番红砗磲生物钟基因Cryptochrome和Period克隆鉴定及光照相关性分析
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国家自然科学基金(42266003)和国家重点研发计划(2022YFD2401301; 2022YFD2401305)共同资助


Cloning, identification, and light response analysis of two circadian clock genes Cryptochrome and Period in boring giant clam Tridacna crocea
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    摘要:

    生物钟是生物体适应地球周期性昼夜变化而演化出体内自持的计时机制,Cryptochrome和Period是生物钟的2个核心基因,其通过形成PER/CRY异二聚体调控生物体节律。本研究应用分子生物学和生物信息学技术对番红砗磲(Tridacna crocea)生物钟基因Cryptochrome和Period的编码区进行了克隆和特征分析,继而开展了其组织特异性和不同光照周期下的表达节律性研究。结果显示,Cryptochrome编码区碱基序列长1 641 bp,编码546个氨基酸,理论等电点为6.08,分子量为62.98 kDa;Period编码区碱基序列长4 386 bp,编码1 461个氨基酸,理论等电点为6.14,分子量为164.99 kDa;Hdock互作模式研究结果显示,2个蛋白可形成异二聚体,结合能为–279.88 kcal/mol。组织表达分析显示,Cryptochrome和Period在7个组织中均有表达,其中,外外套膜、内外套膜、鳃和闭壳肌相对表达量较高(Cryptochrome在心脏中亦有较高表达量)。Cryptochrome和Period基因在外外套膜、内外套膜、鳃和闭壳肌中均昼夜变化,并呈现振荡表达的规律。在内、外外套膜中,Cryptochrome和Period基因分别在光照后1 h和光照前2 h表达量达到最高,且均随光照周期的推移(光照时间07:00~19:00推迟至09:00~21:00)而推移;在鳃组织中,正常光周期处理的Cryptochrome和Period基因均在光照前1 h表达量达到最高,当光照延迟2 h后,2个基因的最高表达时间推至光照后1 h;与之相反,闭壳肌中Cryptochrome和Period基因在08:00左右表达量最高,且未随光照延迟而发生变化。综上,本研究首次克隆获得番红砗磲2个关键生物钟基因Cryptochrome和Period,初步验证了这2个基因在主要组织表达的昼夜节律性,结果对揭示番红砗磲行为、生理节律和光照适应机制具有重要意义。

    Abstract:

    The circadian clock is an intrinsic timing mechanism that has evolved in organisms to adapt to the Earth’s periodic diurnal changes. Cryptochrome and Period genes are pivotal in regulating the circadian system. These form PER/CRY heterodimer complexes, which translocate from the cytoplasm to the nucleus and inhibit the transcription of CLOCK/BMAL1-driven E-box elements, thereby functioning as downstream effector genes. These two genes are also influenced by environmental cues, such as external light, which can remodel the periodic rhythm and ultimately facilitate adaptation to environmental rhythmic changes. The boring giant clam Tridacna crocea is a dominant species within the Tridacnidae family, with notable economic and ecological importance. However, this species is currently classified as a rare and protected animal owing to environmental changes, overfishing and other factors. A defining characteristic of this species is its symbiotic relationship with zooxanthellae, in which light plays a fundamental role in the giant clam-zooxanthellae symbiosis. Additionally, light is a crucial regulatory factor influencing the circadian clock. Consequently, exploring the influence of circadian rhythms on the expression of core clock genes in T. crocea can provide crucial data to support conservation efforts and breeding programs for this species. In this study, the SeqMan software was used to assemble sequences obtained through sequencing. The software SignalP 5.0 Server and SMART 4.0 were used to conduct online analysis and prediction of giant clam Cryptochrome and Period functional domains. The ExPASy Server online analysis software was used to analyze and predict the physicochemical properties of the sequences, DNAMMAN software was used to conduct multiple sequence comparison of the sequencing results, and the BLAST option of NCBI database was used to conduct homology analysis of Cryptochrome and Period sequences. The phylogenetic tree of Cryptochrome and Period was constructed and analyzed using the neighbor-joining method in MEGA 7.0. For homology modeling, I-TASSER was adopted, and Hdock was utilized to investigate the interaction mode between Cryptochrome and Period. The interaction mode of the docking results was analyzed with PyMOL 2.3.0. Furthermore, PCR technology was employed to clone and characterize the coding regions of the Cryptochrome and Period from T. crocea. Subsequently, tissue-specific expression analysis of Cryptochrome and Period was performed, and their expression levels in various tissues were quantified under different photoperiods. The results showed that the coding region of Cryptochrome has a base sequence length of 1 641 bp, encoding 546 amino acids, with a theoretical isoelectric point of 6.08 and a molecular weight of 62.98 kDa; the coding region of Period has a base sequence length of 4 386 bp, encoding 1 461 amino acids, with a theoretical isoelectric point of 6.14 and a molecular weight of 164.99 kDa. The Hdock interaction model showed that these two proteins could form heterodimers with a binding energy of –279.88 kcal/mol. Tissue expression analysis indicated that Cryptochrome and Period were expressed in all seven tissues examined, with relatively high expression levels in the outer mantle, inner mantle, gill and adductor muscle (Cryptochrome showed a high level of expression in the heart). Cryptochrome and Period genes exhibited oscillatory expression patterns that varied with the circadian cycle in the outer mantle, inner and outer mantle, gill, and adductor muscle. Under normal lighting conditions, the expression level of Cryptochrome in the outer mantle, inner and outer mantle, and the adductor muscle all reached the maximum value at 1 h of light treatment, whereas the expression level in the gill reached the maximum value at 5 h of dark treatment, and then showed a decreasing trend with increased light time. When the illumination was delayed for 2 h, the expression level of Cryptochrome in the outer mantle, inner and outer mantle, and gill all reached the maximum value at 1 h illumination. The expression level in the adductor muscle reached the maximum value at 7 h darkness treatment, and then all showed a decreasing trend with increased illumination time. Under normal light conditions, the expression level of Period in the outer mantle, inner and outer mantle, and gill reached the maximum value at 5 h of darkness treatment, whereas the expression levels in the adductor muscle reached the maximum value at 1 h of light treatment, and all showed a trend of first decreasing and then increasing with increased light time. When the illumination was delayed for 2 h, the expression level of Period in outer mantle and adductor muscle reached the maximum value at 7 h of darkness treatment, In contrast, the expression levels in inner and outer mantle and gill reached the maximum value at 1 h of light treatment, and both showed a decreasing trend with an increased illumination time. In conclusion, this study represents the initial successful cloning of two essential circadian clock genes, Cryptochrome and Period, from the species T. crocea. Our preliminary validation of the diurnal rhythmic expression patterns of these genes in key tissues provided valuable insights into the behavioral and physiological rhythms of T. crocea, as well as its mechanisms underlying light adaptation.

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杨文红,南祎晗,李萌,刘春胜,杨毅,於锋,顾志峰.番红砗磲生物钟基因Cryptochrome和Period克隆鉴定及光照相关性分析.渔业科学进展,2025,46(4):153-167

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  • 收稿日期:2024-10-04
  • 最后修改日期:2024-11-09
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  • 在线发布日期: 2025-07-14
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