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鄢慧玲, 褚武英, 吴萍, 汪建华, 刘小燕, 张建社. 鳜肝脏和心脏生物钟基因表达节律分析[J]. 水生生物学报, 2016, 40(2): 243-251. DOI: 10.7541/2016.34
引用本文: 鄢慧玲, 褚武英, 吴萍, 汪建华, 刘小燕, 张建社. 鳜肝脏和心脏生物钟基因表达节律分析[J]. 水生生物学报, 2016, 40(2): 243-251. DOI: 10.7541/2016.34
YAN Hui-Ling, CHU Wu-Ying, WU Ping, WANG Jian-Hua, LIU Xiao-Yan, ZHANG Jian-She. CIRCADIAN RHYTHMICITY OF CLOCK GENES IN LIVER AND HEART OF MANDARIN FISH (SINIPERCA CHUATSI)[J]. ACTA HYDROBIOLOGICA SINICA, 2016, 40(2): 243-251. DOI: 10.7541/2016.34
Citation: YAN Hui-Ling, CHU Wu-Ying, WU Ping, WANG Jian-Hua, LIU Xiao-Yan, ZHANG Jian-She. CIRCADIAN RHYTHMICITY OF CLOCK GENES IN LIVER AND HEART OF MANDARIN FISH (SINIPERCA CHUATSI)[J]. ACTA HYDROBIOLOGICA SINICA, 2016, 40(2): 243-251. DOI: 10.7541/2016.34

鳜肝脏和心脏生物钟基因表达节律分析

CIRCADIAN RHYTHMICITY OF CLOCK GENES IN LIVER AND HEART OF MANDARIN FISH (SINIPERCA CHUATSI)

  • 摘要: 在12h光照、12h黑暗交替(Light-Dark; LD)光制下,研究分析了褪黑素和皮质醇水平在鳜血清中的昼夜变化规律,以及13个生物钟基因(Arntl1、Clock、Cry1a、Cry3、Cry-dash、Npas2、Npas4、Nr1d1、Nr1d2、Per1、Per3、Rora和Tim)在鳜(Siniperca chuatsi)肝脏和心脏中的昼夜表达规律。试验在一昼夜内的ZT0(06:00)、ZT3(09:00),ZT6(12:00),ZT9(15:00),ZT12(18:00),ZT15(21:00),ZT18(24:00),ZT21(03:00,2nd d),ZT24(06:00,2nd d) (Zone time,ZT) 9个时间点随机抽取3尾鳜采集其血清、肝脏和心脏。经SPSS 单因素方差分析和Matlab余弦分析,结果显示: 鳜血清中褪黑素和皮质醇含量均呈现出昼夜节律性振荡,褪黑素含量白天显著降低(P0.05),夜间显著上升,皮质醇含量白天缓慢降低,夜间ZT15(21:00)-ZT18(24:00)显著升高,随后开始缓慢降低; 两种激素最低相位都为ZT15(21:00)。在13个生物钟基因中,Cry-dash、Npas4、Nr1d1、Per1和Tim 5个基因在鳜肝脏内具有明显的昼夜节律性,其中Npas4、Nr1d1、Per1、Tim的表达规律相似,皆呈现出光照阶段表达降低,黑暗阶段表达升高的趋势; 但Cry-dash则表现出光照阶段先升高后降低,黑暗阶段先降低后升高的规律。在鳜心脏中,Arntl1、Clock、Cry1a、Npas2、Nr1d1、Nr1d2、Per3、Rora和Tim 9个基因都表现出明显的昼夜节律,表达趋势分为两种: Arntl1、Clock、Nr1d2的表达量在光照阶段降低,黑暗阶段升高; 而Cry1a、Npas2、Nr1d1、Per3、Rora和Tim的表达量在ZT0(06:00)-ZT15(21:00)持续低水平,ZT15(21:00)-ZT18(24:00)表达量显著上升,ZT18(24:00)-ZT21(03:00)表达量降低。研究结果表明: 生物钟基因在鳜肝脏和心脏中所表达的昼夜节律不同。

     

    Abstract: Clock genes are the molecular core of circadian rhythm of vertebrates. In the present study, we investigated the expression of clock genes which contained Arntl1, Clock, Cry1a, Cry3, Cry-dash, Npas2, Npas4, Nr1d1, Nr1d2, Per1, Per3, Rora and Tim in liver and heart of Mandarin fish (Siniperca chuatsi) under the 12h-light and 12h-dark (LD) cycle condition. Melatonin and cortisol levels were also investigated under the same condition. Livers, hearts and plasma of Mandarin fish were collected at ZT0(06:00), ZT3(09:00), ZT6(12:00), ZT9(15:00), ZT12(18:00), ZT15(21:00), ZT18(24:00), ZT21(03:00, 2nd day), ZT24(06:00, 2nd day) (Zone time, ZT) with three fishes at each time point. The total mRNA was extracted from each sample, the semi-quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to determine the temporal changes in mRNA levels. Blood plasma melatonin and cortisol were quantified by competitive ELISA. The data were analyzed both by the One-Way ANOVA (SPSS 17.0) and the cosine function (Matlab 7.0) to investigate the circadian rhythm of these genes. The result indicated that: Cry-dash, Npas4, Nr1d1, Per1 and Tim displayed daily rhythmic expression in the liver. Except for Cry-dash, the expression of the other genes decreased during the light phase and increased in the dark phase. In addition, there were two rhythmic characters of the 9 genes which contained Arntl1, Clock, Cry1a, Npas2, Nr1d1, Nr1d2, Per3, Rora and Tim in the heart of Mandarin fish: 3 genes including Arntl1, Clock and Nr1d2 were reduced at light but increased at dark phase and the mRNA levels of the others sustained low in ZT0 (06:00)-ZT15 (21:00), while increased significantly in ZT15 (21:00)-ZT18 (24:00) and then decreased in ZT18 (24:00)-ZT21 (03:00). Plasma melatonin levels showed a biphasic diurnal pattern, with higher concentrations during the dark phase than the light phase, its lowest level was observed at ZT15 (21:00). Changes in the serum cortisol levels exhibited circadian pattern over a 24h period that the peak detected at ZT18 (24:00) and the trough detected at ZT15 (21:00). In summary, most of the clock genes were expressed in a circadian manner in liver and heart of Mandarin fish (Siniperca chuatsi), with differences in phase and amplitude.

     

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