日本沼虾StAR基因克隆及其低氧胁迫下表达分析
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Q 786; S 917

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国家重点研发计划 (2023YFE0205100);国家自然科学基金 (31672633);宁夏回族自治区重点研发计划 (2022ZDYF0569);上海市科技创新行动计划( 23XD1421600, 22015820700)


Molecular cloning and expression analysis of StAR gene from oriental river pawn (Macrobrachium nipponense) in response to hypoxia
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    摘要:

    为了研究类固醇激素合成急性调节蛋白 (steroidogenic acute regulatory protein, StAR)在日本沼虾应答低氧胁迫过程中所发挥的作用,实验应用cDNA末端快速扩增 (RACE) PCR技术克隆了日本沼虾StAR全长cDNA序列,并利用在线软件对其序列特征进行生物信息学分析,采用半定量RT-PCR方法分析StAR在日本沼虾不同组织的分布情况,采用实时荧光定量PCR(qRT-PCR)与免疫印迹(Western blot)技术对其在不同低氧胁迫阶段中的表达规律进行检测,观察低氧胁迫下精巢组织中StAR蛋白免疫组化定位。结果显示,日本沼虾StAR其cDNA全长3 361 bp (NCBI登录号:MW263908),包括5'-UTR 323 bp,3'-UTR 2 129 bp,开放阅读框909 bp,编码302个氨基酸。氨基酸序列比对及系统进化分析结果显示,日本沼虾StAR基因与三疣梭子蟹等甲壳动物StAR聚类一支,具有最近的亲缘关系。半定量RT-PCR检测结果显示,StAR在日本沼虾不同组织中均有表达,其表达量在肝胰腺和心脏组织中最低,在精巢组织中表达处于较高水平。使用qRT-PCR技术检测日本沼虾精巢中StAR在低氧胁迫下时空表达情况,结果表明,与对照组相比,低氧处理组精巢中StAR基因表达量在1~48 h均显著上调,而在低氧处理组96 h显著降低。此外,本实验对StAR进行了原核表达及抗血清制备,采用Western blot检测StAR蛋白表达丰度基本与基因表达模式相似,免疫组化结果显示,StAR 蛋白在精细胞与间质细胞中均有表达。综上所述,长期低氧胁迫显著降低了StAR基因的转录表达水平,并且该基因可能在类固醇合成路径及精子发生过程中均发挥重要作用。本研究结果为进一步解析日本沼虾类固醇激素合成分子机制奠定基础。

    Abstract:

    Steroid acute regulatory protein (StAR) is an important enzyme in the early stage of steroid hormone synthesis. Recent studies have found that aquatic hypoxia can influence synthesis pathway of steroid hormones in fish as an endocrine disruptor, however, little information was observed in crustaceans. In order to investigate the roles of StAR in the oriental river prawn Macrobrachium nipponense under hypoxia, StAR gene has been cloned and characterized. A full-length cDNA sequence of StAR from M. nipponense was cloned by RACE (rapid amplification of cDNA ends) PCR. We examined the expression levels of StAR gene in different tissues by semi-quantitative RT-PCR, at the same time, the expression profile of StAR gene transcription level and protein expression abundance in testis of M. nipponense under hypoxia were also examined by quantitative real-time PCR (qRT-PCR) and Western blot, respectively. The results showed that the cDNA of StAR gene was 3 361 bp in length (NCBI: MW263908), including 323 bp 5'-untranslated regions (UTR), 2 129 bp 3'-UTR, 909 bp open reading frame (ORF), which encodes 302 amino acids. Based on bioinformatics analysis, phylogenetic tree analysis showed that the StAR gene of M. nipponense was closely related to the StAR gene of Portunus trituberculatus. The semi-quantitative RT-PCR results showed that StAR was highly expressed in testis tissues, but the lowest expressed in hepatopancreas and heart tissues. qRT-PCR results showed that the mRNA expression of StAR in the testis from hypoxia 1- 48 h was significantly higher than those in control group, but there was a significant lower mRNA expression of StAR in hypoxia 96 h group than those in control group. In addition, the recombinant plasmid was constructed and the StAR polyclonal antibody was obtained by immunizing rabbit. The expression abundance of StAR protein was detected by Western blot, which was basically similar to the gene expression pattern. In addition, immunohistochemical results showed that StAR protein was expressed in sperm cells and Leydig cell in testis tissue. Acute hypoxic stress significantly inhibited expression level of StAR, which may play an important role in spermatogenesis. The results of this study lay a foundation for further analysis of the molecular mechanism of steroid hormone synthesis in M. nipponense.

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郑诚,薛程,赵倩倩,孙盛明.日本沼虾StAR基因克隆及其低氧胁迫下表达分析[J].水产学报,2024,48(3):039106

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  • 收稿日期:2022-03-03
  • 最后修改日期:2022-05-16
  • 录用日期:2022-05-19
  • 在线发布日期: 2024-03-19
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