Integrative and Comparative Biology Advance Access originally published online on May 17, 2006
Integrative and Comparative Biology 2006 46(6):931-939; doi:10.1093/icb/icl006
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Analysis of multiple tissue-specific cDNA libraries from the Pacific whiteleg shrimp, Litopenaeus vannamei
* Medical University of South Carolina, Department of Biochemistry and Molecular Biology Charleston, SC 29425, USA
Marine Biomedicine and Environmental Sciences Center, Medical University of South Carolina Charleston SC 29412, USA
Correspondence: 1E-mail: grossp{at}musc.edu
 |
Synopsis |
|---|
Top
 Synopsis IntroductionMaterials and methodsResults and discussionConcluding remarksREFERENCES |
|---|
Multiple small-scale transcriptome studies have been undertaken for various members of the Penaeidae. Penaeid shrimp are important both as members of diverse ecosystems around the world and for their importance as commercial commodities. Of the many shrimps, the most important from this family is the Pacific whiteleg shrimp, Litopenaeus vannamei, as it is the primary shrimp used in worldwide aquaculture. The sequencing and analysis of 13 656 expressed sequence tags (ESTs) from this species is presented. ESTs were derived from multiple tissue-specific cDNA libraries with an emphasis being placed on those tissues with predicted immune function. Assembly of the sequences into non-overlapping clusters yielded 7466 putative unigenes (1981 contigs and 5485 singletons). Multiple approaches were taken to assign putative function to each transcript; sequence homology searches using BLASTX (Basic Local Alignment Search Tool: Translated query versus protein database) of the National Center for Biotechnology Information's (NCBI) GenBank Database and Gene Ontology annotation, and still a significant portion of the shrimp ESTs (62%) had no homology with known proteins in the public databases. The sequence and complete annotation of all ESTs is available at www.marinegenomics.org, a publicly accessible database. In addition to providing the basic resources for microarray construction, transcript profiling, and novel gene discovery, this study constitutes the largest combined analysis of ESTs from any shrimp species and is a prelude to an even larger effort aimed at identifying and depleting highly redundant genes from shrimp cDNA libraries toward the goal of sequencing 100 000 shrimp ESTs.
|
Introduction |
|---|
|
Top
SynopsisIntroductionMaterials and methodsResults and discussionConcluding remarksREFERENCES |
|---|
The conversion of expressed RNA into cDNA libraries gives a "snapshot" of gene expression from a cell, cell line, or tissue and has been especially useful in discerning the functional status of that cell line or tissue, and has led to the development of the field of functional genomics or transcriptomics. These "expressed sequence tag (EST)" libraries have provided a particularly fertile ground for the discovery of new genes, novel alleles, and polymorphisms, which has contributed to comparative genomics and genetics. This is particularly true when the organism being studied is not an established model with a deep molecular-based research history. Thus cDNA library exploitation remains a viable and useful research tool for the rapid accumulation of molecular data for novel model organisms. While a great deal of attention has been paid over the years to developing certain insect (Hexapoda) models, especially Drosophila and Anopheles as exemplars of the arthropod lineage, much less attention has been focused on other members of the Pancrustacea grouping, including the Crustacea.
Transcriptomics in Crustacea
Relatively few comprehensive projects have been undertaken to define a representative transcriptome of any crustacean; however, several smaller, and a few ambitious projects are being undertaken. Most notable among the more ambitious projects are those being undertaken by or in cooperation with the Joint Genome Institute (JGI) at the United States Department of Energy. These include 2 projects sponsored directly by the Community Sequencing Project at JGI. Both projects involve developing large databases of ESTs, the first will be for 2 gammarid amphipod species, Parhyale hawaiensis and Jassa slatteryi (see: www.jgi.doe.gov/sequencing/why/CSP2006/crustaceans.html) and the second will be for the flat porcelain crab, Petrolisthes cinctipes, (see: www.jgi.doe.gov/sequencing/why/CSP2006/porcelaincrab.html). These projects are still pending at JGI as they were selected for EST-sequence development for 2006. A more mature, but equally ambitious, project has been undertaken by the Daphnia Genomics Consortium to generate a large EST core for 2 species of water fleas, Daphnia magna (Watanabe and others 2005
) and D. pulex. This project seeks to develop a suite of genomics tools for these 2 branchiopod species, including full genome sequencing (see: daphnia.cgb.indiana.edu/) and much of the data accumulated has already been made available in a Daphnia genome database under the name wFleaBase (Colbourne and others 2005).
Several smaller transcriptome analyses of various crustaceans have been reported in the literature, most notable of which are analyses of small EST collections from the harpacticoid copepod, Tigriopus japonicus (Lee and others 2005) and Artemia franciscana (Chen and others 2003). The latter study, using A. franciscana, exemplifies the utility of cDNA library construction for the purposes of gene fishing, where the use of the library was not for creating a large catalog of genes but to capture a specific set of ESTs for particular genes of interest, artemin and ferritin, such that the full sequence of these expressed genes could be elucidated as a starting point for their further study (Chen and others 2003). The study of T. japonicus demonstrates the direct utility of the cDNA library, where the sequencing of 686 randomly selected ESTs yielded a number of potential candidate expressed genes that may be of future importance in ecotoxicological studies (Lee and others 2005).
Transcriptomics in Penaeidae
Among Crustacea, by far the greatest number of separate small-scale transcriptome analyses has been undertaken using various members of the Penaeidae. The penaeid shrimp make for an interesting assemblage of species whose members have a quite varied natural history and whose evolutionary relationship in terms of speciation events is widely dispersed throughout time. Some clades separated only recently while other nodes are very ancient. The group shares an important commonality that gives them special importance; virtually all members are of commercial importance as a food commodity. As an aggregate, shrimp are among the largest fisheries industries in the world. The total value of the United States consumer seafood market topped $61 billion in 2003 and shrimp consumption alone made up over 15% of that demand, making it the most eaten seafood in the United States (Johnson 2004). Worldwide wild-capture of shrimp has steadily increased over the past 10 years, reaching an all-time high of over 3 million metric tons in 2000, while world aquaculture of shrimp has been steadily growing over the same period reaching close to 1.3 million metric tons in 2002 (Johnson 2004). Research into the genetics and genomics of shrimp has been gaining in importance over the past decade and the number of small-scale transcriptome projects has followed this trend, with each of the commercially important species receiving some attention. An earliest project focused on the black tiger shrimp, Penaeus monodon (Lehnert and others 1999). That study encompassed sequencing a total of 151 ESTs from 3 tissue-specific cDNA libraries: cephalothorax, eyestalk, and pleopod, and yielded 60 new genes previously unidentified in P. monodon with 49 of these being wholly new to crustaceans. A later study reported on the sequencing of 615 ESTs from a P. monodon hemocyte cDNA library from which several immune-effector genes were identified, 3 of which (2 putative antimicrobial peptides and 1 heat shock protein) were fully sequenced (Supungul and others 2002). A somewhat larger study was published comparing tissue-specific cDNA libraries from 2 tissues, hepatopancreas and hemocytes, from 2 related species of shrimp, the Pacific whiteleg shrimp, Litopenaeus vannamei and the Atlantic white shrimp, Litopenaeus setiferus (Gross and others 2001). In that study, a total of 2045 ESTs were sequenced and comparisons were made with respect to functional differences between tissues. Emphasis was placed on identification of immune-function genes. Sequences from that study were among the first to be deposited in the web-based marine genomics database (www.marinegenomics.org), a clearing-house for genomic and transcriptomic data of marine organisms (McKillen and others 2005). Sequences from this database, combined with ESTs generated de novo from L. stylirostrus and Trachypenaeus birdy were subsequently used to generate a comprehensive set of simple sequence repeat markers across species (Perez and others 2005). More recently, the analysis of cDNA libraries from the eyestalk of the Kuruma prawn, Marsupenaeus japonicus, has been used to examine the expression profile with special emphasis on endocrine control of reproduction. Sequencing of some 1988 ESTs yielded 136 contigs and 1250 singletons of which only 16.7% were found to have significant similarity to previously identified genes (Yamano and Unuma 2006).
Comparative studies examining gene expression in specific tissues of disease-stressed shrimp relative to apparently healthy animals have been used to identify transcripts involved in immune defense. One study used cDNA libraries generated from hemocytes obtained from white spot syndrome virus (WSSV) infected animals and apparently healthy animals and sequenced 635 clones from the normal hemocyte library and 370 clones from the WSSV hemocyte cDNA library. Putative defense genes accounted for 2.7% of EST sequenced from normal animals and 15.7% from WSSV-infected animals (Rojtinnakorn and others 2002). A second study used cDNA libraries constructed from hemocytes of P. monodon infected with Vibrio harveyi and from healthy individuals to identify immune-function genes, resulting in the identification of several homologs of antimicrobial peptides conserved among shrimp (Supungul and others 2004).
It is clear that shrimp have been a major focus for small-scale transcriptomic analyses and several species have been utilized for these studies, all of which have relevance owing in part to their importance as a commercial commodity. In addition, these studies contribute to the understanding of comparative genomics in crustaceans and in arthropods in general. Presented here is a new methodology for cDNA library depletion for the reduction of redundancy within cDNA libraries with the goal of enhancement of rare-gene recovery. This methodology is currently being employed to further enhance the utility of the constructed cDNA libraries analyzed in this study. Furthermore, the data presented here constitute the largest transcriptomic analysis of multiple L. vannamei cDNA libraries derived from several tissues. This includes the collection of 7896 new ESTs from 6 tissues, namely hemocyte, hepatopancreas, gill, lymphoid organ, eyestalk, and ventral nerve cord. Characterization of these new sequences coupled with an integrated analysis of ESTs previously collected from 34 libraries from L. vannamei provides new insights into the transcriptome of shrimp.
|
Materials and methods |
|---|
|
Top
SynopsisIntroductionMaterials and methodsResults and discussionConcluding remarksREFERENCES |
|---|
Tissue collection and RNA isolation
The following tissues were dissected from the Pacific whiteleg shrimp, L. vannamei: hemolymph, gill, hepatopancreas, lymphoid organ, eyestalk, and ventral nerve cord. Hemocytes were isolated from hemolymph by centrifugation. Total RNA was isolated from each tissue using the RNeasy® Miniprep kits (Qiagen, Valencia, CA). Genomic DNA was removed from the RNA preparation by in-column digestion using RNase-free DNase kit (Qiagen®) following the manufacturer's instructions.
cDNA library construction
cDNA libraries were constructed using the SMARTTM cDNA Library Construction Kit (Clontech Laboratories, Palo Alto, CA) according to the manufacturer's instructions. This protocol uses PCR-based methods for cDNA amplification. The amplified and size-fractionated cDNA is ligated into the 
TriplEx2 vector arms and packaged using the Gigapack® Gold (Stratagene, LaJolla, CA) packaging system. For each cDNA library constructed, the TriplEx2 phage constructs were converted to pTriplEx plasmids by mass conversion. The titer of each library was assessed and aliquots of the phage extract containing 
60 000 clones were inoculated into 500 µl of log-phase Escherichia coli, strain BM25.8, and grown at room temperature for 30 min. The culture was then supplemented with 3 ml of LB medium containing 150 µg/ml carbenicillin and grown for 1 h at 31°C with constant shaking. The converted library was either stored in 10% glycerol at –80°C or directly plated for arraying and picking of individual colonies. Subtracted libraries were prepared using the PCR Select cDNA Subtractive Hybridization Kit (Clontech) according to the manufacturer's instructions.
Sequencing of expressed sequence tags (ESTs)
Randomly selected clones from each of the 6 newly constructed cDNA libraries were sequenced either by SeqWright (Houston, TX) or Rexagen (Seattle, WA). All ESTs were sequenced from the 5'-end with the Clontech® pTriplEx2 forward primer (5'-AGCTCCGAGATCTGGACGAGC-3').
Bioinformatic analysis
Sequences were analyzed with the EST analysis pipeline hosted at www.marinegenomics.org (McKillen and others 2005). In brief, EST sequences are extracted from the raw data by filtering out sequences corresponding to vector, poly-A tail, and eliminating any sequence of poor quality. The trimmed sequences then undergo a BLAST against National Center for Biotechnology Information's (NCBI's) GenBank using the BLAST tool (Altschul and others 1990). GenBank databases currently used include the GenBank non-redundant CDS translations protein databases (nr) with BLASTX, the GenBank non-redundant nucleotide databases (nt) with BLASTN, and the GenBank EST databases with BLASTN (dbEST). The sequences also undergo BLAST against all local Marine Genomics databases. Sequences are annotated with Gene Ontology (GO) terms by searching the sequences against the GO database using BLAST (Ashburner and others 2000; Stevens and others 2000). Species-specific GO analyses are currently reported in a pie-chart on each species-entry page and also within each EST-sequence page.
Arraying of cDNA libraries-colony picking and gridding
LB agar containing 50 µg/ml carbenicillin was poured into sterile 22.5 cm2 QTrays (Genetix, Hampshire, UK). The pTriplEx2 libraries were plated onto each tray at a density of 3000–4000 cfu in 500 µl LB media containing 50 µg/ml carbenicillin and grown overnight at 31°C. Individual clones were picked using the Genetix QBot, with a 96 pin (1 mm pins) picking head into 96 well plates containing 150 µl LB media/7.5% glycerol with 50 µg/ml carbenicillin and incubated for 16–18 h at 31°C with agitation. After incubation, clones were transferred to 384 well plates using the QBot.
For the gridding, bacterial clones were spotted onto Performa II positively charged nylon membranes measuring 22.2 cm x 22.2 cm (Genetix, Hampshire, UK) using the QBot. Clones were spotted (9216/membrane) using the 384-Pin Spring-Loaded Gridding Head with 0.25 mm pins. The membranes were transferred to QTrays containing LB agar with 50 µg/ml carbenicillin for overnight incubation at 31°C. After incubation, the membranes were treated with denaturing solution (0.5 M NaOH/ 1.5 M NaCl in water) for 5 min, neutralizing solution (1 M Tris/1.5 M NaCl in water, pH 7.5) for 5 min, then 2x SSC for 5 min. The membranes were then allowed to dry overnight at room temperature and exposed to 120 mJ/cm2 UV light to crosslink the DNA.
Probing cDNA libraries for redundant genes
Prior to hybridization the membranes were washed in a solution of 5x SSC/0.5% SDS/1 mM EDTA, pH 8.0 in water to remove excess cell debris. The membranes were then placed in hybridization solution (6x SSC/50% deionized formamide/0.5% SDS/ 1% Blocking solution in water) at 42°C overnight. The membranes were hybridized using digoxigenin (DIG) labeled probes representing the most highly redundant genes detected from initial sequencing and detected using anti-digoxigenin AP and CSPD according to a protocol adapted from Roche Diagnostics. Probes were labeled with alkali-labile DIG-dUTP (Roche Diagnostics, Indianapolis, IN) by PCR and then added to fresh hybridization solution for overnight incubation at 42°C. After hybridization the membranes were washed twice in 2x SSC/0.1% SDS for 5 min at room temperature with gentle rocking. Next, they were washed twice in (pre-heated) 0.2x SSC/0.1% SDS for 15 min at 65°C with gentle rocking then washed in wash buffer (0.1 M maleic acid/0.15 M NaCl in water, pH 7.5 containing 0.3% Tween-20) for 5 min at room temperature. The membranes were then blocked in 1x maleic acid buffer containing 1% blocking solution (Blocking Reagent, Roche Diagnostics, in maleic acid buffer) for 30 min at room temperature with gentle rocking. After blocking, anti-DIG-AP (Roche Diagnostics, Indianapolis, IN) was added in fresh blocking buffer, diluted 1:10 000, and incubated for 30 min at room temperature. Membranes were then washed twice in wash buffer for 15 min at room temperature and twice in detection buffer (100 mM Tris/100 mM NaCl in water, pH 9.5) for 5 min at room temperature. Then the membranes were treated with 3 ml of a 1:100 dilution of CSPD (Roche Diagnostics, Indianapolis) in detection buffer. The membranes were sealed in hybridization bags and gently massaged for 5 min at room temperature. Excess CSPD solution was removed and the membranes were placed at 37°C for 15 min. Finally, the membranes were exposed, colony side up, to Kodak BioMax Light (Kodak, Rochester, NY) film at room temperature from 30 min to overnight, depending on the signal intensity. The film was developed using the Kodak X-OMAT 1000A processor.
|
Results and discussion |
|---|
|
Top
SynopsisIntroductionMaterials and methodsResults and discussionConcluding remarksREFERENCES |
|---|
EST sequencing and assembly
A total of 13 656 ESTs were sequenced from multiple cDNA libraries made from 6 different tissues from the Pacific white shrimp L. vannamei. Of these sequences, 7896 were randomly selected from non-normalized cDNA libraries made from the following tissues; hemocyte, hepatopancreas, gill, lymphoid organ, eyestalk, and ventral nerve cord. The remaining 5760 sequences were randomly selected from 34 different suppression subtractive hybridization (SSH) cDNA libraries (hemocyte, gill, and hepatopancreas) specifically designed to enrich for immune-related genes (Table 1). A detailed description of these subtracted libraries will be reported elsewhere, but in brief they were generated from shrimp induced with WSSV, dsRNA, and inactivated microbes. To determine the number of unique genes or unigenes, the raw sequences were first trimmed to remove vector and Poly-A sequence, and then assembled into contiguous overlapping sequence alignments (contigs). This analysis identified 7466 unigenes represented by 1981 contigs and 5485 singletons (50%). The proportion of unigenes found in the entire EST collection that are represented by singletons suggests a high rate of discovery of novel genes. It should be noted, however, that the number of unigenes may have been overestimated. First, many of the sequences are from short transcripts (300–400 bp) derived from subtracted libraries. Second, all sequences were single-pass reads from the 5'-end and therefore many "unique" sequences may actually be non-overlapping regions of the same gene. All ESTs were compiled into the L. vannamei EST database at the Marine Genomics Project website, www.marinegenomics.org, where complete information on each transcript is available including tissue/library origin, contig analysis, and functional annotations (BLASTX, BLASTN, and GO).
|
|
Functional annotation
To assign putative function to each EST, the sequences were submitted for comparison with protein sequences of known genes in the public databases at NCBI (www.ncbi.nlm.nih.gov). Using a BLASTX E-value cutoff of
1 x 10–4, 5162 sequences (38%) matched known sequences within the NCBI protein database. Of the remaining 8494 ESTs (62%), 4407 had low similarity matches (E-value 
1 x 10–4), and 4087 had no similarity to any known gene (Table 2). The percentage of shrimp ESTs with no match to any known protein is high (62%) but compares with EST collections in other crustaceans (Lee and others 2005; Yamano and Unuma 2006). The proportion of singletons from each library (40–50%) suggests that some genes are enriched in a tissue-specific manner. Further functional annotation was assigned to a subset of ESTs with significant BLASTX scores using the GO database (www.geneontology.org). The GO method classifies genes within a hierarchy using a systematic nomenclature of attributes that can be assigned to all gene products. Of the 5162 ESTs with significant BLASTX scores, 3821 could be annotated within the GO hierarchy (Fig. 1). According to the GO classification scheme more than a single attribute can be assigned to a particular gene and indeed many of the shrimp genes mapped to more than 1 of the 3 major GO categories (molecular functions, biological processes, and cellular components). A more detailed look at the breakdown of shrimp ESTs within the whole GO hierarchy reveals a diverse array of functions including metabolic enzymes, structural proteins, and binding proteins. Similar to the BLASTX results, a comparison of the GO annotations across tissues reveals similar profiles of common housekeeping genes along with tissue-specific gene-expression profiles. For example transcripts with homology to known immune genes were more abundant in the hemocyte, gill, and lymphoid organ tissues whereas transcripts involved in homeostasis and metabolism were more abundant in the hepatopancreas. The complete GO mapping for the shrimp ESTs can be accessed at www.marinegenomics.org.
|
Immune gene expression
As previously mentioned, the majority of ESTs in this collection were selected from tissues of expected immune function. This particular focus on immune tissues is designed to facilitate our ongoing research identifying the molecular components that control the shrimp immune response. Nearly 40% of the ESTs were derived from hemocytes, the shrimp blood cells, which are hypothesized to be the primary immune cells of shrimp, and experimental data have shown that hemocytes are involved in phagocytosis, encapsulation, and the synthesis and secretion of antimicrobial peptides (Bachere and others 2004
). Other tissues including gill, hepatopancreas, and lymphoid organ were also examined for immune genes because these tissues are reported to be major sites of bacterial accumulation during infection and are often infiltrated with hemocytes (van de Braak and others 2002; Burgents and others 2003). As previously reported, antimicrobial peptides (AMPs) such as the penaeidins and the crustins (Gross and others 2001) were abundantly expressed in the hemocytes. Transcripts with homology to protease inhibitors, coagulation factors, lysozyme, and heat shock proteins were also identified in the hemocytes (Table 2). A single transcript with homology to the Drosophila immune deficiency (imd) gene, which is a central component of the signal transduction pathway controlling AMP gene expression in flies, was identified in the lymphoid organ library. Anti-LPS factor (ALF) was identified in the hemocytes, gill, and lymphoid organ libraries.
Methodology for enhancing novel gene discovery and future EST sequencing
Comprehensive characterization of the transcriptome of any organism requires large-scale sequencing of ESTs (100 000) from multiple tissues. Toward the goal of characterizing the shrimp transcriptome an effort is underway in our lab to sequence 100 000 shrimp ESTs (100 000 transcripts from both 3' and 5' ends). An important issue to consider before sequencing a large collection of ESTs is how to maintain a high rate of discovery of novel genes. If the complexity of an EST library is low, rare transcripts may not present and there is an increased risk of repeatedly sequencing the same gene. Even if the EST library is complex, a few genes may be overrepresented and the chances of selecting rare transcripts may be hampered by the presence of redundant sequences. In order to gain access to the less abundant components of the shrimp transcriptome an approach was devised that relies on high-throughput selection of novel sequences, which relies on the observation that expressed gene redundancy in unmodified cDNA libraries tends to be steeply Poisson distributed (Gross and others 2001). Thus removal of even only the top few highest redundancy genes will shift random EST selection toward less common genes. For this approach, 60 000 clones were picked from each of the 6 tissue-specific libraries (described above) and arrayed onto nylon membranes. Using the contig analysis of the sequenced ESTs from a pilot sequencing project of 384 clones per library, the most highly redundant genes are selected from each EST library. These selected ESTs are then used to probe the arrayed clones from their respective EST libraries. Positively hybridizing clones are then depleted from the library by "cherry-picking" the non-hybridizing clones, creating a new "depleted" EST library that can be used for large-scale sequencing (Fig. 2).
|
Using this depletion method, the unbiased (original) shrimp hemocyte cDNA library was probed with the 5 most highly redundant genes, which were selected based on contig analysis to identify highly redundant sequences obtained from an initial pilot EST sequencing from this shrimp hemocyte cDNA library. Genes identified as highly redundant were actin, myosin, cuticle protein-DD9B, and cuticle protein-BCS1 (identified at www.marinegenomics.org as MGID# 10298, 10435, 10538, 10284). These ESTs accounted for
12% of the sequences recovered from the hemocyte cDNA library on the first pass. Extrapolating this value to the cDNA library as a whole, it is expected that 12% of all clones spotted on the membrane should be positive for 1 of these 4 ESTs. When 30 000 arrayed hemocyte clones were probed with these 4 genes 9% of the clones hybridized. Control cross-referencing of the hybridized spots that came from clones already sequenced in the initial EST collection indicated that probe-positive sequences on the membrane were indeed those selected for probing. |
Concluding remarks |
|---|
|
Top
SynopsisIntroductionMaterials and methodsResults and discussionConcluding remarksREFERENCES |
|---|
Transcriptomic analysis, especially large-scale sequencing and analysis of ESTs, remains an essential tool for conducting functional genomic research, including microarray construction, novel gene discovery, and transcript profiling. Collections of ESTs also provide the basis for assembling future genomic projects, by facilitating annotation of coding regions, and providing functional context to genomic sequence. The EST collection presented in this study is the most comprehensive to date for the economically important penaeid shrimp and serves as a valuable resource for the crustacean research community. For example, the L. vannamei database at www.marinegenomics.org has been mined by other research groups to identify microsatellite markers that should be useful in breeding programs and other genetic studies (Perez and others 2005
). A selection of genes from the shrimp EST collection presented here has been used to construct a shrimp cDNA microarray, which will serve as a powerful tool for conducting transcriptomic analysis in shrimp (Chapman and others, this issue). This EST collection has allowed the identification of target genes for future functional studies; of particular interest to our laboratory are those genes that may be involved in the shrimp immune response. The EST collection reported in this study is expected to assist a larger shrimp EST project that will include the sequencing of 100 000 ESTs from both the 5'- and 3'-ends (for a total of 200 000 sequences). The current EST collection is being used to identify and deplete, in a high-throughput fashion, the most redundant genes from each cDNA library thus increasing the chances of novel gene discovery in the larger sequencing project.
|
Acknowledgements |
|---|
This research was funded by the National Science Foundation (MCB0516279) and the United States Department of Agriculture (NRICGP-CSREES/AREA 2004-35205-14219 and NRICGP-CSREES 2005-35205-15459). Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the supporting bodies mentioned herein. This is publication #25 for the Marine Genomics Program of the Marine Biomedicine and Environmental Sciences program at MUSC.
Conflict of interest: None declared.
|
Footnotes |
|---|
From the symposium "Genomic and Proteomic Approaches in Crustacean Biology" presented at the annual meeting of the Society for Integrative and Comparative Biology, January 4–8, 2006, at Orlando, Florida.
|
REFERENCES |
|---|
|
Top
SynopsisIntroductionMaterials and methodsResults and discussionConcluding remarksREFERENCES |
|---|
Altschul, SF, W Gish, W Miller, EW Myers, DJ Lipman. 1990. Basic local alignment search tool. J Mol Biol 215:(3)403–10.[CrossRef][Web of Science][Medline]
Ashburner, M, CA Ball, JA Blake, D Botstein, H Butler, JM Cherry, AP Davis, K Dolinski, SS Dwight, JT Eppig. 2000. Gene ontology: Tool for the unification of biology. The gene ontology consortium. Nat Genet 25:(1)25–9.[CrossRef][Web of Science][Medline]
Bachere, E, Y Gueguen, M Gonzalez, J de Lorgeril, J Garnier, B Romestand. 2004. Insights into the anti-microbial defense of marine invertebrates: The penaeid shrimps and the oyster Crassostrea gigas. Immunol Rev 198:149–68.[CrossRef][Web of Science][Medline]
Burgents, JE, KG Burnett, LE Burnett. 2003. Disease resistance of pacific white shrimp, Litopenaeus vannamei, following the dietary administration of a yeast culture food supplement. Aquaculture 231:1–8.[CrossRef]
Chapman, RW, J Robalino, H Trent. 2006. EcoGenomics: Analysis of complex systems. Integr Comp Biol 46:902–911.
Chen, T, R Amons, JS Clegg, AH Warner, TH MacRae. 2003. Molecular characterization of artemin and ferritin from Artemia franciscana. Eur J Biochem 270(1):137–45.[CrossRef]
Colbourne, JK, VR Singan, DG Gilbert. 2005. Wfleabase: The Daphnia genome database. BMC Bioinformatics 6(1):45.
Gross, PS, TC Bartlett, CL Browdy, RW Chapman, GW Warr. 2001. Immune gene discovery by expressed sequence tag analysis of hemocytes and hepatopancreas in the pacific white shrimp, Litopenaeus vannamei, and the Atlantic white shrimp, L. setiferus. Dev Comp Immunol 25(7):565–77.
Johnson, HM. 2004. 2004 Annual report of the United States seafood industry. Jacksonville, OR H.M. Johnson & Associates.
Lee, YM, IC Kim, SO Jung, JS Lee. 2005. Analysis of 686 expressed sequence tags (ESTs) from the intertidal harpacticoid copepod Tigriopus japonicus (crustacea, copepoda). Mar Pollut Bull 51(8–12):757–68.
Lehnert, SA, KJ Wilson, K Byrne, SS Moore. 1999. Tissue-specific expressed sequence tags from the black tiger shrimp Penaeus monodon. Mar Biotechnol 1(5):465–76.
McKillen, DJ, YA Chen, C Chen, MJ Jenny, HF III Trent, J Robalino, DC Jr McLean, PS Gross, RW Chapman, GW, and others Warr. 2005. Marine genomics: A clearing-house for genomic and transcriptomic data of marine organisms. BMC Genomics 6(1):34.
Perez, F, J Ortiz, M Zhinaula, C Gonzabay, J Calderon, FA Volckaert. 2005. Development of EST-SSR markers by data mining in three species of shrimp: Litopenaeus vannamei, Litopenaeus stylirostris, and Trachypenaeus birdy. Mar Biotechnol 7(5):554–69.[CrossRef]
Rojtinnakorn, J, I Hirono, T Itami, Y Takahashi, T Aoki. 2002. Gene expression in haemocytes of kuruma prawn, Penaeus japonicus, in response to infection with WSSV by EST approach. Fish Shellfish Immunol 13(1):69–83.
Stevens, R, CA Goble, S Bechhofer. 2000. Ontology-based knowledge representation for bioinformatics. Brief Bioinform 1(4):398–414.
Supungul, P, S Klinbunga, R Pichyangkura, S Jitrapakdee, I Hirono, T Aoki, A Tassanakajon. 2002. Identification of immune-related genes in hemocytes of black tiger shrimp (Penaeus monodon). Mar Biotechnol 4(5):487–94.
Supungul, P, S Klinbunga, R Pichyangkura, I Hirono, T Aoki, A Tassanakajon. 2004. Antimicrobial peptides discovered in the black tiger shrimp Penaeus monodon using the EST approach. Dis Aquat Organ 61(1–2):123–35.
van de Braak, CB, MH Botterblom, EA Huisman, JH Rombout, WP van der Knaap. 2002. Preliminary study on haemocyte response to white spot syndrome virus infection in black tiger shrimp Penaeus monodon. Dis Aquat Organ 51(2):149–55.
Watanabe, H, N Tatarazako, S Oda, H Nishide, I Uchiyama, M Morita, T Iguchi. 2005. Analysis of expressed sequence tags of the water flea Daphnia magna. Genome 48(4):606–9.[CrossRef]
Yamano, K and T Unuma. 2006. Expressed sequence tags from eyestalk of kuruma prawn, Marsupenaeus japonicus. Comp Biochem Physiol A Mol Integr Physiol 143(2):155–61.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  N. Jayasundara, D. W. Towle, D. Weihrauch, and C. Spanings-Pierrot Gill-specific transcriptional regulation of Na+/K+-ATPase {alpha}-subunit in the euryhaline shore crab Pachygrapsus marmoratus: sequence variants and promoter structure J. Exp. Biol., June 15, 2007; 210(12): 2070 - 2081. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||