生物学杂志

生物学杂志

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蚤状溞caspase-3基因在不同环境条件下的表达分析

  

  1. 1. 宁波大学 海洋学院， 宁波 315211； 2. 宁波大学 应用海洋生物技术教育部重点实验室， 宁波 315211
  • 出版日期:2019-02-18 发布日期:2019-02-18
  • 通讯作者: 王丹丽,教授, 研究方向为水生动物学研究，E-mail:wangdanli@nbu.edu.cn
  • 作者简介:吴乐乐,研究方向为生物技术,E-mail:550576364@qq.com
  • 基金资助:
    国家自然基金项目(31572221)

Expression analysis of caspase-3 gene in Daphnia pulex under different environmental conditions

  1. 1. School of Marine Sciences, Ningbo University, Ningbo 315211; 2. Key Laboratory of Applied Marine Biotechnology, Ministry of Education, Ningbo University, Ningbo 315211, China
  • Online:2019-02-18 Published:2019-02-18

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摘要： 以蚤状溞(Daphnia pulex)为研究对象，采用Real-Time PCR技术分析了蚤状溞caspase-3 mRNA表达水平随着不同培养温度、种群密度和食物种类变化；用Caspase-3的特异性底物Ac-DEVD-AMC测定了Caspase-3酶活在不同培养温度、种群密度和食物种类条件下的变化，并通过整体原位杂交检测了caspase-3基因在蚤状溞不同生殖状态下的定位表达。研究结果：蚤状溞caspase-3 mRNA表达量随着温度和种群密度的增加而增加；以Banta培养液培养的蚤状溞caspase-3 mRNA表达量与小球藻培养的表达量无显著差异（P>0.05）；且caspase-3 mRNA的表达量与Caspase-3酶活的变化规律相一致；整体原位杂交结果显示，caspase-3基因在蚤状溞活体细胞几乎都有表达，在两性溞中表达量较高，孤雌成溞中表达量较低。caspase-3基因的表达与环境有关，而蚤状溞的衰老和caspase-3 mRNA的表达及Caspase-3的酶活有关，但环境因子如何影响caspase-3基因的表达进而调控枝角类生殖转换还需今后进一步的研究。

关键词: 蚤状溞, caspase-3基因, mRNA表达, 酶活, 原位杂交

Abstract: Using Daphnia pulex as the researching object, qPCR was used to analyze the changes of expression level of caspase-3 mRNA in different culture temperatures, population densities and food types; then the enzyme activities of Caspase-3 under three different culture conditions were measured using the specific substrate Ac-DEVD-AMC; in situ hybridization was used to detect the localizations and changes of caspase-3 mRNA in different reproductive states of D. pulex. The main results were as follows: expression levels of caspase-3 mRNA were up-regulated with the increase of temperature and population density, but there was no obvious changes of caspase-3 mRNA expression in the food type group. In addition, qPCR results were in good agreement with the result of enzyme activity assays. Whole-mount in situ hybridization showed that caspase-3 was expressed in living cells of D. pulex at different growth stages and was highly expressed in adult individuals, which was essentially consistent with the result of qPCR. The expression of caspase-3 gene was relevant to changes of environmental factors, while aging of D. pulex was related to the expression of caspase-3 mRNA and the enzyme activity of Caspase-3. Thus, further research is needed to explore how environmental factors affecting the expression of caspase-3 mRNA, which subsequently regulates cyclical parthenogenesis in Daphnia.

Key words: Daphnia pulex, caspase-3, mRNA expression, enzyme activity, in situ hybridization

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