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1.
Major proteins of the holm oak leaf proteome have been previously identified using a combination of 2-DE, MS analysis and BLAST similarity search (Jorge et al., Proteomics 2005, 5, 222-234). That study, conducted with field samples from mature trees, revealed the existence of a great variability in the 2-DE protein map, with qualitative as well as quantitative changes, both analytical and biological. A similar study has been carried out with 2-year-old seedlings to analyze and study: (i) changes in the 2-DE protein profile at different tree developmental stages; (ii) the 2-DE protein map variability between three different Spanish provenances; and (iii) variations in the 2-DE protein profile in response to drought stress. Although the protein profile of leaves from seedlings and mature trees was fairly similar, the biological variance found was lower in the former. In the present study, new proteins have been identified. At least four different protein spots differentiated Spanish provenances, two of them identified as an ATP synthase alpha chain, and a 2,3-bisphosphoglycerate-independent phosphoglycerate mutase. Fourteen different protein spots were qualitatively variable between well-watered and drought-stressed seedlings, with some of them corresponding to enzymes of carbohydrate and protein metabolism. Data presented indicated the mobilization of storage proteins and carbohydrates, as well as photosynthesis inhibition under drought conditions. 相似文献
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《植物学报(英文版)》2010,52(1)
An efficient protein extraction method is crucial to ensure successful separation by two-dimensional electrophoresis(2-DE)for recalcitrant plant species, in particular for grapevine(Vitis vinifera L.). Trichloroacetic acid-acetone(TCA-acetone)and phenol extraction methods were evaluated for proteome analysis of leaves and roots from the Tunisian cultivar 'Razegui'. The phenol-based protocol proved to give a higher protein yield,a greater spot resolution, and a minimal streaking on 2-DE gels for both leaf and root tissues compared with the TCA-based protocol. Furthermore, the highest numbers of detected proteins on 2-DE gels were observed using the phenol extraction from leaves and roots as compared with TCA-acetone extraction. 相似文献
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Evaluation of protein extraction methods for Vitis vinifera leaf and root proteome analysis by two-dimensional electrophoresis 总被引:1,自引:0,他引:1
An efficient protein extraction method is crucial to ensure successful separation by two-dimensional electrophoresis(2-DE)for recalcitrant plant species, in particular for grapevine(Vitis vinifera L.). Trichloroacetic acid-acetone(TCA-acetone)and phenol extraction methods were evaluated for proteome analysis of leaves and roots from the Tunisian cultivar 'Razegui'. The phenol-based protocol proved to give a higher protein yield,a greater spot resolution, and a minimal streaking on 2-DE gels for both leaf and root tissues compared with the TCA-based protocol. Furthermore, the highest numbers of detected proteins on 2-DE gels were observed using the phenol extraction from leaves and roots as compared with TCA-acetone extraction. 相似文献
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To understand the mechanisms of heat stress responses in perennial grasses, differential proteins in leaves and roots of two genotypes of Kentucky bluegrass (Poa pratensis), including heat-tolerant ‘Midnight’ and heat-sensitive ‘Brilliant’, were analyzed with two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS). Plants were exposed to heat stress for 28 days in growth chambers. Under 7–28 days of heat stress, leaf photochemical efficiency declined significantly while electrolyte leakage increased in leaves and roots, and to a lesser extent for heat-tolerant ‘Midnight’ than for heat-sensitive ‘Brilliant’. Compared with leaves, cell membrane damage due to heat stress was more severe in roots. The 2-DE and MS analysis identified 37 heat-responsive proteins in leaves, 28 heat-responsive proteins in roots; 14 proteins in leaves and 9 proteins in roots exhibited differential expression between the two genotypes. The results indicate that proteins involved in metabolism and energy in leaves and those in antioxidant defense in roots are associated with heat tolerance in Kentucky bluegrass. The differential accumulation of these proteins might be the reason for different heat tolerance in two Kentucky bluegrass genotypes in aerial and underground parts. 相似文献
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A reference database of the major soluble proteins of the primary root of the maize inbred line B73 was generated 5 days after germination (DAG) using a combination of 2-DE and MALDI-TOF MS. A total of 302 protein spots were detected with CBB in a pH 4-7 range and 81 proteins representing 74 distinct Genbank accessions were identified. Only 28% of the major proteins identified in 5 DAG primary roots were identified in similarly analyzed 9 DAG primary roots documenting remarkable changes in the accumulation of abundant soluble proteins early in primary root development. 相似文献
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Protein pattern changes in tomato under in vitro salt stress 总被引:2,自引:0,他引:2
F. Amini A. A. Ehsanpour Q. T. Hoang J. Sh. Shin 《Russian Journal of Plant Physiology》2007,54(4):464-471
The investigation of salt-induced changes in the proteome would highlight important genes because of a high resolution of
protein separation by two-dimensional gel electrophoresis (2-DE) and protein identification by mass spectrometry and database
search. Tomato (Lycopersicon esculentum Mill.) is a model plant for studying the mechanisms of plant salt tolerance. Seeds of tomato cv. Shirazy were germinated
on water-agar medium. After germination, seedlings were transferred to Murashige and Skoog nutrient medium supplemented with
0, 40, 80, 120, and 160 mM NaCl. After 24 days, leaf and root samples were collected for protein extraction and shoot dry
weight measurement. Alterations induced in leaf and root proteins under salt stress treatments were studied by one-dimensional
SDS-PAGE. Leaf proteins were also analyzed by 2-DE. With increasing salt concentration in the medium, shoot dry weight decreased.
SDS-PAGE showed induction of at least five proteins with mol wts of 30, 62, and 75 kD in roots and 38 and 46 kD in leaves.
On the 2-DE gel, more than 400 protein spots were reproducibly detected. At least 18 spots showed significant changes under
salt stress. Three of them corresponded to new proteins, while six proteins were up-regulated and five proteins were down-regulated
by salt stress. In addition, salinity inhibited the synthesis of four leaf proteins. Ten spots were analyzed by matrix-assistant
laser desorption/ionization-time of flight (MALDI-TOF), which led to the identification of some proteins, which could play
a physiological role under salt stress. The expression of new proteins(enoyl-CoA hydratase, EGF receptor-like protein, salt
tolerance protein, phosphoglycerate mutase-like protein, and M2D3.3 protein) under salt stress indicates that tomato leaf
cells respond to salt stress by changes in different physiological processes. All identified proteins are somehow related
to various salt stress responses, such as cell proliferation.
Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 4, pp. 526–533.
The text was submitted by the authors in English. 相似文献
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Black spot disease in poplar is a disease of the leaf caused by fungus. The major pathogen is Marssonina brunnea f. sp. multigermtubi. To date, little is known about the molecular mechanism of poplar (M. brunnea) interaction. In order to identify the proteins related to disease resistance and understand its molecular basis, the clone "NL895" (P. euramericana CL"NL895"), which is highly resistant to M. brunnea f. sp. multigermtubi, was used in this study. We used two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) to identify the proteins in poplar leaves that were differentially expressed in response to black spot disease pathogen, M. brunnea f. sp. multigermtubi. Proteins extracted from poplar leaves at 0, 12, 24, 48, and 72 h after pathogen-inoculation were separated by 2-DE, About 500 reproducible protein spots were detected, of which 40 protein spots displayed differential expression in levels and were subjected to Matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) followed by database searching. According to the function, the identified proteins were sorted into five categories, that is, protein synthesis, metabolism, defense response and unclassified proteins. 相似文献
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Knowledge of stress-responsive proteins is critical for further understanding the molecular mechanisms of stress tolerance.
The objectives of this study were to establish a proteomic map for a perennial grass species, creeping bentgrass (A. stolonifera L.), and to identify differentially expressed, salt-responsive proteins in two cultivars differing in salinity tolerance.
Plants of two cultivars (‘Penncross’ and ‘Penn-A4’) were irrigated daily with water (control) or NaCl solution to induce salinity
stress in a growth chamber. Salinity stress was obtained by adding NaCl solution of 2, 4, 6, and 8 dS m−1 in the soil daily for 2-day intervals at each concentration, and then by watering soil with 10 dS m−1 solution daily for 28 days. For proteomic map, using two-dimensional electrophoresis (2-DE), approximately 420 and 300 protein
spots were detected in leaves and roots, respectively. A total of 148 leaf protein spots and 40 root protein spots were excised
from the 2-DE gels and subjected to mass spectrometry analysis. In total, 106 leaf protein spots and 24 root protein spots
were successfully identified. Leaves had more salt-responsive proteins than roots in both cultivars. The superior salt tolerance
in ‘Penn-A4’, indicated by shoot extension rate, relative water content, and cell membrane stability during the 28-day salinity
stress could be mainly associated with its higher level of vacuolar H+-ATPase in roots and UDP-sulfoquinovose synthase, methionine synthase, and glucan exohydrolase in leaves, as well as increased
accumulation of catalase and glutathione S-transferase in leaves. Our results suggest that salinity tolerance in creeping bentgrass could be in part controlled by an
alteration of ion transport through vacuolar H+-ATPase in roots, maintenance of the functionality and integrity of thylakoid membranes, sustained polyamine biosynthesis,
and by the activation of cell wall loosening proteins and antioxidant defense mechanisms. 相似文献
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Besma Sghaier-Hammami Inmaculada Redondo-López Ana M. Maldonado-Alconada Sira Echevarría-Zomeño Jesús V. Jorrín-Novo 《Acta Physiologiae Plantarum》2012,34(3):905-922
The present work is directed at studying changes at the proteome level in Arabidopsis thaliana leaves in response to Pseudomonas syringae virulent (Pst) and avirulent (Pst avrRpt2) strains. Arabidopsis leaves were sampled from challenged plants at 4, 8 and 24 h post inoculation. Proteins were TCA–acetone–phenol
extracted and subjected to 2-DE (5–8 pH range) and MS/MS (MALDI–TOF–TOF) analysis. Out of 800 matched spots on each of the
36 gels analysed, 147 spots were either absent in at least one of the conditions studied (time or treatments; qualitative
variable spots) or differentially accumulated between time and treatments (quantitative variable spots). Out of the 24 proteins
successfully identified over TAIR10 database, 23 have not been reported previously in similar proteomics studies of the Arabidopsis thaliana–Pseudomonas syringae interaction. The exhaustive statistical analysis performed, including principal component and heat map, showed that 24 h
post inoculation can clearly discriminate the challenged plants from the control. The protein change occurred early (4 h post
inoculation) following the virulent pathogen infection, whereas the change occurred later (24 h post inoculation) following
the avirulent pathogen inoculation. Concerning the variable proteins, three behavioural groups can be observed: group 1 (common
protein changes in response to virulent and avirulent pathogen infection), group 2 (protein changes in response to virulent
pathogen infection) and group 3 (protein changes in response to avirulent pathogen infection). Differential identified proteins
following the pathogen infection belonged to different groups including those of oxidative stress defence, enzymes of metabolic
pathways and molecular chaperones. 相似文献
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Petrak J Ivanek R Toman O Cmejla R Cmejlova J Vyoral D Zivny J Vulpe CD 《Proteomics》2008,8(9):1744-1749
After reading many 2-DE-based articles featuring lists of the differentially expressed proteins, one starts experiencing a disturbing déjà vu. The same proteins seem to predominate regardless of the experiment, tissue or species. To quantify the occurrence of individual differentially expressed proteins in 2-DE experiment reports, we compiled the identities of differentially expressed proteins identified in human, mouse, and rat tissues published in three recent volumes of Proteomics and calculated the appearance of the most predominant proteins in the dataset. The most frequently identified protein is a highly abundant glycolytic enzyme enolase 1, differentially expressed in nearly every third experiment on both human and rodent tissues. Heat-shock protein 27 (HSP27) and heat-shock protein 60 (HSP60) were differentially expressed in about 30 percent of human and rodent samples, respectively. Considering protein families as units, keratins and peroxiredoxins are the most frequently identified molecules, with at least one member of the group being differentially expressed in about 40 percent of all experiments. We suggest that the frequent identification of these proteins must be considered in the interpretation of any 2-DE studies. We consider if these commonly observed changes represent common cellular stress responses or are a reflection of the technical limitations of 2-DE. 相似文献
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This study describes the identification of outer membrane proteins (OMPs) of the bacterial pathogen Pasteurella multocida and an analysis of how the expression of these proteins changes during infection of the natural host. We analysed the sarcosine-insoluble membrane fractions, which are highly enriched for OMPs, from bacteria grown under a range of conditions. Initially, the OMP-containing fractions were resolved by 2-DE and the proteins identified by MALDI-TOF MS. In addition, the OMP-containing fractions were separated by 1-D SDS-PAGE and protein identifications were made using nano LC MS/MS. Using these two methods a total of 35 proteins was identified from samples obtained from organisms grown in rich culture medium. Six of the proteins were identified only by 2-DE MALDI-TOF MS, whilst 17 proteins were identified only by 1-D LC MS/MS. We then analysed the OMPs from P. multocida which had been isolated from the bloodstream of infected chickens (a natural host) or grown in iron-depleted medium. Three proteins were found to be significantly up-regulated during growth in vivo and one of these (Pm0803) was also up-regulated during growth in iron-depleted medium. After bioinformatic analysis of the protein matches, it was predicted that over one third of the combined OMPs predicted by the bioinformatics sub-cellular localisation tools PSORTB and Proteome Analyst, had been identified during this study. This is the first comprehensive proteomic analysis of the P. multocida outer membrane and the first proteomic analysis of how a bacterial pathogen modifies its outer membrane proteome during infection. 相似文献
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Increasing efforts are being made to develop more sensitive and faster molecular methodologies at the genomic and proteomic levels for the identification of protein markers after exposure to ionizing radiation (IR). However, few specific protein markers, especially organ-specific markers, have been identified. In this study, we analyzed altered protein expressions in various tissues, namely, brain, lung, spleen, and intestine, from 1 Gy-irradiated mice by employing 2-DE analysis. MALDI-TOF MS and peptide mapping identified 25 proteins that showed greater than twofold expressional changes. In order to confirm significant differences between control and IR-treated samples, ten identified proteins with available commercial antibodies were selected for immunoblotting. Of these, only five showed protein expression patterns that were similar to 2-DE data. These were heat shock protein 5 (HSP 5), HSP 90 kDa β, HSP 1, transaldolase 1 (TA1), and phosphoglycerate kinase 1 (PGK1). In particular, PGK1 was specifically upregulated in mouse intestine, and TA1 was specifically downregulated in brain by irradiation. TA1 expression was unaltered in other tissues. Based on these data, we suggest that TA1 and PGK1 can be considered as candidate tissue-specific protein markers of IR exposure. 相似文献
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Nasal lavage fluid and proteomics as means to identify the effects of the irritating epoxy chemical dimethylbenzylamine. 总被引:3,自引:0,他引:3
The aims of this study were to describe the changes in the nasal lavage fluid (NLF) protein pattern after exposure to the irritating epoxy chemical dimethylbenzylamine (DMBA) and to identify the affected proteins using a proteomic approach. The protein patterns of NLF from six healthy subjects and eight epoxy workers with airway irritation were analysed using two-dimensional gel electrophoresis (2-DE) before and after exposure to 100 microg m(-3) DMBA for 2 h in an exposure chamber. NLF proteins were identified by (i) comparison with a 2-DE NLF reference database; (ii) N-terminal amino acid sequencing; and (iii) mass spectrometry. In NLF from healthy subjects, the levels of immunoglobulin A increased and the levels of Clara cell protein 16 (CC16) decreased after chamber exposure, while in NLF from epoxy workers, alpha(2)-macroglobulin and caeruloplasmin increased. Two previously unidentified proteins decreased in NLF from epoxy workers after exposure; these were identified as statherin and calgranulin B. In addition, the subjects who developed high counts of eosinophils in their nasal mucosa after chamber exposure had significantly lower levels of immunoglobulin-binding factor (IgBF) before exposure than subjects with low eosinophil infiltration. These results show that short-term exposure to DMBA causes distinct changes in NLF proteins. Moreover, three proteins that have previously not been associated with upper airway irritation were identified: statherin, calgranulin B and IgBF. Further studies are needed to investigate whether these proteins may be used as biomarkers of airway irritation and to give new insight into the ways in which occupational exposure to irritants causes inflammation of the airways. 相似文献
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Proteomic analysis of Citrus sinensis roots and leaves in response to long-term magnesium-deficiency
Hao-Yang Peng Yi-Ping Qi Jinwook Lee Lin-Tong Yang Peng Guo Huan-Xin Jiang Li-Song Chen 《BMC genomics》2015,16(1)