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| Methylation Analysis of Promoter of Dmc1 Gene Related to Meiosis in Hybrid Triploid Loach Misgurnus anguillicaudatus |
| MA Kexin, LI Yifan, YANG Yueyao, LI Chuan, GUO Wenxuan, ZHUANG Zixin, WANG Wei, ZHOU He |
| Key Laboratory of Mariculture ＆ Stock Enhancement in North China's Sea, Ministry of Agriculture and Rural Affairs, Key Laboratory of Applied Biology and Aquaculture of Northern Fishes in Liaoning Province, College of Fisheries and Life Science, Dalian Ocean University, Dalian 116023, China |
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Abstract In order to investigate the important role of DNA Meiotic Recombinase 1 (Dmc1) in the infertility mechanism of the hybrid triploid loach Misgurnus anguillicaudatus(4n ♀ × 2n ♂), the Dmc1 gene was cloned from the hybrid triploid loach, and the physicochemical properties and phylogenetic position of the gene were analyzed by bioinformatics. The methylation and expression levels of the promoter region of Dmc1 gene were analyzed by bisulfite sequencing (BSP) and RT-PCR in the gonadal tissues of female and male offspring of loach triploid crosses and their parents. The results showed that the Dmc1 gene had the full length of 3895 bp, and an open reading frame (ORF) of 1029 bp encoding 342 amino acids, with the predicted molecular weight of 37.85 ku, and the theoretical isoelectric point (pI) of 6.95. Multiple sequence alignment showed that the amino acid sequence of Dmc1 in the loach was similar to that of other fish in Cypriniformes and less similar to arthropods. The phylogenetic tree revealed that the loach clustered with other osteichthyans in terms of Dmc1 evolution. BSP analysis showed that the methylation rates of the Dmc1 promoter region were 100% in the tetraploid maternal parent, 80.0% in diploid paternal parent, and 82.2% in triploid offsprings. There was higher methylation rate of the Dmc1 promoter region in the male offspring of triploids than that in the diploid paternal parent, with the significantly lower expression level of the Dmc1 gene than that in the diploid paternal parent. The methylation rate of the Dmc1 promoter region was shown to be lower in the female offspring of triploids than that in the tetraploid maternal parent, with significantly higher expression level of the Dmc1 than that in the tetraploid maternal parent. Pearson correlation coefficient analysis indicated that the methylation rate of the Dmc1 promoter region was negatively correlated with its expression level (r =-0.986, P=0.014). These findings suggested that the expression level of the Dmc1 gene in different ploidy loach was negatively regulated by methylation of the promoter region, leading to the speculation that the DNA methylation rate of the Dmc1 gene was correlated with the meiotic abnormalities and low fertility of triploid loaches. These findings provide theoretical basis for unraveling the molecular mechanisms underlying the infertility of hybrid triploid loaches.
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Received: 19 April 2024
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