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Screening of genes related to sulfide metabolism in Urechis unicinctus (Echiura, Urechidae) using suppression subtractive hybridization and cDNA microarray analysis
Comparative Biochemistry and Physiology, Part D 7 (2012) 254–259
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Screening of genes related to sulfide metabolism in Urechis unicinctus (Echiura, Urechidae) using suppression subtractive hybridization and cDNA microarray analysis Xiaoli Shi 1, Mingyu Shao 1, Litao Zhang, Yubin Ma, Zhifeng Zhang ⁎ Key Laboratory of Marine Genetics and Breeding (Ocean University of China), Ministry of Education, Qingdao 266003, China
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Article history: Received 30 November 2011 Received in revised form 12 April 2012 Accepted 21 April 2012 Available online 28 April 2012 Keywords: cDNA microarray qRT-PCR Sulfide stress Suppression subtractive hybridization Urechis unicinctus
a b s t r a c t Exogenous sulfide can generally induce metabolic injuries in most organisms and even cause death. However, organisms inhabiting intertidal zones, hydrothermal vents, and cold seeps, can tolerate, metabolize, and utilize sulfide. In this study, both suppression subtractive hybridization and cDNA microarray analysis were employed to screen sulfide metabolism-related genes from the body wall in echiuran worm Urechis unicinctus, a marine sediment species. A total of 3456 monoclones were isolated and 82 were identified as differentially expressed genes in worms exposed to 50 μM sulfide for 24 h, compared to controls. The identified genes encoded proteins with multiple processes, including metabolism, cellular process, biological regulation, response to stimulus, multicellular organismal process, localization, development, and cellular component organization. Eight genes, serase, vacuolar protein, src tyrosine kinase, sulfide oxidase-like oxidoreductase, aprataxin, SN-RNP, aminopeptidase, and predicted protein, were selected to verify expression in the worm using qRT-PCR. The agreement of gene expression evaluation was 62.5% between the results of microarray analysis and qRT-PCR. These new data will provide clues for further probing of the molecular mechanism of sulfide metabolism. © 2012 Elsevier Inc. All rights reserved.
1. Introduction Endogenous sulfide, produced in vertebrates (Abe and Kimura, 1996) and invertebrates (Julian et al., 2002, 2005a; Gainey and Greenberg, 2005), plays a role at low concentrations as a cellular signaling molecule. Exogenous sulfide, described as the aggregate of H2S, HS−, and S2−, is a common substance in hydrothermal vents, cold seeps, sewer outfalls, marshes, mudflats, and highly eutrophic freshwater lakes (Nicholls and Kim, 1982), and can cause harm to organisms in a variety of ways, including reversible cytochrome c oxidase inhibition (Evans, 1967; Nicholls, 1975), hemoglobin oxygen affinity decrease (Carrico et al., 1978), sulfhemoglobin formation (Bagarinao, 1992; Kraus et al., 1996), mitochondrial depolarization (Julian et al., 2005b), free radical production (Tapley et al., 1999; Eghbal et al., 2004), oxidative damage to RNA and DNA (Joyner-Matos et al., 2010), coelomocyte death and cell proliferation decrease (Hance et al., 2008), and inhibition of nearly 20 enzymes involved in aerobic metabolism (Bagarinao, 1992). Inhibition of cytochrome c oxidase is believed to be the main mechanism of H2S toxicity (Beauchamp et al., 1984), although alternative mechanisms, including activation of ATP-activated potassium channels and alteration in cell signaling pathways, have also been postulated (Szabó, 2007). ⁎ Corresponding author. Tel./fax: + 86 532 82031647. E-mail address: [email protected] (Z. Zhang). 1 Joint first authors. 1744-117X/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.cbd.2012.04.001
Animals in sulfide-rich habitats typically reduce sulfide toxicity by employing physiological mechanisms (Grieshaber and Völkel, 1998), including sulfide-oxidizing enzymes and sulfide-binding amino acids (Joyner et al., 2003; Brand et al., 2007). In phototrophic sulfur bacteria, sulfide metabolism is a complex network and the processes involved remain incompletely understood. Several enzymes which catalyze sulfide oxidation have been isolated and some, such as flavocytochrome c and sulfide-quinone oxidoreductase (SQR), have been well characterized at the molecular level (Frigaard and Dahl, 2008). Researchers have found that SQR possesses the same mitochondrial electron acceptor in both prokaryotes and some eukaryotes (Theissen et al., 2003; Theissen and Martin, 2008). Furthermore, three consecutive sulfide oxidation reactions based on SQR, sulfur dioxygenase, and sulfur transferase have been reported in rat liver as well as in body wall of the lugworm, Arenicola marina (Hildebrandt and Grieshaber, 2008). Although the key genes in sulfide metabolism have been thoroughly investigated, it is still not clear how the expression of these genes is regulated and whether more genes participate in these biological processes. The Urechis unicinctus is a species of Echiura, Xenopneusta, Urechidae and Urechis and inhabits intertidal and subtidal mudflats in China, Korea, Russia, and Japan. It has been suggested to be a sulfide tolerant species, capable of sulfide metabolism and utilization (Zhang et al., 2006; Ma et al., 2010; Wang et al., 2010). Its SQR has been cloned and characterized (Ma et al., 2011a) and assessed regarding the influence of sulfide exposure on mRNA and protein expression (Ma et al., 2011b). Suppression subtractive hybridization (SSH) and cDNA microarray analysis are two
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useful techniques for rapidly detecting differentially expressed genes at high throughput. In the present study, we used these techniques to screen for sulfide metabolism genes in U. unicinctus and further verified the results using quantitative real-time polymerase chain reaction (qRT-PCR). These data can provide a good foundation for further exploration and understanding of the molecular mechanism of sulfide metabolism.
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Hybridizations and subsequent scanning, visualization, and quantitation were also performed by HGRC. The relative intensity of each spot was represented as the ratio of the Cy3 and Cy5 channels (R/G ratio), and converted to base-2 logarithms (log2). Spots with log2 R/G ratios > 1.0 (i.e., a 2-fold change) were considered upregulated and spots with ratios b −1.0 (i.e., a 0.5-fold change) were considered downregulated.
2. Materials and methods 2.1. Animals and sample collection
2.5. Sequence analysis
U. unicinctus, collected from a coastal intertidal flat in Yantai, China, had a mean fresh mass of 32.7±6.3 g and were maintained for 1 week in an aerated, recirculating seawater aquarium (20±1 °C, pH 8.0, salinity 25‰), and fed microalgae (Chlorella vulgaris and Mtzschia closterium). Feeding was discontinued 24 h prior to experimentation. For sulfide exposure, 20 worms were randomly assigned to a sulfide treated (50 μM sulfide in seawater) group and a control group, and the experiment was conducted in two airproof glass tanks (50 × 30 × 30 cm) with 40 L of seawater. The 50 μM sulfide concentration was produced by adding a sulfide stock solution (10 mM, pH 8.0), prepared using Na2S·9H2O, to the seawater and the sodium sulfide concentration was maintained by further stock addition at 2 h intervals according to spectrophotometric monitoring using a methylene blue method (Cline, 1969). All other conditions were the same as those used for acclimation and no mortality occurred during the experiment. At 24 h after sulfide exposure, seven worms were removed from each tank. Body wall from each worm was excised, frozen in liquid nitrogen, and stored at −80 °C for subsequent experiments.
All differentially expressed sequences were sequenced, submitted for BLAST X analysis, and searched for homology using National Center for Biotechnology Information databases. Matches were considered significant only when the probability (E value) was b1 × 10 − 4 and the sequences were classified by Gene Ontology terms (GO) using Blast2GO for level 2 categories.
2.2. Total RNA and mRNA isolation Total RNA from each body wall was extracted with Trizol (Invitrogen Corp., Beijing, China) according to the manufacturer's protocol. The RNA was quantified by 260 nm absorbance on a spectrophotometer and the quality assessed by electrophoresis, based on the rRNA band integrity. About 1.4 μg of mRNA was isolated from 700 μg of total RNA (each worm yielded similar RNA quantities) using the PolyATtract® mRNA Isolation Systems kit (Promega Corp., Madison, WI, USA). 2.3. Construction of subtractive library SSH was used to generate cDNA libraries enriched with genes differentially expressed under sulfide exposure. cDNA from sulfideexposed worm body wall was used as the tester and cDNA from the control worm was used as the driver. A subtractive cDNA library was constructed using the PCR-Select cDNA Subtraction kit (Clontech Laboratories, Inc., Beijing, China) according to the manufacturer's instructions. The subtracted PCR products were ligated into pMD-18T vector (Takara Bio Inc., Otsu, Japan) and transformed into Escherichia coli DH5α cells, which were then cultured overnight on LB agar plates containing 50 ppm ampicillin. Single colonies were then picked and screened for inserts using PCR, and insert fragments were amplified using primers (M13 (-47) forward and M13 (-48) reverse primers). 2.4. cDNA microarray A cDNA microarray chip containing 3456 cDNA clones was produced by Beijing Huada Institute for Gene Research Center (HGRC, Beijing, China). Inserts were amplified by PCR using adaptor-specific primers in the PCR-Select cDNA Subtraction kit (Clontech Lab, Inc., Beijing, China), the PCR products were purified via ethanol precipitation in 96-well plates, and the PCR-purified products were sent to the HGRC. Two μg samples of total RNA from the tester and the driver were used to produce Cy3/Cy5-labeled probes for hybridization to the microarray.
2.6. qRT-PCR analysis of selected genes Eight genes were selected and their expression levels in body-wall tissue from three worms for each group were evaluated using qRTPCR. RNA isolation was performed as described above and cDNA was synthesized using a reverse transcription system (Takara Bio, Inc.). All cDNAs were diluted (1:10) in nuclease-free water and stored at −20 °C. qRT-PCR was performed on each sample in duplicate using a fluorescence temperature cycler (7500 Real-Time PCR Systems, Applied Biosystems China, Beijing, China) in the presence of SYBR-green. Optimized qRT-PCR reactions were conducted according to the manufacturer's instructions (SYBR Premix Ex Taq, Takara Bio, Inc.), using β-actin as the internal standard. All primer sequences and PCR products are listed in Table 1. Data were analyzed using the 7500 System Sequence Detection Software version 1.4.0.25 (PE Applied Biosystems, Foster City, CA, USA) and the results presented as fold changes in transcription relative to that of the β-actin gene using the 2 − ΔΔCt method (Livak and Schmittgen, 2001).
2.7. Statistical analysis Data from qRT-PCR were calculated as mean±S.E. (n=3 with 2 replicates) and significant differences between the treatment and control groups were evaluated using one-way analysis of variance (ANOVA) followed by the Duncan's method, using the SPSS statistical package (version 18.0) at a significance level of pb 0.05. Table 1 Primers used in qRT-PCR analysis. Gene
Primer sequence (5′–3′)
PCR product (bp)
Serase
F-CTCTGATGCAGCACAACTGG R-TCCCCTAGGAGACATTGCTG F-ACTGCTCTCATGCAGGCTCT R-CACCAACTGGTCCAACACTG F-TCAGCCGAGTCGTCACATAG R-GTGTTGATCAGCCCAACCTT F-GCGGCGGATTATCTGTGT R-GTTCACGGGTGATCCTGATG F-GTGGACCTCATGCTTGGAAT R-ATCCAGAGCTCTTGGTGGAA F-GACATCAGGACCACCTTTCC R-GCCTGGGTGTAGACAGGAGA F-AGCGTCAAAACCACCTGAAC R-TGCTTACACCGTCTGCATTC F-GCCTCTGGCCTTGATAAGTG R-CTTGTCCTGATGGTCGGTTC F-CCCATCTACGAGGGATACGC R-CCTTGATGTCACGGACGATT
184
Vacuolar protein Src tyrosine kinase Sulfite oxidase-like oxidoreductase Aprataxin SN-RNP U1 Predicted protein Aminopeptidase β-actin
225 171 176 241 194 180 160 151
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3. Results 3.1. Construction and screening of cDNA library In the present study, a subtracted cDNA library was constructed from the sulfide-exposed worm body wall and compared to the control using SSH. The subtraction efficiency was 2 15, determined by comparison of the abundance of β-actin between the subtracted and unsubtracted products. Based on the above efficiency, 4200 differential colonies were generated by cloning the SSH cDNA products into a plasmid vector and 3456 monoclones were screened using colony PCR. The size of the inserted fragments ranged from 150 to 650 bp, with most at 300–600 bp. 3.2. Microarray analysis The results of microarray screening based on the 3456 monoclones from SSH showed that, of the 178 spots (differential sequences) obtained and sequenced, 133 spots were upregulated (ratio≥2) and 45 spots were downregulated (ratio≤0.5) in sulfide‐exposed worms. 3.3. Identification of the differentially expressed genes Eighty-two ESTs were identified successfully from the 178 sequences mentioned above and 26% of the assembled sequences (21 genes) shared significant similarity to known genes (E b 1 × 10 − 4) in GenBank, while 64% showed no significant similarity (E > 1 × 10 − 4) to any known genes or proteins in the database (Table 2). The 21 genes were classified into 8 groups based on their biological process category (Fig. 1), including metabolism, cellular process, biological regulation, response to stimulus, multicellular organismal process, localization, development, and cellular component organization. 3.4. Expression of the selected differentially expressed genes Expressions of the 8 differentially expressed genes (serase, vacuolar protein sorting 45, src tyrosine kinase, sulfide oxidase-like oxidoreductase, aprataxin, SN-RNP, aminopeptidase, and predicted protein) detected using qRT-PCR indicated that after sulfide exposure 5 genes (serase, src tyrosine kinase, sulfide oxidase-like oxidoreductase, SN-RNP, predicted protein) showed upregulated expression and the other 3 genes showed downregulated expression (Fig. 2). The correlation between the qRT-PCR data and the microarray data regarding these 5 of the 8 genes indicated that
Fig. 1. Classification of genes in level 2 biological process categories. Sequences having no homology with sequences known in the public database are not shown (E> 1 × 10− 4).
the microarray results were good indicators of overall gene expression changes. In upregulated genes, the predicted protein gene expression was the highest, being 1.34-fold higher than the control (pb 0.05), while serase and SN-RNP were increased less than 1 fold (p>0.05). Among the downregulated genes, a significant decrease was detected in aprataxin and aminopeptidase (pb 0.05), at 0.44‐fold and 0.41-fold lower than the control, respectively. 4. Discussion In this study, we report a work regarding sulfide metabolism-related genes, screened using SSH libraries and cDNA microarray, from sulfideexposed and control body walls of U. unicinctus. The selected differentially expressed gene sequences were classified to eight main gene categories which were related to metabolism, cellular process, biological regulation, response to stimulus, multicellular organismal process, localization, development, and cellular component organization. 4.1. A combined approach using SSH and cDNA microarray SSH is a powerful technique for obtaining differentially expressed genes at a large-scale survey, which enables the comparison of two populations of mRNA by cloning genes that are expressed in one
Table 2 Blast homology search results of significant sequences from microarray analysis. Clone ID Upregulated 22 416 420 126 411 457 225 127 431 334 Downregulated 423 133 230 239 438 118 445 224
No. of clones
Product length (bp)
Highest BlastX match
Species
E value
1 1 1 2 1 1 2 1 1 1
664 406 687 393 454 752 923 373 396 454
Small nuclear ribonucleoprotein Sm D2-like Arginase type I Aprataxin Predicted: zonadhesin-like Predicted protein Hypothetical protein BRAFLDRAFT_81368 RecName: full = arenicin-1; flags: precursor Predicted: zonadhesin-like Usp30 protein Predicted protein
Apis mellifera Strongylocentrotus purpuratus Danio rerio Danio rerio Nematostella vectensis Branchiostoma floridae Arenicola marina Danio rerio Mus musculus Nematostella vectensis
2 × 10− 42 5 × 10− 38 6 × 10− 21 4 × 10− 4 3 × 10− 16 9 × 10− 16 3 × 10− 8 1 × 10− 4 8 × 10− 4 5 × 10− 16
1 2 2 1 1 1 1 1
627 373 373 437 527 660 747 529
Vacular protein sorting 45 Predicted: zonadhesin-like Predicted: zonadhesin-like T-cell receptor beta chain Similar to Serase-1B SN-RNP U1, putative Src-family protein tyrosine kinase Aminopeptidase
Monodelphis domestica Danio rerio Danio rerio Plasmodium berghei strain ANKA Ciona intestinalis Ixodes scapularis Strongylocentrotus purpuratus Lumbricus rubellus
5 × 10− 70 4 × 10− 6 4 × 10− 6 8 × 10− 6 3 × 10− 6 2 × 10− 6 1 × 10− 4 6 × 10− 54
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Fig. 2. Expression of eight genes detected using qRT-PCR of total RNA from body wall of control and sulfide‐exposed worms. The asterisk indicates significant differences at *p b 0.05 determined by ANOVA. n = 3 with 2 replicates; after exposure upregulated genes: predicted protein, 1.34-fold higher; src tyrosine kinase, 0.46-fold; sulfide oxidase-like oxidoreductase, 0.52-fold; SN-RNP, 0.10-fold; and serase, 0.01-fold high; downregulated genes: aprataxin, 0.44-fold lower; aminopeptidase, 0.41-fold; and vacuolar protein, 0.13-fold.
population but not in the other. Currently, the approach has been widely used to identify genes responding to different pathogens and stress (Bayne et al., 2001; Zhang and Gui, 2004; Zhang et al., 2007), however, false positive results frequently occur. The validity of SSH selected sequences can be increased by combining SSH and microarray techniques. The first report of the two methods being successfully combined was in breast cancer research (Yang et al., 1999). A SSH and cDNA microarray combination has been utilized extensively in studies of gene expression (Gracey et al., 2001; Korke et al., 2004). Gracey et al. (2001) used SSH and cDNA microarray to understand the changes in gene expression in a hypoxia-tolerant fish, Gillichthys mirabilis. Their results showed a complex transcriptional response; they found novel differential expressed genes and tissue-specific patterns of expression during hypoxia (Gracey et al., 2001). Korke et al. (2004) examined gene expression profiling of metabolic shift of mammalian cells. They used the two techniques to identify 123 differentially expressed mRNA transcripts related to glucose metabolism from 4972 genes (Korke et al., 2004). In the present study, 3456 differential monoclones, derived from the control and sulfide-exposed body-wall RNA of U. unicinctus, were screened using SSH and 178 different spots were detected using cDNA microarray based on the monoclones. The combination of methods economized both the cost and labor involved in identifying the abundant differentially expressed clones screened using SSH. However, here the positive detection rate of SSH, confirmed by microarray analysis, was ~5.5%. Two reasons for this result were considered. First, if the signal intensity of one gene in two channels was too strong or too weak, the gene could be ignored although it was differentially expressed. And second, with a 24 h sulfide exposure, some genes expressed during the initial part of the exposure may later be inactive or decreased in expression, which caused weak intensity that was not detected. Thus, the cDNA microarray technique was an improvement in the following aspects: (1) elimination of interference by hybrid signals among molecules, (2) increased chip sensitivity, and (3) provided a homogeneous method at a more scientific and reasonable level. A total of 82 differentially expressed genes were identified in this study. However, a high proportion of these genes (64% genes) presented no significant similarity (E > 1 × 10 − 4) to known genes or proteins in the database. Two possible reasons for this result might have been: (1) some genes may represent untranslated regions of known
genes, and (2) no orthologs were registered in the GenBank, in which some unknown genes were detected which may help extend the understanding of their functions in sulfide metabolism. 4.2. The functional classification of genes The application of gene ontology is one method for integrating gene expression data with particular biological processes, although this method has many shortcomings, such as incompletely annotated databases, imprecise information, and the possibility that one gene may be involved in several biological processes, thus confounding interpretation. Here, most of the sequences differentially expressed with sulfide exposure fell into gene categories related to metabolism and cellular process. 4.2.1. Genes related to metabolism Sulfide is a potent toxin as well as a substrate of the mitochondrial respiration (Grieshaber and Völkel, 1998). Thus, animals have had to evolve mechanisms to effectively exploit H2S while simultaneously avoiding poisoning. A mitochondrial pathway catalyzing sulfide oxidation and combining with consume molecular oxygen, has been found in mammals and invertebrates (Hildebrandt and Grieshaber, 2008). Recently, Budde and Roth (2010) have proved that H2S could induce hypoxia-inducible factor-1 (Hif-1) activity in the nematode, Caenorhabditis elegans (Budde and Roth, 2010). In other species, the genes related to hypoxia were also found in H2S exposed (Han et al., 2006; Joyner-Matos et al., 2006; Roberts et al., 2008). In the present study, 29% of the detected differentially expressed genes were involved in metabolism, such as arginase and vps, being changed in expression with sulfide exposure. Here the arginase was up-regulated while the vps (related to vesicle-mediated transport) was down-regulated. Induced high expression and increased activity of arginase could provide more L-proline to support the biosynthesis of collagen, an important element of the tissue matrix (Que et al., 1998). Arginase activity or expression has been found to increase in a variety of cell types with hypoxia, such as in the brain of rats exposed to hypoxic–ischemic insults (Clarkson et al., 2005), in wound-derived rat macrophages (Albina et al., 1995) and in human pulmonary artery smooth muscle cell (Chen et al., 2009).
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4.2.2. Genes related to cellular process In the present experiment, genes associated with DNA repair and RNA splicing, such as aprataxin and snRNP, were more highly expressed with sulfide exposure. The aprataxin gene may play a role in single-stranded DNA repair. Aprataxin could catalyze the nucleophilic release of adenylate groups covalently linked to 5′ phosphate termini, resulting in termini that can again serve as a substrate for DNA ligase (Rass et al., 2007). Sulfide could cause DNA strand breaks and purine ring opening in Chinese hamster ovary cell (CHO) (Attune-Ramos et al., 2007), resulting in oxidative damage to the DNA and RNA in the mudflat polychaete Glycera dibranchiate (Joyner-Matos et al., 2010). Genes involved in DNA repair, which are activated by c-Jun and ATF2, will increase its expression after exposing intestinal epithelial cells (IEC-18) to 0.05 to 5 mmol/L NaHS (Deplancke and Gaskins, 2003). The same phenomenon was also observed in H2S exposed rat nasal respiratory epithelium (Roberts et al., 2008). 4.2.3. Genes related to others Genes involved in stimulus, such as arenicin-1, were also upregulated by sulfide exposure. Arenicin-1 is a novel antimicrobial peptide (AMP) that has been isolated from coelomocytes of the marine polychaete lugworm A. marina (Ovchinnikova et al., 2004). Until now AMPs have been identified in Potamopyrgus antipodarum, Takifugu rubripes and some other species (Osnas and Lively, 2006; Xie et al., 2009; Matsumoto et al., 2011). AMPs are considered to play a key role in invertebrate host defense and vertebrate defense. But its exact mechanism at molecular level is not completely understood. Park and Lee's (2009) study suggests that arenicin-1 is likely to exert fungicidal effect by disturbing fungal membrane. It is an interesting phenomenon that, here, the majority of predicted proteins are involved with zinc ion binding. Zinc ion related proteins have been implicated in sulfide binding (Flores et al., 2005) and function in a variety of processes, including binding DNA and RNA and participating in protein–protein interactions (Coleman, 1992). The comprehensive role may imply that kinds of genes participate in sulfide metabolism. In summary, genes involved in sulfide metabolism and their interactions and relationships remain unclear. In the present study, 82 differentially expressed genes were obtained from sulfide‐exposed U. unicintus, using a combination of SSH with cDNA microarray analysis. These genes were related to several biological processes, such as hypoxia, cellular process, DNA repair, and a majority of unknown sequences (including unknown sulfide-related genes). These results provide new gene-related information for future molecular research into the mechanism of sulfide metabolism. Acknowledgments This work is supported by the National Natural Science Foundation of China (40776074 and 31072191). References Abe, K., Kimura, H., 1996. The possible role of hydrogen sulfide as an endogenous neuromodulator. J. Neurosci. 16, 1066–1071. Albina, J.E., Henry, W.L., Mastrofrancesco, B., Martin, B.A., Reichner, J.S., 1995. Macrophage activation by culture in an anoxic environment. J. Immunol. 155, 4391–4396. Attune-Ramos, M.S., Wagner, E.D., Gaskins, H.R., Plewa, M.J., 2007. Hydrogen sulfide induces direct radical-associated DNA damage. Mol. Cancer Res. 5, 455–459. Bagarinao, T., 1992. Sulfide as an environmental factor and toxicant: tolerance and adaptations in aquatic organisms. Aquat. Toxicol. 24, 21–62. Bayne, C.J., Gerwick, L., Fujiki, K., Nakao, M., Yano, T., 2001. Immune-relevant (including acute phase) genes identified in the livers of rainbow trout, Oncorhynchus mykiss, by means of suppression subtractive hybridization. Dev. Comp. Immunol. 25, 205–217. Beauchamp, R.O., Bus, J.S., Popp, J.A., Boreiko, C.J., Andjelkovich, D.A., 1984. A critical review of the literature on hydrogen sulfide toxicity. CRC Crit. Rev. Toxicol. 13, 25–95. Brand, G.L., Horak, R.V., Bris, N.L., Goffredi, S.K., Carney, S.L., Govenar, B., Yancey, P.H., 2007. Hypotaurine and thiotaurine as indicators of sulfide exposure in bivalves and vestimentiferans from hydrothermal vents and cold seeps. Mar. Ecol. 28, 208–218. Budde, M.W., Roth, M.B., 2010. Hydrogen sulfide increases hypoxia-inducible factor-1 activity independently of von Hippel–Lindau tumor suppressor-1 in C. elegans. Mol. Biol. Cell 21, 212–217.
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Comparative Biochemistry and Physiology, Part D journal homepage: www.elsevier.com/locate/cbpd
Screening of genes related to sulfide metabolism in Urechis unicinctus (Echiura, Urechidae) using suppression subtractive hybridization and cDNA microarray analysis Xiaoli Shi 1, Mingyu Shao 1, Litao Zhang, Yubin Ma, Zhifeng Zhang ⁎ Key Laboratory of Marine Genetics and Breeding (Ocean University of China), Ministry of Education, Qingdao 266003, China
a r t i c l e
i n f o
Article history: Received 30 November 2011 Received in revised form 12 April 2012 Accepted 21 April 2012 Available online 28 April 2012 Keywords: cDNA microarray qRT-PCR Sulfide stress Suppression subtractive hybridization Urechis unicinctus
a b s t r a c t Exogenous sulfide can generally induce metabolic injuries in most organisms and even cause death. However, organisms inhabiting intertidal zones, hydrothermal vents, and cold seeps, can tolerate, metabolize, and utilize sulfide. In this study, both suppression subtractive hybridization and cDNA microarray analysis were employed to screen sulfide metabolism-related genes from the body wall in echiuran worm Urechis unicinctus, a marine sediment species. A total of 3456 monoclones were isolated and 82 were identified as differentially expressed genes in worms exposed to 50 μM sulfide for 24 h, compared to controls. The identified genes encoded proteins with multiple processes, including metabolism, cellular process, biological regulation, response to stimulus, multicellular organismal process, localization, development, and cellular component organization. Eight genes, serase, vacuolar protein, src tyrosine kinase, sulfide oxidase-like oxidoreductase, aprataxin, SN-RNP, aminopeptidase, and predicted protein, were selected to verify expression in the worm using qRT-PCR. The agreement of gene expression evaluation was 62.5% between the results of microarray analysis and qRT-PCR. These new data will provide clues for further probing of the molecular mechanism of sulfide metabolism. © 2012 Elsevier Inc. All rights reserved.
1. Introduction Endogenous sulfide, produced in vertebrates (Abe and Kimura, 1996) and invertebrates (Julian et al., 2002, 2005a; Gainey and Greenberg, 2005), plays a role at low concentrations as a cellular signaling molecule. Exogenous sulfide, described as the aggregate of H2S, HS−, and S2−, is a common substance in hydrothermal vents, cold seeps, sewer outfalls, marshes, mudflats, and highly eutrophic freshwater lakes (Nicholls and Kim, 1982), and can cause harm to organisms in a variety of ways, including reversible cytochrome c oxidase inhibition (Evans, 1967; Nicholls, 1975), hemoglobin oxygen affinity decrease (Carrico et al., 1978), sulfhemoglobin formation (Bagarinao, 1992; Kraus et al., 1996), mitochondrial depolarization (Julian et al., 2005b), free radical production (Tapley et al., 1999; Eghbal et al., 2004), oxidative damage to RNA and DNA (Joyner-Matos et al., 2010), coelomocyte death and cell proliferation decrease (Hance et al., 2008), and inhibition of nearly 20 enzymes involved in aerobic metabolism (Bagarinao, 1992). Inhibition of cytochrome c oxidase is believed to be the main mechanism of H2S toxicity (Beauchamp et al., 1984), although alternative mechanisms, including activation of ATP-activated potassium channels and alteration in cell signaling pathways, have also been postulated (Szabó, 2007). ⁎ Corresponding author. Tel./fax: + 86 532 82031647. E-mail address: [email protected] (Z. Zhang). 1 Joint first authors. 1744-117X/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.cbd.2012.04.001
Animals in sulfide-rich habitats typically reduce sulfide toxicity by employing physiological mechanisms (Grieshaber and Völkel, 1998), including sulfide-oxidizing enzymes and sulfide-binding amino acids (Joyner et al., 2003; Brand et al., 2007). In phototrophic sulfur bacteria, sulfide metabolism is a complex network and the processes involved remain incompletely understood. Several enzymes which catalyze sulfide oxidation have been isolated and some, such as flavocytochrome c and sulfide-quinone oxidoreductase (SQR), have been well characterized at the molecular level (Frigaard and Dahl, 2008). Researchers have found that SQR possesses the same mitochondrial electron acceptor in both prokaryotes and some eukaryotes (Theissen et al., 2003; Theissen and Martin, 2008). Furthermore, three consecutive sulfide oxidation reactions based on SQR, sulfur dioxygenase, and sulfur transferase have been reported in rat liver as well as in body wall of the lugworm, Arenicola marina (Hildebrandt and Grieshaber, 2008). Although the key genes in sulfide metabolism have been thoroughly investigated, it is still not clear how the expression of these genes is regulated and whether more genes participate in these biological processes. The Urechis unicinctus is a species of Echiura, Xenopneusta, Urechidae and Urechis and inhabits intertidal and subtidal mudflats in China, Korea, Russia, and Japan. It has been suggested to be a sulfide tolerant species, capable of sulfide metabolism and utilization (Zhang et al., 2006; Ma et al., 2010; Wang et al., 2010). Its SQR has been cloned and characterized (Ma et al., 2011a) and assessed regarding the influence of sulfide exposure on mRNA and protein expression (Ma et al., 2011b). Suppression subtractive hybridization (SSH) and cDNA microarray analysis are two
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useful techniques for rapidly detecting differentially expressed genes at high throughput. In the present study, we used these techniques to screen for sulfide metabolism genes in U. unicinctus and further verified the results using quantitative real-time polymerase chain reaction (qRT-PCR). These data can provide a good foundation for further exploration and understanding of the molecular mechanism of sulfide metabolism.
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Hybridizations and subsequent scanning, visualization, and quantitation were also performed by HGRC. The relative intensity of each spot was represented as the ratio of the Cy3 and Cy5 channels (R/G ratio), and converted to base-2 logarithms (log2). Spots with log2 R/G ratios > 1.0 (i.e., a 2-fold change) were considered upregulated and spots with ratios b −1.0 (i.e., a 0.5-fold change) were considered downregulated.
2. Materials and methods 2.1. Animals and sample collection
2.5. Sequence analysis
U. unicinctus, collected from a coastal intertidal flat in Yantai, China, had a mean fresh mass of 32.7±6.3 g and were maintained for 1 week in an aerated, recirculating seawater aquarium (20±1 °C, pH 8.0, salinity 25‰), and fed microalgae (Chlorella vulgaris and Mtzschia closterium). Feeding was discontinued 24 h prior to experimentation. For sulfide exposure, 20 worms were randomly assigned to a sulfide treated (50 μM sulfide in seawater) group and a control group, and the experiment was conducted in two airproof glass tanks (50 × 30 × 30 cm) with 40 L of seawater. The 50 μM sulfide concentration was produced by adding a sulfide stock solution (10 mM, pH 8.0), prepared using Na2S·9H2O, to the seawater and the sodium sulfide concentration was maintained by further stock addition at 2 h intervals according to spectrophotometric monitoring using a methylene blue method (Cline, 1969). All other conditions were the same as those used for acclimation and no mortality occurred during the experiment. At 24 h after sulfide exposure, seven worms were removed from each tank. Body wall from each worm was excised, frozen in liquid nitrogen, and stored at −80 °C for subsequent experiments.
All differentially expressed sequences were sequenced, submitted for BLAST X analysis, and searched for homology using National Center for Biotechnology Information databases. Matches were considered significant only when the probability (E value) was b1 × 10 − 4 and the sequences were classified by Gene Ontology terms (GO) using Blast2GO for level 2 categories.
2.2. Total RNA and mRNA isolation Total RNA from each body wall was extracted with Trizol (Invitrogen Corp., Beijing, China) according to the manufacturer's protocol. The RNA was quantified by 260 nm absorbance on a spectrophotometer and the quality assessed by electrophoresis, based on the rRNA band integrity. About 1.4 μg of mRNA was isolated from 700 μg of total RNA (each worm yielded similar RNA quantities) using the PolyATtract® mRNA Isolation Systems kit (Promega Corp., Madison, WI, USA). 2.3. Construction of subtractive library SSH was used to generate cDNA libraries enriched with genes differentially expressed under sulfide exposure. cDNA from sulfideexposed worm body wall was used as the tester and cDNA from the control worm was used as the driver. A subtractive cDNA library was constructed using the PCR-Select cDNA Subtraction kit (Clontech Laboratories, Inc., Beijing, China) according to the manufacturer's instructions. The subtracted PCR products were ligated into pMD-18T vector (Takara Bio Inc., Otsu, Japan) and transformed into Escherichia coli DH5α cells, which were then cultured overnight on LB agar plates containing 50 ppm ampicillin. Single colonies were then picked and screened for inserts using PCR, and insert fragments were amplified using primers (M13 (-47) forward and M13 (-48) reverse primers). 2.4. cDNA microarray A cDNA microarray chip containing 3456 cDNA clones was produced by Beijing Huada Institute for Gene Research Center (HGRC, Beijing, China). Inserts were amplified by PCR using adaptor-specific primers in the PCR-Select cDNA Subtraction kit (Clontech Lab, Inc., Beijing, China), the PCR products were purified via ethanol precipitation in 96-well plates, and the PCR-purified products were sent to the HGRC. Two μg samples of total RNA from the tester and the driver were used to produce Cy3/Cy5-labeled probes for hybridization to the microarray.
2.6. qRT-PCR analysis of selected genes Eight genes were selected and their expression levels in body-wall tissue from three worms for each group were evaluated using qRTPCR. RNA isolation was performed as described above and cDNA was synthesized using a reverse transcription system (Takara Bio, Inc.). All cDNAs were diluted (1:10) in nuclease-free water and stored at −20 °C. qRT-PCR was performed on each sample in duplicate using a fluorescence temperature cycler (7500 Real-Time PCR Systems, Applied Biosystems China, Beijing, China) in the presence of SYBR-green. Optimized qRT-PCR reactions were conducted according to the manufacturer's instructions (SYBR Premix Ex Taq, Takara Bio, Inc.), using β-actin as the internal standard. All primer sequences and PCR products are listed in Table 1. Data were analyzed using the 7500 System Sequence Detection Software version 1.4.0.25 (PE Applied Biosystems, Foster City, CA, USA) and the results presented as fold changes in transcription relative to that of the β-actin gene using the 2 − ΔΔCt method (Livak and Schmittgen, 2001).
2.7. Statistical analysis Data from qRT-PCR were calculated as mean±S.E. (n=3 with 2 replicates) and significant differences between the treatment and control groups were evaluated using one-way analysis of variance (ANOVA) followed by the Duncan's method, using the SPSS statistical package (version 18.0) at a significance level of pb 0.05. Table 1 Primers used in qRT-PCR analysis. Gene
Primer sequence (5′–3′)
PCR product (bp)
Serase
F-CTCTGATGCAGCACAACTGG R-TCCCCTAGGAGACATTGCTG F-ACTGCTCTCATGCAGGCTCT R-CACCAACTGGTCCAACACTG F-TCAGCCGAGTCGTCACATAG R-GTGTTGATCAGCCCAACCTT F-GCGGCGGATTATCTGTGT R-GTTCACGGGTGATCCTGATG F-GTGGACCTCATGCTTGGAAT R-ATCCAGAGCTCTTGGTGGAA F-GACATCAGGACCACCTTTCC R-GCCTGGGTGTAGACAGGAGA F-AGCGTCAAAACCACCTGAAC R-TGCTTACACCGTCTGCATTC F-GCCTCTGGCCTTGATAAGTG R-CTTGTCCTGATGGTCGGTTC F-CCCATCTACGAGGGATACGC R-CCTTGATGTCACGGACGATT
184
Vacuolar protein Src tyrosine kinase Sulfite oxidase-like oxidoreductase Aprataxin SN-RNP U1 Predicted protein Aminopeptidase β-actin
225 171 176 241 194 180 160 151
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3. Results 3.1. Construction and screening of cDNA library In the present study, a subtracted cDNA library was constructed from the sulfide-exposed worm body wall and compared to the control using SSH. The subtraction efficiency was 2 15, determined by comparison of the abundance of β-actin between the subtracted and unsubtracted products. Based on the above efficiency, 4200 differential colonies were generated by cloning the SSH cDNA products into a plasmid vector and 3456 monoclones were screened using colony PCR. The size of the inserted fragments ranged from 150 to 650 bp, with most at 300–600 bp. 3.2. Microarray analysis The results of microarray screening based on the 3456 monoclones from SSH showed that, of the 178 spots (differential sequences) obtained and sequenced, 133 spots were upregulated (ratio≥2) and 45 spots were downregulated (ratio≤0.5) in sulfide‐exposed worms. 3.3. Identification of the differentially expressed genes Eighty-two ESTs were identified successfully from the 178 sequences mentioned above and 26% of the assembled sequences (21 genes) shared significant similarity to known genes (E b 1 × 10 − 4) in GenBank, while 64% showed no significant similarity (E > 1 × 10 − 4) to any known genes or proteins in the database (Table 2). The 21 genes were classified into 8 groups based on their biological process category (Fig. 1), including metabolism, cellular process, biological regulation, response to stimulus, multicellular organismal process, localization, development, and cellular component organization. 3.4. Expression of the selected differentially expressed genes Expressions of the 8 differentially expressed genes (serase, vacuolar protein sorting 45, src tyrosine kinase, sulfide oxidase-like oxidoreductase, aprataxin, SN-RNP, aminopeptidase, and predicted protein) detected using qRT-PCR indicated that after sulfide exposure 5 genes (serase, src tyrosine kinase, sulfide oxidase-like oxidoreductase, SN-RNP, predicted protein) showed upregulated expression and the other 3 genes showed downregulated expression (Fig. 2). The correlation between the qRT-PCR data and the microarray data regarding these 5 of the 8 genes indicated that
Fig. 1. Classification of genes in level 2 biological process categories. Sequences having no homology with sequences known in the public database are not shown (E> 1 × 10− 4).
the microarray results were good indicators of overall gene expression changes. In upregulated genes, the predicted protein gene expression was the highest, being 1.34-fold higher than the control (pb 0.05), while serase and SN-RNP were increased less than 1 fold (p>0.05). Among the downregulated genes, a significant decrease was detected in aprataxin and aminopeptidase (pb 0.05), at 0.44‐fold and 0.41-fold lower than the control, respectively. 4. Discussion In this study, we report a work regarding sulfide metabolism-related genes, screened using SSH libraries and cDNA microarray, from sulfideexposed and control body walls of U. unicinctus. The selected differentially expressed gene sequences were classified to eight main gene categories which were related to metabolism, cellular process, biological regulation, response to stimulus, multicellular organismal process, localization, development, and cellular component organization. 4.1. A combined approach using SSH and cDNA microarray SSH is a powerful technique for obtaining differentially expressed genes at a large-scale survey, which enables the comparison of two populations of mRNA by cloning genes that are expressed in one
Table 2 Blast homology search results of significant sequences from microarray analysis. Clone ID Upregulated 22 416 420 126 411 457 225 127 431 334 Downregulated 423 133 230 239 438 118 445 224
No. of clones
Product length (bp)
Highest BlastX match
Species
E value
1 1 1 2 1 1 2 1 1 1
664 406 687 393 454 752 923 373 396 454
Small nuclear ribonucleoprotein Sm D2-like Arginase type I Aprataxin Predicted: zonadhesin-like Predicted protein Hypothetical protein BRAFLDRAFT_81368 RecName: full = arenicin-1; flags: precursor Predicted: zonadhesin-like Usp30 protein Predicted protein
Apis mellifera Strongylocentrotus purpuratus Danio rerio Danio rerio Nematostella vectensis Branchiostoma floridae Arenicola marina Danio rerio Mus musculus Nematostella vectensis
2 × 10− 42 5 × 10− 38 6 × 10− 21 4 × 10− 4 3 × 10− 16 9 × 10− 16 3 × 10− 8 1 × 10− 4 8 × 10− 4 5 × 10− 16
1 2 2 1 1 1 1 1
627 373 373 437 527 660 747 529
Vacular protein sorting 45 Predicted: zonadhesin-like Predicted: zonadhesin-like T-cell receptor beta chain Similar to Serase-1B SN-RNP U1, putative Src-family protein tyrosine kinase Aminopeptidase
Monodelphis domestica Danio rerio Danio rerio Plasmodium berghei strain ANKA Ciona intestinalis Ixodes scapularis Strongylocentrotus purpuratus Lumbricus rubellus
5 × 10− 70 4 × 10− 6 4 × 10− 6 8 × 10− 6 3 × 10− 6 2 × 10− 6 1 × 10− 4 6 × 10− 54
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Fig. 2. Expression of eight genes detected using qRT-PCR of total RNA from body wall of control and sulfide‐exposed worms. The asterisk indicates significant differences at *p b 0.05 determined by ANOVA. n = 3 with 2 replicates; after exposure upregulated genes: predicted protein, 1.34-fold higher; src tyrosine kinase, 0.46-fold; sulfide oxidase-like oxidoreductase, 0.52-fold; SN-RNP, 0.10-fold; and serase, 0.01-fold high; downregulated genes: aprataxin, 0.44-fold lower; aminopeptidase, 0.41-fold; and vacuolar protein, 0.13-fold.
population but not in the other. Currently, the approach has been widely used to identify genes responding to different pathogens and stress (Bayne et al., 2001; Zhang and Gui, 2004; Zhang et al., 2007), however, false positive results frequently occur. The validity of SSH selected sequences can be increased by combining SSH and microarray techniques. The first report of the two methods being successfully combined was in breast cancer research (Yang et al., 1999). A SSH and cDNA microarray combination has been utilized extensively in studies of gene expression (Gracey et al., 2001; Korke et al., 2004). Gracey et al. (2001) used SSH and cDNA microarray to understand the changes in gene expression in a hypoxia-tolerant fish, Gillichthys mirabilis. Their results showed a complex transcriptional response; they found novel differential expressed genes and tissue-specific patterns of expression during hypoxia (Gracey et al., 2001). Korke et al. (2004) examined gene expression profiling of metabolic shift of mammalian cells. They used the two techniques to identify 123 differentially expressed mRNA transcripts related to glucose metabolism from 4972 genes (Korke et al., 2004). In the present study, 3456 differential monoclones, derived from the control and sulfide-exposed body-wall RNA of U. unicinctus, were screened using SSH and 178 different spots were detected using cDNA microarray based on the monoclones. The combination of methods economized both the cost and labor involved in identifying the abundant differentially expressed clones screened using SSH. However, here the positive detection rate of SSH, confirmed by microarray analysis, was ~5.5%. Two reasons for this result were considered. First, if the signal intensity of one gene in two channels was too strong or too weak, the gene could be ignored although it was differentially expressed. And second, with a 24 h sulfide exposure, some genes expressed during the initial part of the exposure may later be inactive or decreased in expression, which caused weak intensity that was not detected. Thus, the cDNA microarray technique was an improvement in the following aspects: (1) elimination of interference by hybrid signals among molecules, (2) increased chip sensitivity, and (3) provided a homogeneous method at a more scientific and reasonable level. A total of 82 differentially expressed genes were identified in this study. However, a high proportion of these genes (64% genes) presented no significant similarity (E > 1 × 10 − 4) to known genes or proteins in the database. Two possible reasons for this result might have been: (1) some genes may represent untranslated regions of known
genes, and (2) no orthologs were registered in the GenBank, in which some unknown genes were detected which may help extend the understanding of their functions in sulfide metabolism. 4.2. The functional classification of genes The application of gene ontology is one method for integrating gene expression data with particular biological processes, although this method has many shortcomings, such as incompletely annotated databases, imprecise information, and the possibility that one gene may be involved in several biological processes, thus confounding interpretation. Here, most of the sequences differentially expressed with sulfide exposure fell into gene categories related to metabolism and cellular process. 4.2.1. Genes related to metabolism Sulfide is a potent toxin as well as a substrate of the mitochondrial respiration (Grieshaber and Völkel, 1998). Thus, animals have had to evolve mechanisms to effectively exploit H2S while simultaneously avoiding poisoning. A mitochondrial pathway catalyzing sulfide oxidation and combining with consume molecular oxygen, has been found in mammals and invertebrates (Hildebrandt and Grieshaber, 2008). Recently, Budde and Roth (2010) have proved that H2S could induce hypoxia-inducible factor-1 (Hif-1) activity in the nematode, Caenorhabditis elegans (Budde and Roth, 2010). In other species, the genes related to hypoxia were also found in H2S exposed (Han et al., 2006; Joyner-Matos et al., 2006; Roberts et al., 2008). In the present study, 29% of the detected differentially expressed genes were involved in metabolism, such as arginase and vps, being changed in expression with sulfide exposure. Here the arginase was up-regulated while the vps (related to vesicle-mediated transport) was down-regulated. Induced high expression and increased activity of arginase could provide more L-proline to support the biosynthesis of collagen, an important element of the tissue matrix (Que et al., 1998). Arginase activity or expression has been found to increase in a variety of cell types with hypoxia, such as in the brain of rats exposed to hypoxic–ischemic insults (Clarkson et al., 2005), in wound-derived rat macrophages (Albina et al., 1995) and in human pulmonary artery smooth muscle cell (Chen et al., 2009).
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4.2.2. Genes related to cellular process In the present experiment, genes associated with DNA repair and RNA splicing, such as aprataxin and snRNP, were more highly expressed with sulfide exposure. The aprataxin gene may play a role in single-stranded DNA repair. Aprataxin could catalyze the nucleophilic release of adenylate groups covalently linked to 5′ phosphate termini, resulting in termini that can again serve as a substrate for DNA ligase (Rass et al., 2007). Sulfide could cause DNA strand breaks and purine ring opening in Chinese hamster ovary cell (CHO) (Attune-Ramos et al., 2007), resulting in oxidative damage to the DNA and RNA in the mudflat polychaete Glycera dibranchiate (Joyner-Matos et al., 2010). Genes involved in DNA repair, which are activated by c-Jun and ATF2, will increase its expression after exposing intestinal epithelial cells (IEC-18) to 0.05 to 5 mmol/L NaHS (Deplancke and Gaskins, 2003). The same phenomenon was also observed in H2S exposed rat nasal respiratory epithelium (Roberts et al., 2008). 4.2.3. Genes related to others Genes involved in stimulus, such as arenicin-1, were also upregulated by sulfide exposure. Arenicin-1 is a novel antimicrobial peptide (AMP) that has been isolated from coelomocytes of the marine polychaete lugworm A. marina (Ovchinnikova et al., 2004). Until now AMPs have been identified in Potamopyrgus antipodarum, Takifugu rubripes and some other species (Osnas and Lively, 2006; Xie et al., 2009; Matsumoto et al., 2011). AMPs are considered to play a key role in invertebrate host defense and vertebrate defense. But its exact mechanism at molecular level is not completely understood. Park and Lee's (2009) study suggests that arenicin-1 is likely to exert fungicidal effect by disturbing fungal membrane. It is an interesting phenomenon that, here, the majority of predicted proteins are involved with zinc ion binding. Zinc ion related proteins have been implicated in sulfide binding (Flores et al., 2005) and function in a variety of processes, including binding DNA and RNA and participating in protein–protein interactions (Coleman, 1992). The comprehensive role may imply that kinds of genes participate in sulfide metabolism. In summary, genes involved in sulfide metabolism and their interactions and relationships remain unclear. In the present study, 82 differentially expressed genes were obtained from sulfide‐exposed U. unicintus, using a combination of SSH with cDNA microarray analysis. These genes were related to several biological processes, such as hypoxia, cellular process, DNA repair, and a majority of unknown sequences (including unknown sulfide-related genes). These results provide new gene-related information for future molecular research into the mechanism of sulfide metabolism. Acknowledgments This work is supported by the National Natural Science Foundation of China (40776074 and 31072191). References Abe, K., Kimura, H., 1996. The possible role of hydrogen sulfide as an endogenous neuromodulator. J. Neurosci. 16, 1066–1071. Albina, J.E., Henry, W.L., Mastrofrancesco, B., Martin, B.A., Reichner, J.S., 1995. Macrophage activation by culture in an anoxic environment. J. Immunol. 155, 4391–4396. Attune-Ramos, M.S., Wagner, E.D., Gaskins, H.R., Plewa, M.J., 2007. Hydrogen sulfide induces direct radical-associated DNA damage. Mol. Cancer Res. 5, 455–459. Bagarinao, T., 1992. Sulfide as an environmental factor and toxicant: tolerance and adaptations in aquatic organisms. Aquat. Toxicol. 24, 21–62. Bayne, C.J., Gerwick, L., Fujiki, K., Nakao, M., Yano, T., 2001. Immune-relevant (including acute phase) genes identified in the livers of rainbow trout, Oncorhynchus mykiss, by means of suppression subtractive hybridization. Dev. Comp. Immunol. 25, 205–217. Beauchamp, R.O., Bus, J.S., Popp, J.A., Boreiko, C.J., Andjelkovich, D.A., 1984. A critical review of the literature on hydrogen sulfide toxicity. CRC Crit. Rev. Toxicol. 13, 25–95. Brand, G.L., Horak, R.V., Bris, N.L., Goffredi, S.K., Carney, S.L., Govenar, B., Yancey, P.H., 2007. Hypotaurine and thiotaurine as indicators of sulfide exposure in bivalves and vestimentiferans from hydrothermal vents and cold seeps. Mar. Ecol. 28, 208–218. Budde, M.W., Roth, M.B., 2010. Hydrogen sulfide increases hypoxia-inducible factor-1 activity independently of von Hippel–Lindau tumor suppressor-1 in C. elegans. Mol. Biol. Cell 21, 212–217.
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