الفهرس 
Wheat genomeOnly 14 pages are availabe for public view
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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.