Yuan Liu1 and Zhaoxia Cui1,2*
1Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, 266071, China
2National & Local Joint Engineering Laboratory of Ecological Mariculture, Qingdao, 266071, China
Received: 05 March, 2014; Accepted: 04 April, 2015; Published: 06 April, 2015
Zhaoxia Cui, Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao 266071, China; Tel: +86 532 82898509; Fax: +86 532 82898509; Email:
Liu Y, Cui Z (2015) Molecular Cloning and Characterization of Dmc1 from the Chinese Mitten Crab (Eriocheir sinensis). Int J Aquac Fishery Sci 1(1): 024-029. DOI: 10.17352/2455-8400.000005
© 2015 Liu Y, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Eriocheir sinensis; Dmc1; Meiosis; Expression analysis
Dmc1, a member of the RecA/Rad51 superfamily, is essential for meiotic recombination. In this study, a Dmc1 gene (EsDmc1) was identified from screening the larval transcriptomes of Chinese mitten crab Eriocheir sinensis. The full-length cDNA of EsDmc1 was 1478 bp long and contained a 1026 bp open-reading frame encoding 341 amino acids. The genomic fragment of EsDmc1 contained two exons separated by one intron. Several tandem repeats were found in intron. The deduced EsDmc1 protein contained motifs conserved in the RacA/Rad51 superfamily, including the Walker A and B motifs, and L1 and L2 loops. EsDmcl shared 87.3%, 86.5% and 77.4% identity with its homologues in Litopenaeus vannamei, Penaeus monodon and Ixodes scapularis, respectively. Phylogenetic analysis revealed that EsDmc1 had a closer relationship with Dmc1s from arthropods than vertebrates. The EsDmcl transcripts could be detected in all examined larval stages with the highest expression level in the fifth zoeal stage. These results suggest that EsDmc1 could be expressed before reproductive maturity and might have complex functions in crab reproduction.
The Chinese mitten crab Eriocheir sinensis (Henri Milne Edwards 1854) is a commercially important species in Southeast Asia and has been widely farmed in ponds, reservoirs and lakes of China since 1990s . Like other crabs, the mitten crabs exhibit bimodal growth patterns in which males exhibit higher growth rate and larger size than females [2-4]. However, female mitten crabs have greater economic value than males. In addition, sexual precocity has occurred in cultured E. sinensis populations  and caused catastrophic economic losses to crab aquaculture. Understanding the mechanisms involved in reproduction or sexual development and characterization of the related genes in E. sinensis would be helpful for the aquaculture industry.
Dmc1 (disrupted meiotic cDNA), a member of the RecA/Rad51 superfamily that encodes a DNA recombinase, plays a central role in meiotic recombination [6,7]. The Dmc1 gene was initially identified in budding yeast Saccharomyces cerevisiae as a meiosis-specific gene . Afterwards, Dmc1 genes have been isolated from higher eukaryotes, such as human and mouse . In mammals, Dmc1 was also a specifically expressed gene in testicular germ cells and embryonic ovaries in the period of meiosis. Further studies showed that Dmc1 mutations cause meiotic arrest at the zygotene stage without homologue synapsis  or with an occasional synapsis between non-homologues . All of these suggest the crucial role of Dmc1 during meiosis appears to be conserved from yeast to mammals.
Recently, Dmc1 genes have been identified and characterized from several aquatic animals, such as Japanese eel , cyprinid fishes , and only two crustacean species, giant tiger shrimp Penaeus monodon [14,15] and whiteleg shrimp Litopenaeus vannamei . Despite the finding of Dmc1 from testis transcriptome , no study has described the sequence characterization and expression pattern of Dmc1 in E. sinensis. Here, a Dmc1 gene (designated as EsDmc1) showing a predicted amino acid sequence similarity to L. vannamei was found from screening the transcriptomes of larval E. sinensis. The main objectives of the present study are to clone this gene from E. sinensis, detect genomic organization and investigate the expression pattern during larval developmental stages based on transcriptome data.
Materials and Methods
cDNA library construction and gene annotation
Four libraries were conducted from whole bodies of E. sinensis at four larval developmental stages, including the fifth zoeal stage (Z5), the megalopa stages before and after desalination (MB and MA), and the first juvenile instar (J1). Transcriptome sequencing was carried on an Illumina HiSeqTM 2000 platform. A total of 23.48 Gb clean data were generated from larval transcriptomes [18-20]. BLASTx analysis revealed that a unigene (comp19040_c0_seq1) was homologous to Dmc1 in L. vannamei (ADM45305).
Full-length cDNA sequence determination
Gene specific primer (P1), nested primer (P2) and oligo (dT)-adaptor (Table 1) were used to clone the 3' end of EsDmc1cDNA by 3' RACE technique. Initial amplification was carried out with P1 and oligo (dT)-adaptor, followed by nested PCR using P2 and oligo (dT)-adaptor. The amplification was performed in a 25 µl reaction volume containing 17.3 µl sterile distilled H2O, 2.5 µl of 10×PCR buffer, 1.5 µl of MgCl2 (25 mM), 0.5 µl of dNTP (10 mM), 1 µl of each primer (5 µM), 0.2 µl (1 U) of Taq polymerase (TaKaRa), and 1 µl of template. The PCR reactions were performed on TaKaRa PCR Thermal Cycler Dice Model TP600 (Takara Bio Inc.) with an initial denaturation at 94°C for 3 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 56°C for 50 s, extension at 72°C for 1 min, and a final extension at 72°C for 10 min. The PCR products were gel-purified and cloned into pMD18-T simple vector (TaKaRa). After being transformed into the competent cells of Escherichia coli DH5α, the positive recombinants were identified through anti-Amp selection and PCR screening with M13-47 and RV-M primers (Table 1). Three of the positive clones were sequenced to verify the full-length cDNA of EsDmc1.
Genomic DNA amplification
Genomic DNA was extracted from the muscle tissue by standard phenol-chloroform method . To detect the genomic structure of EsDmc1, two gene-specific primers (P3 and P4, see Table 1) were designed according to the obtained cDNA sequence. The PCR was performed in a 25 µl reaction volume containing 17.3 µl sterile distilled H2O, 2.5 µl of 10×PCR buffer, 1.5 µl of MgCl2 (25 mM), 0.5 µl of dNTP (10 mM), 1 µl of each primer (5 µM), 0.2 µl (1 U) of Taq polymerase (TaKaRa), and 1 µl of DNA template (approximately 30 ng). The PCR temperature profile was 94°C for 3 min, followed by 34 cycles of 94°C for 30 s, 55°C for 50 s, 72°C for 1 min, and a final extension at 72°C for 10 min. The PCR products were cloned and sequenced according to the method described above.
Sequence and phylogenetic analysis
Blast algorithm at National Center for Biotechnology Information (http://www.ncbi.nlm.gov/blast/) was used to search the homology of nucleotide and protein sequences. The deduced amino acid sequence was analyzed with the Expert Protein Analysis System (http://www.expasy.org/). SignalP 3.0 program was utilized to predict the presence and location of signal peptide, and the cleavage sites in amino acid sequences (http://www.cbs.dtu.dk/services/SignalP).
Multiple amino acid sequence alignment was performed using the Clustal X with the default settings . A neighbor-joining (NJ) tree with bootstrap values was constructed for phylogenetic analysis using MEGA 4.0 . All the reference sequences for phylogenetic analysis were derived from GenBank.
Expression analyses based on transcriptome data
The reads for EsDmc1 were counted by mapping reads to assembled unigene sequences in the four larval transcriptomes. The expression of EsDmc1 was calculated using the reads per kb per million reads (RPKM) method . The fold change values > 2 and false discovery rates (FDR) adjusted significance values < 0.05 (-log10 (0.05) = 1.3) were considered as the threshold to judge the significance of expression.
Results and Discussion
cDNA cloning and sequence analysis of EsDmc1
The full-length cDNA sequence of EsDmc1 was obtained by overlapping the corresponding unigene with the amplified fragments. It was 1478 bp, and consisted of a 5'-UTR of 213 bp, 3'-UTR of 239 bp and an open-reading frame (ORF) of 1026 bp. A canonical polyadenylation signal-sequence (AATAAA) and poly (A) tail was detected in EsDmc1. The ORF encoded 341 amino acids and no signal peptide was identified (Figure 1). The estimated molecular weight of EsDmc1 was 37.49 kDa and its theoretical isoelectric point was 5.41. The sequence of EsDmc1 was deposited in GenBank under the accession number KP876487.
Genomic organization of EsDmc1
Until now, no genomic sequence of Dmc1 has been identified in crustaceans. The amplified genomic DNA fragment of EsDmc1 was 856 bp and deposited in GenBank under accession number KP876488. By aligning with the corresponding cDNA sequence, the exon-intron boundaries of EsDmc1 were determined (Figure 2). The EsDmc1 genomic sequence consisted of two exons (248 and 355 bp) separated by one intron (253 bp). All splice sites in EsDmc1 followed the canonical GT/AG splicing recognition rule. Several mononucleotide repeats (T)4-7 and one pure dinucleotide repeat (GC)3 were found in the intron (Figure 2). Intronic microsatellites found in EsDmc1 will be helpful in understanding the genetic structure of Dmc1s and studying the possible roles of microsatellites.
Follow us on Academia.edu
Access denied for user 'root'@'localhost' (using password: YES)