Molecular cloning , expression , sequence analysis and in silico comparative mapping of trehalose 6-phosphate gene from Egyptian durum wheat

Trehalose is a non-reducing disaccharide which consists of two glucose units that functions as a compatible solute to stabilize the membrane structures under heat and desiccation stress. Trehalose-6phosphate synthase (TPS) and trehalose-6phosphate phosphatase (TPP) are the key enzymes for trehalose biosynthesize in the plant kingdom. On the basis of bioinformatics prediction, fragment containing an open reading frame of 945 bp was cloned from durum wheat. Sequence comparison and analysis of conserved domains revealed the presence of a TPP domain. Full length of the gene was isolated using gene race technology. Semi-quantitative RT-PCR and real time quantitative PCR indicated that the expression of this gene is up-regulated in response to drought stress. The biochemical assay of the trehalase activity showed that the enzyme's activity decreased under the dehydration stress. The obtained phylogenic tree showed that the isolated TPP protein forms a distinct clad close to the Oryza sativa trehalose-6phosphate phosphatase. In silico and comparative mapping indicated that the isolated TPP gene is localized on rice chromosome 8, durum wheat chromosome 20, bread wheat chromosome 3B, oat linkage group E, sorghum chromosome 4 and barley 5H.


INTRODUCTION
The global food situation is currently being redefined by many driving forces like globalization, urbanization, energy prices, and climate change.According to the report of the food and agriculture organization of the United Nations (FAO) 2010, the number of undernourished *Corresponding authors.E-mail: aymanalidiab@gmail.com.people around the world in 2010 has declined but remains abnormal and unacceptable.The renewed global attention is being given to the role of agriculture and food in development policy.One of the required actions that is suggested by the Egyptian Cabinet, Information and Decision Support Center (IDSC) to solve the food problem in Egypt was to focus on the agricultural research to enhance the capability of crop plants to withstand different abiotic stresses, such as salt, drought, cold and Heat shock which will lead to higher yields by either increasing the crop set and/or by extending crop cultivation in the areas previously denied due to abiotic stresses.
Understanding the gene networks that represent the biological system of plants under abiotic stress and their defense mechanism makes it necessary to characterize the candidate genes that are responsible for the physiological response to the stress.Trehalose is an important building block to build up sugars that create cellular signaling and communication.It is included in the building of a number of cell wall glycolipids.Trehalose is a disaccharide sugar widely distributed in bacteria, fungi, insects, plants and invertebrate animals.In microbes and yeast, trehalose is produced from glucose where trehalose-6-phosphate synthase (TPS) and trehalose-6phosphate phosphatase (TPP) function together as a large complex to synthesize trehalose.Moreover, TPS and TPP serve as sugar storage, metabolic regulator and protect living organisms against abiotic stress (Wiemken, 1990;Strom and Kaasen, 1993).Before 1997, it was thought that trehalose is present only in a few desiccation-tolerant plants.However, its role in plants was not yet fully elucidated.Under osmotic stress, trehalose was shown to accumulate at high levels in resurrection plants such as Selaginalla lepidophylla (Wingler, 2001) and as a sugar reserve or stress protectant in Arabidopsis thaliana (Goddijn and Smeekens, 1998;Vogel et al., 2002;Schluepmann et al., 2003).Interestingly, TPP and TPS genes are broadly found in the genomes of higher plants and form large gene families (Leyman et al., 2001;Schluepmann et al., 2004).Mellor (1992) considered trehalose as a symbiotic determinant between higher plants and microorganisms.However, there is no direct evidence supporting this hypothesis so far.It was found that the precursor of trehalose, trehalose-6-photophate, (T-6-P) is the key regulator in the glycolytic pathway (Blazquez et al., 1998).It targets the initial step of glycolysis to reduce the entrance of glucose into glycolysis.The same role of trehalose in the sugar metabolism was invistigated (Vogel et al., 1998;Paul, 2001;Wingler, 2001;Eastmond and Graham, 2003).From the results of genetic and reverse genetic analysis, trehalose was found to have an essential role in carbohydrate metabolism and development of higher plants.In Arabidopsis, loss of AtTPS1 function is an embryo-lethal phenotype (Eastmond et al., 2002;Schluepmann et al., 2003;Gomez et al., 2006); and a mutation in the maize TPP gene caused abnormalities in the inflorescence architecture (Satoh-Nagasawa et al., 2006).Trehalose content increased in rice as a result of the over expression of fused bacterial TPS and TPP proteins (named TPSP).The bi-functional TPSP protein enhanced the rice tolerance to abiotic stresses (Garg et al., 2002;Jang et al., 2003).Over expression of TPSP had a direct effect on the photosystem II damage under abiotic stress (Garg et al., 2002;Jang et al., 2003).El-Bashiti et al. (2005) reported the possible role of trehalose as osmoprotectant compound in wheat species under salt and drought stress conditions.The accumulation of trehalose in wheat under abiotic stresses was found to be tissue and species specific.
Martı´nez-Barajas and his colleagues (2011) analyzed T6P content and SnRK1 activities in wheat (Triticum aestivum) grain.The data shows a correlation between T6P and sucrose overall that belies a clear effect of developmental stage and tissue type on T6P content, consistent with tissue-specific regulation of SnRK1 by T6P in wheat grain.Homologs of SNF1-related protein kinase1 (SnRK1) marker genes designated in Arabidopsis (Baena-González et al., 2007) was used to prove that regulation of SnRK1 by T6P could operate in vivo, using Wheat Estimated Transcript Server (WhETS; Mitchell et al., 2007).
In long term, the overexpression of trehalose biosynthetic genes in wheat may seem to be promising for improvement of abiotic stress tolerant transgenic wheat.
This work aimed at the isolation, cloning and characterization of functional trehalose-6-phospate phosphatase (TPP) gene from durum wheat under dehydration stress to investigate the trehalose 6 phosphatase (TPP) gene ability for drought tolerant in Durum wheat in order to examine the magnitude of the TPP gene response to drought stress.

Plant materials, growth conditions and stress treatments
Durum wheat plants, (variety Sohag 3) presumably holding genes of resistance to drought were subjected to dehydration stress.Seeds of durum wheat (Triticum turgidum.L. var.durum wheat) were sterilized in 10% sodium hypochloride for 30 min and then rinsed with ddH2O for 1 min.Seeds were planted in soil composed of sand and clay (1:1) for three weeks and watered daily under controlled conditions (28ºC day/25ºC night, 12 h photoperiod, ~500 mol m -2 s -1 photon flux density and 83% relative humidity).Drought treatment was applied as described by Ozturk et al. (2002) where, seedlings were removed from soil, washed carefully and placed on paper towels under the same growing conditions.Leaves were harvested after 2, 4, and 6 h of drought treatment, frozen in liquid nitrogen and stored at -80°C.Control seedlings were planted and grown concurrently in the same conditions without any drought regime (well-watered) then leaves were harvested at the same time and frozen in liquid nitrogen and stored at -80°C.For the estimation of water loss, leaves were weighted at the same time intervals as that used in the dehydration experiments (zero, 2, 4 and 6 h).The ratio of the leaves weight in comparison to the control was used as indication of water loss.

Total RNA isolation
Total RNA was extracted according to Chomczynski (1993) where, 100 mg of the control and drought treated leaves (0, 2, 4 and 6 h) were ground in liquid nitrogen.1 ml of TriPure reagent (Cat.No. 1 667 165, Roche) was added to the fine leave powder and shacked gently.The mixture was left for 5 min at room temperature before adding 0.2 ml of chloroform.The mixture was left at room temperature for 10 min then centrifuged for 15 min at 4°C.Half ml isopropanol was added to the aqueous phase and incubated at room temperature for 10 min.The samples were centrifuged at 12,000 x g for 10 min at 4°C.The RNA pellets were re-suspended in 75% ethanol then centrifuged at 7500 x g for 5 min at 4°C.The RNA pellets were dried and re-suspended in diethylpyrocarbonate (DEPC)-treated RNase-free water and stored at-80°C.

Reverse transcription PCR (RT-PCR)-based cDNA cloning
A pair of primers, (5'-ATGGATTTGAGCAATAGCTC-3' and 5'-ACACTGAGTGCTTCTTCCAT-3') were synthesized and used to perform RT-PCR amplification using ImProm-IITM reverse transcription system (Cat.No. A3800, Promega).According to Liang and Pardee (1995), a cDNA of the TPP gene was generated using a RT-PCR based approach.The PCR cycle condition consists of three segments.The first one was a pre-denaturation for 4 min at 94°C.The second variable segment was consists of 40 cycles each one was 1 min at 94°C, 1.5 min at 55°C and 2 min at 72°C; the last segment was an extension for 10 min at 72°C.The amplified cDNA fragment was purified and cloned for sequencing.

Cloning of PCR product
The PCR products were cloned in pGEM-T Easy plasmid (Promega, USA) and transferred into Escherichia coli DH5α.The white colonies were picked and screened for the presence of the cloned gene of interest through digestion with EcoRI (Sambrook et al., 1989).The pGEM®-T plasmid having TPP cDNA was selected by PCR using T7 and SP6 primers that amplify the 945 bp fragment having the TPP gene.In this reaction, 1.25 units Taq DNA polymerase, 20 pmol primers and 200 mM dNTPS were added to 15 ng of plasmid in a buffer containing 10 mM KCl, 10 mM (NH4)2SO4, 20 mM Tris -HCl-pH 9 and 0.1% Triton® X-100.The PCR cycle condition consists of three segments.The first was a pre-denaturation for 4 min at 94°C.The second variable segment was of 40 cycles each one was 1 minutes at 94°C, 1 min at 55°C and 2 minutes at 72°C; the last segment was an extension for 7 minutes at 72°C.

DNA sequencing and Bioinformatics analysis
The TPP clone was sequenced according to Sanger et al. (1977) using a Big Dye Terminator Cycle sequencing FS Ready Reaction Kit (Applied Biosystems, Foster City, CA) and an ABI PRISM 310 DNA sequencer (Applied Biosystems).A homology search was performed using BLASTX against the NCBI protein database (http://www.ncbi.nlm.nih.gov).Sequences of the trehalose phosphate phosphatase genes that showed similarity to the TPP gene were obtained from the NCBI non-redundant and dbEST data sets using BLASTX or BLASTP ver.2.0.10 (Altschul et al., 1997).The full amino acid sequences of the proteins were aligned using CLUSTAL-X ver.1.8 (Thompson, et al., 1997) and subjected to phylogenetic analysis.Phylogenic trees were constructed using the neighbor-joining (NJ) method (Saitou, and Nei, 1987) with parsimony and heuristic search criteria and 1000 bootstrap replications to assess branching confidence.

Rapid amplification of cDNA PCR (RACE-PCR)
Rapid amplification of cDNA PCR was done to obtain the full length of TPP gene.According to Frohmann (1994), preparation of cDNA and anchor primers was conducted using Roche kit cat No. Diab et al. 11 1734792 (Clontech Lab, Inc).PCR was performed by using SP2 primer from advantage cDNA PCR kit (Clontech) and the primer 5′-CCTCCAGCACTTCGTTTACGAG-3′ designed according to the gene sequence.PCR products were migrated by electrophoresis on 2% (W/V) agarose gel.The glass-milk (BIO 101) was used to recover and purify the DNA fragment, which was then ligated to pGEM®-T easy vector and finally transferred into Escherichia coli DH5α (invetrogen, cat.No.18265-017).the cloned full length gene was sequenced using ABI PRISM big dye terminator cycle sequencing ready reaction kit (PE Applied Biosystem, USA).

Expression patterns using semi-quantitative RT-PCR
Template cDNA was prepared using Super-Script II (Invitrogen) with 1 mg total RNA. 1 µl of cDNA reaction mixture was diluted with 9 µl DEPC treated water, then, 1 µl of diluted mixture was used to perform Semi-quantitative RT-PCR reaction as follows: 1.0 l dNTPS (10 mM), 2.5 l MgCl2 (25 mM), 5.0 l 10X buffer, 5.0 l Forward primer (10 pmol/l), 5.0 l Reverse primer (10 pmol/l) , 1.0 l Template cDNA (25 ng/l) , 0.5 l Taq (5 U/l), up to 50 l dd H2O.The amplification was carried out in Hybrid PCR Express system programmed with specific primers for TPP and 18S (as a control to normalize the amount of cDNA present in each sample) genes as follows: 5 min at 95°C, followed by 35 cycles at 95°C for 45 s, 55°C for 60 s, 72°C for 2 minutes, 72°C for 5 minutes.For each sample, 10 l of the amplification reaction was sizefractionated on a 2% (w/v) agarose gel and stained with ethidium bromide.Bands were detected on UV-transilluminator and photographed by a Gel Documentation system 2000 Bio-Rad to ensure that amplifications were in the linear range, for each template and primer pair.A Gene Ruler TM 1 kb DNA ladder was used as a standard.

Real-time PCR data analysis
Primers of TPP and 18S used for semi-quantitative RT-PCR were used in real time PCR analysis.The most commonly used method for relative quantification is the 2 -ΔΔCt method.Derivation and examples of this method have been described by Livak and Schmittgen (2001).The relative difference in gene expression using the 2 -ΔΔCt method was calculated as follows: Relative fold change in gene expression = 2 -ΔΔCt , Where, ΔΔCt = ΔCt treated -ΔCt untreated and ΔCt = (Ct target gene -Ct reference gene).

Trehalase enzyme assay
Samples weights about 100 mg from drought treated leaves at 0 hr (control), 2, 4, 6 hrs, were ground in liquid nitrogen.The powder was suspended in ice-cold suspension solution containing 0.1 M citrate (Na+), pH 3.7, 1 mM PMSF, 2 mM EDTA and insoluble polyvinylpyrrolidone (10 mg/g dried weight)).2 ml of extraction buffer was added to each 1g dry weight of sample.The homogenate was filtered through two layers of cheesecloth and centrifuged at 31,500 rpm (48,000 g) for 30 min at 4°C in Sorval Combi Plus with T-880 type rotor.The supernatant was used for the enzyme assays.Adapted from Vandercammen et al. (1989).The protein concentration was determined according to Bradford (1976) using bovine serum albumin (BSA) as standard.
Trehalase enzyme activity was measured using glucose oxidaseperoxidase kit (Bicon) according to Müller et al. (1992).The reaction mixture was composed of 10 mM trehalose, 50 mM MES (K + ), pH 6.3 and 0.2 mg crude extract in a final volume of 1 ml.

Time of dehydration shock (h) Loss in water content of leaves (%)
Control 0 0 2 15.79 4 26.36 6 34.5 The reaction was incubated at 37°C for 30 min and then started by the addition of trehalose to the reaction mixture, which was preincubated at 37°C for 10 min.100 µl of samples were taken from the reaction mixture and immediately put in thermostat at 100°C for 3 min to stop the reaction.Precipitates were removed by centrifugation at 8700 rpm for 10 min.For the analysis, 10 µl of the supernatant was mixed with µl of glucose oxidase -peroxidase kit solution, mixed by vortex and then the mixtures were incubated at 37°C for 15 min.The absorbance of the samples was measured at 470 nm in Schimadzu UV-1201 spectrophotometer against blank solution.The increase in the absorbance against time was assumed to be equal to the amount of glucose formed.One unit of trehalase activity is defined as the amount of enzyme that catalyzes the hydrolysis of 1 mmole of trehalose/ minutes at 37°C at pH 6.3.

In silico and comparative mapping of TPP
For in silico mapping, the isolated sequence was compared to rice and oat sequences using BLAST (with an e-value threshold of 1e-1000).The matches were used to identify markers from the genetic linkage map (http://www.tigr.org).The results obtained from this stage were used to construct a comparative map between durum and bread wheat, rice, sorghum, barley and oat to identify the tentative chromosomal location of the gene understudy using comparative mapping strategy (Diab et al., 2007;Abou Ali et al., 2009).

Physiological parameters and dehydration stressspecific transcript profiles
As shown in Table 1, leaves' weight was gradually decreased with dehydration time compared with the control (zero time dehydration).This weight losses indicates the decline in the water content of experimented leaves by 15.79, 26.36 and 34.5% for 2, 4, 6 h dehydration respectively.These results are in agreement with Ozturk et al. (2002) who found that the water content of barley declined by 10% within the initial 4 h, and then more rapidly by 30% (6 h) and 36% at 10 h of stress.Xue et al. (2008) reported that the change in relative leaf water content (LWC) of different genotypes indicated their different susceptibility to water scarcity.The dehydration treatment provides reliable, fast and easy way to detect genes responsible to abiotic stress response in physiological term (Talame et al., 2006).

Molecular cloning of TPP fragment
The RT-PCR reaction produced by TPP1 gene fragment with a length of ≈ 1000 bp is shown in Figure 1.The amplified TPP cDNA fragment was ligated into the pGEM-T easy vector (3015 bp) and transformed in Escherichia coli competent cells.The cloned TPP1 fragment was screened using T7 and SP6 primerd.
Positive colonies having the insert displayed a band about ≈ 1300 bp (TPP fragment with a length of ≈ 1000 bp linked to the region between Sp6 and T7 in native pGEM-T easy plasmid ≈ 300 bp) (Figure 2).

Sequence analysis of trehalose-6-phosphate phosphatase (TPP) fragment
The isolated fragment was sequenced using ABI PRISM. Figure 3 shows the sequence obtained for the TPP fragment.Sequencing of the isolated fragment revealed that the length of Trehalose-6-phosphate phosphatase (TPP) fragment was 945 bp.The obtained sequence was subjected to the BLASTx analysis which proves that the sequence has different degrees of similarity with other TPP genes.The TPP fragment showed similarity to the TPP genes from O. sativa, (EU559275.1)88%, A. thaliana (AY093147.1)66% and Z. mays (NM_001158750.1)66%.

Isolation, cloning and characterization of the fulllength TPP gene
first strand of cDNA was synthesized according to Frohmann (1994) from total RNA using a gene specific cDNA primer SP2, (5'GGACGAACCTCTAAAACCATTC3').The terminal transferase was used to add a homopolymeric A-tail to the 3' end of the cDNA.Since eukaryotic coding sequences and 5'untranslated RNA regions tend to be biased toward G/C residues, the use of a poly (A)-tail decreases the likelihood of inappropriate truncation by the Oligo dT-anchor primer.Additionally, poly(A)-tail was used due to the weaker A/T binding than G/C binding, therefore longer stretches of A residues were required before the Oligo dT-anchor primer will bind to an internal site and truncate the amplification product.As shown in Figure 4, the full length of TPP gene that was obtained by RACE PCR was ≈1500 bp.PCR product was ligated into PGEM-T Easy Vector and then transformed into E. coli.The recombinant plasmid was digested by EcoR1 res-  triction enzyme to release the cloned gene.Two bands were obtained as a result of the digestion reaction of the recombinant plasmid.One was around 3000 bp representing the vector (3015 bp) and the other was around 1500 bp representing the insert (Figure 5).
The TPP gene(s) were isolated before in several studies.Shima et al. (2007) isolated OsTPP1 and OsTPP2 representing the two major trehalose-6-phosphate phosphatase genes expressed in rice, and they found that the rice genome contains nine TPP genes.The OsTPP2 gene encodes a 42.6 kDa protein (382 amino acid residues).The same results were obtained by Pramanik and Imai (2005).They found nine putative TPP genes in the rice genome sequence.In Arabidopsis, 11 TPS and 10 TPP genes have been identified (Leyman et al., 2001;Eastmond and Graham, 2003) The isolated full length gene was sequenced using ABI PRISM (310 Genetic Analyzer); the sequence data is shown in Figure 6.This sequence was utilized to run a homology search using blast tool provided by NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi)(Altschul et al., 1997).The results of the homology research revealed that the isolated gene displayed different degrees of similarities to other TPP genes.The isolated durum wheat TPP showed similarity with the O. sativa, (AB120515.1)TPP by 93%, A. thaliana, AY059840.1 by 68%, Z. mays (NM_001158750.1) by 76%.
According to the open reading frame of the isolated gene, the length of the protein that was expressed from Alignment of the predicted amino acid sequence of TPP with proteins from other species identified several conserved regions are as shown in Figure 7. Pramanik and Imai (2005) reported that the alignment of the OsTPP1protien sequence with other TPP gene products from Saccharomyces cerevisae (ScTPS2), E. coli (EcOtsB) and Arabidopsis (AtTPPA and AtTPPB), revealed that TPP sequences are moderately conserved with exception in the N-terminal region.The two distinct phosphatase boxes that are unique features of phosphatases are highly conserved (Vogel et al., 1998).Eastmond et al. (2002) investigated the TPP and TPS multigene family in plant sequences and suggested that trehalose biosynthesis is highly regulated by environmental changes in plants.Van Dijck et al. (2002) reported that, all TPS proteins in plants contain a conserved Nterminal extension that not found in fungal or bacterial TPS proteins.To determine the evolutionary relatedness of TPP protein to Trehalose 6 phosphate phosphatase proteins isolated from other species, the neighbor joining method (NJ) was used to generate a phylogenic tree based on amino acid sequence homology.The tree showed that TPP protein forms a distinct clad on phylogenetic trees derived from various TPP sequences (Figure 8).Bootstrap analysis placed the durum wheat (Triticum durum) sequence close to O. sativa with a high degree of confidence, demonstrating that the two species descent from common ancestor.

Protein sequence analysis homology modeling
The results of BLAST search against PDB program exhibited a high level of sequence similarity to the crystallized structure for modeling TPP protien (Figure 9).This protein structure is very important to study the mode of action of disaccharides, like trehalose that appear to be one of the most effective stabilizers for dried enzymes  and cell membranes in vitro and in vivo.However, the interaction of trehalose with biological membranes has been studied more than its interactions with other proteins.Carpenter (1993) has reported that trehalose might interact with dry protein by hydrogen bonding to the polar amino acid residues in the protein.On the other hand, the interaction between trehalose and biological membranes indicates that trehalose can replace H 2 O molecules around the polar head groups of the phospholipid in the dry state (Gaber et al., 1986).This hypothesis has been studied by Potts (1994) where trehalose binds to dry phospholipid vesicles.During desiccation, the interaction of trehalose with the biological membrane decreases the melting temperature (Tm) of the membrane to keep its liquid crystalline phase (Crowe et al., 1993).This molding could be used to predict the interaction between the trehalose and other protein candidates in the biological membranes for more understanding of the trehalose mode of action for protecting plants against abiotic stress conditions.

Expression analysis
The results obtained in this work indicate that the expression of the TPP gene was up-regulated under dehydration stress compared to control (Figure 10).The highest expression level of TPP gene under dehydration stress was at 4 h.This up-regulation is important for the synthesis and accumulation of trehalose where, trehalose   is accumulated in large quantities under abiotic stresses (Elbein et al., 2003 andWolf et al., 2003).Reserve transcription combined with the polymerase chain reaction (RT-PCR) has proven to be a powerful method to quantify gene expression according to Murphy et al. (1990).Real-time PCR technology has been adapted to perform quantitative RT-PCR (Heid et al., 1996).The results of the real time PCR of the TPP gene showed that the expression level of TPP was slightly increased (up-regulated) after 4 h of dehydration treatment in leaves of Durum wheat compared with the control (0 h) and the relative fold change calculated by ∆∆CT method, respectively which in agreement with the semi-quantitative PCR results (Figures 11 and 12).The plant's response to dehydration is accompanied by the activation of a group of genes, which are responsible for regulatory proteins that further regulate the transduction of the stress signal and modulate gene expression (Shinozaki and Yamaguchi, 2006).Higo et al. (2006) studied the expression of trehalose gene synthesis (mts and mth, encoding maltooligosyl trehalose synthase and hydrolase) and trehalose hydrolysis (treH) in Anabaena sp.The genes (mts and mth) were up-regulated markedly upon dehydration.Gene disruption of mth resulted in a decrease in the trehalose level and in tolerance during dehydration stress.In contrast, gene disruption of treH resulted in an increase in both the amount of trehalose and tolerance.Trehalose did not stabilize proteins and membranes directly during dehydration; the expression of the two genes, one of which encodes a cofactor of a chaperone DnaK, correlated with trehalose content, a chaperone system induced by trehalose is important for the dehydration tolerance of Anabaena sp.Cumino et al. (2002) found that many other genes, including spsA, encoding sucrose-6-phosphate synthase were up-regulated constantly during dehydration stress.Many genes related to hotosynthesis and ribosomal protein was down-regulated in the early dehydration phase, whereas genes for nitrogen fixation and photosynthesis I was down-regulated in the late dehydration phase.

Determination of trehalase activity under dehydration stress
As shown in Table 2, the activity of trehalase enzyme under dehydration stress was decreased from 1.004 to 0.781 after 2 h and to 0.427 after 4 h.The activity was then elevated after 6 h of dehydration treatment compared to the control (Figure 13).The elevation of trehalse activity after 6 h might be due to internal regulation mechanism in the system biology of the plant to prevent the uncontrolled increase of the trehalose which is important to prevent detrimental effects of trehalose accumulation on the regulation of carbon metabolism (Brodmann, 2002).These results are in agreement with Brodmann (2002) who showed that trehalase activity normally keeps cellular trehalose concentrations low in order to prevent detrimental effects of trehalose accumulation on the regulation of carbon metabolism.The role of trehalase may be of particular importance in interactions of plants with trehalose-producing microorganisms.In support of this hypothesis, expression of the Arabidopsis trehalase gene and trehalose activity were found to be strongly induced by infection of Arabidopsis plants with the trehalose-producing pathogen Plasmodiophora brassicae.Penna (2003) found that trehalose was thought to protect biomolecules from environmental stress, as suggested by its reversible water-absorption capacity to protect biological molecules from desiccationinduced damage.The low levels of trehalose in transgenic plants can be explained by specific trehalase activity, which degrades trehalose; hence, it might be possible to increase trehalose accumulation by down regulating trehalase activity.El-Bashiti et al. (2005) found that trehalase activity in different wheat cultivar was increased under control conditions in both root and shoot of Bolal cultivar compared with salt and drought stress treatments.However, under drought conditions, there was no significant change in trehalase activity of shoot tissues.Trehalase is ubiquitous in higher plants and   single-copy trehalase genes have been identified and functionally characterized from soybean (Glycine max) and Arabidopsis (Aeschbacher et al., 1999;Müller et al., 2001).It is likely that trehalase is the sole route of trehalose breakdown in plants (Müller et al., 2001).Katoh et al. (2004) concluded that although TPS catalyses the transfer of glucose from UDP-glucose to glucose 6phosphate to produce trehalose 6-phosphate and UDP, and TPP catalyses the dephosphorylation of trehalose 6phosphate to trehalose.The low level of accumulation of trehalose may be attributed to the unique gene structure for trehalose metabolism.They investigated the expression of genes for trehalose synthesis, mth (maltooligosyl trehalose hydrolase) and mts (maltooligosyl trehalose synthase), as well as that for trehalose degradation, treH (trehalase), exhibited marked increase upon dehydration.So trehalose did not accumulate so much.

In silico and comparative mapping
Comparative maps can be used to study genome evolu-tion; how the genome has been rearranged through time, and to make inferences about gene organization (Liang et al., 2008).In-silico mapping indicated the matching of the TPP gene sequence with the sequence of rice TPP on chromosome 8 linked to the locus (AQ074215).
Comparative mapping showed that the rice AQ074215 locus on chromosome 8 was also mapped on barley chromosome 5H.The marker (Iwgsc) on bread wheat chromosome 3B was found to be closely liked to the rice locus (AQ074215) on chromosome 8.The results obtained from the comparative mapping showed that the isolated TPP gene is localized on rice chromosome 8, durum wheat chromosome 20, bread wheat chromosome 3B, oat linkage group E, sorghum chromosome 4 and barley 5H (Figures 14 to 16).This work utilizes a comparative analysis of durum and bread wheat, barley, oat, sorghum and rice based on linkage maps and consensus markers across the genome with the goal of linking the complex wheat genome to simpler diploid species such as barley and rice that serve as references.However, more detailed comparisons are needed to veri- Figure (1): Agarose gel shows; (M) 1kb marker, (-ve) negative control, and (TPP) candidate band for TPP fragment.

Figure 14 .
Figure 14.Comparative map showing the locus AQ074215 on Ric e chromosome 8 that is closely linked to other locus on Oat and Tetraploid wheat.

Figure 15 .
Figure 15.Comparative map showing the locus AQ074215 on rice chromosome 8 and maize and sorghum.

Figure 16 .
Figure 16.Comparative map showing the locus AQ074215 on Rice chromosome 8. and its linkage to barley and bread wheat.

Table 1 .
Leaves' weight of durum wheat under dehydration shock treatment.