| Oriental river prawn, Macrobrachium nipponense, is an important shrimp species for freshwater aquaculture in China. There are significant differences of biological and economical traits between male and female shrimp. For example, growth rate of male shrimp is much higher than female one, the weight of male individuals are bigger than female ones with the same age. As a result, studies on sex control of river prawn are theoretically significant as well as high economic value for its aquaculture. Previous study showed the expression of ubiquitin-specific protease9(Usp9), an X-linked gene in Rattus norvegicus, to be significantly higher in the adult female mouse brains than in male brains. Usp9acts to deubiquitinate epsin in nervous system, has been shown to be related to neuronal differentiation and synaptic plasticity. However, cloning and expression analysis of Usp9was not reported in crustaceans.In this study, a highly homologous fragment of Usp9was identified from the testis of orinental river prawn Macrobrachium nipponense, the full-length cDNA sequence was obtained for the first time by the combination of reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The full-length cDNA sequence of Usp9is1217bp, containing an open reading frame of978bp,120bp of5’-untranslated region (UTR),116bp of3’-UTR with a poly (A) tail. The gene encodes a polypeptide of326amino acids with an estimated molecular mass of36.53kD and a theoretical pI of5.32. The deduced amino acid sequence analysis showed that the gene did not have signal peptide and transmembrane region, implying it is the nuclear proteins. The amino acids of Usp9shares including99%,97%and95%similarity to Crassostrea gigas, Bombus impatiens and Monodelphis domestica, respectively, through the comparison with the amino acids of Usp9in other species. The phylogenetic tree based on other species Usp9proteins showed that Usp9from testis of Macrobrachium nipponense shared the closest relationship with Daphnia pulex.In addition, the spatial-temporal expression pattern of Usp9was detected by Quantitative real-time PCR (qRT-PCR) for the first time. qRT-PCRanalysis results, the expression level reached the peak in the5th day of the post-larvae (PL5), when water temperature is28℃. Based on the expression pattern in adult tissues, the highest expression level was observed in brain, followed by abdominal ganglion, testis, ovary, muscle and intestine. The sexually dimorphic expression of Usp9to be significantly higher in the male mouse than female, the M:F ratiosbetween testis and ovary, male brain and female brian were2.86and2.67, the difference were extremely significant (P<0.01).It is the first time to clone the full length cDNA sequence of Usp9gene in Macrobrachium nipponenseand analysed the structure of Usp9gene in this research. Besides, qRT-PCR technique was firstly used to detect the spatial-temporal expression pattern of Usp9gene during embryogenesis, larvae, post-larvae and in various adult tissues. The results shows that Usp9gene of Macrobrachium nipponensemay play important roles in nervous system, and the expressionof Usp9shared the same trend with male-biased gene (M:F>1.5). This research could provide basic information for the function of Usp9gene and references for further studies in genetic regulation mechanism of Macrobrachium nipponense, as well as in other crustacean species. |
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