中文摘要：

Dmrt是性别调控因子Doublesex和Mab-3的相关基因,近年报道了中华绒螯蟹EsDmrt-like只在精巢中表达,为了验证EsDMRT-like蛋白是否在中华绒螯蟹精巢中特异表达及其功能,根据中华绒螯蟹EsDmrt-like基因序列,构建重组质粒pET-32a-EsDmrt-like,转化大肠杆菌BL21,经融合表达和SDS-PAGE分析表明,融合蛋白主要以包涵体形式存在,分子量约为46ku。利用Ni柱亲和纯化融合蛋白免疫家兔,制备获得EsDMRT-like多克隆抗体。Western-blotting检测表明该抗体既能特异地识别重组蛋白,又能特异识别精巢中EsDMRT-like蛋白,并且该抗体仅在精巢中检测到EsDMRT-like蛋白的表达,分子量约为52 ku,为预期单体分子量的二倍。Western-blotting检测变性后精巢总蛋白,该抗体能识别52和34 ku两条条带,证明了二聚体的存在。这一结果暗示EsDMRT-like可能通过形成二聚体形式调控中华绒螯蟹精巢发育。

英文摘要：

Dmrt is a family of genes related to the sexual regulators Doublesex and Mab-3.Our previous studies showed that EsDmrt-like transcripts were detectable only in testis of Chinese mitten crab,Eriocheir sinensis.In order to verify whether EsDMRT-like protein is also specifically expressed in testis,the prokaryotic expression plasmid pET-32a-EsDmrt-like was constructed according to the sequence of EsDmrt-like.The recombinant plasmid was transferred into host bactria Escherichia coli BL21（DE3）,and the recombinant protein Trx-EsDMRT was expressed under the induction of IPTG.SDS-PAGE analysis showed that the fusion protein was highly expressed as inclusion body with the molecular weight of 46 ku.The recombinant protein was purified by affinity for preparing EsDMRT-like antibody.The resultant antibody can specifically recognize the recombinant protein and endogenous EsDMRT-like protein in the testis as revealed by Western-blotting analysis.EsDMRT-like was specifically expressed in the testis,with molecular weight of 52 ku,which is twice as those of the monomer.After denatured treatment by urea and DTT,the protein displayed two forms of polypeptides：52 and 34 ku.Both of them can be recognized by antiserum raised against EsDMRT-like protein,indicating that the form of 34 ku was generated from 52 ku polypeptide and the EsDMRT-like protein exists as a dimer in testis.This result suggested that EsDmrt-like may regulate the male sex development/differentiation by dimerization in the crab.