25 ISJ 16: 25-33, 2019 ISSN 1824-307X RESEARCH REPORT The first identification of a malectin gene (CfMal) in scallop Chlamys farreri: sequence features and expression profiles MQ Wang 1,3 , BJ Wang 1 , M Liu 1 , KY Jiang 1 , L Wang 1,2,4* 1 CAS Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China 2 Laboratory for Marine Biology and Biotechnology, National Laboratory for Marine Science and Technology, Qingdao 266237, China 3 Research Platform for Marine Molecular Biotechnology, National Laboratory for Marine Science and Technology, Qingdao 266237, China 4 CAS Center for Ocean Mega-Science, Chinese Academy of Sciences, Qingdao 266400, China Accepted March 05, 2019 Abstract Malectin is a newly discovered lectin of the endoplasmic reticulum (ER) that might be involved in innate immunity. Information about the roles of malectin in innate immunity is scarce. In the present study, a novel malectin gene (designated as CfMal) from the Zhikong scallop Chlamys farreri was identified and characterized. Sequence features, tissue distribution, and temporal expression profiles were investigated to infer the potential functions of CfMal in innate immunity. The complete cDNA sequence of CfMal comprised 1,111 bp and contained an open reading frame of 909 bp, which encoded 302 amino acid residues. A malectin domain and a transmembrane region were identified in the predicted protein sequence. CfMal mRNA transcripts were detectable in hemocytes, muscle, mantle, gill, hepatopancreas, and gonads. CfMal expression was highest in hemocytes. Stimulation with Vibrio splendidus increased CfMal expression in hemocytes, gill, and hepatopancreas. The mRNA transcripts of CfMal and three related genes, including binding immunoglobulin protein, heat shock protein 90 kDa β member 1 protein and ER degradation enhancing α-mannosidase like protein 1, increased in scallop hemocytes during an artificial ER-stress. Our results indicate that CfMal might not only be involved in ER-stress, but may also play a role in innate immunity of scallops. Key Words: Chlamys farreri; innate immunity; malectin Introduction Lectins are a large family of evolutionally conserved proteins that bind terminal sugars of glycoproteins or polysaccharides; they act as pattern recognition receptors (PRRs) of the innate immune system (Weis and Drickamer, 1996). Based on carbohydrate ligands, subcellular localization, and dependence on divalent cations, animal lectins could be classified into several groups including C-type, F-type, I-type, L-type, P-type, S-type (also known as galectin), and X-type lectins (also known as intelectins), discoidins, and pentraxins (also known as pentaxins) (Dahms and Hancock, 2002; Kilpatrick, 2002; Arasu et al., 2013; Jia et al., 2016; Shao et al., ___________________________________________________________________________ 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, leiwang@qdio.ac.cn 2018; Tian et al., 2018; Wang et al., 2016a; Wei et al., 2018). Lectins localized in the endoplasmic reticulum (ER) are termed as ER-resident lectins (Aebi et al., 2010). Most of these ubiquitous lectins participate in host-pathogen interactions and in immunomodulation (Cambi et al., 2005). Malectin is a highly conserved membrane-anchored ER-resident lectin; it was first identified in Xenopus laevis in 2008 and specifically recognized Glc2Man9GlcNAc2 (G2M9) in newly synthesized glycoproteins (Schallus et al., 2008). Accumulating research shows that malectin is induced by ER-stress and is associated with folding defective glycoproteins to reduce their secretion (Galli et al., 2011; Yang et al., 2018). What’s more, de novo characterization of the spleen transcriptome of the large yellow croaker Pseudosciaena crocea stimulated with polyinosinic:polycytidylic acid (poly IC) revealed that malectin might be involved in antiviral responses (Mu et al., 2014). Moreover, 26 three genes, including binding immunoglobulin protein (BiP, also known as 78 kDa glucose regulated protein), heat shock protein 90 kDa β member 1 protein (also known as 94 kDa glucose-regulated protein, Grp94), and ER degradation enhancing α-mannosidase like protein 1 (EDEM1), were previously reported that exhibited close relationship with Mal during ER stress (Galli et al., 2011; Qin et al., 2012; Merulla et al., 2013). In scallops, Grp94 might play an important role in the innate immune defense of the Yesso scallop Patinopecten yessoensis (also known as Mizuhopecten yessoensis) (Wang et al., 2018c). However, the potential roles of malectin in innate immunity are still unclear. Scallops represent an important aquaculture species with commercial, ecological, and evolutionary importance (Matozzo, 2016; Tascedda and Ottaviani, 2016; Gerdol, 2017; Jielian et al., 2017). As invertebrates, scallops lack clonally derived immunoglobulins and T-lymphocytes based adaptive immunity, and depend on their innate immune system to eliminate non-self-particles and to kill invading pathogens (Song et al., 2015). In the past two decades, many PRRs have been identified in marine scallops, especially in the bay scallop Argopecten irradians and the Zhikong scallop Chlamys farreri. These PRRs include C-type lectin (Mu et al., 2012), galectin (Song et al., 2011), lipopolysaccharide (LPS) and β-1,3-glucan binding proteins (Su et al., 2004), leucine-rich repeat-only proteins (Wang et al., 2017), peptidoglycan recognition proteins (Ni et al., 2007), scavenger receptors (Liu et al., 2011), thioester containing proteins (Zhang et al., 2007), and Toll-like receptors (Wang et al., 2011). These research achievements have enhanced the understanding of the potential functions of these PRRs in invertebrate innate immunity (Song et al., 2015). Therefore, in the current study, we used Zhikong scallops to explore the potential roles of malectin in invertebrate innate immunity. We identified a malectin gene from C. farreri (designated as CfMal) and we analyzed its expression induced by various stimuli, which indicated its potential role in innate immunity. The main purposes of our present work were: (1) to describe the sequence features of CfMal; (2) to investigate the expression profiles of CfMal; and (3) to predict the potential functions of CfMal in innate immunity. Materials and Methods Scallops, in vivo Vibrio stimulation, and sample collection Adult scallops (average 5 cm in shell length) were collected in a local farm in Qingdao, China, in summer; they were maintained in aerated seawater at approximately 20 °C. Vibrio splendidus strain JZ6, which has been proved to be a main kind of pathogens for scallop and widely used for the stimulation (Wang et al., 2019a; Wang et al., 2019b), was cultured in liquid 2216E media (HB0132, HopeBiotech, China) at 28 °C with shaking at 180 rpm overnight. Bacteria were collected by centrifugation at 4000 g for 20 min, and then re-suspended in filtered seawater. Fifteen scallops were immersed for 12 h in filtered water containing live V. splendidus at a final concentration of 1.0 × 10 8 colony forming units per mL at 20 ºC, which constituted the Vibrio stimulation group. Hemocytes, muscle, mantle, gill, hepatopancreas and gonads from both infected and control scallops were collected for CfMal mRNA expression analysis. Primary cultured hemocytes, in vitro ER-stress induction, and sample collection Primary cultures of scallop hemocytes were prepared as previously described (each time point has 5 repetitions, and each repetition was a mixture of 3 individuals, Wang et al., 2014). Briefly, the hemolymph was withdrawn using a sterile syringe from the adductor muscle and diluted (1:3) in modified anticoagulant Alsever's solution (3.36 g·L −1 EDTA, 20.8 g·L −1 glucose, 22.5 g·L −1 NaCl and 8 g·L −1 sodium citrate, pH = 7.0, 1000 mOsm). Approximately 1.0 × 10 5 scallop hemocyte cells were suspended in 200 μL complete Dulbecco’s Modified Eagles Medium (High Glucose, FI101, TransgenBiotech, China) supplemented with 10% TransSerum EQ fetal bovine serum (FS201, TransgenBiotech, China), 10% scallop serum, 100 U·mL −1 penicillin and 100 μg·mL −1 streptomycin (FG101, TransgenBiotech, China). Cells were added to TC-Treated Multiple Well Plates (24 wells, CLS3527, Corning Costar, USA) and incubated for 12 h at 21 °C in 5% CO2. Thapsigargin (Tg, T9033, Sigma-Aldrich, USA), tunicamycin (Tun, 654380, Sigma-Aldrich, USA) and LPS (L2630, Sigma-Aldrich, USA) were added to corresponding wells at a final concentration of 300 ng·mL −1 , 10 μg·mL −1 , and 10 ng·mL −1 , respectively. These stimuli were considered ER-stress induction groups, according to previous reports (Urano et al., 2000; Yoshida et al., 2001; Wang et al., 2015). Among them, Tg specifically could inhibit the fusion of autophagosomes with lysosomes; the last step in the autophagic process. The inhibition of the autophagic process in turn induces stress on the ER which ultimately leads to cellular death (Ganley et al., 2011). Tun is an inhibitor of glycosylation that disturbs protein folding machinery in eukaryotic cells. Tun causes accumulation of unfolded proteins in cell ER and induces ER stress (Namia et al., 2016). While the expression of CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP), which is an ER stress-induced transcription factor, induces apoptosis. And a previous study demonstrated that LPS-induced CHOP expression does not induce apoptosis, but activates a pro-IL-1β activation process (Nakayama et al., 2009). Untreated primary cultures of scallop hemocytes were used as a control. Cells from each experimental group were sampled at 0, 3, 6, 12, 24 and 48 h after stimulation. RNA isolation, cDNA synthesis, and full-length cDNA cloning Total RNA was isolated using TransZol Up (ET111, TransgenBiotech, China). First-strand cDNA was synthesized using EasyScript First-Strand cDNA Synthesis SuperMix (AT301, TransgenBiotech, China) with DNase I (RNase-free, GD201, TransgenBiotech, China). Raw RNA was used as template, and adaptor primer-oligo (dT) as 27 Table 1 Primers used in the present research Primer Sequences (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 qRT-PCR CfEF-1α-qRT-R GGCACAGTTCCAATACCTCCA Internal control for qRT-PCR CfMal-RACE-F1 GCCTCCGATGACACCAGCACC Gene specific primer for RACE CfMal-RACE-F2 CTCTGTAAACTGTAAATATCAGATCAGGGG Gene specific primer for RACE CfMal-RACE-R1 CTTGCATATACACGGCTACACGCCGAC Gene specific primer for RACE CfMal-RACE-R2 GCGAATGTCCAGGAAGTGCGGCTC Gene specific primer for RACE CfMal-CDS-F ATGGCGCTGCGAGCCGCA Gene specific primer for CDS CfMal-CDS-R TTACAGTTTACAGAGGCAGAAGAGGAGAGG Gene specific primer for CDS CfMal-qRT-F AGATTCGCTCAAAGTCGGG Gene specific primer for qRT-PCR CfMal-qRT-R CGCTGAGTGGGATTTCGT Gene specific primer for qRT-PCR CfBiP-qRT-F GGTCTTCTTCAGGTATCAGCAG Gene specific primer for qRT-PCR CfBiP-qRT-R CTTATCTTCCTCAGCAAACATTTCC Gene specific primer for qRT-PCR CfGrp94-qRT-F TCCCAGACGACGAACTTAATCCA Gene specific primer for qRT-PCR CfGrp94-qRT-R GTTACCCATTATTGCCAGAGTGTCC Gene specific primer for qRT-PCR CfEDEM1-qRT-F AGCACCAGTTAAGGATTCTAATGTT Gene specific primer for qRT-PCR CfEDEM1-qRT-R CCACTTCCTCCATACGACTTG Gene specific primer for qRT-PCR M13-47 CGCCAGGGTTTTCCCAGTCACGAC Vector primer for sequencing RV-M GAGCGGATAACAATTTCACACAGG Vector primer for sequencing *The efficiency of CfEF-1α-qRT-F/R, CfMal -qRT-F/R, CfBiP-qRT-F/R, CfGrp94-qRT-F/R and CfEDEM1-qRT-F/R were 98%, 103%, 101%, 97% and 99%, respectively primer (Table 1), according to the manufacturer instructions. Subsequently, a homopolymeric tail was added using terminal deoxynucleotidyl transferase (EP0161, ThermoFisher, USA) and dCTP (10217016, ThermoFisher, USA). We previously identified a malectin homologue sequence of C. farreri by using available public transcriptomic data (Wang et al., 2018b). And it was selected to clone the full-length cDNA sequence of CfMal. Gene-specific primers, CfMal-RACE-R1/2 and CfMal-RACE-F1/2 (Table 1), were designed using Primer Premier 5.00 to obtain the full-length cDNA sequence of CfMal using the rapid-amplification of cDNA ends (RACE) method. All PCR reactions were performed in an MJ Mini Personal Thermal Cycler (Bio-Rad, USA). The PCR products were directly ligated into the pEASY-T3 Cloning Vector (CT301, ThermoFisher, USA). After transformation into phage resistant chemically competent cell Escherichia coli strain Trans1-T1 (CD501, TransgenBiotech, China), positive recombinants were selected using TransCult LB Agar Plates (Ampicillin, CP111, TransgenBiotech, China) and verified by PCR screening with vector primers M13-47 and RV-M (Table 1). Three positive clones were sequenced in a 3730XL automated sequencer (ThermoFisher, USA) by Genscript Biotech (Nanjing) Inc. Bioinformatics analysis of cDNA and protein sequences The protein sequence of CfMal was deduced and analyzed by the EditSeq module of Lasergene 7.1.0.44. CfMal and the three related genes were identified from the genome of C. farreri using BLAST+ 2.8.0, as described previously (Galli et al., 2011; Wang et al., 2007). The protein sequence similarity search was also conducted by BLAST+ 2.8.0. The presence and location of signal peptides and functional domains were predicted using SignalP 4.1 and Simple Modular Architecture Research Tool (SMART) 8.0. A phylogenetic tree was generated with MEGA-X 10.0.1 using the Neighbor-Joining (NJ) method. Bootstrap trials were replicated 1,000 times to derive a confidence value for phylogenetic analysis. Analysis of mRNA expression patterns via quantitative real-time PCR (qRT-PCR) The expression of CfMal was analysed by qPCR in several tissue form control and Vibrio infected animals as well as in hemocytes during induced ER-stress. All qRT-PCR reactions were carried out using TransStart Green qPCR SuperMix UDG (AQ111, TransgenBiotech, China) in a LineGene K FQD-48A Fluorescence Quantitative PCR Detection System (Bioer, China). All the primers 28 Fig. 1 Sequence features and phylogenetic relationships of CfMal. A. Nucleotide and predicted protein sequences of CfMal. The nucleotides and amino acids are numbered on the left margin. The function domain is shaded. The low complexity is boxed. The transmembrane region has a double underline. The stop codon is indicated by asterisks. The polyadenylation signal site (AATAAA) is underlined. B. Phylogenetic tree based on the protein sequences of different malectins. The NJ model was used to infer the evolutionary history. The numbers at the branches indicate the bootstrap value (%). The accession numbers of these sequences are as follows: Apis mellifera, XP_006563359; Aplysia californica, XP_005104301; Biomphalaria glabrata, XP_013067932; Chlamys farreri, AYB71126; Crassostrea gigas, XP_011422439; Cynoglossus semilaevis, XP_016898532; Homo sapiens, NP_055545; Maylandia zebra, XP_004558571; Mizuhopecten yessoensis, XP_021354488; Mus musculus, NP_780612; Oreochromis niloticus, XP_005473062; Pelodiscus sinensis, XP_006114823; Pomacea canaliculata, XP_025090097; Rattus norvegicus, NP_001014005 and Salvelinus alpinus, XP_023857162 29 Fig. 2 Spatial mRNA expression patterns of CfMal. mRNA expression levels in hemocytes, mantle, gill, hepatopancreas, and gonads of five adult scallops were normalized to that of muscle. Vertical bars represent mean ± SD (n = 5); different letters represent statically significant differences (p < 0.05) for qRT-PCR were designed with PerlPrimer 1.1.21 (Table 1). The threshold cycle (CT) slope method, based on serial two-fold dilutions of cDNA, was used to confirm that all pairs of these primers had similar efficiency (Pfaffl et al., 2001; Wang et al., 2018a). For each sample, the expression level of target genes was normalized to that of elongation factor 1 α (CfEF-1α). The relative mRNA abundance of target genes was determined using the comparative CT (2 −ΔΔCt ) method (Schefe et al., 2006; Schmittgen and Livak, 2008). Data are presented as mean ± SD (n = 5, each time point has 5 repetitions, and each repetition was a mixture of 3 individuals); data was subjected to one-way analysis of variance, followed by a multiple comparison using IBM SPSS Statistics software 25.0.0.0. p < 0.05 was considered as statistically significant. Results Molecular features of CfMal and its phylogenetic relationship The full-length cDNA sequence of CfMal obtained via RACE was submitted to GenBank under the accession number MG546685. The complete cDNA sequence of CfMal was 1,111 bp long and consisted of a 40 bp 5’ untranslated region (UTR), a 3’ UTR of 162 bp, and an open reading frame (ORF) of 909 bp. A polyadenylation signal site (AATAAA) was revealed upstream of the polyA tail. The ORF encoded 302 amino acid residues with a predicted molecular mass of 33.665 kDa, and an isoelectric point of 5.210. A malectin domain (from V 61 to I 220 ) and a transmembrane region (from T 278 to C 300 ) were identified in the predicted protein sequence by SMART analysis; no signal peptide was revealed (Figure 1A). BLAST+ search revealed that CfMal shared high identity with its homologues from M. yessoensis (97% identity), Crassostrea virginica (75% identity) and Pomacea canaliculata (68% identity). Phylogenetic analysis showed that CfMal clustered with its counterparts from M. yessoensis and formed a sister branch to their homologue from Crassostrea gigas (Figure 1B). Tissue distribution of CfMal mRNA transcripts The tissue distribution of CfMal mRNA transcripts was detected by qRT-PCR using CfEF-1α as an internal control. CfMal mRNA transcripts were detectable in all the sampled tissues; the highest expression was that of hemocytes, which was 6.98-fold (p < 0.05, relative to muscle), followed by gill (4.23-fold, p < 0.05) and hepatopancreas (3.98-fold, p < 0.05). After stimulation with Vibrio for 12 h, CfMal expression increased significantly in hemocytes, hepatopancreas, and gill (20.07-, 7.36-, and 7.16-fold, respectively, relative to muscle with no stimulation, p < 0.05). No significant differences were observed in muscle, mantle, or gonads, before and after stimulation with Vibrio (Figure 2). Temporal expression of CfMal and related genes during ER-stress induction CfMal and the three related genes (CfBiP, CfGrp94 and CfEDEM1) were identified from the genome of C. farreri using BLAST+ 2.8.0, according to previous description (Wang et al., 2007; Galli et al., 2011). The expression patterns of these genes were 30 Fig. 3 Temporal mRNA expression patterns of CfMal and CfMal-related genes during ER-stress. Vertical bars represent mean ± SD (n = 5); different letters represent statically significant differences (p < 0.05). A. CfBiP B. CfGrp94 C. CfEDEM1 D. CfMal analyzed by qRT-PCR. The expression of these four genes all increased after hemocytes were stimulated with Tg, Tun, or LPS. After 3 h of Tg stimulation, the expression of CfBiP increased significantly (3.82-fold, p < 0.05), reached a peak at 12 h (9.12-fold, p < 0.05), and returned to basal levels after 48 h. After 3 h of Tun stimulation, the expression of CfBiP increased significantly (4.24-fold, p < 0.05), reached a peak at a 6 and 12 h (5.96-fold and 6.19-fold, p < 0.05, respectively), and returned to basal levels after 48 h. After 3 h of LPS stimulation, the expression of CfBiP increased significantly (3.97-fold, p < 0.05), reached a peak at 12 h (8.87-fold, p < 0.05), and decreased at 24 and 48 h after stimulation (3.86- and 4.24-fold, respectively, p < 0.05 for both; Figure 3A). After 6 h of Tg stimulation, the expression of CfGrp94 increased significantly (3.27-fold, p < 0.05), reached a peak at 12 h (6.12-fold, p < 0.05), and returned to basal levels after 24 h. After 6 h of Tun stimulation, the expression of CfGrp94 increased significantly (2.96-fold, p < 0.05), reached a peak at 12 h (8.20-fold, p < 0.05), and returned to basal levels after 24 h. After 3 h of LPS stimulation, the expression of CfGrp94 increased significantly (2.97-fold, p < 0.05), reached a peak at 12 h (6.29-fold, p < 0.05), and returned to basal levels after 48 h (Figure 3B). Expression of CfEDEM1 peaked after 6 h of Tg stimulation (3.17-fold, p < 0.05), and returned to basal levels after 12 h. Expression of CfEDEM1 peaked after 6 h of Tun stimulation (3.31-fold, p < 0.05), and returned to basal levels after 12 h. Expression of CfEDEM1 peaked after 6 h of LPS stimulation (4.96-fold, p < 0.05), and gradually returned to basal levels after 48 h (Figure 3C). Expression of CfMal peaked after 12 h of Tg stimulation (5.17-fold, p < 0.05), decreased significantly after 24 h (2.95-fold, p < 0.05), and returned to basal levels after 48 h. Expression of CfMal increased only after 12 h of Tun stimulation (3.28-fold, p < 0.05). Expression of CfMal peaked after 12 h of LPS stimulation (3.36-fold, p < 0.05) and returned to basal levels after 48 h (Figure 3D). 31 Discussion Malectin is a newly discovered ER-resident lectin, which specifically recognizes G2M9 in newly synthesized glycoproteins (Schallus et al., 2008). Recent research indicates that malectin might play potential roles in innate immunity (Mu et al., 2014; Wang et al., 2018c). However, information about the role of malectin in innate immunity is scarce. In the present study, we identified a novel malectin gene (CfMal) in Zhikong scallop C. farreri. We analyzed CfMal sequence features, its tissue distribution, and temporal expression profiles, in order to predict its potential functions in innate immunity. Bioinformatics analysis revealed that CfMal contained a typical malectin domain, and exhibited high identity with its invertebrate counterparts. Additionally, in the NJ phylogenetic tree CfMal clustered with its homologues from M. yessoensis and C. gigas. The conserved function domain has high similarity with that of other invertebrates. These phylogenetic relationships suggest that CfMal belongs to the invertebrate malectin family. To investigate the potential functions of CfMal in scallops, the tissue distribution of its mRNA transcripts was analyzed. CfMal mRNA transcripts could be detectable in all the sampled tissues; expression was highest in hemocytes, followed by gill and hepatopancreas. Hemocytes play pivotal functions in invertebrate innate immunity (Jia et al., 2017; Jia et al., 2018). Gill is a potential hematopoietic position in mollusks and is the first line of defense against invading microbes in lower animals (Li et al., 2017). The hepatopancreas is considered as main immune organ in crustaceans and mollusks (Wang et al., 2016b). The high abundance of CfMal mRNA transcripts in these tissues indicates that it might be involved in the innate immunity of scallops. CfMal expression in these tissues was significantly up-regulated by Vibrio stimulation, especially in hemocytes, which confirmed this hypothesis. To further investigate the potential roles of CfMal in scallops, the temporal expression profiles of CfMal and three related genes was investigated in hemocytes stimulated with Tg, Tun, or LPS. In previous report, the expression of mammalian BiP, Grp94, EDEM1 and malectin is up-regulated during Tg-induced ER-stress (Galli et al., 2011). In the present study, the expression of CfBiP, CfGrp94, CfEDEM1 and CfMal increased in scallop hemocytes after stimulation with either Tg, Tun, or LPS. Tg and Tun induced similar gene expression modification in hemocytes, which confirmed the hypothesis that CfMal might play a role in ER-stress of scallops. While LPS treated hemocytes showed slightly differences compared to the other stimuli, indicating CfMal might also be involved in innate immunity of scallops. In conclusion, a novel malectin gene (CfMal) was identified and characterized in C. farreri, including sequence features and expression profiles. The expression of CfBiP, CfGrp94, CfEDEM1 and CfMal increased in scallop hemocytes after stimulation with either Tg, Tun, or LPS. The present study provides useful information about the potential functions of CfMal in scallops. Acknowledgements This research was supported by National Natural Science Foundation of China (U1706209), Aoshan Innovation Project in Science and Technology from 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) from the Chinese Academy of Sciences. 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