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中国农学通报 ›› 2015, Vol. 31 ›› Issue (17): 28-37.doi: 10.11924/j.issn.1000-6850.2013-1284

所属专题: 生物技术 水产渔业

• 水产 渔业 • 上一篇    下一篇

青虾泛素特异性蛋白酶9基因Usp9全长cDNA序列的克隆及表达分析

袁晖,周巧,张世勇,傅洪拓   

  • 收稿日期:2013-05-03 修回日期:2013-05-03 接受日期:2013-07-22 出版日期:2015-07-27 发布日期:2015-07-27
  • 通讯作者: 袁晖
  • 基金资助:
    江苏省高技术研究项目“青虾新品系的选育”(BG2007328);中央级科研院所基本科研业务费专项“青虾精巢均一化构建及表达序列标签分析”(2011JBFA02)。

Full-Length cDNA Cloning And Spatial-Temporal Expression Analysis Of Ubiquitin-Specific Protease 9 Gene(Usp9)In Oriental river prawn, Macrobrachium nipponense

  • Received:2013-05-03 Revised:2013-05-03 Accepted:2013-07-22 Online:2015-07-27 Published:2015-07-27

摘要: 本研究首次克隆了青虾的Usp9 (ubiquitin-specific protease 9)基因全长 cDNA序列,并在青虾中使用荧光定量 PCR 技术首次检测了 Usp9 基因在不同组织和发育阶段中的表达变化。青虾 Usp9 基因cDNA全长 1259 bp,包括 162 bp的 5’ UTR, 978 bp的开放阅读框以及 116 bp的 3’ UTR。氨基酸序列比对分析显示,青虾 Usp9蛋白与太平洋牡蛎(Crassostrea gigas)、凤仙花熊蜂(Bombus impatiens)及灰短尾负鼠(Monodelphis domestica)的蛋白相似度分别为 99%、 97%和 95%。应用 MEGA5.1软件对青虾与其他物种的 Usp9氨基酸进行系统进化树分析,结果表明,青虾 Usp9与同为甲壳纲的钩额型蚤状溞聚为一支,具有最近的亲缘关系。采用荧光定量 PCR技术检测了 Usp9基因的时空表达,水温 28℃时, Usp9基因在青虾发育的后幼体发育期第 5天 PL5出现表达高峰。Usp9基因在 6种成体组织中均有表达,表达水平依次为:脑>腹神经>精巢>卵巢>肌肉>肠。而在雌雄组织差异表达分析表明,精巢中Usp9基因表达量比卵巢的比值为 2.86,脑组织中雄虾的表达量比雌虾为 2.27,差异均为极其显著(P<0.01)。这表明Usp9基因的表达和雄性偏向基因趋势是一致的(雄性表达:雌性表达≥1.5)。本研究为进一步开展青虾Usp9基因功能等相关研究奠定了基础,并可为青虾性别的遗传调控机制研究提供参考。

关键词: 评估, 评估

Abstract: In this study, the full-length cDNA sequence was cloned and spatial-temporal expression pattern of Usp9 was detected by quantitative real-time PCR (qRT-PCR) for the first time from the testis of oriental river prawn Macrobrachium nipponense. The full-length cDNA sequence of Usp9 is 1259 bp, containing an open reading frame of 978 bp, 162 bp of 5’ -untranslated region (UTR), 116 bp of 3'-UTR with a poly (A) tail. The deduced amino acid sequence of Usp9 shares 99% , 97% and 95% similarity to Crassostrea gigas, Bombus impatiens and Monodelphis domestica, respectively. The phylogenetic tree based on other species Usp9 proteins showed that Usp9 from testis of M. nipponense shared the closest relationship with Daphnia pulex. qRT-PCR analysis demonstrated that the expression levels of Usp9 in M. nipponense reached the peak in the 5th day of the post- larvae (PL5), when water temperature was 28℃ . 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 Usp9 to be significantly higher in the male mouse than female, the M:F ratios between testis and ovary, male brain and female brain were 2.86 and 2.67, the difference were extremely significant (P<0.01). The result showed that the expression of Usp9 shared the same trend with male-biased gene (M:F≥1.5). This research could provide basic information for the function of Usp9 gene and references for further studies in genetic regulation mechanism of M. nipponense, as well as in other crustacean species.