Molecular cloning of Clock cDNA from the prawn, Macrobrachium rosenbergii
Molecular cloning of Clock cDNA from the prawn, Macrobrachium rosenbergii
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DOI：
10.1016/j.brainres.2005.10.003
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发表时间：
2006-01
期刊：
影响因子：
2.9
通讯作者：
Jin-Shu Yang;Zhong-Min Dai;Fan Yang;Wei-Jun Yang
中科院分区：
文献类型：
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作者：
Jin-Shu Yang;Zhong-Min Dai;Fan Yang;Wei-Jun Yang
Clock属于碱性螺旋-环-螺旋(BHLH)/Per-Arnt-SIM(PAS)超家族转录因子，是动物昼夜节律系统中最重要的蛋白质之一。时钟基因已经从几个物种中克隆出来，包括哺乳动物、昆虫、鸟类、鱼类和两栖动物。在本研究中，我们成功地从罗氏沼虾中分离到一个时钟同源基因(称为Mar-Clock)。该基因全长2949个碱基，包含一个2115个碱基的开放阅读框，编码704个氨基酸的时钟蛋白(称为MAR-Clock)，与其他物种的Clock同源物有很高的同源性(30%-35%)。这是甲壳类生物钟基因的首次报道。Mar-Clock在其C末端有一个非常长的富含谷氨酰胺的结构域(140个氨基酸)，在其他已知的时钟中，该结构域通常在14到57个氨基酸之间，并被认为具有转录激活功能。利用RT-PCR，我们观察到Mar-Clock在所有受试组织中都有表达。用半定量RT-PCR方法研究了光-暗周期中的基因表达谱。结果表明，Mar-Clock基因在中枢神经组织(胸神经节和眼柄)和外周组织(鳃、卵巢、肝胰腺和肌肉)中的表达没有明显的节律性。此外，在去眼柄或恒定暗(DD)的对虾的中枢神经系统(脑、胸和腹神经节)以及去眼柄的鳃中，基因表达有增加的趋势。在恒定光照(LL)和心脏、肌肉中均未见表达变化。
CLOCK, which belongs to the basic helix–loop–helix (bHLH)/PER-ARNT-SIM (PAS) superfamily of transcription factors, is one of the most essential proteins involved in circadian systems of animals. Clock genes have been cloned from several species, including mammals, insects, birds, fish, and amphibians. In the present study, we successfully isolated a Clock homolog (termed Mar-Clock) from the giant prawn, Macrobrachium rosenbergii. The 2949-bp cDNA contained a 2115 bp open reading frame that encoded a putative CLOCK protein of 704 amino acids (termed Mar-CLOCK) exhibiting high identities with CLOCK homologs in other species (30–35%). This is the first report of a circadian clock gene from crustaceans. Mar-CLOCK possessed an exceptionally long glutamine-rich domain (140 amino acids) in its C-terminus, which usually ranges from 14 to 57 amino acids in other known CLOCKs and is supposed to function in transcriptional activation. Using RT-PCR, we observed that Mar-Clock was expressed in all tested tissues. Semiquantitative RT-PCR was performed to investigate the gene expression profile during the light–dark cycle. The results indicated that the expression of the Mar-Clock gene had no significant rhythmicity in central nervous tissues (thoracic ganglia and eyestalk) or peripheral tissues (gill, ovary, hepatopancreas, and muscle). Furthermore, gene expression tended to increase in the central nervous system (brain, thoracic, and abdominal ganglia) of eyestalk-ablated or constant dark (DD) prawns, and in the eyestalk-ablated gill. No expression change was found under constant light (LL) or in heart and muscle.