performance of more advanced studies to characterize the specific roles of signal transduction pathways in abiotic stress. |