Molecular Cloning and Characterization of Dmc1 from the Chinese Mitten Crab (Eriocheir sinensis)

Abstract

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.monodon [14,15] and whiteleg shrimp Litopenaeus vannamei [16]. Despite the finding of Dmc1 from testis transcriptome [17], 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 annotationFour 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 HiSeq TM 2000 platform. A total of 23.48 Gb clean data were generated from larval transcriptomes [18][19][20]. BLASTx analysis revealed that a unigene (comp19040_c0_seq1) was homologous to Dmc1 in L. vannamei (ADM45305). Full-length cDNA sequence determinationGene 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 H 2 O, 2.5 µl of 10×PCR buffer, 1.5 IntroductionThe 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 [1]. Like other crabs, the mitten crabs exhibit bimodal growth patterns in which males e...