RESUMEN
Cyclin B is a regulatory subunit of maturation-promoting factor (MPF), which has a key role in the induction of meiotic maturation of oocytes. MPF has been studied in a wide variety of animal species; however, its expression in crustaceans is poorly characterized. In this study, the complete cDNA sequence of Cyclin B was cloned from the red claw crayfish, Cherax quadricarinatus, and its spatiotemporal expression profiles were analyzed. Cyclin B cDNA (1779 bp) encoded a 401 amino acid protein with a calculated molecular weight of 45.1 kDa. Quantitative real-time PCR demonstrated that Cyclin B mRNA was expressed mainly in the ovarian tissue and that the expression decreased as the ovaries developed. Immunofluorescence analysis revealed that the Cyclin B protein relocated from the cytoplasm to the nucleus during oogenesis. These findings suggest that Cyclin B plays an important role in gametogenesis and gonad development in C. quadricarinatus.
Asunto(s)
Astacoidea/genética , Ciclina B/genética , Regulación del Desarrollo de la Expresión Génica , Factor Promotor de Maduración/genética , Oocitos/metabolismo , Oogénesis/genética , Secuencia de Aminoácidos , Animales , Astacoidea/citología , Astacoidea/crecimiento & desarrollo , Secuencia de Bases , Núcleo Celular/metabolismo , Clonación Molecular , Ciclina B/metabolismo , Citoplasma/metabolismo , ADN Complementario/genética , ADN Complementario/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Femenino , Factor Promotor de Maduración/metabolismo , Meiosis , Datos de Secuencia Molecular , Peso Molecular , Oocitos/citología , Oocitos/crecimiento & desarrollo , Sistemas de Lectura Abierta , Ovario/citología , Ovario/crecimiento & desarrollo , Ovario/metabolismo , Transporte de Proteínas , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de SecuenciaRESUMEN
To better understand the reproductive transformation mechanism of Daphnia carinata, a Doublesex (Dsx) gene was cloned based on rapid amplification of cDNA ends (RACE), and was designated DapcaDsx2. Next, we compared similarities and assumed homology based on deduced amino acid sequences. It showed 97.52, 87.94, and 85.11% identity to orthologous genes in D. magna, D. pulex, and D. galeata respectively. Phylogenetic analysis revealed that DapcaDsx2 clustered in the same class, and was evolutionarily more distant to sequences from other species. qRT-PCR showed that DapcaDsx2 was most abundantly expressed during sexual reproduction (P < 0.05). Using digoxigenin-labeled RNA probes, we studied DapcaDsx2 expression in parthenogenetic and sexual females by whole-mount in situ hybridization. The results revealed that DapcaDsx2 was mainly expressed in the second antennae and several sites of the ventral carapace, whereas higher expression levels were found in sexual than in parthenogenetic females. This suggests that the DapcaDsx2 gene is involved in switching modes of reproduction and in sexual differentiation.