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)

作　　者：鄢慧玲 褚武英[2] 吴萍[2,3] 汪建华[2,4] 刘小燕[1] 张建社[2] 

机构地区：[1]湖南农业大学动物科学技术学院,长沙410128 [2]长沙学院生物与环境工程系,长沙410003 [3]湖南大学生物学院,长沙410082 [4]广西师范大学生命科学学院,桂林541004

出　　处：《水生生物学报》2016年第2期243-251,共9页Acta Hydrobiologica Sinica

基　　金：国家自然科学基金项目(31472256;31230076);湖南省自然科学基金项目(14JJ2135)资助~~

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),ZTl5(21:00),ZT18(24:00),ZT21(03:00,2^(nd)d),ZT24(06:00,2na 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(P≤0.05),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。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, Cry 1 a, Cry3, Cry-dash, Npas2, Npas4, Nrld1, Nrld2, 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, 2no 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, Nrldl, Perl 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 Arntll, Clock, Cryla, Npas2, Nrldl, Nrld2, Per3, Rora and Tim in the heart of Mandarin fish： 3 genes including Arntll, Clock and Nrld2 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 o

关 键 词：鳜 昼夜节律 生物钟基因 褪黑素 皮质醇 

分 类 号：Q344.1[生物学—遗传学]