| The Chinese mitten crab Eriocheir sinensis is one of the most important aquaculture species in China, its culture under facility conditions having started in the early1980's. The annual output has increased during the past decade in China, from200,000tons in the year2000to420,000tons in2004. With the development of intensive culture, various problems have appeared in cultured populations, amongst others, sexual precocity. More and more individual crabs mature when small-sized. After sexual maturation, energy and nutrients are mainly diverted into gonad maturation or reproduction, with little left over for somatic growth, with the consequential devaluation of the commercial product and economic losses. Previous studies have revealed two main external factors as leading to sexual precocity in crabs, the environment and food. Nevertheless, few scientific investigations have been dedicated to elucidating the internal factors inducing sexual precocity. Furthermore, the regulative mechanisms of gonad maturation in E. sinensis at the molecular level are unknown.In the present study, a testis cDNA library of Chinese mitten crab Eriocheir sinensis-was constructed and ESTs were analyzed.30genes related to reproduction and development were discovered. cDNAs of Dmcl was gained basis on expressed sequence tags, followed by analysis of characterization and spatio-temporal expression. The results were as follows:1We constructed a cDNA library from the rapid developmental stage of testis of E. sinensis and sequenced3,388randomly picked clones. After processing,2,990high-quality expressed sequence tags (ESTs) were clustered into2,415unigenes including307contigs and2,108singlets, which were then compared to the NCBI non-redundant (nr) protein and nucleotide (nt) database for annotation with Blastx and Blastn, respectively. After further analysis,922unigenes were obtained with concrete annotations and30unigenes were found to have functions possibly related to the process of reproduction in male crabs-six transcripts relevant to spermatogenesis (especially Cyclin K and RecA homolog DMC1), two transcripts involved in nuclear protein transformation, two heat-shock protein genes, eleven transcription factor genes (a series of zinc-finger proteins), and nine cytoskeleton protein-related genes. Our results, besides providing valuable information related to crustacean reproduction, can also serve as a base for future studies of reproductive and developmental biology.2Dmcl is a protein involved specifically in the meiotic recombination during spermiogenesis of vertebrates, however, it is little characterized in lower invertebrates. In this study, a full-length cDNA coding Dmcl (termed EsDmcl) was cloned from testis of Chinese mitten crab Eriocheir sinensis by reverse transcript-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods. The cDNA obtained was1,360bp long with a1,029bp open reading frame (ORF) encoding341amino acids. The putative EsDmcl protein owns the Walker A (GPESSGKT), B boxes (VTVVD) and two disordered loops (denoted L1and L2motifs). Pairwise alignments demonstrated that EsDmcl had86%,78%,75%,75%and73%identity with its homologues in black tiger shrimp, black-legged tick, mouse, human and zebrafish, respectively. The phylogenetic tree revealed that EsDmcl is more related to invertebrate Dmcl than vertebrate. The EsDmcl transcripts were predominantly expressed in testis. Quantitative RT-PCR was used to determined the expression difference of EsDmcl gene in testis at earlier, middle, later and end stages of E. Sinensis. EsDmcl gene expression was highest at earlier development stage, and the next were middle and later stages. At end development stage, the expression level of EsDmcl gene was lowest. Results suggesting that differential expression of EsDmcl is closely related to spermatocyte meiotic maturation. Therefore, EsDmcl probably performs critical functions in the spermiogenesis of E. Sinensis.3In the present study, we performed suppression subtractive hybridization (SSH) experiments in the crab, Eriocheir sinensis, and constructed the forward subtractive cDNA library with cDNAs from the earlier stage (tester) and the later stage (driver) of the E. sinensis testis. The reverse subtractive cDNA library was constructed with cDNAs from testis during the later stage (tester) and the earlier stage (driver). The subtractive cDNA libraries were obtained including850and700clones respectively. By PCR detection, the length of the inserted fragments was250-1500bp on average. Further results indicated that the cDNA library quality are exactly within SSH library standard and could be used in further studies, also would provide reference for studying the differentially expressed genes to explore the molecular mechanism of testis, and regulation and control of spermatogenesis in E. sinensis.
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