| Clock genes play an pivotal role in the rhythm maturation of the fish ovary. In order to explore the role of clock genes in the rhythm ovarian development and maturation of Tongue Sole (Cynoglossus semilaevis), we used qRT-PCR(real-time fluorescent quantitative PCR) technique to analyze the expressions of Clock1a (circadian locomotor output cycles kaput 1a), Bmal1a (brain and muscle arntlike 1a), Cry1a (cryptochrome 1a), Cry2 (cryptochrome 2) and Per2 (period 2) at the ovarian stages Ⅱ, Ⅲ, Ⅳ, Ⅴ and Ⅵ.
In this study, the cDNA sequences of five clock genes were cloned and the CDS (the coding DNA sequence) of these genes were phylogenetic analyzed. It is found that the CDS sequence length of Clock1a gene coding region is 1620 bp, encoding 539 amino acids, and the encoded amino acid sequence of Clock1a has a predicted molecular weight of 81.9 kDa. There are functional domain PASD1 consisting of 64 amino acids and functional domain PAS11 consisting of 103 amino acids in the sequence. The CDS sequence length of Bmal1a gene coding region is 1881 bp, encoding 626 amino acids, and the encoded amino acid sequence of Bmal1a has a predicted molecular weight of 68.9 kDa. In the 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 gene coding region is 1896 bp, encoding 631 amino acids, and the encoded amino acid sequence of Cry1a has a predicted molecular weight of 71.4 kDa, and there is a functional domain FAD7 composed of 199 amino acids in the sequence. The CDS sequence length of Cry2 gene coding region is 2007 bp, encoding 669 amino acids, and the encoded amino acid sequence of Cry2 has a predicted molecular weight of 76.0 kDa, and the sequence contains a functional domain FAD7 consisting of 199 amino acids. The CDS sequence length of Per2 gene coding region is 4248 bp, encoding 1415 amino acids, and the encoded amino acid sequence of Per2 has a predicted molecular weight of 154.0 kDa. In the sequence, there is a functional domain PeriodC consisting of 295 amino acids and a functional domain PAS11 consisting of 102 amino acids. In this study, 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 is 60% ~ 79%, 94% ~ 100%, 85% ~ 91%, 84% ~ 94% and 70% ~ 84%, respectively. Therefore, this study believes 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 respectively, indicating a close relationship between C. semilaevis and other bony fishes in the long evolutionary process. In addition, 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 are strongly conserved during the evolution of C. semilaevis.
In present study, we found that the expression levels of five clock genes were higher in stage Ⅱ and stage Ⅲ, which were equivalent to the non-breeding season (p<0.05), but lower in stage Ⅳ and stage Ⅴ, which were equivalent to the breeding season (p<0.05). Therefore, the expression profiles of Clock1a, Bmal1a, Cry1a, Cry2 and Per2 in ovary of C. semilaevis also have seasonal characteristic. 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, temperature and melatonin 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 of this study can enrich the theory of ovarian development of C. semilaevis and other bony fish, and provide a theoretical basis for improving breeding technology and seedling efficiency of C. semilaevis. |
