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Ashoub A.,Biodiversity and Climate Research Center | Ashoub A.,Agricultural Genetic Engineering Research Institute AGERI | Berberich T.,Biodiversity and Climate Research Center | Beckhaus T.,Goethe University Frankfurt | Bruggemann W.,Goethe University Frankfurt
Electrophoresis | Year: 2011

The efficient extraction of high-quality proteins is a key factor for a successful proteomic analysis approach. In the method suggested here, absolute ethanol containing 10mM DTT was used to precipitate the proteins in plant tissue homogenates followed by their resuspension in a urea-/thiourea- and NP-40-containing solution. Protein profiles were examined on pH 3-11 non-linear IEF strips and SDS-PAGE and compared with extracts using the established method of acetone-10% TCA/0.07% 2-mercaptoethanol precipitation (V. Méchin et al., Methods Mol. Biol. 2006, 355, 1-8). In addition to protein profile similarity for the two extracts, the acidic part of the acetone containing 10% TCA/0.07% 2-mercaptoethanol extraction showed protein spots with high molecular weight in the range of 250-150kDa, while the ethanol containing 10mM DTT extracts indicated extra proteins spots at the basic part of the gels with molecular weights in the range of 25-15kDa. The MALDI-TOF-MS of differential spots from acetone containing 10% TCA/0.07% 2-mercaptoethanol precipitation method and absolute ethanol containing 10mM DTT indicated no similarity, ruling out the possibility that the two clusters shown represent identical proteins. The described method is easy in implementation, chemicals used are less toxic and proteins are easier to resuspend therefore presents an additional choice to implement towards finding the optimum method for extraction. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Jedmowski C.,Goethe University Frankfurt | Jedmowski C.,Biodiversity and Climate Research Center | Ashoub A.,Biodiversity and Climate Research Center | Ashoub A.,Agricultural Genetic Engineering Research Institute AGERI | And 2 more authors.
Acta Physiologiae Plantarum | Year: 2013

Landraces of barley and of Sorghum bicolor from Egypt were evaluated for their tolerance to drought stress (DS) using the OJIP test of the chl fluorescence fast induction curve. Water was withheld from 4-week-old, pot-grown plants for 8-10 days, until the volumetric soil water content decreased from 30 to below 5 vol% and the leaves reached relative water contents of >60 %. The plants were rewatered and recovery measurements were taken 24 h later. Comparative studies of the most sensitive and the most tolerant lines of both cereals, as evaluated by their Performance Indices (PIabs), revealed a similar behavior in the sensitive lines, i. e., inhibiting effects of DS on PS II connectivity (occurrence of an L band), oxygen evolving complex (occurrence of a K band) and on the J step of the induction curves, associated with an inhibition of electron transport from QA to QB. These effects persisted or were even enhanced in the rewatered plants, which resulted in similar deviations of spider plots of the OJIP parameters in the sensitive lines of both species. In the most tolerant barley accession, drought effects on "early" events (i. e., L, K bands) were much smaller or negligible, and there was no pronounced effect on the J step. However, distinct increases of the I step occurred, pointing to inhibited electron flow to the intersystem electron carriers and beyond PS I. The most tolerant Sorghum line, in contrast, revealed nearly no effects of the DS and recovery treatment on the fluorescence induction curves and OJIP parameters. © 2012 Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków.

Ashoub A.,Biodiversity and Climate Research Center | Ashoub A.,Agricultural Genetic Engineering Research Institute AGERI | Ashoub A.,Goethe University Frankfurt | Beckhaus T.,Goethe University Frankfurt | And 4 more authors.
Planta | Year: 2013

The adaptive response of Egyptian barley land races to drought stress was analyzed using difference gel electrophoresis (DIGE). Physiological measurements and proteome alterations of accession number 15141, drought tolerant, and accession number 15163, drought sensitive, were compared. Differentially expressed proteins were subjected to MALDI-TOF-MS analysis. Alterations in proteins related to the energy balance and chaperons were the most characteristic features to explain the differences between the drought-tolerant and the drought-sensitive accessions. Further alterations in the levels of proteins involved in metabolism, transcription and protein synthesis are also indicated. © 2012 Springer-Verlag Berlin Heidelberg.

El-Menofy W.,Agricultural Genetic Engineering Research Institute AGERI | Osman G.,Agricultural Genetic Engineering Research Institute AGERI | Osman G.,University of Umm Al - Qura | Assaeedi A.,University of Umm Al - Qura | Salama M.,Ain Shams University
Biological Procedures Online | Year: 2014

Baculoviruses have been genetically modified to express foreign genes under powerful promoters in order to accelerate their speed of killing. In this study a truncated form of cry1Ab gene derived from Bacillus thuringinsis (Bt) subsp. aegypti isolate Bt7 was engineered into the genome of the baculovirus Autographa californica multiple nuclearpolyhedrosis wild type virus, in place of the polyhedrin gene by using homologous recombination in Spodoptera frugiperda (Sf) cells between a transfer vector carrying the Bt gene and the wild type virus linearized DNA. Recombinant wild type virus containing the cry1Ab gene was detected as blue occlusion-negative plaques in monolayers of Sf cells grown in the presence of X-Gal. In Sf cells infected with plaque-purified recombinant virus, the cry1Ab gene was expressed to yield a protein of approximately 82-kDa, as determined by immunoblot analysis. The toxicity of the recombinant virus expressing the insecticidal crystal protein (ICP) was compared to that of the wild-type virus. Infected-cell extract was toxic to cotton leaf worm Spodoptera littoralis second instar larvae and the estimated LC50 was 1.7 μg/ml for the recombinant virus compared with that of wild-type virus which was 10 μg/ml. © 2014 El-menofy et al.; licensee BioMed Central Ltd.

Reyes L.H.,Texas A&M University | Abdelaal A.S.,Agricultural Genetic Engineering Research Institute AGERI | Kao K.C.,Texas A&M University
Applied and Environmental Microbiology | Year: 2013

Cross-tolerance and antagonistic pleiotropy have been observed between different complex phenotypes in microbial systems. These relationships between adaptive landscapes are important for the design of industrially relevant strains, which are generally subjected to multiple stressors. In our previous work, we evolved Escherichia coli for enhanced tolerance to the biofuel n-butanol and discovered a molecular mechanism of n-butanol tolerance that also conferred tolerance to the cationic antimicrobial peptide polymyxin B in one specific lineage (green fluorescent protein [GFP] labeled) in the evolved population. In this work, we aim to identify additional mechanisms of n-butanol tolerance in an independent lineage (yellow fluorescent protein [YFP] labeled) from the same evolved population and to further explore potential cross-tolerance and antagonistic pleiotropy between n-butanol tolerance and other industrially relevant stressors. Analysis of the transcriptome data of the YFP-labeled mutants allowed us to discover additional membrane-related and osmotic stress-related genes that confer n-butanol tolerance in E. coli. Interestingly, the n-butanol resistance mechanisms conferred by the membrane-related genes appear to be specific to n-butanol and are in many cases antagonistic with isobutanol and ethanol. Furthermore, the YFP-labeled mutants showed cross-tolerance between n-butanol and osmotic stress, while the GFP-labeled mutants showed antagonistic pleiotropy between n-butanol and osmotic stress tolerance. © 2013, American Society for Microbiology.

Low-stringency single specific primer polymerase chain reaction (LSSP)-PCR was assessed for its suitability in detecting the genotoxic effect of paranitrophenol (PNP) in the dwarf bean (Phaseolus vulgaris) exposed to different concentrations of PNP. DNA was extracted from both PNP-treated and non-treated shoots that was amplified by specific PCR, using universal primers of maturase K chloroplast DNA. PCR products of approximately 776 bp were subsequently used as a template for LSSP-PCR analysis. We detected the genotoxic effect based on LSSP-PCR profiles of the DNA generated in PNP-treated over the non-treated control of bean shoots. A complex electrophoretic pattern consisting of many bands was obtained from control and treated samples. Surprisingly, DNA sequencing data revealed that the homology among the maturase gene amplified from PNP-treated vs. non-treated samples of dwarf beans are comparable. These results showed that the use of LSSP-PCR analysis is not a proper tool to detect genotoxic effect in bean, at least in bean shoots that were exposed to PNP.

Hussein G.M.,Agricultural Genetic Engineering Research Institute AGERI
GM crops & food | Year: 2013

Low transformation efficiency and long generation time for production of transgenic Gerbera jemosonii plants leads to vulnerable gene function studies. Thus, transient expression of genes would be an efficient alternative. In this investigation, a transient expression system for gerbera petals based on the Agrobacterium infiltration protocol was developed using the reporter genes β-glucuronidase (gus) and green florescence protein (gfp). Results revealed the incapability of using the gfp gene as a reporter gene for transient expression study in gerbera flowers due to the detection of green fluorescent color in the non-infiltrated gerbera flower petals. However, the gus reporter gene was successfully utilized for optimizing and obtaining the suitable agroinfiltration system in gerbera flowers. The expression of GUS was detectable after three days of agroinfiltration in gerbera cultivars "Express" and "White Grizzly" with dark pink and white flower colors, respectively. The vacuum agroinfiltration protocol has been applied on the cultivar "Express" for evaluating the transient expression of the two genes involved in the anthocyanin pathway (iris-dfr and petunia-f3' 5'h), which is responsible for the color in flowers. In comparison to the control, transient expression results showed change in the anthocyanin pigment in all infiltrated flowers with color genes. Additionally, blue color was detected in the stigma and pollen grains in the infiltrated flowers. Moreover, blue colors with variant intensities were observed in produced calli during the routine work of stable transformation with f3' 5'h gene.

Ismail R.M.,Agricultural Genetic Engineering Research Institute AGERI
GM crops & food | Year: 2013

Five genetically modified insect resistant sugarcane lines harboring the Bt Cry 1AC gene to produce insecticidal proteins were compared with non-transgenic control by using three types of molecular marker techniques namely, RAPD, ISSR and AFLP. These techniques were applied on transgenic and non-transgenic plants to investigate the genetic variations, which may appear in sugarcane clones. This variation might demonstrate the genomic changes associated with the transformation process, which could change important molecular basis of various biological phenomena. Genetic variations were screened using 22 different RAPD primers, 10 ISSR primers and 13 AFLP primer combinations. Analysis of RAPD and ISSR banding patterns gave no exclusive evidence for genetic variations. Meanwhile, the percentage of polymorphic bands was 0.45% in each of RAPD and ISSR, while the polymorphism generated by AFLP analysis was 1.8%. The maximum percentage of polymorphic bands was 1.4%, 1.1% and 5.5% in RAPD, ISSR and AFLP, respectively. These results demonstrate that most transgenic lines showed genomic homogeneity and verified minor genomic changes. Dendrograms revealing the relationships among the transgenic and control plants were developed from the data of each of the three marker types.

Adawy S.S.,Agricultural Genetic Engineering Research Institute AGERI
GM crops & food | Year: 2013

Cotton is the world's leading natural fiber and second most important oilseed crop and has been a focus of genetic, systematic and breeding research. The genetic and physiological bases of some important agronomic traits in cotton were investigated by QTL mapping through constructing of genetic map with chromosomal assignment. A segregating F2 population derived from an interspecific cross (G. barbadense x G. hirsutum) between two genotypes, cvs. "Giza 83" and "Deltapine" was used in this study. Different molecular markers including SSR, EST, EST-SSR, AFLP and RAPD were employed to identify markers that reveal differences between the parents. In total 42 new markers were merged with 140 previously mapped markers to produce a new map with 182 loci covering a total length of 2370.5 cM. Among these new markers, some of them were used to assign chromosomes to the produced 26 linkage groups. The LG2, LG3, LG11 and LG26 were assigned to chromosomes 1, 6, 5 and 20 respectively. Single point analysis was used to identify genomic regions controlling traits for plant height, number of nodes at flowering time, bolling date, days to flowering and number of bolls. In total 40 significant QTL were identified for the five traits on 11 linkage groups (1, 2, 3, 4, 5, 10, 11, 12, 18, 19 and 23). This work represents an improvement of the previously constructed genetic map in addition to chromosomal assignment and detection of new significant QTL for the five traits in Egyptian cotton. The Significant QTLs detected in this study can be employed in marker assisted selection for molecular breeding programs aiming at developing cotton cultivars with improved agronomic traits.

Mahfouz M.M.,King Abdullah University of Science and Technology | Mahfouz M.M.,Agricultural Genetic Engineering research institute AGERI
Plant Signaling and Behavior | Year: 2010

Epigenetic RNA based gene silencing mechanisms play a major role in genome stability and control of gene expression. Transcriptional gene silencing via RNA-directed DNA methylation (RdDM) guides the epigenetic regulation of the genome in response to disease states, growth, developmental and stress signals. RdDM machinery is composed of proteins that produce and modify 24-nt-long sirNAs, recruit the RdDM complex to genomic targets, methylate DNA and remodel chromatin. The final DNA methylation pattern is determined by either DNA methyltransferase alone or by the combined action of DNA methyltransferases and demethylases. The dynamic interaction between RdDM and demethylases may render the plant epigenome plastic to growth, developmental and environmental cues. The epigenome plasticity may allow the plant genome to assume many epigenomes and to have the right epigenome at the right time in response to intracellular or extracellular stimuli. This review discusses recent advances in RdDM research and considers future perspectives. © 2010 Landes Bioscience.

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