广西师范大学学报（自然科学版） ›› 2025, Vol. 43 ›› Issue (3): 201-212.doi: 10.16088/j.issn.1001-6600.2024041603

• 分子生物学与生物技术 • 上一篇    下一篇

棘胸蛙Clock基因克隆及其转录表达分析

袁鸿1,2, 汪小冬1,2,3, 魏秀英1,2, 王加品1,2, 陈以芳1,2, 姚红艳1,2, 陈敦学1,2*   

  1. 1.贵州大学动物科学学院,贵州贵阳 550025;
    2.贵州白云棘胸蛙科技小院(贵州大学), 贵州贵阳 510080;
    3.贵州农业职业学院畜牧水产系,贵州贵阳 551400
  • 收稿日期:2024-04-16 修回日期:2024-05-23 出版日期:2025-05-05 发布日期:2025-05-14
  • 通讯作者: 陈敦学(1986—),男,湖北公安人,贵州大学副教授,博士。E-mail:chendunxue@126.com
  • 基金资助:
    贵州省科技计划项目(黔科合支撑〔2021〕一般269号);贵州省农业科技支撑项目(黔科合支撑〔2019〕2344号)

Molecular Characterization of Circadian Clock Gene from Giant Spiny Frogs (Quasipaa spinosa) and Its Transcriptional Regulation During Different Developmental Stages

YUAN Hong1,2, WANG Xiaodong1,2,3, WEI Xiuying1,2, WANG Jiapin1,2, CHEN Yifang1,2, YAO Hongyan1,2, CHEN Dunxue1,2*   

  1. 1. College of Animal Science, Guizhou University, Guiyang Guizhou 550025, China;
    2. Guizhou Baiyun Quasipaa spinosa Science and Technology Small Hospital (Guizhou University), Guiyang Guizhou 510080, China;
    3. Department of Animal Husbandry and Fisheries, Guizhou Vocational Collegeof Agriculture, Guiyang Guizhou 551400, China)
  • Received:2024-04-16 Revised:2024-05-23 Online:2025-05-05 Published:2025-05-14

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摘要： 生物钟在生物体内出现昼夜周期性震荡,影响着生物的生长发育。棘胸蛙Quasipaa spinosa作为一种重要的两栖类动物,蝌蚪期主要在白天活动,而变态后则主要在夜间活动,出现相反的昼夜节律特征。目前关于棘胸蛙Clock基因的昼夜变化特征尚不清楚。因此,本研究克隆棘胸蛙的Clock基因,发现其蛋白序列含有1个HLH结构域、2个PAS结构域和1个PAC结构域,且这些结构域在不同物种中高度保守。进化分析表明,鱼类Clock基因可以分为2个不同的组:Clock A组和Clock B组,两栖类Clock基因不分亚型地聚在一起,其中棘胸蛙与高山倭蛙Clock基因紧密聚在一起。为了探索节律基因在棘胸蛙体内的转录特征,选择5个关键节律基因(Clock、Bmal1、Per2、Cry1和RoRα)研究它们不同发育阶段和不同组织中的表达谱。结果显示:Clock基因在所有组织和所有发育阶段中均存在本底表达,且在变态阶段的表达水平最高。节律表达谱显示Clock和Bmal1基因的表达较为一致,均在夜间观察到表达峰值,Per2和Cry1都在一天开始的时候出现表达高峰。但Per2和Cry1的表达模式具有组织特异性,Cry1基因在T4阶段的肌肉、脑、肝和心脏组织中呈现另一个短暂的表达峰,这可能与夜间褪黑素水平的增加或实验动物的行为模式有关。实验结果有助于深刻理解两栖类动物错综复杂的昼夜节律系统。

关键词: 棘胸蛙, Clock基因, 昼夜节律, 进化, 表达谱, 协同作用

Abstract: The circadian clock governs intrinsic daily oscillations in organisms. Nevertheless, the circadian fluctuations of the Clock constituents in Quasipaa spinosa have yet to be characterized. The frog primarily exhibits diurnal behavior during its tadpole stage, but undergoes a transition to nocturnal activity upon reaching the frogling stage. To comprehend the shift from diurnal to nocturnal behavior in the evolutionary progression, it is crucial to acquire profound understanding of the light-dark circadian system exhibited by amphibians. In current study, we conducted a comprehensive analysis of the circadian clock regulation of the Clock gene in this frog. The Qsclock protein features a pivotal HLH domain, two PAS domains, and a PAC domain that demonstrates heightened conservation across various species. Evolutionary analysis revealed that the Clock gene could be classified into two distinct groups: group A and group B. Five key daily rhythmicity genes (Clock, Bmal1, Per2, Cry1, and RoRα) were specifically chosen to comprehensively investigate the transcriptional regulation involved in promoting growth in frogs throughout different tissues and developmental stages. The tissue distribution analyses revealed the ubiquitous expression of the Clock gene across all tissues and all development stages, with the highest levels of expression observed during the metamorphosis stage in all examined tissues. The analysis of cooperative regulation of rhythm revealed a synchronization in Clock and Bmal1 genes, characterized by peak levels observed during nighttime. Furthermore, both Per2 and Cry1 exhibited an early peak expression at the onset of the day. However, the expression patterns of Per2 and Cry1 exhibited tissue specificity with Cry1 genes displayed another transient peak of expression in muscle, brain, liver, and heart tissues during the T4 stage. This could potentially be linked to the increased levels of melatonin during nighttime or the behavioral patterns observed in experimental animals. The findings provide valuable insights into the intricate light-dark circadian system of amphibians.

Key words: Quasipaa spinosa, Clock gene, circadian rhythm, evolutionary, expression profile, synchronization

中图分类号:  S917.4

[1] IMSLAND A K, HANGSTAD T A, JONASSEN T M, et al. The use of photoperiods to provide year round spawning in lumpfish Cyclopterus lumpus[J]. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 2019, 228: 62-70. DOI: 10.1016/j.cbpa.2018.11.004.
[2]TAHARA Y, KURODA H, SAITO K, et al. In vivo monitoring of peripheral circadian clocks in the mouse[J]. Current Biology, 2012, 22(11): 1029-1034. DOI: 10.1016/j.cub.2012.04.009.
[3]ZHANG E E, KAY S A. Clocks not winding down: unravelling circadian networks[J]. Nature Reviews Molecular Cell Biology, 2010, 11(11): 764-776. DOI: 10.1038/nrm2995.
[4]SÁNCHEZ-VÁZQUEZ F J, LÓPEZ-OLMEDA J F, VERA L M, et al. Environmental cycles, melatonin, and circadian control of stress response in fish[J]. Frontiers in Endocrinology, 2019, 10: 279. DOI: 10.3389/fendo.2019.00279.
[5]QIN C J, SHAO T. The clock gene clone and its circadian rhythms in Pelteobagrus vachelli[J]. Chinese Journal of Oceanology and Limnology, 2015, 33(3): 597-603. DOI: 10.1007/s00343-015-4167-x.
[6]BASS J, TAKAHASHI J S. Circadian integration of metabolism and energetics[J]. Science, 2010, 330(6009): 1349-1354. DOI: 10.1126/science.1195027.
[7]DE BUNDEL D, GANGAROSSA G, BIEVER A, et al. Cognitive dysfunction, elevated anxiety, and reduced cocaine response in circadian clock-deficient cryptochrome knockout mice[J]. Frontiers in Behavioral Neuroscience, 2013, 7: 152. DOI: 10.3389/fnbeh.2013.00152.
[8]ZHANG R, LAHENS N F, BALLANCE H I, et al. A circadian gene expression Atlas in mammals: implications for biology and medicine[J]. Proceedings of the National Academy of Sciences of the United States of America, 2014, 111(45): 16219-16224. DOI: 10.1073/pnas.1408886111.
[9]XU H Y, SHI C, YE Y F, et al. Photoperiod-independent diurnal feeding improved the growth and feed utilization of juvenile rainbow trout (Oncorhynchus mykiss) by inducing food anticipatory activity[J]. Frontiers in Marine Science, 2022, 9: 1029483. DOI: 10.3389/fmars.2022.1029483.
[10]GILANNEJAD N, MOYANO F J, MARTÍNEZ-RODRÍGUEZ G, et al. Feeding protocol modulates the digestive process in Senegalese sole (Solea senegalensis) juveniles[J]. Frontiers in Marine Science, 2021, 8: 698403. DOI: 10.3389/fmars.2021.698403.
[11]BOLTON C M, BEKAERT M, EILERTSEN M, et al. Rhythmic clock gene expression in Atlantic salmon parr brain[J]. Frontiers in Physiology, 2021, 12: 761109. DOI: 10.3389/fphys.2021.761109.
[12]BETANCOR M B, SPRAGUE M, ORTEGA A, et al. Central and peripheral clocks in Atlantic bluefin tuna (Thunnus thynnus L.): daily rhythmicity of hepatic lipid metabolism and digestive genes[J]. Aquaculture, 2020, 523: 735220. DOI: 10.1016/j.aquaculture.2020.735220.
[13]ALMAIDA-PAGÁN P F, ORTEGA-SABATER C, LUCAS-SÁNCHEZ A, et al. Impact of a shift work-like lighting schedule on the functioning of the circadian system in the short-lived fish Nothobranchius furzeri[J]. Experimental Gerontology, 2018, 112: 44-53. DOI: 10.1016/j.exger.2018.08.010.
[14]WANG X D, XIE Y G, HU W, et al. Transcriptome characterization and SSR discovery in the giant spiny frog Quasipaa spinosa[J]. Gene, 2022, 842: 146793. DOI: 10.1016/j.gene.2022.146793.
[15]MARTÍN-ROBLES Á J, WHITMORE D, SÁNCHEZ-VÁZQUEZ F J, et al. Cloning, tissue expression pattern and daily rhythms of Period1, Period2, and Clock transcripts in the flatfish Senegalese sole,Solea senegalensis[J]. Journal of Comparative Physiology B, 2012, 182(5): 673-685. DOI: 10.1007/s00360-012-0653-z.
[16]CAVALLARI N, FRIGATO E, VALLONE D, et al. A blind circadian clock in cavefish reveals that opsins mediate peripheral clock photoreception[J]. PLoS Biology, 2011, 9(9): e1001142. DOI: 10.1371/journal.pbio.1001142.
[17]SANCHEZ R E A, KALUME F, DE LA IGLESIA H O. Sleep timing and the circadian clock in mammals: past, present and the road ahead[J]. Seminars in Cell & Developmental Biology, 2022, 126: 3-14. DOI: 10.1016/j.semcdb.2021.05.034.
[18]ZHU H S, LARUE S, WHITELEY A, et al. The Xenopus Clock gene is constitutively expressed in retinal photoreceptors[J]. Molecular Brain Research, 2000, 75(2): 303-308. DOI: 10.1016/S0169-328X(99)00309-5.
[19]COWAN M, AZPELETA C, LÓPEZ-OLMEDA J F. Rhythms in the endocrine system of fish: a review[J]. Journal of Comparative Physiology B, 2017, 187(8): 1057-1089. DOI: 10.1007/s00360-017-1094-5.
[20]KLEIN S L, STRAUSBERG R L, WAGNER L, et al. Genetic and genomic tools for Xenopus research: The NIH Xenopus initiative[J]. Developmental Dynamics, 2002, 225(4): 384-391. DOI: 10.1002/dvdy.10174.
[21]ARISTAKESYAN E A, KARMANOVA I G. Effect of photostimulation on the wakefulness-sleep cycle in the common frog Rana temporaria[J]. Journal of Evolutionary Biochemistry and Physiology, 2007, 43(2): 208-214. DOI: 10.1134/S0022093007020093.
[22]CEINOS R M, CHIVITE M, LÓPEZ-PATIÑO M A, et al. Differential circadian and light-driven rhythmicity of clock gene expression and behaviour in the turbot,Scophthalmus maximus[J]. PLoS One, 2019, 14(7): e0219153. DOI: 10.1371/journal.pone.0219153.
[23]QIN C J, SUN J X, JUN W, et al. Discovery of differentially expressed genes in the intestines of Pelteobagrus vachellii within a light/dark cycle[J]. Chronobiology International, 2020, 37(3): 339-352. DOI: 10.1080/07420528.2019.1690498.
[24]SANCHEZ S E, PETRILLO E, BECKWITH E J, et al. A methyl transferase links the circadian clock to the regulation of alternative splicing[J]. Nature, 2010, 468(7320): 112-116. DOI: 10.1038/nature09470.
[25]CHEN S H, QIAO H, FU H T, et al. Molecular cloning, characterization, and temporal expression of the clock genes period and timeless in the oriental river prawn Macrobrachium nipponense during female reproductive development[J]. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 2017, 207: 43-51. DOI: 10.1016/j.cbpa.2017.02.011.
[26]VELARDE E, HAQUE R, IUVONE P M, et al. Circadian clock genes of goldfish,Carassius auratus: cDNA cloning and rhythmic expression of period and cryptochrome transcripts in retina, liver, and gut[J]. Journal of Biological Rhythms, 2009, 24(2): 104-113. DOI: 10.1177/0748730408329901.
[27]KRYLOV V V, IZVEKOV E I, PAVLOVA V V, et al. Circadian rhythms in zebrafish (Danio rerio) behaviour and the sources of their variability[J]. Biological Reviews, 2021, 96(3): 785-797. DOI: 10.1111/brv.12678.
[28]NISHIDE S Y, HASHIMOTO K, NISHIO T, et al. Organ-specific development characterizes circadian clock gene Per2 expression in rats[J]. American Journal of Physiology. Regulatory, Integrative and Comparative Physiology, 2014, 306(1): R67-R74. DOI: 10.1152/ajpregu.00063.2013.
[29]凌洁彬.中华大蟾蜍(Bufo bufo gargarigans)繁殖峰期和末期生物钟基因表达和激素水平的节律性改变[D].金华:浙江师范大学,2017.
[30]MAZZOCCOLI G, PAZIENZA V, VINCIGUERRA M. Clock genes and clock-controlled genes in the regulation of metabolic rhythms[J]. Chronobiology International, 2012, 29(3): 227-251. DOI: 10.3109/07420528.2012.658127.
[31]ALLADA R, WHITE N E, SO W V, et al. A mutant Drosophila homolog of mammalian Clock disrupts circadian rhythms and transcription of period and timeless[J]. Cell, 1998, 93(5): 791-804. DOI: 10.1016/s0092-8674(00)81440-3.
[32]ARYAL R P, KWAK P B, TAMAYO A G, et al. Macromolecular assemblies of the mammalian circadian clock[J]. Molecular Cell, 2017, 67(5): 770-782.e6. DOI: 10.1016/j.molcel.2017.07.017.
[33]肖锋.中华大蟾蜍(Bufo bufo gargarigans)生物钟基因表达及外周激素水平的昼夜节律研究[D].金华:浙江师范大学,2014. DOI: 10.7666/d.Y2665062.
[34]HERRERO M J, LEPESANT J M J. Daily and seasonal expression of clock genes in the pituitary of the European sea bass (Dicentrarchus labrax)[J]. General and Comparative Endocrinology, 2014, 208: 30-38. DOI: 10.1016/j.ygcen.2014.08.002.
[35]MAZUR M, MARKOWSKA M, CHADZINSKA M, et al. Changes of the clock gene expression in central and peripheral organs of common carp exposed to constant lighting conditions[J]. Chronobiology International, 2023, 40(2): 145-161. DOI: 10.1080/07420528.2022.2157734.
[36]VERA L M, NEGRINI P, ZAGATTI C, et al. Light and feeding entrainment of the molecular circadian clock in a marine teleost (Sparus aurata)[J]. Chronobiology International, 2013, 30(5): 649-661. DOI: 10.3109/07420528.2013.775143.
[37]THRAYA M, HAMMOUD M, HEATH D, et al. Testing the expression of circadian clock genes in the tissues of Chinook salmon,Oncorhynchus tshawytscha[J]. Chronobiology International, 2019, 36(8): 1088-1102. DOI: 10.1080/07420528.2019.1614019.
[38]BO Y, ZHANG W X, LU J, et al. Rhythmic expressions of biological clocks and metabolic genes in marine medaka (Oryzias melastigma)[J]. Aquaculture Research, 2022, 53(9): 3541-3552. DOI: 10.1111/are.15860.
[39]WANG S H, ZHANG T X, HUANG H P, et al. Cloning, tissue distribution, mRNA expression and functional analysis of circadian clock gene per2 from the high-latitude Amur minnow (Phoxinus lagowskii)[J]. Aquaculture International, 2024, 32(3): 2401-2425. DOI: 10.1007/s10499-023-01277-3.
[40]WITT-ENDERBY P A, SLATER J P, JOHNSON N A, et al. Effects on bone by the light/dark cycle and chronic treatment with melatonin and/or hormone replacement therapy in intact female mice[J]. Journal of Pineal Research, 2012, 53(4): 374-384. DOI: 10.1111/j.1600-079X.2012.01007.x.
[41]WEST A C, IVERSEN M, JØRGENSEN E H, et al. Diversified regulation of circadian clock gene expression following whole genome duplication[J]. PLoS Genetics, 2020, 16(10): e1009097. DOI: 10.1371/journal.pgen.1009097.
[42]MISHRA I, KUMARV. Circadian basis of seasonal timing in higher vertebrates[J]. Biological Rhythm Research, 2017, 48(5): 723-738. DOI: 10.1080/09291016.2017.1345447.
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