文章摘要
罗氏沼虾(Macrobrachium rosenbergii)细胞色素P450家族CYP302a1基因克隆及其在蜕皮周期中的表达研究
Cloning and characterization of cytochrome P450 302a1 (CYP302a1) during molting stages in Macrobrachium rosenbergii
投稿时间:2019-07-07  修订日期:2019-09-25
DOI:
中文关键词: 罗氏沼虾  蜕皮周期  基因克隆  CYP302a1  组织表达
英文关键词: Macrobrachium rosenbergii  Molting cycle  CYP302a1  Gene cloning  Expression
基金项目:国家自然科学基金
作者单位E-mail
杨光 华南师范大学生命科学学院 1357501238@qq.com 
秦真东 仲恺农业工程学院动物科技学院  
赵丽娟 仲恺农业工程学院动物科技学院  
沈海洋 华南师范大学生命科学学院  
张梦兰 华南师范大学生命科学学院  
卢志杰 华南师范大学生命科学学院  
叶成凯 华南师范大学生命科学学院  
李凤麟 仲恺农业工程学院动物科技学院  
潘淦 华南师范大学生命科学学院  
林蠡 仲恺农业工程学院动物科技学院 linli@zhku.edu.cn 
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中文摘要:
      甲壳动物的蜕皮过程与其生长发育密切相关。细胞色素P450(CYP)302a1是昆虫蜕皮激素合成通路中的关键酶之一。本研究克隆了罗氏沼虾CYP302a1基因(命名为Mr-CYP302a1),其cDNA全长1859 bp,开放阅读框(ORF)为1629 bp,编码543个氨基酸(aa),分子量大小为61.09 kDa,等电点为8.42。氨基酸序列分析显示CYP302a1基因的保守结构域含有5个P450基因家族特征保守区域:heme-binding、helix-K、helix-C、helix-I及PERF。进化树分析发现Mr-CYP302a1首先与绿虾CYP302a1聚为一支,然后与南美白对虾及三疣梭子蟹等十足目甲壳动物的CYP302a1聚为一支。对Mr-CYP302a1基因进行了克隆和多克隆抗体制备。实时荧光定量PCR(qRT-PCR)和蛋白印迹法(WB)检测表明Mr-CYP302a1在罗氏沼虾的多个组织中均有表达,其中在Y-器官中的表达量最高,性腺中次之。此外,Mr-CYP302a1基因在罗氏沼虾的蜕皮后期表达量很低,蜕皮间期表达量开始上升,在蜕皮前期达峰值。以上结果表明该基因在罗氏沼虾的蜕皮和繁殖过程中扮演着十分重要的角色。
英文摘要:
      The molting process in crustaceans is closely related to their developmental stages. Cytochrome P450(CYP)302a1 is the key enzyme which plays a critical role in the synthesis of molting hormone. Here we present the cloning and characterization of CYP302a1 gene from Macrobrachium rosenbergii (named Mr-CYP302a1). The full-length cDNA of Mr-CYP302a1 gene was 1859 bp with the open reading frame of 1629 bp that encodes 543 amino acids with a molecular weight of 61.09 kDa and an isoelectric point of 8.42. The amino acid sequence analysis revealed that there were five P450 characteristic conserved regions,i.e.,heme-binding, helix-K, helix-C, helix-I, and PERF. Phylogenetic analysis demonstrated that Mr-CYP302a1 was closely related to the CYP302a1 of Neocaridina denticulata, and then clustered with the CYP302a1 from Decapoda crustaceans such as Penaeus vannamei and Portunus trituberculatus. Mr-CYP302a1 was expressed and its polyclonal antibody was generated. Real-time quantitative PCR (qRT-PCR) and Western Blotting (WB) results showed that Mr-CYP302a1 was significantly expressed in various tissues of M. rosenbergii, especially higher expression level was observed in the Y-organ and gonads, indicating that it was involved in the propagation of M. rosenbergii. On the other hand, the expression of Mr-CYP302a1 was significantly lower at the postmolt stage, and it was increased gradually at the intermolt, significantly enhanced and reached the maximal level at the premolt. In summary, our results indicate that Mr-CYP302a1 may play an important role in molting and propagation of M. rosenbergii.
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