Protein gel band method for improving the identification of stem proteins
Ontology highlight
ABSTRACT:
INSTRUMENT(S): LCQ Classic
ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)
TISSUE(S): Stem
SUBMITTER: 
Shihui Yang
LAB HEAD: Hui Wei
PROVIDER: PXD001852 | Pride | 2015-05-11
REPOSITORIES: Pride
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Project description:Identifying the intracellular and cell wall-ionically bound glycoside hydrolases (GHs) carbohydrate esterases (CEs) at the late growth stage of Arabidopsis stems is important for understanding the mechanisms regulating the cell wall integrity. The fact that whole plant stems are used as biomass feedstocks and the mixing of intracellular proteins with the cell wall proteome caused by an increasing proportion of broken cells of stems at the late growth stage pose a challenge in identifying these GHs and CEs. Here, we used a CaCl2-extraction procedure to isolate non-structural proteins from Arabidopsis whole stems, followed by protein solution method for direct identification of stem proteins, and by SDS-PAGE separation and protein gel band method for improving the identification of stem proteins, using Nano-LC-MS/MS analysis. Totally, 75 and 236 stem proteins were identified by using these two methods, respectively, confirming the later method (i.e. protein gel method) identified three time more stem proteins. Among these proteins, based on cell wall protein databases and data mining analyses, 6 and 22 proteins were identified as cell wall proteins from the late growth stage Arabidopsis stems, by using these two methods respectively.
2015-05-11 | PXD001851 | Pride
Project description:Alfalfa, [Medicago sativa (L.) sativa], a widely-grown perennial forage has potential for development as a cellulosic ethanol feedstock. The application of genomic approaches would advance development of alfalfa as a cellulosic feedstock. However, the genomics of alfalfa, a non-model species, is still in its infancy. The recent advent of RNA-Seq, a massively parallel sequencing method for transcriptome analysis, provides an opportunity to expand the identification of alfalfa genes and polymorphisms, and conduct in-depth transcript profiling. Cell walls in stems of alfalfa genotype 708 have higher cellulose and lower lignin concentrations compared to cell walls in stems of genotype 773. Using the Illumina GA-II platform, a total of 198,861,304 expression sequence tags (ESTs, 76 bp in length) were generated from cDNA libraries derived from elongating stem (ES) and post-elongation stem (PES) internodes of 708 and 773. These ESTs were de novo assembled into 132,153 unique sequences. By combining the de novo assembled ESTs (132,153 sequences) with our previously identified EST sequences (341,984 sequences, unpublished data), and the ESTs available from GenBank (12,371 sequences), we built the first Alfalfa Gene Index (MSGI 1.0). MSGI 1.0 contains 124,025 unique sequences including 22,729 tentative consensus sequences (TCs), 22,315 singletons and 78,981 pseudo-singletons. We identified a total of 1, 294 simple sequence repeats (SSR) among the sequences in MSGI 1.0. In addition, a total of 10,826 single nucleotide polymorphisms (SNPs) were predicted between the two genotypes. Transcript profiling of stem internodes of genotypes 708 and 773 was conducted by quantifying the number of Illumina EST reads that were mapped to sequences in MSGI 1.0. We identified numerous candidate genes that may play a role in stem development as well as candidate genes that may contribute to the differences in cell wall composition in stems of the two genotypes. Our results demonstrate that RNA-Seq can be successfully used for gene identification, polymorphism detection and transcript profiling in alfalfa, a non-model, allogamous, autotetraploid species. The alfalfa gene index (MSGI 1.0) assembled in this study, and the SNPs, SSRs and candidate genes identified can be used to improve alfalfa as a cellulosic feedstock. Examination of 2 different tissue types at different developmental stages (Elongating vs. post-elongation stem internodes) in two alfalfa genotypes (708 and 773) with divergent cell wall composition in stems.
2011-04-21 | E-GEOD-26757 | biostudies-arrayexpress
Project description:Water soluble carbohydrates (WSC, composed of mainly fructans, sucrose, glucose and fructose) deposited in wheat stems are important carbon sources for grain filling. Variation in stem WSC concentrations among wheat genotypes is one of the genetic factors influencing grain weight and yield under water-limited environments. Here, we describe the molecular dissection of carbohydrate metabolism in stems, at the WSC accumulation phase, of recombinant inbred SB (Seri/Babax) lines of Triticum aestivum differing in stem WSC concentrations. Affymetrix GeneChip analysis of carbohydrate metabolic enzymes revealed that the mRNA levels of two fructan synthetic enzyme families (sucrose:sucrose 1-fructosyltransferase and sucrose:fructan 6-fructosyltransferase) in the stem were positively correlated with stem WSC and fructan concentrations, while the mRNA levels of enzyme families involved in sucrose hydrolysis (sucrose synthase and soluble acid invertase) were inversely correlated with WSC concentrations. Differential regulation of the mRNA levels of these sucrose hydrolytic enzymes in SB lines resulted in genotypic differences in these enzyme activities. Down-regulation of sucrose synthase and soluble acid invertase in high WSC lines was accompanied by significant decreases in the mRNA levels of enzyme families related to sugar catabolic pathways (fructokinase and mitochondrion pyruvate dehydrogenase complex) and enzyme families involved in diverting UDP-glucose to cell wall synthesis (UDP-glucose 6-dehydrogenase, UDP-glucuronate decarboxylase and cellulose synthase), resulting in a reduction in cell wall polysaccharide contents (mainly hemicellulose) in the stem of high WSC lines. These data suggest that differential carbon partitioning in the wheat stem is one mechanism that contributes to genotypic variation in WSC accumulation. We used Affymetrix GeneChip to dissect genotypic variation in carbohydrate metabolism related to water soluble carbohydrate accumulation in stems of wheat. Experiment Overall Design: 8 genotypes of recombinant inbred lines Seri M82 x Babax with 2 biological replicates per genotype. Grown in the field under rain-fed conditions
2008-10-26 | E-GEOD-9767 | biostudies-arrayexpress
Project description:The barley brittle stem mutants, fs2, designated X054 and M245, have reduced levels of cellulose compared with their isogenic parents Ohichi and Shiroseto. A custom-designed microarray, based on Agilent technology and including genes involved in cell wall metabolism, was used to compare transcript levels in the mutant and parental lines. For both mutants, the microarray revealed a marked decrease in mRNA for the HvCesA4 cellulose synthase gene in specific zones of stem internodes, and this was confirmed by quantitative PCR. Genes expression was measured for the upper and lower zones from the 4th internodes of stems. Plant were at Zadocks developmental stage 49. Gene expression was compared between mutants and their wildtype parents.
2010-07-23 | E-GEOD-17873 | biostudies-arrayexpress
Project description:Stem internodes of grasses function in mechanical support, transport, and, in some species are a major sink organ for carbon in the form of cell wall polymers. To establish the rice elongating internode as a model for secondary cell wall development, we conducted cell wall composition, proteomic and metabolomic analyses of the second rice internode at booting stage. We measured major secondary cell wall components along eight segments of an elongating internode. Cellulose, lignin, and xylose increase as a percentage of cell wall material from the younger to the older internode segments, indicating active cell wall synthesis. With the whole elongating internode, we measured peptides via liquid-chromatography mass spectrometry (LC-MS) following trypsin digestion of size fractionated proteins. This identified a total of 3249 protein groups with at least two unique peptides, including many glycosyltransferases, acyltransferases, glycohyrolases, cell wall-localized proteins, and protein kinases that have or may have functions in cell wall biosynthesis or remodeling. In addition, GO over-representation analysis and KO pathway analysis indicate many proteins involved in biosynthetic processes, especially the synthesis of secondary metabolites such as phenylpropanoids, flavonoids, and tepenoids. Therefore, we also used LC-MS to measure methanol-extracted secondary metabolites from the whole internode at the elongation stage and three post-elongation stages, and from leaf and root at the second post-elongation stage. The results indicate secondary metabolites in stems are distinct from those of roots and leaves, and show different profiles during stem development. This study fills a void of knowledge of proteomics and metabolomics data for grass stems, especially of rice, and provides baseline knowledge for more detailed studies of cell wall synthesis and other biological processes during internode development. This and future work is aimed at optimizing stem development and cell wall composition of grasses to improve agronomic properties and biofuel production.
2017-08-04 | PXD003676 | Pride
Project description:microRNAs can play a crucial role in stress response in plants, including biotic stress. Some miRNAs are known to respond to bacterial infection. This work has addressed the role of miRNAs in Manihot esculenta (cassava)-Xanthomonas axonopodis pv. manihotis (Xam) interaction. Illumina sequencing was used for analyzing small RNA libraries from cassava tissue infected and non-infected with Xam. Cassava variety MBRA685 (resistant to Xam-CIO151) Six-week-old plants were inoculated with 36h-old cultures of the aggressive Xanthomonas axonopodis pv. manihotis strain CIO151 in both leaves and stems. Leaves were inoculated by piercing six holes in the mesophyll and placing a 5µL drop of a liquid Xam-MgCl2 culture calibrated at OD600nm = 0.002 (1 x108cfu/ml). Two leaflets per leaf and three leaves per plant were inoculated. Stems were inoculated by puncture in the stems as described previously (24). At least three plants per collection time were inoculated. Leaves and stems were collected from inoculated plants (0 hours post inoculation -hpi, 6hpi, 24hpi, 2 days post-inoculation -dpi, 5dpi, 7dpi and 15dpi) and non-inoculated plants. RNA extractions were made using a LiCl-acid phenol:chloroform method.
2011-12-01 | E-GEOD-29379 | biostudies-arrayexpress
Project description:modENCODE_submission_3221 This submission comes from a modENCODE project of Michael Snyder. For full list of modENCODE projects, see http://www.genome.gov/26524648 Project Goal: We are identifying the DNA binding sites for 300 transcription factors in C. elegans. Each transcription factor gene is tagged with the same GFP fusion protein, permitting validation of the gene's correct spatio-temporal expression pattern in transgenic animals. Chromatin immunoprecipitation on each strain is peformed using an anti-GFP antibody, and any bound DNA is deep-sequenced using Solexa GA2 technology. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf EXPERIMENT TYPE: CHIP-seq. BIOLOGICAL SOURCE: Strain: OP174(official name : OP174 genotype : unc-119(ed3); wgIs174(ces-1::TY1 EGFP FLAG;unc-119) outcross : 3 mutagen : Bombard tags : GFP::3xFlag description : This strain's transgene was constructed by Mihail Sarov at the Max Planck Institute for Cell Biology in Tubiginen using Tony Hyman's recombineering pipeline. The resulting plasmid was used for biolistic transformation of an unc-119(ed3) strain. The CES-1::EGFP fusion protein is expressed in the correct ces-1 spatio-temporal expression pattern. This strain was used for ChIP-seq experiments to map the in vivo binding sites for the CES-1 transcription factor. made_by : S. Kim ); Developmental Stage: L4; Genotype: unc-119(ed3); wgIs174(ces-1::TY1 EGFP FLAG;unc-119); Sex: Hermaphrodite; EXPERIMENTAL FACTORS: Developmental Stage L4; Target gene ces-1; Strain OP174(official name : OP174 genotype : unc-119(ed3); wgIs174(ces-1::TY1 EGFP FLAG;unc-119) outcross : 3 mutagen : Bombard tags : GFP::3xFlag description : This strain's transgene was constructed by Mihail Sarov at the Max Planck Institute for Cell Biology in Tubiginen using Tony Hyman's recombineering pipeline. The resulting plasmid was used for biolistic transformation of an unc-119(ed3) strain. The CES-1::EGFP fusion protein is expressed in the correct ces-1 spatio-temporal expression pattern. This strain was used for ChIP-seq experiments to map the in vivo binding sites for the CES-1 transcription factor. made_by : S. Kim ); temp (temperature) 20 degree celsius
2011-05-24 | E-GEOD-29467 | biostudies-arrayexpress
Project description:modENCODE_submission_3081 This submission comes from a modENCODE project of Michael Snyder. For full list of modENCODE projects, see http://www.genome.gov/26524648 Project Goal: We are identifying the DNA binding sites for 300 transcription factors in C. elegans. Each transcription factor gene is tagged with the same GFP fusion protein, permitting validation of the gene's correct spatio-temporal expression pattern in transgenic animals. Chromatin immunoprecipitation on each strain is peformed using an anti-GFP antibody, and any bound DNA is deep-sequenced using Solexa GA2 technology. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf EXPERIMENT TYPE: CHIP-seq. BIOLOGICAL SOURCE: Strain: OP174(official name : OP174 genotype : unc-119(ed3); wgIs174(ces-1::TY1 EGFP FLAG;unc-119) outcross : 3 mutagen : Bombard tags : GFP::3xFlag description : This strain's transgene was constructed by Mihail Sarov at the Max Planck Institute for Cell Biology in Tubiginen using Tony Hyman's recombineering pipeline. The resulting plasmid was used for biolistic transformation of an unc-119(ed3) strain. The CES-1::EGFP fusion protein is expressed in the correct ces-1 spatio-temporal expression pattern. This strain was used for ChIP-seq experiments to map the in vivo binding sites for the CES-1 transcription factor. made_by : S. Kim ); Developmental Stage: L3; Genotype: unc-119(ed3); wgIs174(ces-1::TY1 EGFP FLAG;unc-119); Sex: Hermaphrodite; EXPERIMENTAL FACTORS: Developmental Stage L3; Target gene ces-1; Strain OP174(official name : OP174 genotype : unc-119(ed3); wgIs174(ces-1::TY1 EGFP FLAG;unc-119) outcross : 3 mutagen : Bombard tags : GFP::3xFlag description : This strain's transgene was constructed by Mihail Sarov at the Max Planck Institute for Cell Biology in Tubiginen using Tony Hyman's recombineering pipeline. The resulting plasmid was used for biolistic transformation of an unc-119(ed3) strain. The CES-1::EGFP fusion protein is expressed in the correct ces-1 spatio-temporal expression pattern. This strain was used for ChIP-seq experiments to map the in vivo binding sites for the CES-1 transcription factor. made_by : S. Kim ); temp (temperature) 20 degree celsius
2012-01-31 | E-GEOD-35277 | biostudies-arrayexpress
Project description:modENCODE_submission_3857 This submission comes from a modENCODE project of Michael Snyder. For full list of modENCODE projects, see http://www.genome.gov/26524648 Project Goal: We are identifying the DNA binding sites for 300 transcription factors in C. elegans. Each transcription factor gene is tagged with the same GFP fusion protein, permitting validation of the gene's correct spatio-temporal expression pattern in transgenic animals. Chromatin immunoprecipitation on each strain is peformed using an anti-GFP antibody, and any bound DNA is deep-sequenced using Solexa GA2 technology. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf EXPERIMENT TYPE: CHIP-seq. BIOLOGICAL SOURCE: Strain: OP174(official name : OP174 genotype : unc-119(ed3); wgIs174(ces-1::TY1 EGFP FLAG;unc-119) outcross : 3 mutagen : Bombard tags : GFP::3xFlag description : This strain's transgene was constructed by Mihail Sarov at the Max Planck Institute for Cell Biology in Tubiginen using Tony Hyman's recombineering pipeline. The resulting plasmid was used for biolistic transformation of an unc-119(ed3) strain. The CES-1::EGFP fusion protein is expressed in the correct ces-1 spatio-temporal expression pattern. This strain was used for ChIP-seq experiments to map the in vivo binding sites for the CES-1 transcription factor. made_by : S. Kim ); Developmental Stage: embryo; Genotype: unc-119(ed3); wgIs174(ces-1::TY1 EGFP FLAG;unc-119); Sex: mixed Male and Hermaphrodite population; EXPERIMENTAL FACTORS: Developmental Stage embryo; Target gene ces-1; Strain OP174(official name : OP174 genotype : unc-119(ed3); wgIs174(ces-1::TY1 EGFP FLAG;unc-119) outcross : 3 mutagen : Bombard tags : GFP::3xFlag description : This strain's transgene was constructed by Mihail Sarov at the Max Planck Institute for Cell Biology in Tubiginen using Tony Hyman's recombineering pipeline. The resulting plasmid was used for biolistic transformation of an unc-119(ed3) strain. The CES-1::EGFP fusion protein is expressed in the correct ces-1 spatio-temporal expression pattern. This strain was used for ChIP-seq experiments to map the in vivo binding sites for the CES-1 transcription factor. made_by : S. Kim ); temp (temperature) 20 degree celsius
2013-07-11 | E-GEOD-48723 | biostudies-arrayexpress
Project description:modENCODE_submission_3076 This submission comes from a modENCODE project of Michael Snyder. For full list of modENCODE projects, see http://www.genome.gov/26524648 Project Goal: We are identifying the DNA binding sites for 300 transcription factors in C. elegans. Each transcription factor gene is tagged with the same GFP fusion protein, permitting validation of the gene's correct spatio-temporal expression pattern in transgenic animals. Chromatin immunoprecipitation on each strain is peformed using an anti-GFP antibody, and any bound DNA is deep-sequenced using Solexa GA2 technology. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf EXPERIMENT TYPE: CHIP-seq. BIOLOGICAL SOURCE: Strain: OP174(official name : OP174 genotype : unc-119(ed3); wgIs174(ces-1::TY1 EGFP FLAG;unc-119) outcross : 3 mutagen : Bombard tags : GFP::3xFlag description : This strain's transgene was constructed by Mihail Sarov at the Max Planck Institute for Cell Biology in Tubiginen using Tony Hyman's recombineering pipeline. The resulting plasmid was used for biolistic transformation of an unc-119(ed3) strain. The CES-1::EGFP fusion protein is expressed in the correct ces-1 spatio-temporal expression pattern. This strain was used for ChIP-seq experiments to map the in vivo binding sites for the CES-1 transcription factor. made_by : S. Kim ); Developmental Stage: fed L1; Genotype: unc-119(ed3); wgIs174(ces-1::TY1 EGFP FLAG;unc-119); Sex: Hermaphrodite; EXPERIMENTAL FACTORS: Developmental Stage fed L1; Target gene ces-1; Strain OP174(official name : OP174 genotype : unc-119(ed3); wgIs174(ces-1::TY1 EGFP FLAG;unc-119) outcross : 3 mutagen : Bombard tags : GFP::3xFlag description : This strain's transgene was constructed by Mihail Sarov at the Max Planck Institute for Cell Biology in Tubiginen using Tony Hyman's recombineering pipeline. The resulting plasmid was used for biolistic transformation of an unc-119(ed3) strain. The CES-1::EGFP fusion protein is expressed in the correct ces-1 spatio-temporal expression pattern. This strain was used for ChIP-seq experiments to map the in vivo binding sites for the CES-1 transcription factor. made_by : S. Kim ); temp (temperature) 20 degree celsius
2012-05-07 | E-GEOD-37803 | biostudies-arrayexpress