Cloning and Expression Analysis of Clock Genes During Ovarian Development and Maturation of Tongue Sole (Cynoglossus semilaevis)

Abstract

Clock genes play a pivotal role in the rhythm maturation of bony fish ovaries. To explore the role of clock genes in the rhythmic ovarian development and maturation of tongue sole (Cynoglossus semilaevis), we used real-time fluorescent quantitative PCR technique to analyze the expression profiles of circadian locomotor output cycles Kaput 1a (Clock1a), brain and muscle Arntlike 1a (Bmal1a), Cryptochrome 1a (Cry1a), Cryptochrome 2 (Cry2), and Period 2 (Per2) at the ovarian stages Ⅱ, Ⅲ, Ⅳ, Ⅴ, and Ⅵ.The coding DNA sequence (CDS) of five clock genes were cloned and phylogenetically analyzed. We found that the CDS sequence length of Clock1a was 1 620 bp and encoded 539 amino acids, with the encoded amino acid sequence of Clock1a having a predicted molecular weight of 81.9 kDa. Clock1a has the functional domain PASD1 (consisting of 64 amino acids) and PAS11 (consisting of 103 amino acids). The CDS sequence length of Bmal1a was 1 881 bp and encoded 626 amino acids, with the encoded amino acid sequence of Bmal1a having a predicted molecular weight of 68.9 kDa. In the Bmal1a sequence, the functional domain PASD3 is composed of 63 amino acids, and the functional domain PAS11 is composed of 103 amino acids. The CDS sequence length of Cry1a was 1 896 bp and encoded 631 amino acids, with the encoded amino acid sequence of Cry1a having a predicted molecular weight of 71.4 kDa. Its functional domain FAD7 was composed of 199 amino acids. The CDS sequence length of Cry2 was 2 007 bp and encoded 669 amino acids, with the encoded amino acid sequence of Cry2 having a predicted molecular weight of 76.0 kDa. The Cry2 sequence contains a functional domain FAD7 consisting of 199 amino acids. The CDS sequence length of Per2 was 4 248 bp, encoding 1 415 amino acids, and the encoded amino acid sequence of Per2 has a predicted molecular weight of 154.0 kDa. In the Per2 sequence, the functional domain PeriodC consists of 295 amino acids and a functional domain PAS11 consists of 102 amino acids.The neighbor-joining method was used to analyze the Clock1a, Bmal1a, Cry1a, Cry2, and Per2 phylogenetic relationships between C. semilaevis and other bony fish, amphibians, birds, and mammals. The homology of Clock1a, Bmal1a, Cry1a, Cry2, and Per2 with other bony fish was 60%–79%, 94%–100%, 85%–91%, 84%–94%, and 70%–84%, respectively. Therefore, we believe that these five amino acid sequences show strong conserved property. In addition, the homology of Cry1a and Cry2 was 62%, indicating that Cry1a and Cry2 evolved differently during the evolution of C. semilaevis. In the constructed phylogenetic tree, Clock1a, Bmal1a, Cry1a, Cry2, and Per2 of C. semilaevis were clustered together with other bony fishes, indicating a close relationship between C. semilaevis and other bony fish in the evolutionary process. Moreover, the homology of Clock1a between C. semilaevis and mammals, birds, and amphibians is low, indicating that there are evolutionary differences in the evolutionary process of Clock1a. The high homology of Bmal1a, Cry1a, Cry2, and Per2 with mammals, birds, and amphibians suggests that these four clock genes were strongly conserved during the evolution of C. semilaevis.In the present study, we found that the expression levels of five clock genes were high in stagesⅡ and Ⅲ, which were equivalent to the non-reproductive season (P < 0.05). However, the expression levels of five clock genes were low in stages Ⅳ and Ⅴ, which were equivalent to the reproductive season (P < 0.05). Therefore, the expression profiles of Clock1a, Bmal1a, Cry1a, Cry2, and Per2 in the ovaries of C. semilaevis also have seasonal characteristics. The ovarian development and maturation of C. semilaevis goes through stages Ⅱ, Ⅲ, Ⅳ, Ⅴ, and Ⅵ, and then reaches stage Ⅱ again and starts a new reproductive cycle. The variation patterns of seasonal factors, such as light and temperature, in fish ovaries were consistent with the annual expression patterns of the five clock genes in this study. Therefore, it can be considered that the expression of clock genes in C. semilaevis has an annual cycle. The findings presented in this study can enrich the theory of ovarian development and maturation of C. semilaevis and provide a theoretical basis for improving breeding technology and seedling efficiency.

Keywords

• clock gene
• cynoglossus semilaevis
• ovary
• expression profiles