Number Of Expressed Sequence Tags Research Articles

Initial Description of the Genome of Aeluropus littoralis, a Halophile Grass.

The use of wild plant species or their halophytic relatives has been considered in plant breeding programs to improve salt and drought tolerance in crop plants. Aeluropus littoralis serves as halophyte model for identification and isolation of novel stress adaptation genes. A. littoralis, a perennial monocot grass, grows in damp or arid areas, often salt-impregnated places and wasteland in cultivated areas, can survive periodically high water salinity, and tolerate high salt concentrations in the soil up to 1,100 mM sodium chloride. Therefore, it serves as valuable genetic resource to understand molecular mechanisms of stress-responses in monocots. The knowledge can potentially be used for improving tolerance to abiotic stresses in economically important crops. Several morphological, anatomical, ecological, and physiological traits of A. littoralis have been investigated so far. After watering with salt water the grass is able to excrete salt via its salt glands. Meanwhile, a number of ESTs (expressed sequence tag), genes and promoters induced by the salt and drought stresses were isolated, sequenced and annotated at a molecular level. Transfer of stress related genes to other species resulted in enhanced stress resistance. Here we describe the genome sequence and structure of A. littoralis analyzed by whole genome sequencing and histological analysis. The chromosome number was determined to be 20 (2n = 2x = 20). The genome size was calculated to be 354 Mb. This genomic information provided here, will support the functional investigation and application of novel genes improving salt stress resistance in crop plants. The utility of the sequence information is exemplified by the analysis of the DREB-transcription factor family.

Drought Stress Mitigating Morphological, Physiological, Biochemical, and Molecular Responses of Guava (Psidium guajava L.) Cultivars.

Guava (Psidium guajava L.), a major fruit crop of the sub-tropical region, is facing a production decline due to drought stress. Morphophysiological responses to drought stress and underlying transcriptional regulations in guava are, largely, unknown. This study evaluated the drought stress tolerance of two guava cultivars, viz. “Gola” and “Surahi,” at morphological and physiological levels regulated differentially by ESTs (Expressed Sequence Tags). The treatments comprises three moisture regimes, viz. To = 100% (control), T1 = 75%, and T2 = 50% of field capacity. There was an overall decrease in both morphological and physiological attributes of studied guava cultivars in response to drought stress. Nonetheless, the water use efficiency of the “Surahi” cultivar increased (41.86%) speculating its higher drought tolerance based on enhanced peroxidase (402%) and catalase (170.21%) activities under 50% field capacity (T2). Moreover, higher proline and flavonoid contents reinforced drought stress retaliation of the “Surahi” cultivar. The differential expression of a significant number of ESTs in “Surahi” (234) as compared to “Gola” (117) cultivar, somehow, regulated its cellular, biological, and molecular functions to strengthen morphophysiological attributes against drought stress as indicated by the upregulation of ESTs related to peroxidase, sucrose synthase (SUS), alcohol dehydrogenase (ADH), and ubiquitin at morphological, biochemical, and physiological levels. In conclusion, the drought stress acclimation of pear-shaped guava cultivar “Surahi” is due to the increased activities of peroxidase (POD) and catalase (CAT) complimented by the upregulation of related ESTs.

New EST–SSR markers of Coffea arabica: transferability and application to studies of molecular characterization and genetic mapping

Microsatellite markers (SSR) have broad utility in genetic studies due to a high rate of polymorphisms, a codominant nature and multiallelism. EST–SSRs are markers derived from the expressed sequences of a genome and represent transcribed genes. Despite the importance of the genus Coffea, only a small number of EST–SSR markers are currently available. Thus, this study was designed to mine and develop a new set of EST–SSR markers from the Brazilian Coffee Genome Project. We investigated 130,792 expressed sequence tags (ESTs), from which 24,031 DNA sequences with microsatellites were identified. After stability and amplification testing, 101 new EST–SSR markers were developed and analyzed in different coffee species. The average rate of transferability was 88 %, showing that these markers are useful in genetic studies across the genus Coffea. Polymorphism levels and the degree of diversity were consistent with the evolutionary history of the species. All coffee genotypes were discriminated, even the C. arabica genotypes that have known narrow genetic basis. It was also possible to locate 14 EST–SSRs into different linkage groups of the C. arabica genetic map, which demonstrate that these markers can be useful in QTL mapping studies and in molecular-assisted selection.

Transcriptome Analysis of the Barley-Rhynchosporium secalis Interaction

Leaf scald caused by the infection of Rhynchosporium secalis, is a worldwide crop disease resulting in significant loss of barley yield. In this study, a systematic sequencing of expressed sequence tags (ESTs) was chosen to obtain a global picture of the assembly of genes involved in pathogenesis. To identify a large number of plant ESTs, which are induced at different time points, an amplified fragment length polymorphism (AFLP) display of complementary DNA (cDNA) was utilized. Transcriptional changes of 140 ESTs were observed, of which 19 have no previously described function. Functional annotation of the transcripts revealed a variety of infection-induced host genes encoding classical pathogenesis-related (PR) or genes that play a role in the signal transduction pathway. The expression analyses by a semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) revealed that Rar1 and Rpg4 are defense inducible genes, and were consistent with the cDNA-AFLP data in their expression patterns. Hence, the here presented transcriptomic approach provides novel global catalogue of genes not currently represented in the EST databases.

Characterization and comparison of EST-SSRs in Salix, Populus, and Eucalyptus

Simple sequence repeats (SSRs) are molecular markers with great potential for plant genetic studies. With the development of high-throughput sequencing techniques, transcriptome sequencing projects provide valuable resources of expressed sequence tags (ESTs) for developing SSRs. To investigate the variation of EST-SSRs in different trees, three large EST databases of genera Salix, Populus, and Eucalyptus were compared for EST-SSR characteristics. The SSR-containing ESTs took account of 4.3, 8.4, and 12.2 % of the total number of ESTs for Salix, Populus, and Eucalyptus, respectively. The correlation between the repeat number and the motif type was negative, while that of the repeat number with the total SSR length or the number of SSRs was positive. Tri-nucleotide repeats were the most abundant motif type for Salix (47.7 %) and Eucalyptus (44.5 %), whereas di-nucleotide was the most common for Populus (38.8 %). The AG/CT repeat was the most abundant motif detected in all these trees. The most abundant tri- and tetra-nucleotide repeat motifs were AAG/CTT and AAAG/CTTT, respectively, for both Salix and Populus, while those were CCG/CGG and AGCG/CGCT for Eucalyptus. The EST-SSRs tended to occur in A/T-rich regions, and their repeat motif length appeared to decrease in variation with repeat numbers. These results may provide a deep insight into the characteristics of EST-SSRs in Salix, Populus, and Eucalyptus.

Transcriptome analysis of mistletoe (Viscum album) haustorium development

The haustorium is a specific organ for penetration of host tissue in parasitic plants. To better understand the molecular mechanisms of the establishment of host-parasite relationships using gene expression profiles, early stage haustorium germination from mature mistletoe seeds was examined. We have generated a cDNA library that is an excellent source of expressed sequence tags (ESTs) specifically related to haustorium development and host penetration. We analyzed 4,771 ESTs derived from a mistletoe (Viscum album) haustorium cDNA library. Cluster analysis identified 727 contigs (23.88%) and 2,317 singletons (76.12%), yielding a total of 3,044 unique genes. Among these genes, 1,760 clones (57.8%) showed significant similarity to known genes; the remaining 1,284 clones were novel. Annotation of genes with Gene Ontology indicated that the most abundant ESTs reveals that defense against biotic / abiotic stresses, primary metabolic processes, cell wall loosening and modification is critical for haustorium development and establishment of the host-parasite connection. Especially, it is likely that xyloglucan endotransglycosylases (XETs), glucanase, expansins and other cell wall hydrolases cooperate in cell wall modification during the stages of host-parasite connection. Functional expression of full-length forms corresponding to target ESTs was applied to characterize haustorium genes. This EST information will help gene discovery and characterization and provide promising targets for genetic engineering. The EST analysis presented here represents the first reported transcriptome data for V. album var. coloratum. Although the number of ESTs analyzed may not be sufficient to completely elucidate the mechanism of the host-parasite connection, the integrated approaches reported here represent an essential step toward understanding haustorium development in parasitic plants and provide a valuable resource for defining molecular mechanisms in the host-parasite connection.

CDNA microarray analysis of disk abalone genes in gills and hemocytes after viral hemorrhagic septicemia virus (VHSV) challenge

A disk abalone Haliotis discus discus 4.2 K cDNA microarray was designed by selecting abalone expressed sequence tags (ESTs). Transcriptional profiles in gills and hemocytes were analyzed upon abalone challenged with viral hemorrhagic septicemia virus (VHSV) in order to select candidates for screening of immune response genes. Among the 4188 genes analyzed, 280 (6.6%) transcripts were changed their expression level in gills and hemocytes against VHSV challenge compared to control animals. Total of 88 and 65 genes were up-regulated in gills and hemocytes, respectively. These genes can be grouped under various immune-functional categories such as transcription factors (Krüppell-like factor; ETS-family transcription factor), inflammatory and apoptosis related genes (TNF superfamily members, Fas ligand), IFN regulatory proteins (IFN-44 like, interferon gamma-inducible lysosomal thiol reductase) and detoxification proteins (glutathione peroxidase). In contrast, 25 and 102 genes were shown down-regulation in gills and hemocytes, respectively. Among the differentially expressed transcripts, considerably higher numbers of ESTs were represented as either hypothetical (unknown) proteins or no GenBank match suggesting those may be novel genes associated with internal defense of abalone.

Comparative deep transcriptional profiling of four developing oilseeds

Transcriptome analysis based on deep expressed sequence tag (EST) sequencing allows quantitative comparisons of gene expression across multiple species. Using pyrosequencing, we generated over 7 million ESTs from four stages of developing seeds of Ricinus communis, Brassica napus, Euonymus alatus and Tropaeolum majus, which differ in their storage tissue for oil, their ability to photosynthesize and in the structure and content of their triacylglycerols (TAG). The larger number of ESTs in these 16 datasets provided reliable estimates of the expression of acyltransferases and other enzymes expressed at low levels. Analysis of EST levels from these oilseeds revealed both conserved and distinct species-specific expression patterns for genes involved in the synthesis of glycerolipids and their precursors. Independent of the species and tissue type, ESTs for core fatty acid synthesis enzymes maintained a conserved stoichiometry and a strong correlation in temporal profiles throughout seed development. However, ESTs associated with non-plastid enzymes of oil biosynthesis displayed dissimilar temporal patterns indicative of different regulation. The EST levels for several genes potentially involved in accumulation of unusual TAG structures were distinct. Comparison of expression of members from multi-gene families allowed the identification of specific isoforms with conserved function in oil biosynthesis. In all four oilseeds, ESTs for Rubisco were present, suggesting its possible role in carbon metabolism, irrespective of light availability. Together, these data provide a resource for use in comparative and functional genomics of diverse oilseeds. Expression data for more than 350 genes encoding enzymes and proteins involved in lipid metabolism are available at the ‘ARALIP’ website (http://aralip.plantbiology.msu.edu/).

The spectrum of low molecular weight alpha-amylase/protease inhibitor genes expressed in the US bread wheat cultivar Butte 86

BackgroundWheat grains accumulate a variety of low molecular weight proteins that are inhibitors of alpha-amylases and proteases and play an important protective role in the grain. These proteins have more balanced amino acid compositions than the major wheat gluten proteins and contribute important reserves for both seedling growth and human nutrition. The alpha-amylase/protease inhibitors also are of interest because they cause IgE-mediated occupational and food allergies and thereby impact human health.ResultsThe complement of genes encoding alpha-amylase/protease inhibitors expressed in the US bread wheat Butte 86 was characterized by analysis of expressed sequence tags (ESTs). Coding sequences for 19 distinct proteins were identified. These included two monomeric (WMAI), four dimeric (WDAI), and six tetrameric (WTAI) inhibitors of exogenous alpha-amylases, two inhibitors of endogenous alpha-amylases (WASI), four putative trypsin inhibitors (CMx and WTI), and one putative chymotrypsin inhibitor (WCI). A number of the encoded proteins were identical or very similar to proteins in the NCBI database. Sequences not reported previously included variants of WTAI-CM3, three CMx inhibitors and WTI. Within the WDAI group, two different genes encoded the same mature protein. Based on numbers of ESTs, transcripts for WTAI-CM3 Bu-1, WMAI Bu-1 and WTAI-CM16 Bu-1 were most abundant in Butte 86 developing grain. Coding sequences for 16 of the inhibitors were unequivocally associated with specific proteins identified by tandem mass spectrometry (MS/MS) in a previous proteomic analysis of milled white flour from Butte 86. Proteins corresponding to WDAI Bu-1/Bu-2, WMAI Bu-1 and the WTAI subunits CM2 Bu-1, CM3 Bu-1 and CM16 Bu-1 were accumulated to the highest levels in flour.ConclusionsInformation on the spectrum of alpha-amylase/protease inhibitor genes and proteins expressed in a single wheat cultivar is central to understanding the importance of these proteins in both plant defense mechanisms and human allergies and facilitates both breeding and biotechnology approaches for manipulating the composition of these proteins in plants.

Transcriptional interactions during barley susceptible genotype infection with Cochliobolus sativus

A systematic sequencing of expressed sequence tags (ESTs) was used to obtain a global picture of the assembly of barley genes differentially expressed during the hypersensitive reaction of a susceptible genotype in response to an incompatible Cochliobolus sativus pathovar. To identify a large number of plant ESTs, which are induced at different time points, an amplified fragment length polymorphism (AFLP) display of complementary DNA (cDNA) was ulilized. Significant transcriptional changes in the host plant occurred already 4 h post inoculation. Four hundred and fifty six ESTs have been generated, of which 17 (c. 53% up-regulated, 47% down-regulated) have no previously described function. On one hand, the majority of EST-annotations showed protein synthesis, but genes related to signal transduction pathway were also identified. This study provides novel global catalogue ofgene regulations involved in C. sativus-barley interaction not currently represented in EST databases.

EST analysis on adipose tissue of rainbow trout Oncorhynchus mykiss and tissue distribution of adiponectin

Although energy metabolism in mammals is critically regulated by adipokines from adipocytes, it is unclear whether this is the case in fish as well. In this study, over 30,000 expressed sequence tags (ESTs) were obtained from adipose tissue in rainbow trout Oncorhynchus mykiss peritoneal cavity and searched for genes possibly related to lipid metabolism. Large numbers of ESTs encoded digestive enzymes and hormones usually found in the pancreas in higher vertebrates, consistent with the fact that pancreatic cells are dispersed in the adipose tissue. Many ESTs encoded apolipoprotein C-I, fatty acid-binding proteins and lymphocyte G0/G1 switch protein 2, which function in lipid transport, fatty acid accumulation and adipocyte differentiation, respectively. None of the ESTs encoded adipokines. We therefore obtained a cDNA encoding adiponectin, an adipokine that regulates oxidation of glucose and lipids in peripheral tissues, using rainbow trout ESTs in the public database. Real-time RT-PCR analyses revealed that its transcript levels were high in muscle and quite low in adipose tissue. These results strongly suggest that adipocytes of rainbow trout and possibly other fish species, unlike those of mammals, are not involved in the production of adipokines.

EST analysis of functional genes associated with cell wall biosynthesis and modification in the secondary xylem of the yellow poplar (Liriodendron tulipifera) stem during early stage of tension wood formation

A cDNA library was constructed from secondary xylem in the stem of a 2-year-old yellow poplar after being bent for 6h with a 45° configuration to isolate genes related to cell wall modification during the early stages of tension wood formation. A total of 6,141 ESTs were sequenced to generate a database of 5,982 high-quality expressed sequence tags (ESTs). These sequences were clustered into 1,733 unigenes, including 822 contigs and 911 singletons. Homologs of the genes regulate many aspects of secondary xylem development, including those for primary and secondary metabolism, plant growth hormones, transcription factors, cell wall biosynthesis and modification, and stress responses. Although there were only 1,733 annotated ESTs (28.9%), the annotated ESTs obtained in this study provided sequences for a broad array of transcripts expressed in the stem upon mechanical bending, and the majority of them were the first representatives of their respective gene families in Liriodendron tulipifera. In the case of lignin, xylem-specific COMTs were identified and their expressions were significantly downregulated in the tension wood-forming tissues. Additionally, the majority of the auxin- and BR-related genes were downregulated significantly in response to mechanical bending treatment. Despite the small number of ESTs sequenced in this study, many genes that are relevant to cell wall biosynthesis and modification have been isolated. Expression analysis of selected genes allow us to identify the regulatory genes that may perform essential functions during the early stages of tension wood formation and associated cell wall modification.

Identification of differentially expressed genes in the citrus epiphytic-yeast Pichia guilliermondii during interaction with Penicillium digitatum

To gain insight into the antagonistic molecular mechanisms displayed by the biocontrol epiphytic-yeast Pichia guilliermodii (isolate LCBG-03), its patterns of gene expression were evaluated during direct and indirect induction with Penicillium digitatum EfiA, a well-known fungal phytopathogen during the postharvest processing of citrus. Assays of in vitro antagonistic activity indicated a strong inhibitory effect on fungal growth and spore germination by the yeast, even when yeast–fungus interaction was performed on a rich medium. Antagonist gene expression was evaluated in induced condition as well as in direct interaction using differential expressed sequence tags (ESTs) obtained by two methodologies; suppression subtractive hybridization (SSH) and differential display (DD). We obtained the genetic response of the yeast under three different specific metabolic conditions: starvation by carbon source competence, sensing of extracellular metabolites produced by active mycelium of P. digitatum (membrane system) and induction by fungal cell walls. As a result, we observed just one EST, associated as expected to energy metabolism in starvation conditions; for the membrane system, seven ESTs were obtained by the SSH methodology, all related with some of the following metabolic networks: energy, nitrogen, cell cycle, ABC transporters, response to stress and one unknown sequence. The induced (fungal cell walls) system produced the highest number of ESTs, with a total of 22, including all the metabolic networks mentioned above for the membrane system plus ESTs associated with signal transduction.

Next‐generation sequencing for comparative transcriptomics of perennial ryegrass (Lolium perenne L.) and meadow fescue (Festuca pratensis Huds.) during cold acclimation

AbstractPerennial ryegrass (PR; Lolium perenne L.) and meadow fescue (MF; Festuca pratensis Huds.) are important grass species for forage production around the world. These species are genetically related but show different responses to biotic and abiotic stresses such as freezing: PR is severely damaged by cold stress, but MF is not. Recently, next‐generation sequencing technology (e.g. 454 sequencing) has been used to profile the genome‐wide transcriptomics of various plant species. To clarify the genetic differences behind the different cold responses of MF and PR, we developed an effective method for comparing transcriptome data during cold acclimation. The expressed sequence tags (ESTs) from cold‐acclimated MF and PR were obtained by means of minimum‐scale 454 sequencing runs. After trimming based on quality scores, these redundant ESTs were assembled into a total of 1527 contigs and singletons for MF and 1060 for PR. Subsequently, we clustered the contigs and singletons into 1082 (MF) and 661 (PR) clusters based on sequence similarity, and we used the EST numbers for the corresponding clusters to compare levels of gene expression. We observed four clusters (wheat germ agglutinin isolectin, γ‐thionin, jasmonate‐induced protein homolog and germin) with markedly different numbers of ESTs between MF and PR. The species‐specific expression of genes corresponding to these four clusters was confirmed by means of reverse‐transcriptase polymerase chain reaction analysis.

Transcriptome analysis of the oil-rich seed of the bioenergy crop Jatropha curcas L

BackgroundTo date, oil-rich plants are the main source of biodiesel products. Because concerns have been voiced about the impact of oil-crop cultivation on the price of food commodities, the interest in oil plants not used for food production and amenable to cultivation on non-agricultural land has soared. As a non-food, drought-resistant and oil-rich crop, Jatropha curcas L. fulfils many of the requirements for biofuel production.ResultsWe have generated 13,249 expressed sequence tags (ESTs) from developing and germinating Jatropha seeds. This strategy allowed us to detect most known genes related to lipid synthesis and degradation. We have also identified ESTs coding for proteins that may be involved in the toxicity of Jatropha seeds. Another unexpected finding is the high number of ESTs containing transposable element-related sequences in the developing seed library (800) when contrasted with those found in the germinating seed library (80).ConclusionsThe sequences generated in this work represent a considerable increase in the number of sequences deposited in public databases. These results can be used to produce genetically improved varieties of Jatropha with increased oil yields, different oil compositions and better agronomic characteristics.

Integration of transcriptomic and proteomic data from a single wheat cultivar provides new tools for understanding the roles of individual alpha gliadin proteins in flour quality and celiac disease

The complement of alpha gliadins expressed in a single wheat cultivar was examined by assembling expressed sequence tags (ESTs) from Triticum aestivum cv. Butte 86. Twelve of 19 resulting contigs encoded complete proteins, but only two were identical to proteins reported previously. Eight contained various combinations of epitopes important in celiac disease (CD), while four lacked typical CD epitopes. One alpha gliadin contained an additional cysteine residue that could allow incorporation of the protein into glutenin polymers. In addition, two new types of alpha gliadins ending in GFFGTN and GIMSTN were identified. Based on the number of ESTs, genes encoding proteins that contained the greatest numbers of CD epitopes were expressed at the highest levels. Proteins corresponding to 12 contigs were distinguished in Butte 86 flour by tandem mass spectrometry (MS/MS). Unique peptide tags for individual alpha gliadins, including some that lack CD epitopes and the protein containing the extra cysteine, are reported. The ability to distinguish closely related alpha gliadins by MS/MS makes it possible to explore the roles of individual proteins in flour quality, better define relationships between specific proteins and celiac disease, and devise strategies to reduce the immunogenic potential of wheat flour for patients with CD.

Pyrosequencing the Manduca sexta larval midgut transcriptome: messages for digestion, detoxification and defence

The tobacco hornworm Manduca sexta is an important model for insect physiology but genomic and transcriptomic data are currently lacking. Following a recent pyrosequencing study generating immune related expressed sequence tags (ESTs), here we use this new technology to define the M. sexta larval midgut transcriptome. We generated over 387,000 midgut ESTs, using a combination of Sanger and 454 sequencing, and classified predicted proteins into those involved in digestion, detoxification and immunity. In many cases the depth of 454 pyrosequencing coverage allowed us to define the entire cDNA sequence of a particular gene. Many new M. sexta genes are described including up to 36 new cytochrome P450s, some of which have been implicated in the metabolism of host plant-derived nicotine. New lepidopteran gene families such as the beta-fructofuranosidases, previously thought to be restricted to Bombyx mori, are also described. An unexpectedly high number of ESTs were involved in immunity, for example 39 contigs encoding serpins, and the increasingly appreciated role of the midgut in insect immunity is discussed. Similar studies of other tissues will allow for a tissue by tissue description of the M. sexta transcriptome and will form an essential complimentary step on the road to genome sequencing and annotation.

A novel expression profile of the Loxosceles intermedia spider venomous gland revealed by transcriptome analysis

Spiders of the Loxosceles genus are cosmopolitan, and their venom components possess remarkable biological properties associated with their ability to act upon different molecules and receptors. Accidents with Loxosceles intermedia specimens are recognized as a public health problem in the south of Brazil. To describe the transcriptional profile of the L. intermedia venom gland, we generated a wide cDNA library, and its transcripts were functionally and structurally analyzed. After initial analyses, 1843 expressed sequence tags (ESTs) produced readable sequences that were grouped into 538 clusters, 281 of which were singletons. 985 reads (53% of total ESTs) matched to known proteins. Similarity searches showed that toxin-encoding transcripts account for 43% of the total library and comprise a great number of ESTs. The most frequent toxins were from the LiTx family, which are known for their insecticidal activity. Both phospholipase D and astacin-like metalloproteases toxins account for approximately 9% of total transcripts. Toxins components such as serine proteases, hyaluronidases and venom allergens were also found but with minor representation. Almost 10% of the ESTs encode for proteins involved in cellular processes. These data provide an important overview of the L. intermedia venom gland expression scenario and revealed significant differences from profiles of other spiders from the Loxosceles genus. Furthermore, our results also confirm that this venom constitutes an amazing source of novel compounds with potential agrochemical, industrial and pharmacological applications.

Identification of enzyme genes in the liver of the Bleeker’s squid Loligo bleekeri by expressed sequence tag analysis

Expressed sequence tag (EST) analyses were performed with the aim of identifying enzyme genes in the liver of the Bleeker’s squid Loligo bleekeri. Of the 768 ESTs identified and sequenced, 669 were grouped into 324 clusters. Of these clusters, 123 comprising 245 ESTs were found to be homologous to genes reported to date. Among these, 43 clusters were annotated as enzymes according to the Enzyme Commission (EC) numbering system. Two EC groups, oxidoreductases and hydrolases, possessed a large number of ESTs. A cluster homologous to the glutathione peroxidase, an enzyme in the oxidoreductase group, contained 16 ESTs, which accounted for 2.4% of the total ESTs sequenced. There are three serine proteases, three cathepsins, two triacylgricerol lipases, and two chitinases among the clusters homologous to the enzymes in the hydrolase group. Since the squid liver functions in the digestive process, these enzymes would be involved in food digestion. Our data provide information on the various types of enzymes expressed in the squid liver and may provide a useful basis for further characterization of these enzymes.

Differential gene expression profiles in the venom gland/sac of Orancistrocerus drewseni (Hymenoptera: Eumenidae)

To determine differential gene expression profiles in the venom gland and sac (gland/sac) of a solitary hunting wasp species, Orancistrocerus drewseni Saussure (1857), a subtractive cDNA library was constructed by suppression subtractive hybridization. A total of 498 expressed sequence tags (EST) were clustered and assembled into 205 contigs (94 multiple sequences and 111 singletons). About 65% (134) of the contigs had matched BLASTx hits (E< or =10(-4)). Among these, 115 contigs had similarity to proteins with assigned molecular function in the Gene Ontology database, and most of them (112 contigs, 83%) were homologous to genes from Hymenoptera, particularly to Apis mellifera (98 contigs). The contigs encoding hyaluronidase and phospholipase A2, known to be main components of wasp venoms, were found in high frequencies (27 and 4%, respectively, as judged by the number of ESTs) in the gene ontology category of catalytic activity. Full-length open reading frames of hyaluronidase and phospholipase A2 were characterized and their abundance in the venom gland/sac was confirmed by quantitative real-time PCR. Several contigs encoding enzymes, including zinc-metallopeptidases that are likely involved in the processing and activation of venomous proteins or peptides, were also identified from the library. Discovery of venom gland/sac-specific genes should promote further studies on biologically active components in the venom of O. drewseni.