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Cloning, expression profiling and promoter functional analysis of bone morphogenetic protein 2 in the tongue sole (Cynoglossus semilaevis)
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Qian MA1, 2, Yanjun FAN1, Zhimeng ZHUANG1, 3, *, Shufang LIU1, 2
Acta Oceanologica Sinica | 2018, 37(2) : 76 - 84
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Acta Oceanologica Sinica | 2018, 37(2): 76-84
• Marine Biology •
Cloning, expression profiling and promoter functional analysis of bone morphogenetic protein 2 in the tongue sole (Cynoglossus semilaevis)
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Qian MA1, 2, Yanjun FAN1, Zhimeng ZHUANG1, 3, *, Shufang LIU1, 2
Affiliations
  • 1 Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China
  • 2 Function Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266200, China
  • 3 Function Laboratory for Marine Biology and Biotechnolgy, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266200, China
Published: 2018-02-25 doi: 10.1007/s13131-018-1164-x
Outline
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BMP2 plays crucial roles in vertebrate developmental process and acts as a bone inducer during osteogenesis. We present here the molecular cloning of bmp2 cDNA from the marine flatfish Cynoglossus semilaevis, and the analysis of bmp2 expression profiling and promoter function. The full length of bmp2 cDNA sequence is 2 048 bp, which encodes a protein of 422 amino acids. Tissue expression distribution of bmp2 was examined in 14 tissues of mature individuals by quantitative real time PCR (qRT-PCR). The results revealed that bmp2 was expressed ubiquitously, and the highest expression level was detected in the spinal cord. Moreover, bmp2 expression levels were detected at 15 sampling time points of early developmental stages (egg, larva, juvenile and fingerling stages). The highest expression level of bmp2 was observed at the gastrula stage, which was about ten times higher than those at the other three embryo stages. Whole-mount in situ hybridization showed that the bmp2 signal was strongly detected at the location of the crown-like larval fin, heart and liver, and slightly expressed in the notochord at one day post hatch (dph); then the expression of bmp2 started to be concentrated in notochord at three dph. Subsequently, we characterized the 5′-flanking region of bmp2 by testing the promoter activity by Luciferase reporter assays. Positive regulatory region was detected at the location of –179 to +109. The predicted transcription factor binding sites (E-box binding factors, zinc finger transcription factor, etc.) in this region might participate in the transcriptional regulation of the bmp2 gene.

cloning  /  gene expression pattern  /  promoter transcriptional activity  /  bone morphogenetic protein  /  Cynoglossus semilaevis  /  early developmental stages
Qian MA, Yanjun FAN, Zhimeng ZHUANG, Shufang LIU. Cloning, expression profiling and promoter functional analysis of bone morphogenetic protein 2 in the tongue sole (Cynoglossus semilaevis)[J]. Acta Oceanologica Sinica, 2018 , 37 (2) : 76 -84 . DOI: 10.1007/s13131-018-1164-x
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1 Introduction
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Bone morphogenetic proteins (BMPs), belonging to the transforming growth factor beta (TGF-β) superfamily, were initially discovered by their ability to induce endochondral osteogenesis in vivo (Urist et al., 1979). To date, more than 30 members of the BMP family have been identified; numerous studies have demonstrated the efficacies of these BMP members in inducing bone formation (Kang et al., 2004; Cheng et al., 2003; Ducy and Karsenty, 2000). BMP2 has been extensively studied due to its predominant position within the BMP family and essential role in chondrogenesis and osteogenesis. BMP2 is required for mammalian osteogenesis, inducing osteoblast differentiation (Huang et al., 2002). For instance, Burkus et al. (2003) reported the positive effect of BMP2 on human long bone fracture repair, which includes formation of both cartilage and bone. Endogenous BMP2 was shown to play a pivotal role in formation of the mouse long bone (Kugimiya et al., 2005). The effect of BMP2 in chondrocyte and osteoblast differentiation and maturation has also been demonstrated in lower vertebrates. Quint et al. (2002) showed the involvement of BMP2 in lepidotrichia differentiation. It has been reported by Smith et al. (2006) that misexpression of bmp2b could induce ectopic bone formation within the regenerate of zebrafish caudal fin.
In addition to bone formation, BMPs have been demonstrated to be pleiotropic factors involving various growth and differentiation processes (Balemans and Van Hul, 2002; Hogan, 1996). The fundamental function of BMP2 in vertebrate embryonic development is directly involved in processes of dorsal–ventral axis specification (Graff, 1997). In zebrafish, bmp2 participated in the induction and maintenance of ventrolateral cell fate during early development (Kishimoto et al., 1997). Furthermore, Hammond et al. (2009) also demonstrated a critical late role for bmp2b in the morphogenesis of semicircular canals in the zebrafish inner ear.
The important role of BMP2 in growth and development processes have contributed to the growing interest in understanding regulatory mechanism of cartilage and bone development in marine fish. So far the bmp2 gene has been cloned from various teleost species, however, studies regarding the role of bmp2 in development have been rarely reported except for the very few organisms such as zebrafish, senegalese sole and gilthead sea bream (Fernández et al., 2011; Marques et al., 2014; Marques et al., 2016). In terms of the activities of BMP2 during growth and development in fish, the aim of the work was to provide new insights into the evolutionary relationship between bmp2 and other bmp members, and to investigate bmp2 gene expression profiles and protein functions.
In this study, our goal is to gain insight into the genetic characteristics and functions of BMP2 in the tongue sole (Cynoglossus semilaevis). As the first step, the full length bmp2 cDNA was cloned by homology cloning and the rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR). Spatial- and temporal-expression analysis of bmp2 during early developmental stages was performed by using qRT-PCR and whole-mount in situ hybridization. In order to explore transcriptional regulatory mechanism of the elaborated control of bmp gene expression, promoter activity of 5′-flanking region of bmp2 gene was tested by Luciferase reporter assays. Relative results will provide new information to clarify the role of bmp transcription regulation in accomplishing functions related to skeletal development. Our findings would help understanding the role of bmp2 in regulating early development of this flatfish at the molecular level.
2 Materials and methods
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2.1 Experiment design and sample source
A total of 12 three-year old adult fish (six females and six males) were used to detect the bmp2 tissue expression distribution. After being rapidly dissected from these live individuals, 14 tissues (brain, cartilage, dorsal fin, gill, gonad, heart, intestine, kidney, liver, muscle, skin (with scales), spleen, spinal cord and stomach) were immediately frozen in liquid nitrogen and kept at –80°C until use.
In order to examine the ontogenetic expression pattern of bmp2 at early developmental stages, embryos and immature fish (larvae, juveniles and fingerlings) were used to supply samples for quantitative real time PCR (qRT-PCR) and whole-mount in situ hybridization analysis. The cultivation temperature for fertilized eggs was kept at 22°C. Fish samples were collected under the condition of empty stomach in the early morning. Samples for qRT-PCR are shown in Table 1, i.e., 20 eggs were collected at each egg stage (Multi-cell, Gastrula, Embryonic and Pre-hatching stage) and developmental stages were identified according to Wan et al. (2004), six individuals at each early developmental stage (larvae, juveniles and fingerlings) were also randomly collected. The whole body of these samples was frozen in liquid nitrogen for RNA extraction. Samples for whole-mount in situ hybridization (newly hatched, one and three dph) were collected and fixed in 4% paraformaldehyde (PFA) for 12–16 h, dehydrated with solutions with different ratios of ethanol (30%, 50% and 70%), and stored in 70% ethanol at 4°C.
2.2 The first-strand cDNA synthesis
Total RNA was extracted from frozen tissues of adult fish as well as samples from different early developmental stages using TRIzol Reagent (Invitrogen, USA) according to the manufacturer′s instructions. The isolated RNA samples were suspended in DEPC-treated water, quantified using NanoVueTM (GE Healthcare) at A260 nm and A280 nm, and then analyzed for integrity on agarose gel. The first-strand cDNA was synthesized from total RNA using PrimeScriptTM RT reagent Kit with gDNA Eraser (Takara Bio., China) following the manufacturer′s instructions. The cDNA synthesis included a genomic DNA elimination reaction and the RT Primer Mix contained both Oligo dT Primer and Random 6 mers.
2.3 Cloning of bmp2 cDNA
According to the conserved sequences of bmp2 gene from other teleost species, a pair of degenerate primers, BMP2-F/R (Table 2), was designed to enable cloning of the corresponding partial fragments of bmp2 cDNA. PCR amplification was performed in a typical reaction; the condition was one initial denaturing step of 3 min at 94°C, followed by 32 cycles of 30 s at 94°C, 30 s at 53–58°C, 30 s at 72°C, and a final 10 min at 72°C.
Based on the obtained partial fragments of bmp2 cDNA, four specific primers, BMP2-5′-OUTER/INNER and BMP2-3′-OUTER/INNER (Table 2) were designed for amplification of the cDNA ends of the bmp2 gene by using the 5′-Full RACE Kit and 3′-Full RACE Core Set Ver.2.0 (Takara Bio., China) following the manufacturer′s instructions.
All the amplified fragments of the expected sizes were purified with a E.Z.N.A. Gel Extraction Kit (OMEGA, USA), cloned into a pMD18-T vector (Takara Bio., China), then transformed into Escherichia coli DH5α and sequenced by the Beijing Genomics Institute (BGI, Shenzhen, China).
2.4 Sequence and phylogenetic analysis
The full length cDNA of bmp2 gene was assembled by aligning the overlapping fragments and the primer sequences. The signal peptides were predicted with Signalp 4.1 (http://genome.cbs.dtu.dk/services/SignalP). Putative domains and possible N-glycosylation sites were identified by PROSITE (http://www.expasy.org/prosite) (de Castro et al., 2006). Promoter prediction of bmp2 gene was performed by BDGP (http://www.fruitfly.org/seq_tools/promoter.html), and putative transcription factor binding sites (TFBS) were identified by Version 8.3 of TRANSFAC (Messeguer et al., 2002) and the MatInspector program (http://www.genomatix.de).
Putative bmp2 amino acid sequences of C. semilaevis and other vertebrates were used to construct a phylogenetic tree using the Neighbor-Joining (NJ) method with MEGA Version 5.1. The ClustalW program was employed to align all the sequences with the default option.
2.5 Promoter activity of the 5′-flanking region of bmp2 gene
2.5.1 Construction of luciferase-reporter gene vector for bmp2 gene promoter
A DNA fragment with the length of 1 863 bp (–1754 to +109) was cloned from the 5′-flanking regions of bmp2 gene (Gene ID: 103381057). Promoter deletion experiment was designed to monitor the promoter activity of the 5′-flanking regions. DNA fragments with a series of nested deletions (gradually truncated 5′-end with the predicted promoter sequence) were generated by using the primers listed in Table 2, which were designed according to C. semilaevis bmp2 sequence as well as the promoter prediction results from TRANSFAC and MatInspector programs. A total of six plasmids were constructed by inserting DNA fragments of various sizes from the 5′-flanking region of bmp2 gene into the region between the HindIII and XhoI sites of the pGL4.10 vector (Promega, E665A). The sequences and orientations of the constructs were verified by nucleotide sequencing.
2.5.2 Cell culture and transient transfection
ZF4 cells (purchased from ATCC) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS, Lifetechnologies), 100 U/mL penicillin (Lifetechnologies) and 100 U/mL streptomycin (Lifetechnologies). Cells were seeded to 96-well plates for 24 h before transfection. When the cell grew to 90%, equivalent amounts of plasmids were transfected into ZF4 cells following the instructions of transfection reagent (Lipofectamine® 2000, Thermo Fisher Scientific). pGL4.51 (Promega, E132A) was used as a reference (positive control), while pGL4.10 (Promega, E665A) without promoter was used as negative control. The ratio of target plasmids to pGL4.51 or pGL4.10 were both 1:1.
2.5.3 Detection of luciferase activity
After transfection for 48 h, luciferase activity was detected by using Luciferase Reporter Gene Assay Kit (Beyotime) in accordance with the manufacturer’s instructions. Three replications were set up for each sample from bmp2 gene, and relative light unit (RLU) was detected by EnVision® Multilabel Reader (PerkinElmer, USA).
2.6 Quantitative real time PCR
The qRT-PCR was conducted to determine bmp2 tissue expression distribution in adult fish as well as their dynamic expression patterns at early developmental stages. The primers BMP2-RT-F/R, EF1A-F/R and +-F/R were used for amplifying the bmp2, elongation factor 1-alpha (ef1a) and 18S rRNA (18s) fragments, respectively (Table 2). The qRT-PCR was conducted on a 7500 ABI Real time PCR system (Applied Biosystems, USA). Amplifications were performed in a 20 μL final volume containing 1 μL cDNA sample, 10 μL SYBR® Premix Ex TaqTM (Perfect Real Time) (Takara Bio., China), 0.4 μL ROXII, 0.4 μL of each primer and 7.8 μL ddH2O. A negative control was always included. PCR amplifications were performed in triplicate using the following conditions: initial denaturing at 95°C for 10 s, followed by 40 cycles of 5 s at 95°C and 34 s at 60°C. A dissociation protocol was always performed after thermocycling to determine target specificity. Expression of ef1a was used as the internal control for bmp2 tissue expression distribution analysis, and 18s was used as the internal control for the bmp2 ontogenetic expression patterns (Ma et al., 2015). The ratio changes in the target genes relative to the control genes (ef1a and 18s) were determined by the 2–△△CT method (Livak and Schmittgen, 2001) and the transcript level was described in terms of its relative concentration (RCtarget/RCcontrol).
2.7 Whole-mount in situ hybridization
For in situ hybridization, plasmids (pGEM-T vector, Promega) containing the fragment of bmp2 cDNA (619 bp) was linearized by restriction endonuclease digestion using NcoI (NEB Biolabs). Sense and antisense digoxigenin (DIG)-labeled RNA probes were synthesized from the plasmids with SP6 or T7 transcriptase by using DIG RNA Labeling Kit (Roche) following the manufacturer′s instructions. Whole-mount in situ hybridization procedures followed the standard protocols according to Holland et al. (1996). Samples at different developmental stages were first rehydrated, washed, treated with Proteinase K, followed by 4% PFA refix. Specimens were incubated for 3 h at 60°C in prehybridization buffer and then hybridized with the labelled probes for 16 h at 60°C. After hybridization, the larvae were washed and blocked with blocking reagent (Roche) at room temperature for 2 h, then incubated with an alkalinephosphatase-conjugated anti-DIG antibody (1:2 000 dilution) over night at 4°C. Finally, excess anti-DIG antibodies were removed by rinsing, and signals were visualized by incubation in NBT/BCIP reagents (Roche) in dark. Specimens were observed and photographed using a Nikon microscope (SMZ1500).
2.8 Statistical analysis
All data were expressed as mean±SD and analyzed by one-way ANOVA (analysis of variance) to determine significant differences between means using SPSS 16.0. Values were considered statistically significant when P<0.05.
3 Results
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3.1 Characteristics and phylogenetic analysis of bmp2 gene
As shown in Fig. 1, C. semilaevis bmp2 cDNA is 2 048 bp and contains a 5′-untranslated region (UTR) of 442 bp, an open reading frame (ORF) of 1 269 bp and a 3′ UTR of 337 bp (GenBank ID: KC422338). The predicted amino acid sequence of the bmp2 cDNA consists of 422 amino acid residues with a 23 amino-acid signal peptide. The predicted molecular weight of BMP2 is 47.47 kDa, and the theoretical isoelectric point (pI) is 8.65. Seven cysteine residues and an RVSR sequence were found in the bmp2 amino acid sequence.
A phylogenetic tree was constructed by the Neighbor-Joining method to investigate relationships of bmp2 genes in the listed species, based on the genetic distances calculated with the Poisson correction model (Fig. 2). Results showed that lineages were composed of two branches: teleost branch and mammal branch. Within the lineage of teleost bmp2 branch, C. semilaevis formed a single branch out of the branch composed of 13 teleost species (five from Cichlidae, two from Tetraodontidae, and one each from Pomacentridae, Sciaenidae, Nothobranchiidae, Sparidae, Carangidae and Paralichthyidae). Basically, the phylogenetic relationship result among vertebrates was consistent with the traditional taxonomy.
3.2 Functional analysis of the 5′-flanking region of bmp2 gene
The DNA fragment with the total length of 1 873 bp was obtained from 5′-flanking region of the bmp2 gene. To test the promoter activity of the 5′-flanking region, gradual deletions were performed according to promoter prediction results. As a result, a total of six promoter fragments were obtained and inserted into the pGL4.10 vector, sequencing results showed that all these sequences were correctly inserted into the vector. Luciferase reporting assay showed that activities of bmp2 promoter with the total length of 1 873 bp could be successfully detected. No significant variation in promoter activity was detected by the gradual deletion of 5′ ends of the promoter fragments of bmp2 gene (Fig. 3), which minimize the positive regulatory region of bmp2 to –179 to +109.
A TATA box was found –1 505 bp upstream from the start codon of the bmp2 gene by TRANSFAC. MatInspector was used for searching for transcription factor binding sites in the positive regulatory region of bmp2 gene (–179 to +109). The results indicated that several factors, such as E-box binding factors, zinc finger transcription factor and binding site for a Pbx1/Meis1 heterodimer were located in the 5′-flanking region of C. semilaevis bmp2 (Fig. 4).
3.3 Gene expression pattern of C. semilaevis bmp2
3.3.1 Tissue expression distribution of bmp2
Tissue expression distribution of bmp2 in C. semilaevis adults was determined by qRT-PCR. As shown in Fig. 5, the highest bmp2 expression level was detected in the spinal cord, similar high expression levels were found in the spleen, skin (with scales), intestine, gill, cartilage and brain; medium expression levels were detected in the liver, stomach, kidney, gonad and muscle; the lowest expression level was detected in the heart.
3.3.2 Temporal-expression of bmp2 during early development
As indicated in Fig. 6, bmp2 was highly expressed at the gastrula stage during early embryo development, and the expression level was about ten times higher in comparison with those at other egg stages. An increasing trend of bmp2 expression was detected from late embryo stages to early larva stages. The bmp2 expression levels stayed constant after 30 dph.
3.3.3 Spatial-expression of bmp2 at early larva stages
Expression patterns of C. semilaevis bmp2 at early larva stages were examined by whole-mount in situ hybridization. As shown in Fig. 7, the bmp2 expression in newly hatched larvae was quite low. At one dph, bmp2 started to be strongly expressed in the crown-like larval fin, heart and liver; slightly expressed in the notochord. The expression of bmp2 started to be concentrated in notochord but much less expressed in other locations at three dph.
4 Discussion
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4.1 Characteristics of C. semilaevis bmp2 gene
In general, members of the BMP family exhibit similar gene characteristics. For instance, the C-terminal (comprising seven cysteine residues) was found conserved among BMP members, similar cleavage sites RXXR (related to sequential cleavage of the synthesized nonactive precursor protein and release of mature ligand) could also be found at certain location of the BMP amino acid sequences (Bragdon et al., 2011; Heng et al., 2010). In this study, the cleavage site RVSR was observed in C. semilaevis bmp2 amino acid sequence. Similarly, our previous study showed that cleavage sites (RTTR and RSIR) were found in C. semilaevis bmp6 and bmp7 genes, and their locations were conserved in the sequences (Ma et al., 2017).
The presence of two BMP2 isoforms (BMP2a and BMP2b) were observed in Astyanax mexicanus, Danio rerio and Oryzias latipes, while only one has been reported in the genome of other vertebrates including C. semilaevis. As shown in Fig. 2, the three bmp2b genes clustered with other bmp2 genes to form a teleost bmp2 lineage, and the D. rerio bmp2a formed a single branch out of the teleost bmp2 cluster. These results supported the view that bmp2b would represent the bmp2 orthologous gene (Marques et al., 2016). One possible reason for the presence of bmp2a and bmp2b isoforms might be the gene duplication event in fish (D. rerio and other closely related species) (Sato and Nishida, 2010; Talbot and Hopkins, 2000; Taylor et al., 2001).
Transcription regulation is one of the most important steps in the regulation of gene expression. However, limit is known about transcriptional regulation of BMP2 by bone- and cartilage-related transcription factors (TFs). In addition, predicted function determined from their primary structure is not sufficient to understand the mode of gene expression regulation at the transcriptional level. In this study, we analyzed the promoter activity of 5′-flanking region of bmp2 gene, since promoter is the best-characterized transcriptional regulatory element. Positive regulatory region of C. semilaevis bmp2 was detected at the location of –179 to +109. In comparison with our previous results of C. semilaevis bmp6 and bmp7, these three bmp genes displayed different promoter features, i.e., the positive regulatory region of the bmp6 gene located at –272 to +28, while bmp7 contained one potential region fairly far upstream of the start codon (–740 to –396).
Regulatory elements and transcription factors involved in the control of bmp2 expression were also identified in this study. A TATA box was found in the 5′-flanking region of the bmp2 gene. However, the investigation of TATA box around the transcription initiation sites was not performed in this study. As reported by Dathe et al. (2009), the bmp2 gene was shown to be flanked by long regions without nearby genes that may contain important regulatory elements. The long-range regulators controlling BMP2 transcription have also been reported by Chandler et al. (2007). A survey of bmp2 genes in teleosts (such as zebrafish, gilthead seabream and spotted gar) has failed to identify TATA boxes upstream the TSS, suggesting that the presence of TATA-less promoters in BMP2 genes is a common feature (Marques et al., 2016). Lack of consensus TATA box around the transcription initiation sites in bmp genes has been demonstrated (Kawai and Sugiura, 2001; Simon et al., 2002), including human and mouse bmp2 genes (Ghosh-Choudhury et al., 2001; Sugiura, 1999). It is now clear that only 10%–20% of mammalian promoters contain a functional TATA box, and TATA-less promoters can also regulate tissue-specific expression (Hochheimer and Tjian, 2003; Ling et al., 2009; Sandelin et al., 2007).
4.2 Expression patterns of bmp2 gene
The central role of bmp2 subfamily in calcified tissues, particularly in scales, has been reviewed by Marques et al. (2016). High expression levels of bmp2 gene in calcified tissues (i.e., bone, scales and caudal fin) were detected in Solea senegalensis and Sparus aurata (Marques et al., 2014; Rafael et al., 2006), which supported the well documented role of bmp2 in tissue mineralization (Alexander et al., 2011; Wang et al., 1990). Similarly, bmp2 exhibited the highest expression level in the mixed tissue (skin with scales) in this study, which contained a mixture of soft and calcified tissues. However, low level of bmp2 in dorsal fin and high level in cartilage were detected in mature C. semilaevis, revealing an inconsistent pattern in tissue distribution.
High expression of bmp2 in the brain, spinal cord and intestine of C. semilaevis and D. rerio, supported the important role of this gene in central (particularly in the enteric) nervous system formation (Chalazonitis and Kessler, 2012; Sato et al., 2010). Among the other tissues with high bmp2 expression, gill was known to be important for processes such as osmoregulation and respiration, which the BMP signaling was reported to be involved with (Kültz, 2012). As reported by Rafael et al. (2006), high expression of BMP2 in the liver of S. aurata suggested a possible role for this protein in liver cell trans-differentiation. In this study, high expression of bmp2 was not observed in the liver and heart of C. semilaevis adults, however whole-mount in situ hybridization assessed bmp2 expression at these locations in two days old larvae. These results suggested that bmp2 could be involved in early development of soft tissues such as heart and liver.
BMP2 has been implicated to be essential for appropriate embryonic dorsoventral patterning of zebrafish embryos during gastrulation (Xue et al., 2014). In this study, a significant increment of bmp2 expression was detected in 13 hpf (hours post fertilization) embryos, corresponding to C. semilaevis gastrula stage. Similar results were also detected in S. aurata, revealing that the bmp2 expression was strongly and transiently up-regulated at 10 hpf (gastrulation) (Rafael et al., 2006). Taking the previous demonstration that bmp2b represents the orthologous bmp2 gene in zebrafish, significantly higher expression of bmp2b was also detected at pregastrula and gastrula stages when comparing to other embryo stages (Martínez-Barberá et al., 1997). In agreement with the observations in other higher vertebrates such as mouse and chicken, BMP2 was also detected at pregastrula and gastrula stages, when calcified structures were not yet formed (Schlange et al., 2000; Ying and Zhao, 2001). These findings suggested the important role of BMP2 in fish development at the onset of gastrulation, which is a decisive stage for cell fate and embryonic patterning (Rafael et al., 2006).
The bmp2 expression presented in one-day old C. semilaevis was at levels similar to that observed in pre-hatching embryos (Fig. 6). Subsequently, highest bmp2 mRNA levels were respectively detected at four and two days post hatching. Under this circumstance, preliminary investigation of bmp2 location in C. semilaevis early larvae was performed by whole-mount in situ hybridization. The results revealed the potential role of bmp2 in fin growth, as well as development of heart, liver and notochord.
5 Conclusions
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The isolation of bmp2 in this study, as well as the identification of transcription factors involves in the control of bmp2 expression, provides encouragement for research of the bmp members in teleosts. According to expression pattern of the bmp2 gene, we reported the potential role of bmp2 in fin growth and development of notochord, which may help elucidating the role of bmp2 in regulating bone formation and growth of C. semilaevis.
Funding
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  • The Special Scientific Research Funds for Central Non-profit Institutes, Chinese Academy of Fishery Sciences under contract No. 2016RC-LX02; the National Natural Science Foundation of China under contract No. 31201981.
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doi: 10.1007/s13131-018-1164-x
  • Receive Date：2017-03-03
  • Online Date：2026-04-13
  • Published：2018-02-25
Article Data
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History
  • Received：2017-03-03
  • Accepted：2017-06-05
Funding
The Special Scientific Research Funds for Central Non-profit Institutes, Chinese Academy of Fishery Sciences under contract No. 2016RC-LX02; the National Natural Science Foundation of China under contract No. 31201981.
Affiliations
    1 Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China
    2 Function Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266200, China
    3 Function Laboratory for Marine Biology and Biotechnolgy, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266200, China

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表12种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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