Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the colon and rectum with intervals of acute exacerbation. Its etiology remains unknown, although recent studies suggest that pro-inflammatory cytokines initiate the inflammatory responses. In this study, we investigated the ameliorative effects of YB-21 (new product containining Lactobacillus plantarum, Barley Bran and Nymphaeaceae) in a dextran sulfate sodium (DSS)-induced colitis in mice. The experimental groups were divided into 6 groups (n = 10) of ICR mice: normal control(NC), DSS-treated(PC, positive control), DSS+latic-L-treated (3×107CFU/ml), DSS+latic-M-treated (3×109CFU/ml), DSS+latic-H-treated (3×1011CFU/ml), and DSS+YB-21-treated. Histological examinations indicated that YB-21 suppressed edema, mucosal damage, and the loss of crypts induced by DSS. Moreover, YB-21 reduced serum level expressions of, tumor necrosis factor-α (TNF-α). These results suggest that YB-21 has an anti-colitic effect by the suppression of intestinal inflammatory responses in DSS-induced colitis in mice.

Mycoplasma (M.) felis and M. canis is related with pneumonia or conjunctivitis in domestic cats and several diseases in a variety of other animals, including lower respiratory tract disease or pleuritis. Polymerase chain reaction (PCR) assays has been reported as an easy and useful method. It could be conducted even on nasal swab samples as a non-invasive rapid testing tools for large numbers of Mycoplasma species. However, PCR assays have to conduct multiple assays because of a lot of Mycoplasma species. Therefore, it need to perform several tests and reveal time consuming procedures. In this study, we developed a sensitive and specific multiplex polymerase chain reaction (PCR) assay that detects simultaneously two species like as M. felis and M. canis. The simultaneous detection of M. felis and M. canis primers were used to differentiate two mycoplasma species. The target DNA fragments were specifically amplified M. felis and M. canis PCR with 16S ribosomal DNA primers. Single and mixed Mycoplasma species DNA templates were submitted to validate the specificity of the multiplex PCR. The corresponding specific DNA products were amplified for each pathogen. The detection limit of the developed multiplex PCR is 102 pg with M. felis and M. canis DNA. Furthermore, the developed multiplex PCR detected successfully M. felis in feline nasal specimens. The multiplex PCR assay provides a novel tool for simultaneous detection and differentiation of M. felis and M. canis in cats.

Heat shock transcription factors(HSFs) are the major heat shock factors regulating the heat stress response. They participate in regulating the expression of heat shock proteins (HSPs), which are critical in the protection against stress damage and many other important biological processes. In this study, a genome-wide analysis was carried out to identify all HSFs soybean genes. Twenty six nonredundant HSF genes(GmHsf) were identified in the latest soybean genome sequence. Chromosomal location, protein domain and motif organization of GmHsfs were analyzed in soybean genome. The phylogenetic relationships, gene duplications and expression profiles of GmHsf genes were also presented in this study. According to their structural features, the predicted members were divided into the previously defined classes A–C, as described in Arabidopsis. Using RT-PCR, the expression patterns of 26 GmHsf genes were investigated under heat stress. The data revealed that these genes presented different expression levels in response to heat stress conditions. Real-time (q)RT-PCR was performed to investigate transcript levels of five GmHsfs in response to multiple abiotic stresses. Differential expression of five GmHsfs implies their role during abiotic stresses. Subcellular localization using GFP-fusion protein demonstrated that GmHsf12 and GmHsf34 were restricted to the nucleus and GmHsf28 was localized in the nucleus and cytoplasm in plant. The results provide a fundamental clue for understanding of the complexity of the soybean HSF gene family and cloning specific function genes in further studies and applications.

The plant-specific NAC (NAM, ATAF, and CUC)-domain proteins play important roles in plant development and stress responses. Comparative time-course expression analyses were carried out to analyze the expression levels of 62 soybean NAC genes during drought stress in order to search for the stress-inducible NAC genes. Ten GmSNAC (Glycine max stress-inducible NAC) genes having the significant differential expression in response to the drought stress and abscisic acid (ABA) hormone application were further investigated for their expression profiles with various stresses such as drought, high salinity, cold and with ABA treatments by the quantitative real-time PCR analyses. In this research, the full-length cDNAs of eight GmSNAC were isolated for the further studies. Eight GmSNAC proteins were tested for their transcription activation in the yeast assay system. Two GmSNAC proteins showed the very high transcriptional activities and the other two GmSNAC proteins displayed moderate levels of transactivation while the remaining four GmSNAC proteins lacked transactivation in yeast. Subcellular localization of eight GmSNAC proteins was analyzed via the green fluorescent protein-GmSNAC fusion protein in tobacco plant cell. Three GmSNAC proteins with the C-terminal transmembrane domain were localized to the nucleus and cytoplasmic fractions. The other five GmSNAC proteins were targeted to the nucleus. The function of GmSNAC49 gene was further investigated using the overexpression transgenic Arabidopsis. Germination rate in transgenic plants over-expressing GmSNAC49 was delayed in the media supplemented with mannitol or ABA compared with that of wild-type (WT) plants. The 35S:GmSNAC49 transgenic Arabidopsis displayed improved tolerance to drought stress compared to the WT. The results of this systematic analysis of the GmSNAC family responsive to abiotic stress will provide novel tools and resources for the development of improved drought tolerant transgenic soybean cultivars

Comparative time-course expression analyses were carried out to analyze the expression levels of 60 soybean WRKY genes during abiotic stress in order to search for the stress-inducible WRKY genes. Five GmWRKY(Glycine max WKRY) genes having the significant differential expression in response to the drought stress and abscisic acid(ABA) hormone application were further investigated for their expression profiles with various stresses such as drought, high salinity, cold and with ABA treatments by the quantitative real-time PCR analyses. In this research, the full-length cDNAs of five GmWRKY were isolated for the further studies. Five GmWRKY proteins were tested for their transcription activation in the yeast assay system. GmWRKY3 proteins showed the very high transcriptional activities and the other two GmWRKY proteins displayed moderate levels of transactivation while the remaining two GmWRKY proteins lacked transactivation in yeast. Subcellular localization of five GmWRKY proteins was analyzed via the green fluorescent protein-GmWRKY fusion protein in tobacco plant cell and all of GmWRKY proteins were targeted to the nucleus. In order to analyze the function of GmWRKY genes in plant, 35S:GmWRKY overexpression(OE) transgenic Arabidopsis were generated. Root growth and germination rates in transgenic OE plants were investigated in the media supplemented with mannitol, NaCl or ABA compared with that of wild-type(WT) plants. The 35S:GmWRKY42 transgenic Arabidopsis displayed reduced tolerance to drought stress compared to the WT. The results of this systematic analysis of the GmWRKY family responsive to abiotic stress will provide novel tools and resources for the development of improved drought tolerant transgenic soybean cultivars

Arabidopsis atDjC53 and atDjC32 gene DnaJ-like protein homologous to DnaJ-like protein was characterized for the functional analysis of DnaJ-like protein. It was shown that atDjC53 and atDjC32 RNA expression is induced by heat shock stress and atDjC53- and atDjC32-GFP was targeted to the nucleus of protoplasts. The atDjC53 and atDjC32 promoter (1 kb) was isolated and fused to the GUS reporter gene to investigate gene regulation of atDjC53 and atDjC32 specific to heat shock stress or to developmental organ in the transgenic lines. RNAi and overexpression construct was employed to generate atDjC53 and atDjC32 knock-out plants for the study of their function. Molecular function of atDjC53 and atDjC32 is discussed in relation to heat shock and also developmental stages in Arabidopsis.

Heat shock transcription factors (HSFs) are the major heat shock factors regulating the heat stress response. They participate in regulating the expression of heat shock proteins (HSPs), which are critical in the protection against stress damage and many other important biological processes. In this study, a genome-wide analysis was carried out to identify all HSFs soybean genes. Twenty six nonredundant HSF genes (GmHsf) were identified in the latest soybean genome sequence. Chromosomal location, protein domain and motif organization of GmHsfs were analyzed in soybean genome. The phylogenetic relationships, gene duplications and expression profiles of GmHsf genes were also presented in this study. According to their structural features, the predicted members were divided into the previously defined classes A–C, as described in Arabidopsis. Using RT-PCR, the expression patterns of 26 GmHsf genes were investigated under heat stress. The data revealed that these genes presented different expression levels in response to heat stress conditions. Real-time (q)RT-PCR was performed to investigate transcript levels of five GmHsfs in response to multiple abiotic stresses. Differential expression of five GmHsfs implies their role during abiotic stresses. Subcellular localization using GFP-fusion protein demonstrated that GmHsf12 and GmHsf34 were restricted to the nucleus and GmHsf28 was localized in the nucleus and cytoplasm in plant. The results provide a fundamental clue for understanding of the complexity of the soybean HSF gene family and cloning specific function genes in further studies and applications.

Salinity stress severely affects plant growth and development causing crop loss worldwide. Suaeda asparagoides is a salt-marsh euhalophyte widely distributed in southwestern foreshore of Korea. To isolate salt tolerance genes from S. asparagoides, we constructed a cDNA library from leaf tissues of S. asparagoides that was treated with 200 mM NaCl. A total of 1,056 clones were randomly selected for EST sequencing, and 932 of them produced readable sequence. By sequence analysis, we identified 538 unigenes and registered each in National Center for Biotechnology Information. The 80 salt stress related genes were selected to study their differential expression. Reverse Transcriptase-PCR and Northern blot analysis revealed that 23 genes were differentially expressed under the high salinity stress conditions in S. asparagoides. They are functionally diverse including transport, signal transduction, transcription factor, metabolism and stress associated protein, and unknown function. Among them dehydrin (SaDhn) and RNA binding protein (SaRBP1) were examined for their abiotic stress tolerance in yeast (Saccharomyces cerevisiae). Yeast overexpressing SaDhn and SaRBP1 showed enhanced tolerance to osmotic, freezing and heat shock stresses. This study provides the evidence that SaRBP1 and SaDhn from S.asparagoides exert abiotic stress tolerance in yeast. Information of salt stress related genes from S. asparagoides will contribute for the accumulating genetic resources to improve osmotic tolerance in plants.

The ubiquitin conjugating enzyme E2 (UBC E2) mediates selective ubiquitination, acting with E1 and E3 enzymes to designate specific proteins for subsequent degradation. In the present study, we characterized the function of the mung bean VrUBC1 gene (Vigna radiata UBC 1). RNA gel-blot analysis showed that VrUBC1 mRNA expression was induced by either dehydration, high salinity or by the exogenous abscisic acid (ABA), but not by low temperature or wounding. Biochemical studies of VrUBC1 recombinant protein and complementation of yeast ubc4/5 by VrUBC1 revealed that VrUBC1 encodes a functional UBC E2. To understand the function of this gene in development and plant responses to osmotic stresses, we overexpressed VrUBC1 in Arabidopsis (Arabidopsis thaliana). The VrUBC1-overexpressing plants displayed highly sensitive responses to ABA and osmotic stress during germination, enhanced ABA- or salt-induced stomatal closing, and increased drought stress tolerance. The expression levels of a number of key ABA signaling genes were increased in VrUBC1-overexpressing plants compared to the wild-type plants. Yeast two-hybrid and bimolecular fluorescence complementation demonstrated that VrUBC1 interacts with AtVBP1 (A. thaliana VrUBC1 Binding Partner 1), a C3HC4-type RING E3 ligase. Overall, these results demonstrate that VrUBC1 plays a positive role in osmotic stress tolerance through transcriptional regulation of ABA-related genes and possibly through interaction with a novel RING E3 ligase.

Arabidopsis atDjC53 and atDjC32 gene DnaJ-like protein homologous to DnaJ-like protein was characterized for the functional analysis of DnaJ-like protein. It was shown that atDjC53 and atDjC32 RNA expression is induced by heat shock stress and atDjC53- and atDjC32-GFP was targeted to the nucleus of protoplasts. The atDjC53 and atDjC32 promoter (1 kb) was isolated and fused to the GUS reporter gene to investigate gene regulation of atDjC53 and atDjC32 specific to heat shock stress or to developmental organ in the transgenic lines. RNAi and overexpression construct was employed to generate atDjC53 and atDjC32 knock-out plants for the study of their function. Molecular function of atDjC53 and atDjC32 is discussed in relation to heat shock and also developmental stages in Arabidopsis.

Heat shock transcription factors (HSFs) are the major heat shock factors regulating the heat stress response. They participate in regulating the expression of heat shock proteins (HSPs), which are critical in the protection against stress damage and many other important biological processes. In this study, a genome-wide analysis was carried out to identify all HSFs soybean genes. Twenty six nonredundant HSF genes (GmHsf) were identified in the latest soybean genome sequence. Chromosomal location, protein domain and motif organization of GmHsfs were analyzed in soybean genome. The phylogenetic relationships, gene duplications and expression profiles of GmHsf genes were also presented in this study. According to their structural features, the predicted members were divided into the previously defined classes A–C, as described in Arabidopsis. Using RT-PCR, the expression patterns of 26 GmHsf genes were investigated under heat stress. The data revealed that these genes presented different expression levels in response to heat stress conditions. Real-time (q)RT-PCR was performed to investigate transcript levels of five GmHsfs in response to multiple abiotic stresses. Differential expression of five GmHsfs implies their role during abiotic stresses. Subcellular localization using GFP-fusion protein demonstrated that GmHsf12 and GmHsf34 were restricted to the nucleus and GmHsf28 was localized in the nucleus and cytoplasm in plant. The results provide a fundamental clue for understanding of the complexity of the soybean HSF gene family and cloning specific function genes in further studies and applications.

Salinity stress severely affects plant growth and development causing crop loss worldwide. Suaeda asparagoides is a salt-marsh euhalophyte widely distributed in southwestern foreshore of Korea. To isolate salt tolerance genes from S. asparagoides, we constructed a cDNA library from leaf tissues of S. asparagoides that was treated with 200 mM NaCl. A total of 1,056 clones were randomly selected for EST sequencing, and 932 of them produced readable sequence. By sequence analysis, we identified 538 unigenes and registered each in National Center for Biotechnology Information. The 80 salt stress related genes were selected to study their differential expression. Reverse Transcriptase-PCR and Northern blot analysis revealed that 23 genes were differentially expressed under the high salinity stress conditions in S. asparagoides. They are functionally diverse including transport, signal transduction, transcription factor, metabolism and stress associated protein, and unknown function. Among them dehydrin (SaDhn) and RNA binding protein (SaRBP1) were examined for their abiotic stress tolerance in yeast (Saccharomyces cerevisiae). Yeast overexpressing SaDhn and SaRBP1 showed enhanced tolerance to osmotic, freezing and heat shock stresses. This study provides the evidence that SaRBP1 and SaDhn from S.asparagoides exert abiotic stress tolerance in yeast. Information of salt stress related genes from S. asparagoides will contribute for the accumulating genetic resources to improve osmotic tolerance in plants.

We have isolated wound-inducible genes from soybean using suppression subtractive hybridization (SSH) method and were able to obtain the full-length clone of GmDjp1 gene encoding DnaJ-like protein. The full-length cDNA of GmDjp1 is 689 bp with an open reading frame (ORF) consisting of 163 amino acid (aa). Genomic southern blot confirmed that soybean genome has two copies of GmDjp1 gene. Northern blot analysis showed that the RNA expression of GmDjp1 gene is specifically induced by heat, NaCl, wounding and drought stresses. It was demonstrated that GmDjp1-GFP was targeted to the nucleus in tobacco cell. GmDjp1 overexpression plants showed more susceptible to salt and heat stress compared to WT. RNA expression level of Hsp18.2 and Hsp25.3-P was lower than that of WT during recovery after heat hock in plants. This indicates that GmDjp1 may play a negative regulator to stress responses in plants.

A low temperature-inducible cDNA designated as VrUBC1 from mungbean (Vigna radiata) was isolated by subtractive hybridization method. By rapid amplification of cDNA end technique, the full-length cDNA of VrUBC1 was obtained. The full-length cDNA of VrUBC1 contains an open reading frame of 444 nucleotides in length and capable of specifying a 16.5-kDa protein of 148 amino acids (aa) with an isoelectric point of 7.72. VrUBC1 mRNA was induced by NaCl and ABA, but not by wounding and low temperature stress. It was shown that VrUBC1-GFP was localized to the cytoplasm in tobacco cell. To examine the function of VrUBC1, VrUBC1 was expressed in Escherichia coli as His-fusion protein. Purified VrUBC1-His recombinant protein was shown to have ubiquitination activity in vitro. For the in vivo functional analysis of VrUBC1, VrUBC1 was expressed in yeast ubc4/5 double mutant. Stress tolerance was tested in the VrUBC1 overexpressing Arabidopsis transgenic plants. We propose that VrUBC1 play an important role in protein degradation processes during abiotic stress in plants.

For purposes of studying intron structures and predicting consensus splice motifs, a total of 102 legume species were used to isolate introns across the family. Of 196 gene-targeted PCR primer pairs, we successfully amplified 118 intron-containing genes (60.2%) and obtained a total of 1,870 introns with an average size of 143 nucleotides. Species-based compilation of 5’- and 3’-splicing motifs showed lineage-specific conservation in each splicing motif. Compilation of the entire intron set permitted prediction of the consensus sequences of splicing signal motifs in legumes, AYGWGTABABGH and TVNC/TAGGHTV for the 5’- and 3’-splicing motifs, respectively. Interestingly, these consensus motifs are very similar to the corresponding splicing signals of two model systems, Arabidopsis and rice. This result is suggestive of conservation of pre-mRNA splicing mechanisms in higher plants. Multiple alignments of CALTL introns demonstrated that the region from the branch point to 3’ splice site was relatively more conserved than the region from5’ splice site to the branch point. Phylogenetic analysis demonstrated that each of three splicing motifs, 5’-splice sites, 3’-splice sits, and branch site, was relevant to evolutionary divergence of species and phylogenetically informative, suggesting that splice signal sequences would be useful as a potential tool for the molecular phylogenetic analysis.

A core genetic map of the legume Medicago truncatula has been established by analyzing the segregation of 288 sequence-characterized genetic markers in an F2 population composed of 93 individuals. These molecular markers correspond to 141 ESTs, 80 BAC end sequence-tags, and 67 resistance gene analogs, covering 513 cM. In the case of EST-based markers we used an intron-targeted marker strategy, with primers designed to anneal in conserved exon regions and amplify across intron regions. Polymorphisms were significantly more frequent in intron vs exon regions, thus providing an efficient mechanism to map transcribed genes. Genetic and cytogenetic analysis produced eight well-resolved linkage groups, which have been previously correlated with eight chromosomes by means of FISH with mapped BAC clones. We anticipated that mapping of conserved coding regions would have utility for comparative mapping among legumes; thus 60 of the EST-based primer pairs were designed to amplify orthologous sequences across a range of legume species. As an initial test of this strategy, we used primers designed against M. truncatula exon sequences to rapidly map genes in Medicago sativa. The resulting comparative map, which includes 68 bridging markers, indicates that the two Medicago genomes are highly similar, and establishes the basis for a “Medicago” composite map.

Cross-species translation of genomic information may play a pivotal role in applying biological knowledge gained from one species to other genomes. Abiotic stress-responsive genes in Arabidopsis have been translated to a legume model system, Medicago truncatula. A total of 1,370 Arabidopsis genes were identified by searching TAIR database, expression profiling data and literatures. For purposes of cross-genome identification of orthologous genes, tBlastX or BlastP were employed between these two model systems. Candidate genes potentially associated with abiotic stress responses were classified into 18 functional criteria and corresponding genomic locations were analyzed by Circos program. To do this, user-friendly bioinformatic analysis platform was established. In order to discover abiotic stress-associated genes, gene network and/or interactome analyses were conducted using a combination of AraNet web-based platform and CytoScape program. As a result, we could identify 240 key genes that appeared to play an important role within central gene networks. We anticipate that these genes may impact molecular breeding programs by developing them into genetic markers and discovering trait-associated nucleotide variations.

We isolated two low temperature-inducible cDNAs designated as MLT5 and MLT31 from mungbean by subtractive suppression hybridization method. By rapid amplification of cDNA end technique, the full-length cDNAs designated MLT5 and MLT31 were obtained. The full-length cDNA of MLT5 contains an open reading frame of 324 nucleotides coding for 107 amino acids. The full-length cDNA of MLT31 contains an open reading frame of 444 nucleotides in length and capable of specifying a 16.5-kDa protein of 148 amino acids (aa) with an isoelectric point of 7.72. RNA expression of MLT5 was strongly induced by low temperature in the early time and also by wounding, NaCl and ABA. MLT31 mRNA was induced by NaCl and ABA but not by wounding and low temperature stress. MLT5 encodes a protein of unknown function, of which targeting site is predicted to be chloroplast membrane protein. To examine the localization of MLT5, MLT5-GFP was expressed in tobacco cells. It was shown that MLT5-GFP was localized to surface of chloroplast in tobacco cell. To examine the function of MLT31, MLT31 was expressed in Escherichia coli as His-fusion protein. Purified MLT31-His recombinant protein will be tested for ubiquitination activity in vitro. In addition, for the in vivo functional analysis of MLT31, MLT31 will be expressed in yeast ubc9 knock-out mutant. We propose that MLT5 and MLT31 play an important role in signal pathway of abiotic stress in plants.

Photorespiration reduces carbon fixation rate, but is essential process in plant. Photorespiration involves reactions in chloroplasts, peroxisomes, and mitochondria. In photorepiratory peroxisome, alanine glyoxylate aminotransferase (AGT) catalyzes alanine and glyoxylate into glycine and pyruvate. We isolated a low temperature-inducible cDNA encoding AGT from mungbean leaves. The full-length cDNA, designated as MLT9, contains an open reading frame of 1,203 nucleotides coding for a protein of 401 amino acids. Genomic DNA blotting showed that the mungbean genome has one copy of MLT9. MLT9 mRNA was induced not only by low temperature but also by drought stress, but ABA and NaCl did not induce RNA expression of MLT9. In mungbean, AGT activity was higher in the non-stressed leaves compared to the low-temperature treated leaves. Based on GFP/RFP targeting experiment, GFP-MLT9 fusion protein and SKL-RFP, a peroxisome marker, were colocalized to peroxisome in tobacco protoplasts. This suggests that peroxisomal MLT9 plays a role in photorespiratory metabolism in response to low temperature and drought stress.

We isolated wound-inducible genes using suppression subtractive hybridization (SSH) method and were able to obtain to clone W3 gene encoding dnaJ like protein. The full-length cDNA of W3 is 689 bp with an open reading frame (ORF) consisting of 163 amino acid (aa). Genomic southern blot confirmed that soybean genome has two copies of W3 gene. Northern blot analysis was also carried out for the gene expression during heat, NaCl, drought, wounding stresses. The expression of W3 gene specifically induced by heat, NaCl, wounding and drought stress. Using GFP fusion vector, W13-GFP was targeted both to nucleus. For the functional analysis of W3, His-tagged W3 recombinant protein was heterologously expressed in E. coli. The W3 recombinant cells showed enhanced heat tolerance compared to that of vector control cells. We suggest that dnaJ-like W3 protein function as molecular chaperone in the nucleus of the plant cell during various stresses.