Analysis of expressed genes in ethylene-treated potato leaves using expressed sequence tags

To isolate useful and interesting plant genes in large quantities, random sequencing of cDNA clones from potato leaf library treated with ethylene was performed. Partial sequences of randomly selected 210 clones with the insert of longer than 500 base pair (bp) as well as poly (A) tail have been compared with sequences in GeneBank, EMBL and DDBJ nucleic acid databases and fostered 193 expressed sequence tags (ESTs). The 210 cDNA clones identified are related to various aspect of metabolic pathways such as glycolysis, amino acid synthesis, translation mechanism, ribosome synthesis, hormone response, stress response, regulation of gene expression, and signal transduction. Among the 193 ESTs, 12 ESTs (29 cDNA clones) appeared more than once and 181 ESTs appeared once regarded as a solitary group. Out of 210 clones, 29 clones (13.8%) have no similarity to the known nucleotide sequences and could serve as a potentially useful resource for plant molecular biology referring to particular genes. Nucleotide sequencing to generate more ESTs from ethylene-induced as well as non-induced potato leaf is in progress as well.     

Comparative characterization of sweetpotato antioxidant genes from expressed sequence tags of dehydration-treated fibrous roots under different abiotic stress conditions

Drought stress is one of the most adverse conditions for plant growth and productivity. The plant antioxidant system is an important defense mechanism and includes antioxidant enzymes and low-molecular weight antioxidants. Understanding the biochemical and molecular responses to drought is essential for improving plant resistance to water-limited conditions. Previously, we isolated and characterized expressed sequence tags (ESTs) from a full-length enriched cDNA library prepared from fibrous roots of sweetpotato subjected to dehydration stress (Kim et al. in BMB Rep 42:271–276, [5]). In this study, we isolated and characterized 11 sweetpotato antioxidant genes from sweetpotato EST library under various abiotic stress conditions, which included six intracellular CuZn superoxide dismutases (CuZnSOD), ascorbate peroxidase, catalase, glutathione peroxidase (GPX), glutathione-S-transferase, thioredoxin (TRX), and five extracellular peroxidase genes. The expression of almost all the antioxidant genes induced under dehydration treatments occurred in leaves, with the exception of extracellular swPB6, whereas some antioxidant genes showed increased expression levels in the fibrous roots, such as intracellular GPX, TRX, extracellular swPA4, and swPB7 genes. During various abiotic stress treatments in leaves, such as exposure to NaCl, cold, and abscisic acid, several intracellular antioxidant genes were strongly expressed compared with the expression of extracellular antioxidant genes. These results indicated that some intracellular antioxidant genes, especially swAPX1 and CuZnSOD, might be specifically involved in important defense mechanisms against oxidative stress induced by various abiotic stresses including dehydration in sweetpotato plants.     

Identification of genes related to Parkinson's disease using expressed sequence tags.

In a search for novel target genes related to Parkinson's disease (PD), two full-length cDNA libraries were constructed from a human normal substantia nigra (SN) and a PD patient's SN. An analysis of the gene expression profiles between them was done using the expressed sequence tags (ESTs) frequency. Data for the differently expressed genes were verified by quantitative real-time RT-PCR, immunohistochemical analysis and a cell death assay. Among the 76 genes identified with a significant difference (P > 0.9), 21 upregulated genes and 13 downregulated genes were confirmed to be differentially expressed in human PD tissues and/or in an MPTP-treated mice model by quantitative real-time RT-PCR. Among those genes, an immunohistochemical analysis using an MPTP mice model for alpha-tubulin including TUBA3 and TUBA6 showed that the protein levels are downregulated, as well as the RNA levels. In addition, MBP, PBP and GNAS were confirmed to accelerate cell death activity, whereas SPP1 and TUBA3 to retard this process. Using an analysis of ESTs frequency, it was possible to identify a large number of genes related to human PD. These new genes, MBP, PBP, GNAS, SPP1 and TUBA3 in particular, represent potential biomarkers for PD and could serve as useful targets for elucidating the molecular mechanisms associated with PD.     

Generation of 919 expressed sequence tags from immature flower buds and gene expression analysis using expressed sequence tags in the model plant Lotus japonicus

Lotus japonicus has received increased attention as a potential model legume plant. In order to study gene expression in reproductive organs and to identify genes that play a crucial function in sexual reproduction, we constructed a cDNA library from immature flower buds containing anthers at the stage of developing tapetum cells in L. japonicus, and characterized 919 expressed sequence tags (ESTs) randomly selected from a cDNA library of the immature flower buds. The 919 ESTs analyzed were clustered into 821 non-redundant EST groups. As a result of a database search, 436 groups (53%) out of the 821 groups showed sequence similarity to genes registered in the public database. Out of these 436 groups, 109 groups showed similarity to genes encoding hypothetical proteins whose function had not yet been estimated. Three hundred eighty five groups (47%) showed no significant homology to known sequences and were classified as novel sequences. A comparison of 821 non-redundant EST sequences and EST sequences derived from the whole plant L. japonicus revealed that 474 EST sequences derived from immature flower buds were not found in the EST sequences of the whole plant. In order to confirm the expression pattern of potential reproductive-organ specific EST clones, nine clones, which were not matched to ESTs derived from the whole plant, were selected, and RT-PCR analysis was performed on these clones. As a result of RT-PCR, we found two novel anther specific clones. One clone was homologous to a gene encoding human cleft lip and palate associated transmembrane protein (CLPTM1) like protein, and the other clone did not show a significant similarity to any genes deposited in the public database. These results indicate that ESTs analyzed here represent a valuable resource for finding reproductive-organ specific genes in Lotus japonicus.     

Differential expression of expressed sequence tags in alfalfa roots under aluminum stress

To gain a better understanding of differentially expressed sequence tags (ESTs) for aluminum (Al) tolerance and to investigate the molecular mechanisms of Al toxicity, cDNA subtraction libraries were generated from Al-stressed roots of alfalfa (Medicago sativa L.) compared with no Al-stressed ones, employing suppression subtractive hybridization. Using differential screening technique in which the probes were labeled with DIG, we identified 45 non-redundant ESTs in Al-stressed alfalfa root tips with significantly altered expression. Among the up-regulated ESTs, we have found genes encoding identified proteins, including malate dehydrogenase, 6-phosphogluconate dehydrogenase, peroxidase, and an ABC transporter, while the down-regulate genes included ATPase, secretory carrier membrane protein 2, pectinesterase inhibitor. In addition, two novel ESTs, EW678752 and EY976957, up- and down-regulated by Al stress were sequenced. Analyzed by real-time PCR, the expressions of EST EW678718, EW678739, EY976969 and EW678728, which encode for ABC transporter, malate dehydrogenase, peroxidase and 6-phosphogluconate dehydrogenase correspondingly, increased 1.64-, 2.75-, 3.27- and 6.54-folds, respectively, and the expression of EY976957 encoding for ATPase decreased 3.27 folds. The expression of EST EW678752 increased 34.54-fold, while that of EY976957 decreased 16.68 folds. It suggested that the two novel ESTs maybe play a significant role in the aluminum tolerance of alfalfa.     

Structural and functional analyses of the wheat genomes based on expressed sequence tags (ESTs) related to abiotic stresses.

To gain insights into the structure and function of the wheat (Triticum aestivum L.) genomes, we identified 278 ESTs related to abiotic stress (cold, heat, drought, salinity, and aluminum) from 7671 ESTs previously mapped to wheat chromosomes. Of the 278 abiotic stress related ESTs, 259 (811 loci) were assigned to chromosome deletion bins and analyzed for their distribution pattern among the 7 homoeologous chromosome groups. Distribution of abiotic stress related EST loci were not uniform throughout the different regions of the chromosomes of the 3 wheat genomes. Both the short and long arms of group 4 chromosomes showed a higher number of loci in their distal regions compared with proximal regions. Of the 811 loci, the number of mapped loci on the A, B, and D genomes were 258, 281, and 272, respectively. The highest number of abiotic stress related loci were found in homoeologous chromosome group 2 (142 loci) and the lowest number were found in group 6 (94 loci). When considering the genome-specific ESTs, the B genome showed the highest number of unique ESTs (7 loci), while none were found in the D genome. Similarly, considering homoeologous group-specific ESTs, group 2 showed the highest number with 16 unique ESTs (58 loci), followed by group 4 with 9 unique ESTs (33 loci). Many of the classified proteins fell into the biological process categories associated with metabolism, cell growth, and cell maintenance. Most of the mapped ESTs fell into the category of enzyme activity (28%), followed by binding activity (27%). Enzymes related to abiotic stress such as beta-galactosidase, peroxidase, glutathione reductase, and trehalose-6-phosphate synthase were identified. The comparison of stress-responsive ESTs with genomic sequences of rice (Oryza sativa L.) chromosomes revealed the complexities of colinearity. This bin map provides insight into the structural and functional details of wheat genomic regions in relation to abiotic stress.     

Analysis of expressed sequence tags of the water flea Daphnia magna.

To study gene expression in the water flea Daphnia magna we constructed a cDNA library and characterized the expressed sequence tags (ESTs) of 7210 clones. The EST sequences clustered into 2958 nonredundant groups. BLAST analyses of both protein and DNA databases showed that 1218 (41%) of the unique sequences shared significant similarities to known nucleotide or amino acid sequences, whereas the remaining 1740 (59%) showed no significant similarities to other genes. Clustering analysis revealed particularly high expression of genes related to ATP synthesis, structural proteins, and proteases. The cDNA clones and EST sequence information should be useful for future functional analysis of daphnid biology and investigation of the links between ecology and genomics.     

The molecular ecologist's guide to expressed sequence tags

Genomics and bioinformatics have great potential to help address numerous topics in ecology and evolution. Expressed sequence tags (ESTs) can bridge genomics and molecular ecology because they can provide a means of accessing the gene space of almost any organism. We review how ESTs have been used in molecular ecology research in the last several years by providing sequence data for the design of molecular markers, genome-wide studies of gene expression and selection, the identification of candidate genes underlying adaptation, and the basis for studies of gene family and genome evolution. Given the tremendous recent advances in inexpensive sequencing technologies, we predict that molecular ecologists will increasingly be developing and using EST collections in the years to come. With this in mind, we close our review by discussing aspects of EST resource development of particular relevance for molecular ecologists.