De novo transcriptome analysis of Perna viridis highlights tissue-specific patterns for environmental studies

BMC Genomics

Kenneth MY Leung⁴  Leo L Chan⁴  Chris KC Wong³  Paul KS Lam¹  Jian Wen Qiu³  Sarah ST Mak²  Jack CH Ip²  Priscilla TY Leung² 

[1] Department of Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong, China;The Swire Institute of Marine Science and School of Biological Sciences, The University of Hong Kong, Pokfulam, Hong Kong, China;Department of Biology, Hong Kong Baptist University, Kowloon, Hong Kong, China;State Key Laboratory in Marine Pollution, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong, China

关键词: Biomonitor;    Tissue specificity;    RNA-seq;    Mussel;    Perna viridis;    De novo transcriptome;   

Others  :  1139557
DOI  :  10.1186/1471-2164-15-804

 received in 2014-05-21, accepted in 2014-08-26,  发布年份 2014

【 摘 要 】

Background

The tropical green-lipped mussel Perna viridis is a common biomonitor throughout the Indo-Pacific region that is used for environmental monitoring and ecotoxicological investigations. However, there is limited molecular data available regarding this species. We sought to establish a global transcriptome database from the tissues of adductor muscle, gills and the hepatopancreas of P. viridis in an effort to advance our understanding of the molecular aspects involved during specific toxicity responses in this sentinel species.

Results

Illumina sequencing results yielded 544,272,542 high-quality filtered reads. After de novo assembly using Trinity, 233,257 contigs were generated with an average length of 1,264 bp and an N50 length of 2,868 bp; 192,879 assembled transcripts and 150,111 assembled unigenes were obtained after clustering. A total of 93,668 assembled transcripts (66,692 assembled genes) with putative functions for protein domains were predicted based on InterProScan analysis. Based on similarity searches, 44,713 assembled transcripts and 25,319 assembled unigenes were annotated with at least one BLAST hit. A total of 21,262 assembled transcripts (11,947 assembled genes) were annotated with at least one well-defined Gene Ontology (GO) and 5,131 assembled transcripts (3,181 assembled unigenes) were assigned to 329 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The quantity of assembled unigenes and transcripts obtained from male and female mussels were similar but varied among the three studied tissues, with the highest numbers recorded in the gills, followed by the hepatopancreas, and then the adductor muscle. Multivariate analyses revealed strong tissue-specific patterns among the three different tissues, but not between sexes in terms of expression profiles for annotated genes in various GO terms, and genes associated with stress responses and degradation of xenobiotics. The expression profiles of certain selected genes in each tissue type were further validated using real-time quantitative polymerase chain reaction assays and a similar tissue-specific trend was seen.

Conclusions

The extensive sequence data generated from this study will provide a valuable molecular resource for facilitating environmental studies with P. viridis, and highlight the importance of tissue-specific approaches in the future.

【 授权许可】

   
2014 Leung et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

【 参考文献 】

• [1]Goldberg ED, Bowen VT, Farrington JW, Harvey G, Martin JH, Parker PL, Risebrough RW, Robertson W, Schneider E, Gamble E: Mussel watch. Environ Conserv 1978, 5(2):101-125.
• [2]Livingstone DR, Chipman JK, Lowe DM, Minier C, Mitchelmore CL, Moore MN, Peters LD, Pipe RK: Development of biomarkers to detect the effects of organic pollution on aquatic invertebrates: recent molecular, genotoxic, cellular and immunological studies on the common mussel (Mytilus edulis L.) and other mytilids. Int J Environ Pollut 2000, 13(1–6):56-91.
• [3]Nicholson S, Lam PKS: Pollution monitoring in Southeast Asia using biomarkers in the mytilid mussel Perna viridis (Mytilidae : Bivalvia). Environ Int 2005, 31(1):121-132.
• [4]Yap CK, Shahbazi A, Zakaria MP: Concentrations of heavy metals (Cu, Cd, Zn and Ni) and PAHs in Perna viridis collected from seaport and non-seaport waters in the Straits of Johore. Bull Environ Contam Toxicol 2012, 89(6):1205-1210.
• [5]Shahbazi A, Zakaria MP, Yap CK, Tan SG, Surif S, Mohamed CAR, Sakari M, Bakhtiari AR, Bahry PS, Chandru K, Mirsadeghi SA: Use of different tissues of Perna viridis as biomonitors of polycyclic aromatic hydrocarbons (PAHs) in the coastal waters of Peninsular Malaysia. Environ Forensics 2010, 11(3):248-263.
• [6]Fang JKH, Wu RSS, Zheng GJ, Lam PKS, Shin PKS: Seasonality of bioaccumulation of trace organics and lysosomal integrity in green-lipped mussel Perna viridis. Sci Total Environ 2010, 408(6):1458-1465.
• [7]Tang CH, Wang WH: Butyltin accumulation in two marine bivalves along a pollution gradient. Environ Toxicol Chem 2008, 27(10):2179-2185.
• [8]Shek WM, Murphy MB, Lam JCW, Lam PKS: Polycyclic musks in green-lipped mussels (Perna viridis) from Hong Kong. Mar Pollut Bull 2008, 57(6–12):373-380.
• [9]Rajagopal S, Venugopalan VP, van der Velde G, Jenner HA: Greening of the coasts: a review of the Perna viridis success story. Aquat Ecol 2006, 40(3):273-297.
• [10]Wang Z, Gerstein M, Snyder M: RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet 2009, 10(1):57-63.
• [11]Craft JA, Gilbert JA, Temperton B, Dempsey KE, Ashelford K, Tiwari B, Hutchinson TH, Chipman JK: Pyrosequencing of Mytilus galloprovincialis cDNAs: tissue-specific expression patterns. PLoS One 2010, 5(1):e8875.
• [12]Bettencourt R, Pinheiro M, Egas C, Gomes P, Afonso M, Shank T, Santos RS: High-throughput sequencing and analysis of the gill tissue transcriptome from the deep-sea hydrothermal vent mussel Bathymodiolus azoricus. BMC Genomics 2010, 11:559. BioMed Central Full Text
• [13]Milan M, Coppe A, Reinhardt R, Cancela LM, Leite RB, Saavedra C, Ciofi C, Chelazzi G, Patarnello T, Bortoluzzi S, Bargelloni L: Transcriptome sequencing and microarray development for the Manila clam, Ruditapes philippinarum: genomic tools for environmental monitoring. BMC Genomics 2011, 12:234. BioMed Central Full Text
• [14]Hou R, Bao ZM, Wang S, Su HL, Li Y, Du HX, Hu JJ, Hu XL: Transcriptome sequencing and de novo analysis for Yesso scallop (Patinopecten yessoensis) using 454 GS FLX. PLoS One 2011, 6(6):e21560.
• [15]Kinoshita S, Wang N, Inoue H, Maeyama K, Okamoto K, Nagai K, Kondo H, Hirono I, Asakawa S, Watabe S: Deep sequencing of ESTs from nacreous and prismatic layer producing tissues and a screen for novel shell formation-related genes in the pearl oyster. PLoS One 2011, 6(6):e21238.
• [16]Zhang GF, Fang XD, Guo XM, Li L, Luo RB, Xu F, Yang PC, Zhang LL, Wang XT, Qi HG, Xiong ZQ, Que HY, Xie YL, Holland PWH, Paps J, Zhu YB, Wu FC, Chen YX, Wang JF, Peng CF, Meng J, Yang L, Liu J, Wen B, Zhang N, Huang ZY, Zhu QH, Feng Y, Mount A, Hedgecock D: The oyster genome reveals stress adaptation and complexity of shell formation. Nature 2012, 490(7418):49-54.
• [17]Zhao XL, Yu H, Kong LF, Li Q: Transcriptomic responses to salinity stress in the Pacific oyster Crassostrea gigas. PLoS One 2012, 7(9):e46244.
• [18]Leung PTY, Park TJ, Wang Y, Che CM, Leung KMY: Isoform-specific responses of metallothioneins in a marine pollution biomonitor, the green-lipped mussel Perna viridis, towards different stress stimulations. Proteomics 2014, 14(15):1796-1807.
• [19]Li ZZ, Chen R, Zuo ZH, Mo ZP, Yu A: Cloning, expression and identification of two glutathione S-transferase isoenzymes from Perna viridis. Comp Biochem Physiol B Biochem Mol Biol 2013, 165(4):277-285.
• [20]Zhao H, Sagert J, Hwang DS, Waite JH: Glycosylated hydroxytryptophan in a mussel adhesive protein from Perna viridis. J Biol Chem 2009, 284(35):23344-23352.
• [21]Guerette PA, Hoon S, Seow Y, Raida M, Masic A, Wong FT, Ho VHB, Kong KW, Demirel MC, Pena-Francesch A, Amini S, Tay GZ, Ding D, Miserez A: Accelerating the design of biomimetic materials by integrating RNA-seq with proteomics and materials science. Nat Biotechnol 2013, 31(10):908-915.
• [22]Cao YY, Li ZB, Li QH, Chen XJ, Chen L, Dai G: Characterization of eight novel microsatellite markers in the green-lipped mussel Perna viridis (Mytilidae). Genet Mol Res 2013, 12(1):344-347.
• [23]Ong CC, Yusoff K, Yap CK, Tan SG: Genetic characterization of Perna viridis L. in peninsular Malaysia using microsatellite markers. J Genet 2009, 88(2):153-163.
• [24]Li XL, Wu XY, Yu ZN: Complete mitochondrial genome of the Asian green mussel Perna viridis (Bivalvia, Mytilidae). Mitochondr DNA 2012, 23(5):358-360.
• [25]Iqbal A, Khan MS, Goswami U: Cytogenetic studies in green mussel, Perna viridis (Mytiloida : Pteriomorphia), from West Coast of India. Mar Biol 2008, 153(5):987-993.
• [26]Johnson BR, Atallah J, Plachetzki DC: The importance of tissue specificity for RNA-seq: highlighting the errors of composite structure extractions. BMC Genomics 2013, 14:586. BioMed Central Full Text
• [27]Coppe A, Bortoluzzi S, Murari G, Marino IAM, Zane L, Papetti C: Sequencing and characterization of striped venus transcriptome expand resources for clam fishery genetics. PLoS One 2012, 7(9):e44185.
• [28]Men LN, Yan SC, Liu GJ: De novo characterization of Larix gmelinii (Rupr.) Rupr. transcriptome and analysis of its gene expression induced by jasmonates. BMC Genomics 2013, 14:548. BioMed Central Full Text
• [29]Meng XL, Liu M, Jiang KY, Wang BJ, Tian X, Sun SJ, Luo ZY, Qiu CW, Wang L: De novo characterization of Japanese scallop Mizuhopecten yessoensis transcriptome and analysis of its gene expression following cadmium exposure. PLoS One 2013, 8(5):e64485.
• [30]Feder ME, Hofmann GE: Heat-shock proteins, molecular chaperones, and the stress response: Evolutionary and ecological physiology. Annu Rev Physiol 1999, 61:243-282.
• [31]Leung PTY, Wang Y, Mak SST, Ng WC, Leung KMY: Differential proteomic responses in hepatopancreas and adductor muscles of the green-lipped mussel Perna viridis to stresses induced by cadmium and hydrogen peroxide. Aquat Toxicol 2011, 105(1–2):49-61.
• [32]Zhang ZH, Zhang QZ: Molecular cloning, characterization and expression of heat shock protein 70 gene from the oyster Crassostrea hongkongensis responding to thermal stress and exposure of Cu2+ and malachite green. Gene 2012, 497(2):172-180.
• [33]Woo S, Jeon HY, Kim SR, Yum S: Differentially displayed genes with oxygen depletion stress and transcriptional responses in the marine mussel, Mytilus galloprovincialis. Comp Biochem Physiol Part D Genomics Proteomics 2011, 6(4):348-356.
• [34]Meng J, Zhu QH, Zhang LL, Li CY, Li L, She ZC, Huang BY, Zhang GF: Genome and transcriptome analyses provide insight into the euryhaline adaptation mechanism of Crassostrea gigas. PLoS One 2013, 8(3):e58563.
• [35]Negri A, Oliveri C, Sforzini S, Mignione F, Viarengo A, Banni M: Transcriptional response of the mussel Mytilus galloprovincialis (Lam.) following exposure to heat stress and copper. PLoS One 2013, 8(6):e66802.
• [36]Rewitz KF, Styrishave B, Lobner-Olesen A, Andersen O: Marine invertebrate cytochrome P450: Emerging insights from vertebrate and insect analogies. Comp Biochem Physiol C Toxicol Pharmacol 2006, 143(4):363-381.
• [37]Nelson DR: Progress in tracing the evolutionary paths of cytochrome P450. Biochim Biophys Acta 2011, 1814(1):14-18.
• [38]Baldwin WS, Marko PB, Nelson DR: The cytochrome P450 (CYP) gene superfamily in Daphnia pulex. BMC Genomics 2009, 10:169. BioMed Central Full Text
• [39]Zanette J, Goldstone JV, Bainy ACD, Stegeman JJ: Identification of CYP genes in Mytilus (mussel) and Crassostrea (oyster) species First approach to the full complement of cytochrome P450 genes in bivalves. Mar Environ Res 2010, 69:S1-S3.
• [40]Guo HH, Bao ZM, Du HX, Zhang LL, Wang S, Sun LY, Mou XY, Hu XL: Identification of Cytochrome P450 (CYP) genes in Zhikong scallop (Chlamys farreri). J Ocean Univ 2013, 12(1):97-102.
• [41]Sole M, Livingstone DR: Components of the cytochrome P450-dependent monooxygenase system and 'NADPH-independent benzo a pyrene hydroxylase' activity in a wide range of marine invertebrate species. Comp Biochem Physiol C Toxicol Pharmacol 2005, 141(1):20-31.
• [42]Wan PJ, Shi XQ, Kong Y, Zhou LT, Guo WC, Ahmat T, Li GQ: Identification of cytochrome P450 monooxygenase genes and their expression profiles in cyhalothrin-treated Colorado potato beetle. Leptinotarsa decemlineata Pest Biochem Physiol 2013, 107(3):360-368.
• [43]Guzov VM, Unnithan GC, Chernogolov AA, Feyereisen R: CYP12A1, a mitochondrial cytochrome P450 from the house fly. Arch Biochem Biophys 1998, 359(2):231-240.
• [44]Sheehan D, Meade G, Foley V, Dowd C: Structure, function and evolution of glutathione transferases: implications for classification of non-mammalian members of an ancient enzyme superfamily. Biochem J 2001, 360:1-16.
• [45]Hoarau P, Damiens G, Romeo M, Gnassia-Barelli M, Bebianno MJ: Cloning and expression of a GST-pi gene in Mytilus galloprovincialis. Attempt to use the GST-pi transcript as a biomarker of pollution. Comp Biochem Physiol C Toxicol Pharmacol 2006, 143(2):196-203.
• [46]Wang C, Zhao J, Mu C, Wang Q, Wu H, Wang C: cDNA cloning and mRNA expression of four glutathione S-transferase (GST) genes from Mytilus galloprovincialis. Fish Shellfish Immunol 2013, 34(2):697-703.
• [47]Yang HL, Zeng QY, Li EQ, Zhu SG, Zhou XW: Molecular cloning, expression and characterization of glutathione S-transferase from Mytilus edulis. Comp Biochem Physiol B Biochem Mol Biol 2004, 139(2):175-182.
• [48]Boutet I, Tanguy A, Moraga D: 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 2004, 146(1):53-64.
• [49]Lin Q, Liang XF, Hu YL, Wang L, Liu XX: Cloning and Sequence Analysis of Four Classes of Glutathione S-Transferase Genes in Jinjiang Oyster (Crassostrea ariakensis). Asian J Ecotoxicol 2009, 4(2):237-243.
• [50]Xu C, Pan L, Liu N, Wang L, Miao J: Cloning, characterization and tissue distribution of a pi-class glutathione S-transferase from clam (Venerupis philippinarum): response to benzo alpha pyrene exposure. Comp Biochem Physiol C Toxicol Pharmacol 2010, 152(2):160-166.
• [51]Zhang LB, Qiu LH, Wu HF, Liu XL, You LP, Pei D, Chen LL, Wang Q, Zhao JM: Expression profiles of seven glutathione S-transferase (GST) genes from Venerupis philippinarum exposed to heavy metals and benzo a pyrene. Comp Biochem Physiol C Toxicol Pharmacol 2012, 155(3):517-527.
• [52]Umasuthan N, Revathy KS, Lee Y, Whang I, Choi CY, Lee J: A novel molluscan sigma-like glutathione S-transferase from Manila clam, Ruditapes philippinarum: Cloning, characterization and transcriptional profiling. Comp Biochem Physiol C Toxicol Pharmacol 2012, 155(4):539-550.
• [53]Kim M, Ahn I-Y, Cheon J, Park H: Molecular cloning and thermal stress-induced expression of a pi-class glutathione S-transferase (GST) in the Antarctic bivalve Laternula elliptica. Comp Biochem Physiol A Mol Integr Physiol 2009, 152(2):207-213.
• [54]Park H, Ahn I-Y, Kim H, Lee J, Shin SC: Glutathione S-transferase as a biomarker in the Antarctic bivalve Laternula elliptica after exposure to the polychlorinated biphenyl mixture Aroclor 1254. Comp Biochem Physiol C Toxicol Pharmacol 2009, 150(4):528-536.
• [55]Feng X, Singh BR: Molecular identification of glutathione S-transferase gene and cDNAs of two isotypes from northern quahog (Mercenaria mercenaria). Comp Biochem Physiol B Biochem Mol Biol 2009, 154(1):25-36.
• [56]Yang J, Wei X, Xu J, Yang D, Liu X, Yang J, Fang J, Hu X: A sigma-class glutathione S-transferase from Solen grandis that responded to microorganism glycan and organic contaminants. Fish Shellfish Immunol 2012, 32(6):1198-1204.
• [57]Miao J, Pan L, Liu N, Xu C, Zhang L: Molecular cloning of CYP4 and GSTpi homologues in the scallop Chlamys farreri and its expression in response to Benzo a pyrene exposure. Mar Genom 2011, 4(2):99-108.
• [58]Feldmeyer B, Wheat CW, Krezdorn N, Rotter B, Pfenninger M: Short read Illumina data for the de novo assembly of a non-model snail species transcriptome (Radix balthica, Basommatophora, Pulmonata), and a comparison of assembler performance. BMC Genomics 2011, 12:317. BioMed Central Full Text
• [59]Yi AX, Leung KMY, Lam MHW, Lee JS, Giesy JP: Review of measured concentrations of triphenyltin compounds in marine ecosystems and meta-analysis of their risks to humans and the environment. Chemosphere 2012, 89(9):1015-1025.
• [60]Barsiene J, Syvokiene J, Bjornstad A: Induction of micronuclei and other nuclear abnormalities in mussels exposed to bisphenol A, diallyl phthalate and tetrabromodiphenyl ether-47. Aquat Toxicol 2006, 78:S105-S108.
• [61]Wong SWY, Leung PTY, Djurisic AB, Leung KMY: Toxicities of nano zinc oxide to five marine organisms: influences of aggregate size and ion solubility. Anal Bioanal Chem 2010, 396(2):609-618.
• [62]Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, Adiconis X, Fan L, Raychowdhury R, Zeng QD, Chen ZH, Mauceli E, Hacohen N, Gnirke A, Rhind N, di Palma F, Birren BW, Nusbaum C, Lindblad-Toh K, Friedman N, Regev A: Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol 2011, 29(7):644-U130.
• [63]Li WZ, Godzik A: Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences. Bioinformatics 2006, 22(13):1658-1659.
• [64]Conesa A, Gotz S, Garcia-Gomez JM, Terol J, Talon M, Robles M: Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 2005, 21(18):3674-3676.
• [65]Zdobnov EM, Apweiler R: InterProScan - an integration platform for the signature-recognition methods in InterPro. Bioinformatics 2001, 17(9):847-848.
• [66]Li B, Dewey CN: RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics 2011, 12:323. BioMed Central Full Text
• [67]Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(T)(−Delta Delta C) method. Methods 2001, 25(4):402-408.
• [68]Sheir SK, Handy RD, Henry TB: Effect of pollution history on immunological responses and organ histology in the marine mussel Mytilus edulis exposed to cadmium. Arch Environ Contam Toxicol 2013, 64(4):701-716.