Abstract
The Agamaki clam (Sinonovacula constricta) is an economically important shellfish in Asia. However, genomic research on this species is still in its infancy, and genomic resources are largely unavailable. The objective of this study was to generate expressed sequence tags (ESTs) from a normalized liver complementary DNA library and to identify genes that function in immune defense. A total of 5,296 ESTs were sequenced, from which 540 contigs and 3,473 singletons were identified. BLAST homology analysis indicated that only 20.7% of these ESTs were homologues of known genes while the remaining 79.3% appeared to be novel sequences. Based on sequence similarities, 43 putative immune genes were identified that encode proteases and protease inhibitors, adhesive proteins, stress proteins, lysosomal enzymes, and signal transduction regulators. Our study thus provides both a large collection of novel transcripts and a detailed annotation of immune genes for an important bivalve species.
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Adams MD, Kelley JM, Gocayne JD, Dubnick M, Polymeropoulos MH, Xiao H, Merril CR, Wu A, Olde B, Moreno RF et al (1991) Complementary DNA sequencing: expressed sequence tags and human genome project. Science 252:1651–1656
Adams MD, Kerlavage AR, Fleischmann RD, Fuldner RA, Bult CJ, Lee NH, Kirkness EE et al (1995) Initial assessment of human gene diversity and expression patterns based upon 83 million nucleotides of cDNA sequence. Nature 377(6547 Suppl):3–174
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410
Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402
Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT et al (2000) Gene Ontology: tool for the unification of biology. Nat Genet 25:25–29
Bai ZY, Yin YX, Hu SN, Wang GL, Zhang XW, Li JL (2009) Identification of genes involved in immune response, microsatellite, and SNP markers from expressed sequence tags generated from hemocytes of freshwater pearl mussel (Hyriopsis cumingii). Mar Biotechnol 11:520–530
Beck G, Ellis TW, Habicht GS, Schluter SF, Marchalonis JJ (2002) Evolution of the acute phase response: iron release by echinoderm (Asterias forbesi) coelomocytes, and cloning of an echinoderm ferritin molecule. Dev Comp Immunol 26:11–26
Birnboin HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523
Boutet I, Tanguy A, Moraga D (2004) Characterisation and expression of four mRNA sequences encoding glutathione S-transferases pi, mu, omega and sigma classes in the Pacific oyster Crassostrea gigas exposed to hydrocarbons and pesticides. Mar Biol 146:53–64
Boutet I, Moraga D, Marinovic L, Obreque J, Chavez-Crooker P (2008) Characterization of reproduction-specific genes in a marine bivalve mollusc: Influence of maturation stage and sex on mRNA expression. Gene 407:130–138
Cheng W, Hsiao IS, Hsu CH, Chen JC (2004) Change in water temperature on the immune response of Taiwan abalone Haliotis diversicolor supertexta and its susceptibility to Vibrio parahaemolyticus. Fish Shellfish Immunol 17:235–243
Contreras-Vergara CA, Harris-Valle C, Sotelo-Mundo RR, Yepiz-Plascencia G (2004) A mu-class glutathione S-transferase from the marine shrimp Litopenaeus vannamei: molecular cloning and active-site structural modeling. J Biochem Molec Toxicol 18:245–252
Ewing B, Hillier L, Wendl MC, Green P (1998a) Base-calling of automated sequencer traces using phred. I. Accuracy assessment. Genome Res 8:175–185
Ewing B, Hillier LD, Wendl MC, Green P (1998b) Base-calling of automated sequencer traces using Phred. II. Error probabilities. Genome Res 8:186–194
Frova C (2006) Glutathione transferases in the genomics era: new insights and perspectives. Biomol Eng 23:149–169
Gong NP, Ma ZJ, Li Q, Li Q, Yan ZG, Xie LP, Zhang RQ (2008) Characterization of calcium deposition and shell matrix protein secretion in primary mantle tissue culture from the marine pearl oyster Pinctada fucata. Mar Biotechnol 10:457–465
Gueguen Y, Cadoret JP, Flament D, Barreau-Roumiguière C, Girardot AL, Garnier J, Hoareau A, Bachère E, Escoubas JM (2003) Immune gene discovery by expressed sequence tags generated from hemocytes of the bacteria-challenged oyster, Crassostrea gigas. Gene 303:139–145
Hanash SM, Pitteri SJ, Faca VM (2008) Mining the plasma proteome for cancer biomarkers. Nature 452:571–579
Hayes JD, Flanagan JU, Jowsey IR (2005) Glutathione transferases. Annu Rev Pharmacol Toxicol 45:51–88
Hedgecock D, Gaffney PM, Goulletquer P, Guo XM, Reece K, Warr GW (2005) The case for sequencing the oyster genome. J Shellfish Res 24:429–442
Ish-Horowicz D, Burke JF (1981) Rapid and efficient cosmid cloning. Nucleic Acids Res 9:2989–2998
Jenny MJ, Ringwood AH, Lacy ER, Lewitus AJ, Kempton JW, Gross PS, Warr GW, Chapman RW (2002) Potential indicators of stress response identified by expressed sequence tag analysis of hemocytes and embryos from the eastern oyster, Crassostrea virginica. Mar Biotechnol 4:81–93
Kang YS, Kim YM, Park KI, Kim Cho SK, Choi KS, Cho M (2006) Analysis of EST and lectin expressions in hemocytes of Manila clams (Ruditapes philippinarum) (Bivalvia: Mollusca) infected with Perkinsus olseni. Dev Comp Immunol 30:1119–1131
Medzhitov R, Janeway CJ (2000) Innate immune recognition: mechanisms and pathways. Immunol Rev 173:89–97
Myrnes B, Nilsen IW (2007) Glutathione S-transferase from the lcelandic scallop (Chlamys islandica): isolation and partial characterization. Comp Biochem Physiol 144:403–407
Nair SV, Del Valle H, Gross PS, Terwilliger DP, Smith LC (2005) Macroarray analysis of coelomocyte gene expression in response to LPS in the sea urchin. Identification of unexpected immune diversity in an invertebrate. Physiol Genomics 22:33–47
Nilsen IW, Øverbø K, Sandsdalen E, Sandaker E, Sletten K, Myrnes K (1999) Protein purification and gene isolation of chlamysin, a cold-active lysozyme-like enzyme with antibacterial activity. FEBS Lett 464:153–158
Niu DH, Li JL, Wang GL, Jiang ZY, Zhang WB, Shen YB, Feng BB (2007) The genetic diversity of mitochondrial 16 S rRNA gene fragment in six populations of Sinonovacula constricta. Journal of Shanghai Fisheries University 16:1–6
Niu DH, Li JL, Liu DB (2008a) Polymorphic microsatellite loci for population studies of the razor clam, Sinonovacula constricta. Conserv Genet 9:1393–1394
Niu DH, Li JL, Shen HD, Jiang ZY (2008b) Sequence variability of mitochondrial DNA-COI gene fragment and population genetic structure of six Sinonovacula constricta populations. Acta Ocean Sin 30:109–116
Niu DH, Li JL, Zheng RL (2008c) Isolation and Sequence Characterization of Microsatellite DNA in Razor Clam (Sinonovacula constricta). Periodical of Ocean University of China 38:733–738
Oakley AJ (2005) Glutathione transferases: new functions. Curr Opin Struct Biol 15:716–723
Okubo K, Hori N, Matoba R, Niiyama T, Fukushima A, Kojima Y, Matsubara K (1992) Large scale cDNA sequencing for analysis of quantitative and qualitative aspects of gene expression. Nat Genet 2:173–179
Peatman EJ, Wei X, Feng J, Liu L, Kukuktas H, Li P, He C, Rouse D, Wallace R, Dunhan R et al (2004) Development of expressed sequence tags (ESTs) from eastern oyster (Crassostrea virginica): lessons learned from previous efforts. Mar Biotechnol 6:S491–S496
Quilang J, Wang SL, Li P, Abernathy J, Peatman E, Wang YP, Wang LL, Shi YH, Wallace R, Guo XM, Liu ZJ (2007) Generation and analysis of ESTs from the eastern oyster. Crassostrea virginica Gmelin and identification of microsatellite and SNP markers. BMC Genomics 8:157
Reusch TBH, Veron AS, Preuss C, Weiner J, Wissler L, Beck A, Klages S, Kube M, Reinhardt R, Bornberg-Bauer E (2008) Comparative analysis of expressed sequence tag (EST) libraries in the seagrass Zostera marina subjected to temperature stress. Mar Biotechnol 10:297–309
Rhee JS, Raisuddin S, Hwang DS, Horiguchi T, Cho HS, Lee JS (2008) A Mu-class glutathione S-transferase (GSTM) from the rock shell Thais clavigera. Comp Biochem Physiol C Comp Pharmacol Toxicol 148:195–203
Roberts SB, Goetz FW (2003) Expressed sequence tag analysis of genes expressed in the bay scallop, Argopecten irradians. Biol Bull 205:227–228
Roberts S, Goetz G, White S, Goetz F (2009) Analysis of genes isolated from plated hemocytes of the Pacific oyster, Crassostreas gigas. Mar Biotechnol 11:24–44
Saavedra C, Bachère E (2006) Bivalve genomics. Aquaculture 256:1–14
Sarropoulou E, Nousdili D, Magoulas A, Kotoulas G (2008) Linking the genomes of nonmodel teleosts through comparative genomics. Mar Biotechnol 10:227–233
Smith LC, Chang L, Britten RJ, Davidson EH (1996) Sea urchin genes expressed in activated coelomocytes are identified by expressed sequence tags. Complement homologues and other putative immune response genes suggest immune system homology within the deuterostomes. J Immunol 156:593–602
Song LS, Xu W, Li CH, Li HL, Wu LT, Xiang JH, Guo XM (2006) Development of expressed sequence tags from the bay scallop, Argopecten irradians irradians. Mar Biotechnol 6:713–728
Suzuki T, Shin IT, Kohara Y, Kasahara M (2004) Transcriptome analysis of hagfish leukocytes: a framework for understanding the immune system of jawless fishes. Dev Comp Immunol 28:993–1003
Tanguy A, Guo XM, Ford SE (2004) Discovery of genes expressed in response to Perkinsus marinus challenge in Eastern (Crassostrea virginica) and Pacific (C. gigas) oysters. Gene 338:121–131
Tanguy A, Bierne N, Saavedra C, Pina B, Bachère E, Kube M, Bazin E, Bonhomme F et al (2008) Increasing genomic information in bivalves through new EST collections in four species: development of new genetic markers for environmental studies and genome evolution. Gene 408:27–36
von Schalburg KR, Leong J, Cooper GA, Robb A, Beetz-Sargent MR, Lieph R, Holt RA, Moore R, Ewart KV, Driedzic WR, ten Hallers BFH, Zhu B, de Jong PJ, Davidson WS, Koop BF (2008) Rainbow smelt (Osmerus mordax) genomic library and EST resources. Mar Biotechnol 10:487–491
Wang RC, Zheng XD (2004) Progress of marine shellfishes culture in China and its prospect. Periodical of Ocean University of China 34:775–780
Wynne JW, O'Sullivan MG, Cook MT, Stone G, Nowak BF, Lovell DR, Elliott NG (2008) Transcriptome analyses of amoebic gill disease-affected Atlantic salmon (Salmo salar) tissues reveal localized host gene suppression. Mar Biotechnol 10:338–403
Yu J, Farjo R, Macnee SP, Baehr W, Stambolian DE, Swaroop A (2003) Annotation and analysis of 10, 000 expressed sequence tags from developing mouse eye and adult retina. Genome Biol 4:R65
Zhang B, Jin W, Zeng Y, Su ZX, Hu SN, Yu J (2004) EST-based analysis of gene expression in the porcine brain. Genomics Proteomics Bioinformatics 2:237–244
Zdobnov EM, Apweiler R (2001) InterProScan—an integration platform for the signature-recognition methods in InterPro. Bioinformatics 17:847–848
Zheng MG, Sun XQ, Zhang JX (2007) Construction of cDNA library of liver and kidney from abalone (Haliotis discus Hannai) and preliminary analysis of immune gene. High Technol Lett 17:319–324
Acknowledgments
This study was supported by grants from “863” Hi-tech research and development program of China (2006 AA10 A410) and Shanghai Leading Academic Discipline Project (Y1101).
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B. Feng and L. Dong contributed equally to this study.
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Feng, B., Dong, L., Niu, D. et al. Identification of Immune Genes of the Agamaki Clam (Sinonovacula constricta) by Sequencing and Bioinformatic Analysis of ESTs. Mar Biotechnol 12, 282–291 (2010). https://doi.org/10.1007/s10126-009-9216-z
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DOI: https://doi.org/10.1007/s10126-009-9216-z