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OTUB1是哺乳动物雌激素信号通路中的一个关键因子,它可以使雌激素受体α(ERα)去泛素化并抑制其转录活性。本研究从实验室拟穴青蟹(Scylla paramamosain)转录组测序数据库中筛选出OTUB基因片段,利用SMART RACE技术克隆得到Sp-OTUB的cDNA全长。该序列全长1 261 bp,包括5’非编码区(5’-UTR)40 bp,开放阅读框(ORF)804 bp和3’非编码区(3’-UTR)417 bp。其ORF编码267个氨基酸,预测其分子量为31 ku。该蛋白序列中包含一个OTU样的半胱氨酸蛋白酶催化结构域和一个由半胱氨酸(C)、天冬氨酸(D)和组氨酸(H)组成的催化三联体。实时定量PCR结果显示,Sp-OTUB在成熟拟穴青蟹各组织器官中均有表达,其中在血淋巴中的表达量最大,精巢中表达量最低。在卵巢发育过程中,Sp-OTUB的表达量在卵巢增值期为最大,与其他各时期具有显著差异;在精巢发育各阶段,Sp-OTUB表达量在精子细胞期达到最大,显著高于精母细胞期和成熟精子期。同源系统进化分析Sp-OTUB与大多数物种的OTUB1聚为一支,与肩突硬蜱(Ixodes scapularis)和紫海胆(Strongylocentrotuspurpuratus)亲缘关系最近。实验结果初步显示Sp-OTUB在拟穴青蟹生殖调控方面可能发挥重要作用。
OTUB1 is a key factor in the mammalian estrogen signaling pathway that can deubiquitinate and inhibit the transcriptional activity of the estrogen receptor alpha (ERa). In this study, the OTUB gene fragment was screened from the Scylla paramamosain transcriptome sequencing database, and the full-length cDNA of Sp-OTUB was cloned by SMART RACE technology. The sequence was 1 261 bp in length, including 40 bp in 5 ’untranslated region (ORF), 804 bp in open reading frame (ORF) and 417 bp in 3’ untranslated region (3’-UTR). Its ORF encodes 267 amino acids and its molecular weight is predicted to be 31 ku. This protein sequence contains an OTU-like cysteine protease catalytic domain and a catalytic triad of cysteine (C), aspartic acid (D) and histidine (H). The results of real-time quantitative PCR showed that Sp-OTUB was expressed in all tissues and organs of mature crab, and the expression was the highest in hemolymph and the lowest in testis. During ovary development, the expression of Sp-OTUB was the largest in ovarian proliferation stage, which was significantly different from other stages. The expression of Sp-OTUB reached the maximum during spermatogenesis in all stages of testicular development, which was significantly higher than that of sperm Cell stage and mature sperm stage. Homologous phylogenetic analysis Sp-OTUB clustered together with most species of OTUB1, most closely related to Ixodes scapularis and Strongylocentrotus purpuratus. The preliminary results show that Sp-OTUB may play an important role in the reproductive regulation of Scylla serrata.