拟穴青蟹OTUB的分子克隆及其在性腺发育过程中的表达  

Molecular Cloning and Expression Analysis of OTUB during Gonadal Development in Scylla paramamosain

作　　者：高洁[1] 邹志华[1] 贾锡伟[1] 林鹏[1] 张子平 王艺磊[1] 

机构地区：[1]集美大学水产学院厦门,中国361021 [2]西东大学生物系,美国新泽西州07079

出　　处：《动物学杂志》2013年第3期407-416,共10页Chinese Journal of Zoology

基　　金：国家自然科学基金项目(No.31072200);集美大学创新团队基金项目(No.2010A001)

摘　　要：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 of the estrogen signaling pathway in mammals. It has been proved that OTUB1 is a novel ERa-interacting protein with capability of deubiquitinating ERa and then repressing its transcriptional activity. In this study, expressed sequence tag （EST） of Scylla paramamosain OTUB gene（ OTU-domain Ub- aldehyde-binding protein） was identified from transcriptome sequencing library and cloned by using SMART Rapid Amplification of cDNA end （RACE） technique. The full-length cDNA of Sp-OTUB is 1 261 bp, including 40 bp in 5＇-UTR, 804 bp in open-reading frame （ORF） and 417 bp in 3＇-UTR. The ORF encodes 365 amino acids with a predicted molecular weight of 31 ku, and the translated protein contains a typical OTU- like cysteine protease catalytic domain and a putative catalytic triad which was composed of conserved cysteine,histidine, and possibly the aspartate residues. Real-time quantitative PCR showed that Sp-OTUB was expressed in all detected tissues of mature crabs with the highest expression in haemolymph and the lowest expression in testis. In the process of ovarian development, Sp-OTUB showed the highest expression in proliferation stage compared to other development stages. In secondary spermatocyte stage, the expression of Sp-OTUB reached the highest and was significantly higher than that of primary spermatocyte stage or spermatid stage. Phylogenetic analysis based on the OTUB protein homologue sequences indicated that Sp-OTUB was separated together with OTUB1 in most other species, and Sp-OTUB had the closest genetic relationship with that of lxodes scapularis or Strongylocentrotus purpuratus. The preliminary experimental results showed that Sp-OTUB may play an important role in reproductive regulation.

关 键 词：拟穴青蟹 组织表达 性腺发育 包含OTU结构域的泛素乙醛结合蛋白 

分 类 号：S917.4[农业科学—水产科学]