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。
Using Daphnia pulex as the researching object,q PCR 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,q PCR 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 q PCR. 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-3mRNA,which subsequently regulates cyclical parthenogenesis in Daphnia.
引文
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