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Expression and functional analysis of the Akt gene from Daphnia pulex.
Comparative Biochemistry and Physiology B: Biochemistry & Molecular Biology ( IF 1.9 ) Pub Date : 2020-06-13 , DOI: 10.1016/j.cbpb.2020.110462
Xuanyu Zhou 1 , Shun Cheng 2 , Chunpu Zhao 1 , Chongyuan Lin 1 , Danli Wang 1 , Yunlong Zhao 3
Affiliation  

Daphnia pulex is a nutrient-rich freshwater crustacean with two different reproduction methods. Akt is a serine/threonine protein kinase that plays an important role in cell growth, survival, and lifespan regulation. To explore the function of Akt in the growth and aging process of Daphnia pulex, we cloned the cDNA sequence of the open reading frame (ORF) of the akt gene based on the bioinformatic analysis of the transcriptome data of D. pulex, and analyzed the structural features of the Akt protein. Gene silencing was performed using RNA interference (RNAi), and the expression of the Akt gene and protein before and after interference were analyzed using qPCR and western blotting. The results showed that the expression of akt in D. pulex at different ages showed a “W” pattern, being significantly higher at 20 days than at 10 days and 15 days (P < .05). The expression trend of Akt protein and mRNA were similar, with lower expression at a younger age (1–5 day), after which expression gradually increased from 10 days age, and showed no significant change after 25 days, which might be caused by a lag of protein translation. RNAi reduced the expression of the Akt gene and protein by at least 76%, and the survival rate and reproductive capacity of D. pulex were significantly lower in the RNAi group compared with those in the control group. This study provides a better understanding of the function of the akt gene in D. pulex.



中文翻译:

水蚤(Daphnia pulex)Akt基因的表达和功能分析。

水蚤(Daphnia pulex)是一种营养丰富的淡水甲壳动物,具有两种不同的繁殖方法。Akt是一种丝氨酸/苏氨酸蛋白激酶,在细胞生长,存活和寿命调节中起重要作用。为了探索Akt在水蚤的生长和衰老过程中的功能,我们基于对D. pulex转录组数据的生物信息学分析,克隆了akt基因开放阅读框(ORF)的cDNA序列,并对其进行了分析。 Akt蛋白的结构特征。使用RNA干扰(RNAi)进行基因沉默,并使用qPCR和Western blotting分析干扰前后Akt基因和蛋白质的表达。结果表明akt的表达D.状溞不同年龄呈“W”的模式,在比第10天20天及15天(即显著更高P <  0.05)。Akt蛋白和mRNA的表达趋势相似,在较低的年龄(1-5天)表达较低,此后从10天开始逐渐增加,而在25天后无明显变化,这可能是由Akt引起的。蛋白质翻译滞后。RNAi使Akt基因和蛋白质的表达降低了至少76%,并且与对照组相比,RNAi组中的D. pulex的存活率和生殖能力显着降低。这项研究更好地了解了D. pulex中akt基因的功能

更新日期:2020-06-26
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