斑马鱼基因的表达模式以及青蛙启动子在斑马鱼视杆光感受器中的转录活性。

Expression patterns of zebrafish genes and the transcriptional activity of the frog promoter in zebrafish rod photoreceptors.

作者信息

Yang Xiaojun, Fu Jinling, Wei Xiangyun

机构信息

Neuroscience Center, Shantou University Medical College, Shantou, China.

Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, PA.

出版信息

Mol Vis. 2017 Dec 30;23:1039-1047. eCollection 2017.

PMID:29386877

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC5757853/

Abstract

PURPOSE

Daily modulation of gene expression is critical for the circadian rhythms of many organisms. One of the modulating mechanisms is based on nocturnin, a deadenylase that degrades mRNA in a circadian fashion. The genes are expressed broadly, but their tissue expression patterns differ between mice and the frog ; this difference suggests that the extent of the regulation of gene expression varies among species. In this study, we set out to characterize the expression patterns of two zebrafish genes; in addition, we asked whether a frog promoter has transcriptional activity in zebrafish.

METHODS

We used reverse transcription (RT)-PCR, quantitative real-time PCR (qRT-PCR), and rapid amplification of cDNA ends (RACE) analysis to determine whether the and genes are expressed in the eye, in situ hybridization to determine the cellular expression pattern of the gene in the retina, and confocal microscopy to determine the protein expression pattern of the transgenic reporter green fluorescent protein (GFP) driven by the frog promoter.

RESULTS

We found that the amino acid sequences of zebrafish and are highly similar to those of frog, mouse, and human nocturnin homologs. Only is expressed in the eye. Within the retina, mRNA was expressed at higher levels in the retinal photoreceptors layer than in other cellular layers. This expression pattern echoes the restricted photoreceptor expression of in the frog. We also found that the frog promoter can be specifically activated in zebrafish rod photoreceptors.

CONCLUSIONS

The high level of similarities in amino acid sequences of human, mouse, frog, and zebrafish nocturnin homologs suggest these proteins maintain a conserved deadenylation function that is important for regulating retinal circadian rhythmicity. The rod-specific transcriptional activity of the frog promoter makes it a useful tool to drive moderate and rod-specific transgenic expression in zebrafish. The results of this study lay the groundwork to study nocturnin-based circadian biology of the zebrafish retina.

摘要

目的

基因表达的日常调节对许多生物体的昼夜节律至关重要。其中一种调节机制基于夜蛋白，它是一种去腺苷酸化酶，以昼夜节律的方式降解信使核糖核酸（mRNA）。这些基因广泛表达，但它们在小鼠和青蛙中的组织表达模式有所不同；这种差异表明基因表达的调控程度在不同物种间存在差异。在本研究中，我们着手对两个斑马鱼夜蛋白基因的表达模式进行表征；此外，我们还探究了青蛙夜蛋白启动子在斑马鱼中是否具有转录活性。

方法

我们使用逆转录（RT）-聚合酶链反应（PCR）、定量实时PCR（qRT-PCR）以及cDNA末端快速扩增（RACE）分析来确定夜蛋白α和夜蛋白β基因是否在眼睛中表达，使用原位杂交来确定夜蛋白α基因在视网膜中的细胞表达模式，并使用共聚焦显微镜来确定由青蛙夜蛋白启动子驱动的转基因报告绿色荧光蛋白（GFP）的蛋白质表达模式。

结果

我们发现斑马鱼夜蛋白α和夜蛋白β的氨基酸序列与青蛙、小鼠和人类夜蛋白同源物的序列高度相似。只有夜蛋白α在眼睛中表达。在视网膜内，夜蛋白α信使核糖核酸在视网膜光感受器层中的表达水平高于其他细胞层。这种表达模式与青蛙中夜蛋白α在光感受器中的局限性表达相呼应。我们还发现青蛙夜蛋白启动子可在斑马鱼视杆光感受器中被特异性激活。