Molecular Cloning of Full-Length Dmrt1 cDNA of Reeves Turtle (Chinemys reevesii)

Abstract

ABSTRACT. We cloned and sequenced a full-length open reading frame turtle dmrt1 cDNA (Crdmrt1) that was 1,504 bp in length and encoded 371 amino acid residues. RT-PCR analysis in different tissues of adult male turtle showed that the Crdmrt1 cDNA fragment was only detected in the testis. The amino acid sequence derived from Crdmrt1 demonstrated high homology to sequences from dmrt1 of Pelodiscus sinensis (92% identities and 93% positives) and Elaphe quadrivirgata (75% identities and 83% positives). The deduced amino acid from Crdmrt1 contained a conserved DM domain, a male-specific motif, and a P/S-rich region. In DMRT1 from reptiles, birds, mammals, amphibians, and fish, the amino acid identities and positives for DM domains were 85-100% and 88-100%, respectively, those for male-specific motifs were 47-100% and 60-100%, and those for P/S-rich regions were 24-100% and 35-100%. The module consisted of intertwined CCHC and HCCC Zn 2+ -binding sites in the DM domain and was conserved in all 11 species analyzed in this study. Amino acid sequences of Crdmrt1 and previously reported DMRT1s, DMRT2s, and DMRT3s were subjected to phylogenetic analysis. The resulting tree showed that CrDMRT1 belongs to DMRT1, and turtles are a sister group to a cluster of birds and snakes. This is the first study of the cloning of full-length dmrt1 from a reptile.

Effects of Incubation Temperature on the Expression of Sex-Related Genes in the Chinese Pond Turtle, <b><i>Mauremys reevesii</i></b>

“…For example, transcriptome analysis revealed that genes involved in steroid enzyme synthesis and brain development were sexually dimorphic expressed prior to TSP in T. scripta [Czerwinski et al, 2016], similar to what is observed for Sf1 , Wt1 , and Dax1 involved in gonadal development in C. picta [Valenzuela et al, 2006;Valenzuela, 2008a, b], and it also revealed that Cirbp (cold-inducible RNAbinding protein) is involved in sex determination in C. serpentina [Schroeder et al, 2016]. But there are few studies on the molecular networks of TSD reptiles in other regions such as Asia [Hoshi and Nakao, 2008;Inamdar et al, 2015]. Thus, molecular studies across TSD reptiles representing other turtle families, reptilian lineages, and geographic regions are overdue and necessary if we are to fully understand the evolution of TSD.…”

“…The purified fragments were cloned into the pEASY-T1 vector (Transgen) and sequenced using M13F and M13R primers. For Dmrt1 , we used the full length sequence (GenBank: AB365876.1) previously cloned by others [Hoshi and Nakao, 2008].…”

“…To our knowledge, this is the first such study in a chelonian of the family Geomydidae. Specifically, we cloned full open reading frames (ORFs) of sex-related genes such as Cyp19a1 ( aromatase ), Foxl2 , Rspo1 , Sf1 , and Sox9 and investigated the expression patterns of these genes as well as of the previously characterized Dmrt1 [Hoshi and Nakao, 2008] during the TSP at different incubation temperatures to assess the potential roles of these genes in the sex determination of M. reevesii . These genes were chosen because they play critical roles in sex determination in other vertebrates, including TSD reptiles.…”

The roles of Dmrt (Double sex/Male-abnormal-3 Related Transcription factor) genes in sex determination and differentiation mechanisms: Ubiquity and diversity across the animal kingdom

“…In these two species, there is no observable difference in Dmrt1 expression at male-and female-promoting temperature in the early stages of development, but as development progresses, the expression of Dmrt1 increases at a male-promoting temperature (26 8C) and decreases to an undetectable level at a female-promoting temperature (33 8C) [62]. In the Reeves turtle (Mauremys reevesii), the expression of Dmrt1 was studied only in male individuals, where it was found to be expressed in testes but not in liver, spleen, brain or kidney [64]. Finally, DM genes have been isolated from other turtle species, but none has yet been subjected to a functional analysis [65].…”

Differential <i>DMRT1</i> Expression in the Gonads of <i>Podarcis sicula</i> (Reptilia: Lacertidae)

“…Its expression is upregulated at male-producing temperatures in turtles, alligators and salamanders [Kettlewell et al, 2000;Torres Maldonado et al, 2002;Rhen et al, 2007;Ferguson-Smith, 2007;Hoshi et al, 2008]. DMRT1 downregulation, together with upregulation of the P450 aromatase gene (CYP19A1) in species with temperature-dependent sex determination such as salamander, produces sex reversal from genetic males to phenotypic females [Murdock and Wibbels, 2006;Sakata et al, 2006].…”