The Molecular Characterzation And Functional Analysis Of Sexual Develonment Related Genes Sxl And Dmrt In The Prawn,Macrobrachium Rosenbergii

The giant freshwater prawn, Macrobrachium rosenbergii, is an important economic animal in aquaculture, and distributed widely in tropic and subtropic areas of the Pacific Ocean. The prawn has high commercial values because of its fast growth, great size and dietary omnivority. Although substantial progress has been achieved on breeding and culture technologies of prawn farming, the molecular mechanisms underlying the sexual phenotypic differentiation remain largely unclear. In the present study, we focus on two key genes of the most conservative and important sex determination pathway in metazoans, Sxl and Dmrt from the prawn Macrobrachium rosenbergii, reveal their structural properties and try to elucidate their potential function in the sexual development and reproduction in this economic species.Part one, molecular cloning and functional characterization of the Sxl genes in M. rosenbergii. First, a partial Sxl fragment was amplified and determined with a degenerate nested PCR. With this fragment as a DNA probe, we screened a central nervous system cDNA-library of the prawn and sequenced all the positive clones. The sequencing revealed at least four Sxl isoforms existing in M. rosenbergii. The four structural variants were named as MroSxl1, MroSxl2, MroSxl3and MroSxl4with cDNA full lengths of2054,1977,1897and1031bp, respectively. Both results of Northern blotting and isoform-specific RT-PCR identified the authentically transcription of the four variants in the prawn. Southern blotting was performed to decide the copy number of MroSxl gene at the genomic level, where the result implicated that a single Sxl locus was present in the M. rosenbergii genome. Second, to identify whether the sex-specific expressing mechanism was applied to MroSxl, we performed isoform-specific RT-PCR and Western blotting. The results implied that each variant existed in both males and females. However, the expressed level of MroSXL3/4was much higher in ganglion, hepatopancreas and ovary of females. Further analysis of the protein distribution in bisexual gonads was performed with immunohistochemistry, where the results showed that the MroSXL protein was prominently localized in the spermatocyte but no expression in the sperm; while in the previtellogenic oocyte, MroSXL was prominently localized in the cytoplasm; when the oocyte differentiated into the vitellogenic stage the MroSXL protein was trans-imported into the nucleus.Part two, molecular cloning and functional characterization of the Dmrt genes in M. rosenbergii. First, we performed degenerated RT-PCR and RACE method to obtain two entire cDNAs of Dmrt genes, where one (2073bp in length) contains a1614-bp open reading frame (ORP) encoding a537-aa protein, while the other906-bp cDNA contains a618-bp ORF that encodes a205-aa protein. Alignments of amino-acid sequences revealed that both the genes contained the DM domain, an unusual zinc finger module which is well conserved among all DMRT subfamilies. Further phylogenetic analysis undoubtedly placed the present two deduced M. rosenbergii DM domain proteins into two Arthropoda-specific subfamilies, DMRT11E and DMRT99B, according to which we named them MroDmrtllE and MroDmrt99B, respectively. Second, we used RT-PCR to investigate tissue distributions and temporal expression during embryogenesis of the two MroDmrt genes. The results implied a sexually dimorphic expression pattern of the two MroDmrt genes in the prawn:MroDmrt11E transcription was prominent in testis, moderate in bisexual ganglion, while much lower in ovary (over ten times lower than in testis, P<0.01). On the other hand, MroDmrt99B was exclusively expressed in testis but dispensable in ovary and other tissues, although its expression level in testis was relatively low. Additionally, during the embryogenesis, the expression pattern of two MroDmrt transcripts tended to higher levels, and MroDmrt99B exhibited a later transcription.The present results reveal the conservation and specificity of the core sex determination pathway and its key molecules in crustaceans. Our study provides not only substantial evidence to elucidate the molecular mechanisms of sex differentiation and regulation in economic crustaceans like the prawn, but also important basic data for application on manual sex control in aquaculture.