الفهرس 
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Abstract
Wheat is one of the most important and the most grown cereal crop in the
world. Its importance derived from many properties and uses of its kernels, which
make it a stable food for the world’s population. In Egypt, wheat production is
considered the most important item to the government and population. Abiotic
stresses, especially temperature and drought, are the primary causes of plant loss
worldwide. The wheat plants exposure to hot wind (up to 30/25°C day/night) even
for a short time and drought stress after anthesis during grain-filling period
reduced grain yield by reducing weight per grain, rather than grain size, number,
and quality. The Heat shock proteins buffer this environmental variation is
important in relation to stress resistance and adaptation to the environment.
Therefore, they are important factors for the maintenance of homeostasis across
environmental regimes. Understanding the role of HSPs in relation to stress
resistance in a more applied perspective as a potential indicator of stress is
important. This study aims to support the plant breeder with important information
about the heat shock protein genes which could be used for wheat improvement
for heat stress tolerance.
This study was carried out in the Laboratory of the Molecular Biology
Department at the Genetic Engineering and Biotechnology Research Institute
(GEBRI), Minoufiya University, Sadat city, Egypt, during the period from 2008 to
2012. Two Egyptian wheat cultivars, one Mexican and one Chinese wheat
cultivars have been chosen in order to carry out this study. The wheat cultivars
have been selected depending upon their background for heat tolerance. Heat
shock protein gene expression was studied at three levels. The first level was
protein level using SDS-PAGE. The expression of these proteins was studied at
RNA level using both RT-PCR heat shock protein genes amplification and
differential display analysis. Finally, DNA fingerprint was made for all cultivars
under study using SRAP and TRAP molecular markers.
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Summary
Surface-sterilized grains from each cultivar were germinated in sterilized
Petri-dishes containing sterilized filter papers moistened with tap water. Ten-day
old seedlings of each cultivar were exposed to heat stress at different degrees, 31,
34, 37, 40, and 45°C for 2 hours inside water baths and at different lengths of
incubation periods at 37°C for indicated time periods (0.5, 1, 2, 4, and 24 hours),
in a controlled incubator. Dehydration treatment was performed by leaving whole
ten-day-old seedlings on the light bench for two hours. ABA treatment was also
carried out using the excised leaves which were float on the light bench for 24
hours on a 5<I 0-5 M ABA solution. Total protein (after the heat shock,
dehydration and ABA treatments) was extracted and separated on SDS-PAGE to
analyze the Heat Shock Protein (HSP) synthesis in both leaves and roots of a heat
tolerant and susceptible wheat cultivars.
Total RNA was isolated and RNA quantity and integrity were estimated.
Heat Shock Protein genes have been amplified using the heat shock protein gene
family specific primers (HSP70a, HSP70b, HSPlOla, and HSPlOlb) by RT-PCR.
RNA fingerprinting was performed by using differential display analysis using
three arbitrarily primers as forward primers and four anchored primers as reverse
primers. The members of wheat HSP gene families were identified by modified
differential display analysis using three HSP genes specific primers as forward
primer and four anchored primers as reverse primers.
Genomic DNA was isolated from leaves of all cultivars by using CTAB
modified method. Ten SRAP and eight TRAP primers were used to generate
fingerprints of four wheat genotypes. Some obtained heat shock protein genes
cDNA fragments were excised, eluted from agarose gel and sequenced. The
cDNA nucleotide sequence was analyzed, and searched their homologies with the
sequences published in GenBank/EMBL DNA databases. The obtained cDNA
nucleotide sequence was submitted in the National Center of Biotechnology
Information (NCBI) server.
123
Summary
The presence of high molecular weight HSP in wheat leaf started to appear
after heat shock at different treatments in the four wheat cultivars. HSP wasn’t
observed only at control treatment but also at 31 and 34°C, while it was detected
early after 0.5 hour at 37°C and still observed at all other heat shock, dehydration
and ABA treatments in all cultivars under study. No consistent cultivar-dependent
differences in the accumulation protein belonging to HSP family were observed
expect, HSP 60 was observed during 40°C and 45°C only in ’Gemmeiza 7’
cultivar but it was observed in all cultivars at heat shock (at 37°C for 4 and 24
hours), dehydration, and ABA treatments. Some small heat shock protein bands
(sHSP) have been obtained in the heat tolerant cultivars (i.e. ’Giza168’ and ’SERI
82’) where not observed in the heat susceptible cultivars (’Gemmeiza 7’ and
’Chinese Spring’) like sHSP16.9 at 34 and 40°C for two hours. The results
indicate that high molecular weight HSP and low molecular weight HSP
expression differed at different temperature degrees in all cultivars and most HSPs
have been detected in wheat plant subjected to heat shock at 37°C for two hours.
Drastic increase in both hHSP and sHSP expression was observed in all tested
cultivars subjected to dehydration treatment and their gene expression can be
induced by ABA treatment indicating potential involvement of HSP in plant
osmotic stress responses. Little difference was obtained between four wheat
cultivars in total extracted protein leaf analysis pattern
Low protein concentration was observed in wheat roots tissue in comparing
to leaf tissue. Significant accumulation of HSP family protein that probably
resulted from translation of the different members of the HSP group of mRNAs in
the roots of wheat seedlings subjected to heat shock, dehydration and ABA
treatment was observed. HSP 100 band wasn’t visible at low temperature but
became intense at 34°C in ’SERI 82’ cultivar only, while it was observed at higher
temperature 40°C in ’Giza 168’, ’SERI 82’ and ’Chinese Spring’ cultivars and
45°C in all tested cultivars. Meanwhile, its expression was detected at 37°C after
two hours in ’Giza 168’ and ’SERI 82’ cultivars , disappeared after 4 and 24 hours
124
Summary
of heat shock at 37°C and was observed only in ’Giza 168’ cultivar seedlings
subjected to dehydration treatment.
The total amount of isolated RNA differed from sample to sample
according to the treatment and to the number of expressed genes. The amount of
total isolated RNA from both treatments 37°C for one hour and 37°C for two hours
was higher than the other samples while the isolated mRNA was slightly higher in
the control samples other than the treatments. Heat shock protein gene expression
differed in the quantity and quality during all abiotic treatments in our study. Two
fragments of the HSP 16.9 gene family have been amplified representing different
members of that gene family at molecular weight of 900 and 700 bp, respectively
in all cultivars during all treatment with different degrees of gene expression. The
obtained 394 bp cDNA fragment sequence (sequencing results of 700 bp cDNA
fragment) consist of 5’UTR from the first base till no. 56 bp, CDS segment was
located between base no. 57 bp to no. 307 bp and 3’ from no. 308 to no. 394 bp.
BLASTN pairwise alignment analysis of nucleotide sequence of obtained HSP
16.9 cDNA fragment (HSP16.9 sam 2) showed that fragment sequence shared
high significant identity (83%) with other heat shock protein 16.9 genes sequence
and other low molecular weight heat shock protein database sequences. The amino
acid conceptual translation of obtained cDNA HSP 16.9 fragment was 83 amino
acids length. More than two putative conserved domains have been detected in our
predicted protein sequence such as Alpha crystallin domain (ACD). Our results
strongly suggest that obtained HSP 16.9 cDNA fragment may be a big part of a
new gene of wheat heat shock protein 16.9 which belong to the LMW HSP class I
multigene family. We have submitted this HSP16.9 cDNA fragment nucleotide
sequence by direct submission of sequence data to NCB I GenBank with HSP16.9
sam 2 as a name and we have get back the GenBank accession number of our
submission as (JQ809331).