Dearworth James R, Selvarajah Brian P, Kalman Ross A, Lanzone Andrew J, Goch Abraham M, Boyd Alison B, Goldberg Laura A, Cooper Lori J
Department of Biology and Neuroscience Program, Lafayette College, 311 Kunkel Hall, Easton, PA 18042, USA.
Vision Res. 2011 Jan 28;51(2):288-95. doi: 10.1016/j.visres.2010.10.025. Epub 2010 Nov 2.
A mammalian-like melanopsin (Opn4m) has been found in all major vertebrate classes except reptile. Since the pupillary light reflex (PLR) of the fresh water turtle takes between 5 and 10 min to achieve maximum constriction, and since photosensitive retinal ganglion cells (ipRGCs) in mammals use Opn4m to control their slow sustained pupil responses, we hypothesized that a Opn4m homolog exists in the retina of the turtle. To identify its presence, retinal tissue was dissected from seven turtles, and total RNA extracted. Reverse transcriptase-polymerase chain reactions (RT-PCRs) were carried out to amplify gene sequences using primers targeting the highly conserved core region of Opn4m, and PCR products were analyzed by gel electrophoresis and sequenced. Sequences derived from a 1004-bp PCR product were compared to those stored in GenBank by the basic local alignment search tool (BLAST) algorithm and returned significant matches to several Opn4ms from other vertebrates including chicken. Quantitative real-time PCR (qPCR) was also carried out to compare expression levels of Opn4m in different tissues. The normalized expression level of Opn4m in the retina was higher in comparison to other tissue types: iris, liver, lung, and skeletal muscle. The results suggest that Opn4m exists in the retina of the turtle and provides a possible explanation for the presence of a slow PLR. The turtle is likely to be a useful model for further understanding the photoreceptive mechanisms in the retina which control the dynamics of the PLR.
除爬行动物外,在所有主要脊椎动物类别中均发现了一种类似哺乳动物的黑视蛋白(Opn4m)。由于淡水龟的瞳孔光反射(PLR)需要5到10分钟才能达到最大收缩程度,并且由于哺乳动物中的光敏感视网膜神经节细胞(ipRGCs)利用Opn4m来控制其缓慢持续的瞳孔反应,我们推测在龟的视网膜中存在Opn4m的同源物。为了确定其存在,从七只龟中解剖出视网膜组织,并提取总RNA。使用针对Opn4m高度保守核心区域的引物进行逆转录聚合酶链反应(RT-PCR)以扩增基因序列,通过凝胶电泳分析PCR产物并进行测序。通过基本局部比对搜索工具(BLAST)算法将来自1004 bp PCR产物的序列与GenBank中存储的序列进行比较,结果显示与包括鸡在内的其他脊椎动物的几种Opn4m有显著匹配。还进行了定量实时PCR(qPCR)以比较Opn4m在不同组织中的表达水平。与其他组织类型(虹膜、肝脏、肺和骨骼肌)相比,视网膜中Opn4m的标准化表达水平更高。结果表明Opn4m存在于龟的视网膜中,并为缓慢PLR的存在提供了一种可能的解释。龟可能是进一步了解视网膜中控制PLR动态的光感受机制的有用模型。