RESUMEN
The growth arrest and DNA-damage-inducible protein 45 gamma (Gadd45g) is known to play a major role in embryonic development and sex determination. In this study, two Gadd45g genes were isolated from half-smooth tongue sole (Cynoglossus semilaevis). Using chromosomal fluorescence in situ hybridization (FISH), Gadd45g1 and Gadd45g2 were located on the W and Z chromosomes, respectively. The full-length cDNA sequences of Gadd45g1 (1270bp) and Gadd45g2 (1181bp) were predicted to contain a 480-bp coding sequence that could encode a protein of 159 amino acids residues. A phylogenetic tree showed that the predicted Gadd45g1 and Gadd45g2 amino acid sequences clustered closely in one branch. It is proposed that Gadd45g1 and Gadd45g2 are paralogous genes derived from the divergence of the sex chromosome. Ka/Ks ratios indicated that Gadd45g1 and Gadd45g2 may have undergone a high number of mutations and have a divergence time of only about 68,000years, although Gadd45g homologs are highly conserved. The qRT-PCR demonstrated that Gadd45g1 and Gadd45g2 were highly expressed in ovary, and negligibly expressed in testis of male and neo-male. During development of the ovary (from 80 to 150days), the expression levels of both genes reached high levels. Gadd45g1 was also highly expressed at 50days, the stage just before gonad differentiation in C. semilaevis. All these findings imply functional divergence of the two Gadd45g homologs; Gadd45g1 may be necessary for sex differentiation in the early stage of gonad development, and then Gadd45g1 and Gadd45g2 maintain ovary development and the female character of half-smooth tongue sole.
Asunto(s)
Peces Planos/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Evolución Molecular , Femenino , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Datos de Secuencia Molecular , Ovario/metabolismo , Filogenia , Procesos de Determinación del Sexo , Diferenciación Sexual , Testículo/metabolismo , Proteinas GADD45Asunto(s)
Carpas/genética , Clonación Molecular , Antígenos de Histocompatibilidad Clase II/genética , Polimorfismo Genético , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN , Cartilla de ADN , Antígenos de Histocompatibilidad Clase II/química , Datos de Secuencia Molecular , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de AminoácidoRESUMEN
Cathepsin D is a lysosomal aspartic proteinase which participates in various degradation functions within the cell. In this current study, we cloned and characterized the complete cDNA of grass carp cathepsin D through 5'- and 3'-RACE. The cathepsin D contained a 56 bp 5' terminal untranslated region (5'-UTR), a 1197 bp open reading frame encoding 398 amino acids, and a 394 bp 3'-UTR. Grass carp cathepsin D shared high similarity with those from other species, and showed the highest amino acid identity of 91% to Danio rerio. Unlike many other organisms, the grass carp cathepsin D contains only one N-glycosylation site closest to the N-terminal. Real-time quantitative RT-PCR demonstrated that Cathepsin D expressed in all twelve tissues (bladder, brain, liver, heart, gill, muscle, fin, eye, intestines, spleen, gonad and head kidney). The relative expression levels of Cathepsin D in gonad and liver were 26.58 and 24.95 times as much as those in fin, respectively. The expression level of Cathepsin D in muscle approximately 16-fold higher, in intestines and spleen were 12-fold higher. The cathepsin D expression showed an upward trend during embryonic development. After challenged with Aeromonas hydrophil, the expression of grass carp cathepsin D gene showed significant changes in the four test tissues (liver, head kidney, spleen and intestines). The fact that the bacterial infection can obviously improve the cathepsin D expression in immune-related organs, may suggest that cathepsin D plays an important role in the innate immune response of grass carp.