294 ISJ 15: 294-301, 2018 ISSN 1824-307X RESEARCH REPORT Molecular characterization of a defender against apoptotic cell death 1 gene (CfDAD1) from the mollusk Chlamys farreri MQ Wang1,3, BJ Wang1, M Liu1, KY Jiang1, L Wang1,2,4* 1CAS Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China 2Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266237, China 3Research Platform for Marine Molecular Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266237, China 4Center for Ocean Mega-Science, Chinese Academy of Sciences, Qingdao 266400, China Accepted August 27, 2018 Abstract The defender against apoptotic cell death 1 (DAD1) was a negative regulator to inhibit the apoptosis process. In the present study, a DAD1 gene (designated as CfDAD1) was identified in Zhikong scallop Chlamys farreri. Its full-length cDNA sequence of CfDAD1 contained a 342 bp open reading frame (ORF), which encoded a mature protein of 113 amino acids. A DAD domain was revealed from the deduced protein sequence of CfDAD1. The mRNA transcripts of CfDAD1 could be detectable in all the investigated tissues, including hemocytes, muscle, mantle, gill, hepatopancreas and gonad, among which the maximum expression level were found in hemocytes. Being infected with Vibrio splendidus, Staphylococcus aureus or Yarrowia lipolytica, the mRNA expression levels of CfDAD1 in hemocytes were all significantly up-regulated. Moreover, the apoptosis level of CfDAD1-suppressed group was significant higher than those of control groups. All these results indicated that CfDAD1 was efficient negative regulator of apoptosis and involved in the innate immune responses of scallop. Key Words: Apoptosis; Chlamys farreri; Innate immunity Introduction Apoptosis, also termed as programmed cell death (PCD), was one of the most fundamental processes essential for normal growth and regular development in multicellular organisms (Nagata, 2018). The main characteristic morphological features of apoptosis included cellular shrinkage, chromatin condensation, chromosomal DNA fragmentation, global mRNA decay, membrane blebbing and nuclear condensation (Banfalvi, 2017). Apoptosis was considered as a pivotal component of various biological processes, such as chemical induced cell death, embryonic development, hormone dependent atrophy, normal cell turnover, proper functioning of the immune defense system, and so on (Lockshin, 2016). Several factors required ___________________________________________________________________________ Corresponding author: Lei W ang CAS Key Laboratory of Experimental Marine Biology Institute of Oceanology Chinese Academy of Sciences Qingdao 266071, China E-mail: wanglei@qdio.ac.cn for execution or regulation of apoptosis were rather conserved in evolution, including caspases (Galluzzi et al., 2016), inhibitor of apoptosis proteins (IAPs) (Kocab and Duckett, 2016), and also defender against apoptotic cell death 1 gene (DAD1) (Zhang et al., 2016). However, the activation of apoptotic pathways and the interaction between the key genes differ between the main taxonomic groups of invertebrates, and it is important to investigate the diversity of the apoptotic pathways found in the animal kingdom (Estévez-Calvar et al., 2013). DAD1, a regulatory protein to inhibit the apoptosis process, was first described and characterized as a negative regulator of mammalian cell death that may act downstream of the B-cell lymphoma 2 (BCL2) protein (Nakashima et al., 1993). The function of DAD1 has been well investigated in multicellular model organisms, for examples, mutants in mouse DAD1 exhibited aberrant morphology, developmental delay and increased apoptosis during embryogenesis (Nishii et al., 1999; Hong et al., 2000), while over expression of DAD1 inhibited developmental apoptosis during 295 embryogenesis in Caenorhabditis elegans (Zhang et al., 2016). These research achievements indicated that DAD1 would play irreplaceable roles in regulating apoptosis and cell viability. Compared with the extensive identification and investigation of DAD1 in vertebrates, although DAD1 has already been identified and primary characterized in the bay scallop Argopecten irradians (Zhu et al., 2008), black tiger shrimp Penaeus monodon (Molthathong et al., 2008) and kuruma shrimp Marsupenaeus japonicas (Zheng et al., 2016), the information on DAD1 in marine invertebrate is still rare and fragmentary. Zhikong scallop Chlamys farreri (Mollusca; Bivalvia; Pteriomorphia) is an important and representative species of mollusk, not only for its economic and ecological importance, but also for its increasing value in the invertebrate innate immunity investigation (Matozzo, 2016; Tascedda and Ottaviani, 2016; Gerdol, 2017). The research on DAD1 in C. farreri would enhance the understanding of its potential functions in apoptosis process and invertebrate innate immunity. However, no information on DAD1 was available in Zhikong scallop yet. To bridge this gap, a novel DAD1 gene (designated as CfDAD1) has been identified and investigated in C. farreri, and the main objectives of the present study were (1) to characterize the molecular features of CfDAD1 (2) to validate the spatial and temporal expression pattern of its mRNA transcripts, and (3) to confirm its function via double strand RNA (dsRNA) mediated RNA interference (RNAi). Material and Method Scallops, in vivo experimental infection and samples collection Adult scallops (average 50 mm in shell length) were purchased from a local farm in Qingdao, China, and maintained in aerated seawater at about 20 °C. Approximately two hundred scallops were used for in vivo experimental infection assay according to our previous report (Wang et al., 2017). Animals were infected by bath exposure using different bacteria and yeast. Being acclimated for two weeks, fifty scallops were transferred to tanks containing live bacteria Vibrio splendidus strain JZ6 at a final concentration of 1.0 × 108 colony forming units (CFUs) per 1 mL, defined as Gram-negative bacterial infection group. Another fifty scallops were transferred and kept in the tanks containing live bacteria Staphylococcus aureus strain 33025 (SanYao, China) at a final concentration of 1.0 × 108 CFUs mL-1, defined as Gram-positive bacterial infection group. The third fifty scallops were maintained in the fungi-containing tanks with live yeast Yarrowia lipolytica strain N11b at a final concentration of 1.0 × 108 CFUs mL-1, defined as fungi infection group. And the last fifty scallops were treated as the control group. Five scallops from every group were randomly sampled at 0, 3, 6, 12, 24 and 48 h post stimulation, respectively. The hemolymphs were collected and centrifuged at 800 g, 4 °C for 10 min to harvest the hemocytes for RNA isolation. Hemocytes, muscle, mantle, gill, hepatopancreas and gonad from five untreated scallops (one animal was employed as one repeat for one tissue sample) were collected to determine the spatial expression pattern of CfDAD1 mRNA transcripts. RNA isolation and cDNA synthesis Raw RNA was isolated using RNAiso Plus (9108, Takara, Japan). The SuperScript IV Reverse Transcriptase (18090010, Thermo Fisher Scientific, USA) was employed to synthesize the first-strand cDNA synthesis using RQ1 DNase I (M6101, Promega, USA) treated raw RNA as template and adaptor primer-oligo (dT) as primer (Table 1). The reaction were carried out at 55 °C for 1 h, terminated by heating at 80 °C for 5 min, then a homopolymeric tail was added with dCTP (4028, Takara, Japan) and terminal deoxynucleotidyl transferase (TdT, 2230, Takara, Japan) and then stored at -80 °C till use. Cloning the complete cDNA sequence of CfDAD1 In our previous works (Wang et al., 2018b), a transcript sequence homologues to previous identified DAD1s was generated via assembling and screening public available transcriptomic data and expression sequence tags (ESTs) in C. farreri. And this transcript sequence was selected for further cloning the complete cDNA sequence of CfDAD1. Four gene-specific primers, CfDAD1-RACE-R1/2 and CfDAD1-RACE-F1/2, were designed based on this sequence for 5` and 3` rapid amplification of cDNA ends (RACE) technique, respectively (Table 1). All PCR amplification was carried out in a MJ Mini Personal Thermal Cycler (Bio-Rad, USA), and PCR products were gel-purified using Monarch DNA Gel Extraction Kit (T1020S, NEB, USA), ligated into the pMD18-T simple vector (D103A, Takara, Japan), and then transformed into the competent cells Escherichia coli strain DH5α (CB101, Tiangen, China). The positive recombinants were identified via anti-ampicillin selection and verified by PCR using M13-47 and RV-M as primers (Table 1). Five of the positive clones were sequenced using a PRISM 3730XL automated sequencer (Thermo Fisher Scientific, USA). Bioinformatical analysis of CfDAD1 cDNA and deduced amino acid sequences The blast+ 2.7.1 was employed to perform search for protein sequence similarity. The deduced protein sequences of CfDAD1 were analyzed by Lasergene suite 7.1.0.44 using the EditSeq module. The presence and location of signal peptide and the function domains were predicted with SignalP 4.1 and Simple Modular Architecture Research Tool (SMART) 7.0, respectively. Multiple sequence alignments were generated using Clustal Omega 1.2.4 combined with Sequence Manipulation Suite 2.0. A Neighbor-Joining (NJ) phylogenic tree was generated using MEGA-X 10.0.1. Bootstrap trials were replicated 1000 times to derive confidence value for the phylogeny analysis. Quantitative real-time PCR analysis of CfDAD1 mRNA expression profiles The spatial and temporal mRNA expression profiles of CfDAD1 in hemocytes of scallops infected by various microbes were detected by quantitative real-time PCR (qRT-PCR). All qRT-PCR reactions 296 Table 1 Oligonucleotide primers used in the experiments Primers Sequence (5`-3`) Brief information adaptor primer GGCCACGCGTCGACTAGTAC Anchor primer for 3’ RACE adaptor primer-oligo (dG) GGCCACGCGTCGACTAGTACG10HN Anchor primer for 5’ RACE adaptor primer-oligo (dT) GGCCACGCGTCGACTAGTACT17VN Olido (dT) for cDNA synthesizing CfEF-1α-qRT-F* ATCCTTCCTCCATCTCGTCCT Internal control for real-time PCR CfEF-1α-qRT-R* GGCACAGTTCCAATACCTCCA Internal control for real-time PCR CfDAD1-CDS-F ATGCCTGATAGTTTATTTTCCGTGGTG Gene specific primer for CDS CfDAD1-CDS-R CTATCCAATGAAGTTGATAACAACTAA Gene specific primer for CDS CfDAD1-dsRNA-Basic-F TTATATTCTGTCATTCACTTC Gene specific primer CfDAD1-dsRNA-Basic-R TGTAAAATAATATGAGCAAAAATAAAG Gene specific primer CfDAD1-dsRNA-T7-F GGATCCTAATACGACTCACTATAGGGATCCTTATATTCTGTCATTCACTTC Gene specific primer with T7 promoter CfDAD1-dsRNA-T7-R GGATCCTAATACGACTCACTATAGGGATCCTGTAAAATAATATGAGCAAAAATAAAG Gene specific primer with T7 promoter CfDAD1-qRT-F* CGGCTACAAGTGAATCCACAG Gene specific primer for real-time PCR CfDAD1-qRT-R* TCCTTCCCATCACCATTTCCA Gene specific primer for real-time PCR CfDAD1-RACE-F1 CATTGGATAGTATGTTATGGAAATGGT Gene specific primer for RACE CfDAD1-RACE-F2 ATTCTTCAACAGTACAGATGTGTATAT Gene specific primer for RACE CfDAD1-RACE-R1 GGATGACGCCTGTCATCAGGATGTAGA Gene specific primer for RACE CfDAD1-RACE-R2 TTTTTGCAAAATAAAATCAACGTGGCA Gene specific primer for RACE EGFP-dsRNA-Basic-F CGACGTAAACGGCCACAAGT GFP primer EGFP-dsRNA-Basic-R CTTGTACAGCTCGTCCATGC GFP primer EGFP-dsRNA-T7-F GGATCCTAATACGACTCACTATAGGGATCCGACGTAAACGGCCACAAGT GFP primer incorporated with T7 promoter EGFP-dsRNA-T7-R GGATCCTAATACGACTCACTATAGGGATCCTTGTACAGCTCGTCCATGC GFP primer incorporated with T7 promoter M13-47 CGCCAGGGTTTTCCCAGTCACGAC Sequencing primer RV-M GAGCGGATAACAATTTCACACAGG Sequencing primer * The efficiency of CfEF-1α-qRT-F/R and CfDAD1-qRT-F/R were 98% and 101%, respectively. were carried out with the SYBR premix ExTaq (RR420, Takara, Japan) in a LineGene K FQD-48A (A4) Fluorescence Quantitative PCR Detection System (Bioer, China) using 100 ng cDNA as template. All the primer information for qRT-PCR was listed in Table 1. The efficiency of qRT-PCR primers were checked using serial two-fold dilutions of cDNA (Pfaffl, 2001), and the efficiency of CfEF-1α-qRT-F/R and CfDAD1-qRT-F/R were almost the same (98% and 101%, respectively). The mRNA expression of CfDAD1 was normalized to that of elongation factor 1 α (EF-1α) for each sample. The relative mRNA expression levels of CfDAD1 were calculated using comparative CT method (2-ΔΔCT method) (Schmittgen and Livak, 2008) as mean ± S.D. (n = 5). The data were tested with one-way analysis of variance (ANOVA) followed by a multiple comparison using IBM SPSS Statistics 23.0.0.0, and the p values less than 0.05 were considered statistically significant. Knock-down of CfDAD1 gene in vivo via dsRNA mediated RNA interference and apoptotic assay T7 promoter adapted primers CfDAD1-dsRNA-T7-F/R and EGFP-dsRNA-T7-F/R (Table 1) were used to amplify cDNA fragments from CfDAD1 and enhanced green fluorescent protein (EGFP), and the PCR products were gel-purified and used as templates to synthesize dsRNA. The dsRNA products were synthesized via in vitro transcription according to the our previously reports (Wang et al., 2011; Wang et al., 2016a,b; Wang et al., 2018a), and its concentration was quantified using Nanodrop Lite (Thermo Fisher Scientific, USA) by the absorbance at 260 nm and adjusted to a final concentration of 1 mg mL-1. One hundred micrograms of CfDAD1 dsRNA was injected into the adductor of each scallop, and the control groups received an injection of 100 μg EGFP dsRNA or PBS, while the untreated scallops were employed as blank group. Hemocytes from five randomly sampled scallops of each treatment were collected every 12 h (0, 12, 24, 36, 48, 60, 72, 84, 96, 108 and 120 h post dsRNA injection) and used for total RNA isolation and cDNA synthesis. The effect of dsRNA-mediate RNAi for CfDAD1 in scallops was verified via qRT-PCR, and 70% inhibition of mRNA abundance after dsRNA injection was a threshold for an effective RNAi assay (Krueger et al., 2007). Five individuals were randomly sampled at 0, 48, 72 and 120 h post dsRNA injection, and the hemolymphs were collected and centrifuged at 800 g, 4 °C for 10 min to harvest the hemocytes for apoptotic assay. The apoptosis level of scallops was assayed via caspase-3 colorimetric assay kit (KGA204, KeyGEN BioTECH, China). All data were given in terms of OD450 as means ± S.D (n=5). The data were tested with one-way analysis of variance (one-way ANOVA) followed by a multiple comparison (S-N-K) using IBM SPSS Statics 23.0.0.0, and the p values less than 0.05 were considered statistically significant. 297 Fig. 1 Sequence features, multiple alignments and phylogenetic analysis of CfDAD1. A: Nucleotide and deduced protein sequences of CfDAD1. The nucleotides and amino acids were numbered along the left margin. The function domain was in shade. The transmembrane segments were underlined. The stop codon was indicated by asterisks. B: Multiple alignments of CfDAD1 with previous known ones. The black shadow region indicated positions of same amino acid residues shared by all sequences. Similar amino acid residues were shaded in grey. Gaps were indicated by dashes to improve the alignment. C: Consensus phylogenetic tree based on the amino acid sequences of different DAD1s. Neighbor-Joining method was selected to infer the evolutionary history. The numbers at the forks stood for the bootstrap value (%). The sequences and their accession numbers are as follows: Argopecten irradians, AAX56947; Gallus gallus, AAC60276; Haliotis discus, ANF99571; Haliotis diversicolor, AGA92566; Homo sapiens, AAB58540; Marsupenaeus japonicas, AON76443; Mus musculus, CAA73779; Orseolia oryzae, ALH18096; Palaemon carinicauda, AGJ03553; Penaeus monodon, ABU54835; Sus scrofa, BAA13115 Results The molecular feathers and phylogeny relationship of CfDAD1 The complete cDNA sequence of CfDAD1 was obtained via 5` and 3` RACE, and then submitted to GenBank with the accession number KU361830. It comprised 660 bp, containing a 78 bp 5` untranslated regions (UTR), a 240 bp 3` UTR with a poly A tail and a 342 bp open reading frame (ORF). This ORF encoded a mature protein of 113 amino acid residues with a theoretical molecular mass at about 12.66 kDa and a calculated isoelectric point (pI) of 7.99. No signal peptide could be predicted in the deduced protein sequence. The deduced amino acid sequence of CfDAD1 contained a DAD domain (from L5 to G113). Additionally, three transmembrane segments (from A30 to F51, from F56 to N78, and from A93 to I112) were also revealed (Figure 1A). An alignment of the protein sequence of CfDAD1 with those previously identified ones was shown in Figure 1B. The deduced amino acid sequence of CfDAD1 exhibited high similarity with previously identified ones, such as 99% identity with that of A. irradians (AAX56947), 94% with Mizuhopecten yessoensis (OWF34780), and 81% with Haliotis diversicolor (AGA92566). A NJ phylogenetic tree based on protein sequences from multiple DAD1s was positioned separately into two main branches, sequences form invertebrates and vertebrates were separated. Within the invertebrates, CfDAD1 was closer to similar sequences from another mollusc such as bay scallop A. irradians and far from those from crustaceans (Figure 1C). 298 Fig. 2 Spatial and temporal mRNA expression patterns of CfDAD1. A: Spatial mRNA expression pattern of CfDAD1. The mRNA expression level in hemocytes, mantle, gill, hepatopancreas and gonad of five adult scallops was normalized to that of muscle. B-D: Temporal mRNA expression patterns of CfDAD1 in hemocytes at 0, 3, 6, 12, 24 and 48 h post microbe infection (B: V. splendidus, C: S. aureus, D: Y. lipolytica). Vertical bars represented mean ± S.D. (n = 5), and bars with different characters were statically significant (p < 0.05) The spatial and temporal expression profile of CfDAD1 mRNA transcripts The qRT-PCR was employed to detect the spatial and temporal mRNA expression profiles of CfDAD1 with EF-1α as internal control. The CfDAD1 mRNA was detectable in all the tested tissues. The maximum level was observed in hemocytes, which was 5.33-fold (p < 0.05) of that in muscle, followed by gill and hepatopancreas, which were 3.29-fold and 3.16-fold of that in muscle (p < 0.05), respectively. And the mRNA expression levels in mantle and gonads were 1.05-fold and 1.12-fold of that in muscle (p > 0.05), respectively (Figure 2A). The mRNA expression levels of CfDAD1 were all up-regulated post different in vivo experimental infection. The mRNA expression level of CfDAD1 significantly up-regulated at 3 h post V. splendidus infection (4.04-fold compared with the origin level, p < 0.05), and reached to the peak level at 12 h (14.08-fold, p < 0.05, Figure 2B). In the S. aureus infection group, its mRNA expression level was significantly up-regulated at 6 h post infection (5.36-fold, p < 0.05), reached the peak level at 12 h post infection (9.38-fold, p < 0.05), maintained at a high level at 24 h (5.71-fold, p < 0.05) and then decreased to the origin level at 48 h (Figure 2C). Being infected by Y. lipolytica, its mRNA transcripts were significantly induced at 6 h post infection (3.17-fold, p < 0.05) and reached the maximum level at 12 h post infection (5.98-fold, p < 0.05), maintained at a high level at 24 h (3.32-fold, p < 0.05) and then decreased to the origin level at 48 h (Figure 2D). The apoptotic levels in CfDAD1-suppressed scallops In the present study, the effect of dsRNA-mediate RNAi was verified via qRT-PCR, and the mRNA expression level of CfDAD began to decrease at 24 h post dsRNA injection (0.66-fold, p < 0.05) and kept at a rather low level (less than 0.3-fold of the normal expression level) during 36 to 84 h post RNAi (Figure 3A). After dsRNA injection, the apoptosis level of CfDAD1-suppressed scallops lagged behind the expression change of CfDAD1 and was obviously higher than those of other groups at 48, 72 and 120 h post injection (Figure 3B). 299 Fig. 3 RNAi of CfDAD1 and apoptosis level. A: The relative abundance of CfDAD1 mRNA during RNAi. Each values was shown as mean ± S.D. (n = 5), and bars with different characters indicated statically significant (p < 0.05). B: Gene silencing of CfDAD1 increased apoptosis level. Each values was presented as OD405 and shown as mean ± S.D. (n = 5), and bars with different characters indicated statically significant (p < 0.05) Discussion DAD1 was a negative regulatory protein to inhibit the apoptosis process (Kelleher and Gilmore, 1997; Hong et al., 2000; Lockshin, 2016; Nagata, 2018). In the present study, the complete cDNA sequence of CfDAD1 was identified and characterized in C. farreri. Its sequence feathers, high similarity and close phylogenetic relationship to previously identified ones collectively suggested that it was a novel member of invertebrate DAD1 family and may have similar functions. DAD1 it has been observed to be ubiquitously expressed in various tissues in transcriptional level in marine invertebrates (Molthathong et al., 2008; Zhu et al., 2008; Zheng et al., 2016), and in the present study, the CfDAD1 mRNA could be also detected in all the tested tissues. The highest mRNA expression level was found in hemocytes, followed with gill and hepatopancreas, and the variable spatial distribution of CfDAD1 mRNA transcripts was speculated to be related with tissue function. The low apoptotic activity in hemocytes compared to other tissues has been reported in other bivalves 300 such as the Mytilus galloprovincialis (Romero et al., 2011), which was consist with our observation, for the function of CfDAD1, the higher gene expression the lower apoptotic activity. Moreover, the hepatopancreas was believed to be the central immune related organ in crustaceans and mollusks (Chai et al., 2010), while gill was regarded as the first defense line against invading microbes in fish and invertebrates (Ellis, 2001; Wang et al., 2016c). The high mRNA expression level of CfDAD1 in these three tissue indicated that DAD1 might be involved in the innate immune responses of scallop. It has been reported that DAD1 could respond to various stimuli in transcriptional level. For examples, MjDAD1 showed both dose-dependent and time-dependent responses to nitrite stress in M. japonicas (Zheng et al., 2016), while the mRNA expression levels of AiDAD1 gene of hemolymph were significantly high after injury in A. irradians (Zhu et al., 2008). In the present study, CfDAD1 mRNA transcripts in hemocytes could be significantly induced by the stimulation of three typical microbes. One potential speculation would be that the increase of CfDAD1 could reduce the apoptotic activity in hemocytes, for the low apoptotic level could favor the phagocytic activity of hemocytes to engulf invading pathogens as more as possible before its dead (Canesi et al., 2002). So, the rapid responses of CfDAD1 to various invading pathogens indicated that apoptosis and its regulators could play pivotal role in the innate immune response of scallops. The response of DAD1s to inhibit apoptosis has been well studied in an array of model organisms. For examples, mutants of DAD1 could increase cell apoptosis during embryogenesis in mouse (Hong et al., 2000), while the over expression of DAD1 would inhibit developmental apoptosis in C. elegans (Sugimoto et al., 1995). For neither gene mutation technique nor gene overexpression method was available in marine mollusk yet, dsRNA-mediated RNAi technique was applied to inhibit the expression of CfDAD1 to investigate its potential effects. In our previous reports, this dsRNA-mediated RNAi technique has been successfully applied to inhibit the expression of several target genes in scallop. In the present study, this protocol was employed as a powerful tool to analyse more in detail the apoptosis. After dsRNA injection, the apoptosis level of CfDAD1-suppressed scallops was obviously higher than those of other groups, suggesting the negative regulating roles of CfDAD1 during apoptosis in scallop. In conclusion, the complete cDNA sequence of a novel DAD1 gene was identified and characterized in C. farreri. Its mRNA expression levels significantly increased post microbe infection. The CfDAD1-suppressed scallops exhibited higher apoptosis level. All these results indicated that CfDAD1 was efficient negative regulator of apoptosis and involved in the innate immunity. Acknowledgement This research was supported by the National Natural Science Foundation of China (U1706209), Aoshan Innovation Project in Science and Technology from Qingdao National Laboratory for Marine Science and Technology (2016ASKJ07), Science and Technology Service Network Plan (STS) Major Deployment Project (KFZD-SW-106) and STS Regional Centre Project (Fujian province) from Chinese Academy of Sciences. 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