Cloning and Expression Response of PcCyc Gene to Photoperiod in Red Swamp Crayfish Procambarus clarkii
XIE Wei1, JIANG Qichen2, SUN Bin1, YANG Ying1, LI Peng1, YAN Jie1, ZHOU Kaiya1
1.Jiangsu Key Laboratory for Biodiversity & Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing 210023, China; 2.Jiangsu Province Fisheries Research Institute, Nanjing 210017, China
Abstract：In this study, a partial cDNA sequence of cycle gene (PcCyc), one of the core genes of the circadian clock, was cloned from red swamp crayfish Procambarus clarkii with body length of 9—11 cm by rapid amplification of cDNA ends (RACE) technology, and then the mRNA expression levels of PcCyc gene were detected in brain, eye stalk and hepatopancreas of the red swamp crayfish exposed to various photoperiods of 12L∶12D, 0L∶24D, and 0L∶72D under full-spectrum fluorescent lamps at 7:00, 11:00, 15:00, 19:00, 23:00, 03:00 and 7:00 the next day by real-time quantitative PCR. The results showed that the mRNA of PcCyc gene had a complete open reading frame (ORF) of 2010 base pairs encoding 669 amino acids, and the amino acid sequence of PcCYC had the maximal similarity (94%) to that of the red claw crayfish Cherax quadricarinatus, with the protein molecular weight of about 73.963 ku, the theoretical isoelectric point of 6.56, which is a hydrophobic non-secreted protein with no signal peptide. The structural domain analysis revealed that there were a BHLH/PAS domain, including one HLH domain and two PAS domains in the PcCYC protein, with secondary structure of 11 alpha-helices, 12 beta-sheets, and 24 random coils. The PcCyc gene was shown to be 24-hour rhythm fluctuation in both eyestalk and hepatopancreas, no matter what photoperiod was. However, the rhythmic fluctuation was broken in the brain as the photoperiod changed. The findings provide the foundation for further exploration of the role and mechanism of the Cyc gene in circadian clock of the red swamp crayfish.
[1]Woelfle M A, Yan O Y, Phanvijhitsiri K, et al. The adaptive value of circadian clocks: an experimental assessment in cyanobacteria[J]. Current Biology,2004,14(16):1481-1486. [2]Silver A C, Arjona A, Walker W E, et al. The circadian clock controls toll-like receptor 9-mediated innate and adaptive immunity[J]. Immunity,2012,36(2):251-261. [3]Gekakis N, Staknis D, Nguyen H B, et al. Role of the CLOCK protein in the mammalian circadian mechanism[J]. Science,1998,280(5369):1564-1569. [4]Hermann-Luibl C, Helfrich-Frster C. Clock network in Drosophila[J]. Current Opinion in Insect Science,2015,7:65-70. [5]Sangoram A M, Saez L, Antoch M P, et al. Mammalian circadian autoregulatory loop:a timeless ortholog and mPer1 interact and negatively regulate CLOCK-BMAL1-induced transcription[J]. Neuron,1998,21(5):1101-1113. [6]Wang G L, Jiang B H, Rue E A, et al. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension[J]. Proceedings of the National Academy of Sciences of the United States of America,1995,92(12):5510-5514. [7]Ikeda M, Nomura M. cDNA cloning and tissue-specific expression of a novel basic helix-loop-helix/PAS protein (BMAL1) and identification of alternatively spliced variants with alternative translation initiation site usage[J]. Biochemical and Biophysical Research Communications,1997,233(1):258-264. [8]Zhang L, Hastings M H, Green E W, et al. Dissociation of circadian and circatidal timekeeping in the marine crustacean Eurydice pulchra[J]. Current Biology,2013,23(19):1863-1873. [9]Ikeno T, Numata H, Goto S G. Molecular characterization of the circadian clock genes in the bean bug, Riptortus pedestris, and their expression patterns under long-and short-day conditions[J]. Gene,2008,419(1/2):56-61. [10]Wu D L, Potluri N, Lu J P, et al. Structural integration in hypoxia-inducible factors[J]. Nature,2015,524(7565):303-308. [11]Sadacca L A, Lamia K A, DeLemos A S, et al. An intrinsic circadian clock of the pancreas is required for normal insulin release and glucose homeostasis in mice[J]. Diabetologia,2011,54(1):120-124. [12]Nakahata Y, Kaluzova M, Grimaldi B, et al. The NAD+-dependent deacetylase SIRT1 modulates CLOCK-mediated chromatin remodeling and circadian control[J]. Cell,2008,134(2):329-340. [13]戈贤平. 我国淡水虾蟹养殖业的现状及发展方向[G]//可持续水产养殖——资源、环境、质量——2003水产科技论坛论文集. 北京:中国水产科学研究院,2003:81-89. [14]Foley L E, Gegear R J, Reppert S M. Human cryptochrome exhibits light-dependent magnetosensitivity[J]. Nature Communications,2011,2:356. [15]Marcheva B, Ramsey K M, Buhr E D, et al. Disruption of the clock components CLOCK and BMAL1 leads to hypoinsulinaemia and diabetes[J]. Nature,2010,466(7306):627-631. [16]倪银华,吴涛,金黎,等. 哺乳动物营养代谢的时间生物学研究进展[J]. 中国细胞生物学学报,2011,33(2):190-196. [17]Strauss J, Dircksen H. Circadian clocks in crustaceans:identified neuronal and cellular systems[J]. Frontiers in Bioscience,2010,15(1):1040-1074. [18]Nelson-Mora J, Prieto-Sagredo J, Loredo-Ranjel R, et al. Putative pacemakers in the eyestalk and brain of the crayfish Procambarus clarkii show circadian oscillations in levels of mRNA for crustacean hyperglycemic hormone[J]. PLoS One,2013,8(12):e83937. [19]Fanjul-Moles M L, Prieto-Sagredo J. The circadian system of crayfish:a developmental approach[J]. Microscopy Research and Technique,2003,60(3):291-301. [20]Grabek K R, Chabot C C. Daily rhythms of PERIOD protein in the eyestalk of the American lobster, Homarus americanus[J]. Marine and Freshwater Behaviour and Physiology,2012,45(4):269-279. [21]Manfrin C, Tom M, De Moro G, et al. The eyestalk transcriptome of red swamp crayfish Procambarus clarkii[J]. Gene,2015,557(1):28-34. [22]Shen H S, Hu Y C, Ma Y C, et al. In-depth transcriptome analysis of the red swamp crayfish Procambarus clarkii[J]. PLoS One,2014,9(10):e110548. [23]Dunlap J C. Molecular bases for circadian clocks[J]. Cell,1999,96(2):271-290. [24]O′Grady J F, Hoelters L S, Swain M T, et al. Identification and temporal expression of putative circadian clock transcripts in the amphipod crustacean Talitrus saltator[J]. Peer J,2016,4:e2555. [25]Biscontin A, Wallach T, Sales G, et al. Functional characterization of the circadian clock in the Antarctic krill,Euphausia superba[J]. Scientific Reports,2017,7(1):17742. [26]Rudic R D, Mcnamara P, Curtis A M, et al. BMAL1 and CLOCK,two essential components of the circadian clock,are involved in glucose homeostasis[J]. PLoS Biology,2004,2(11):e377. [27]Ripperger J A, Schibler U. Rhythmic CLOCK-BMAL1 binding to multiple E-box motifs drives circadian Dbp transcription and chromatin transitions[J]. Nature Genetics,2006,38(3):369-374. [28]Shirai H, Oishi K, Ishida N. Bidirectional CLOCK/BMAL1-dependent circadian gene regulation by retinoic acid in vitro[J]. Biochemical and Biophysical Research Communications,2006,351(2):387-391. [29]Miyakawa H, Toyota K, Hirakawa I, et al. A mutation in the receptor Methoprene-tolerant alters juvenile hormone response in insects and crustaceans[J]. Nature Communications,2013,4:1856. [30]Soüanez-Organis J G, Racotta I S, Yepiz-Plascencia G. Silencing of the hypoxia inducible factor 1-HIF-1-obliterates the effects of hypoxia on glucose and lactate concentrations in a tissue-specific manner in the shrimp Litopenaeus vannamei[J]. Journal of Experimental Marine Biology and Ecology,2010,393(1/2):51-58. [31]Escamilla-Chimal E G, Velázquez-Amado R M, Fior-delisio T, et al. Putative pacemakers of crayfish show clock proteins interlocked with circadian oscillations[J]. The Journal of Experimental Biology,2010,213(Pt 21):3723-3733. [32]Kloss B, Price J L, Saez L, et al. The Drosophila clock gene double-time encodes a protein closely related to human casein kinase Iepsilon[J].Cell,1998,94(1):97-107. [33]Kloss B, Rothenfluh A, Young M W, et al. Phosphorylation of PERIOD is influenced by cycling physical associations of DOUBLE-TIME PERIOD, and TIMELESS in the Drosophila clock[J]. Neuron,2001,30(3):699-706. [34]Price J L, Blau J, Rothenfluh A, et al. Double-time is a novel Drosophila clock gene that regulates PERIOD protein accumulation[J]. Cell,1998,94(1):83-95. [35]温敏. 海参皂苷对小鼠生物钟及脂质代谢基因节律的影响[D]. 青岛:中国海洋大学,2014.
2021, Vol. 40 
