[Cloning of four members of giant panda Dmrt genes]

Sex determining genes Mab-3 of C. elegans and Doublesex of Drosophila contain a common DNA binding motif called DM (Doublesex and Mab-3) domain, both of which regulate similar aspects of sexual development. Human Doublesex-related gene DMRT1 has been identified, which also contains the conserved DM-related DNA-binding domain and plays an essential role in gonadal differentiation. We amplified genomic DNA of the giant panda using the DM degenerate primers and detected two bands, approximately 140 bp and 250 bp. After cloned into T-easy vector and sequenced, four sequences showed high homology with the DM domain. Amino acid sequence of the first clone is 100% identical with the Dmrt1 of human, mouse and pig, hence we named it as pDmrt1. The second clone is 96% identical with human DMRTB1, and the third one 100% with the Dmrt3 of mouse and medaka, which were named as pDmrtb1 and pDmrt3 respectively. The last sequence contains an intron of 116 bp within the DM domain, which encodes an amino acid sequence 100% identical with human DMRTC2, accordingly we named it as pDmrtc2. Based on similarities of amino acid sequences of the DM domain, Dmrt protein sequences from human, mouse and giant panda were included in a phylogenetic tree. They revealed seven distinct subgroups: Dmrt1, Dmrt2, Dmrt3, Dmrt4 (DMRTA1), Dmrt5 (DMRTA2), Dmrt6 (DMRTB1) and Dmrt7 (DMRTC2). Our results further reveal the unexpected complexity and the evolutionary conservation of the DM domain gene family in both invertebrates and vertebrates.