| The Dmrt(Doublesex and mab-3 related transcription factor)gene family is a superfamily of genes which encode transcriptional regulatory factors involved in the processes of sex differentiation,gonadal development,gametogenesis,tissue organ formation and function maintenance in vertebrates.It encodes transcription factors with a conserved DM functional domain,depending on zinc finger structure to binding with DNA.The Dmrt gene family is the most crucial gene family in vertebrate research for sex determination and differentiation,and Dmrtl gene is also the most studied and closely gene related to sexual development and differentiation.Amphibians,is a large vertebrate taxon in habitats transition from aquatic to terrestrial,with a complex life cycles and unique breeding mode represent a taxon which need to pay high attention.However,the research on sex determination and differentiation in amphibians obviously lags behind compared to other vertebrate taxa.Therefore,we selected six representative species of amphibians,Caudata are Ranodon shihi and Tylototriton shanjing,Anura are Rhacophorus omeimontis?Hyla annectans?Vibrissaphora leishanensis and Bufo gargarizans,as research subjects for cloning and evolutionary analysis of amphibians Dmrt related genes.The results as following:(1)The DM domain sequences of the Dmrt1 gene we cloned in six species,Dmrt2 gene in Vibrissaphora leishanensis and Ranodon shihi,Dmrt3 gene of Hyla annectans were cloned respectively,the homology of the DM domain in three genes was over 80%through sequence alignment,and phylogenetic relationship has shown to be evolutionarily conservative.(2)The full-length cDNA sequences of the Dmrt1 gene in six species were successfully obtained using SMART-RACE technology.All of them obtained a complete open reading frame and related functional domains,allowing structural,functional and evolutionary analysis.Among them,the full-length sequence of Dmrt1 in Ranodon shihi is 1,656 bp,including 1,020 bp open reading frame encoding 339 amino acid sequences.The full-length of Dmrt1 in Tylototriton shanjing is 1,686 bp which contains 1,032 bp open reading frame encoding 343 amino acid sequences.The full-length of Dmrt1 in Rhacophorus omeimontis is 1,917 bp,including 918 bp open reading frame,encoding 305 amino acid sequences.The full-length of Dmrt1 in Hyla annectans is 1,938 bp,containing 1,023 bp open reading frame,encoding 340 amino acid sequences.The full-length of Dmrt1 in Vibrissaphora leishanensis is 1,194 bp,including an open reading frame of 945 bp,which encodes 314 amino acid sequences.For Bufo gargarizans,it is 1,141 bp,including 1,044 bp open reading frame,encoding 347 amino acid sequences.Functional domain analysis showed that all Dmrt1 genes have DM conserved domain and Dmrt1 functional domain,signal peptide detection did not reveal relevant signal peptide cleavage sites,and Dmrt1 is a non-secretory protein and hydrophilic protein.Protein secondary structure including ?-helix,extended strand and random coil,and the best matching template for DM domain tertiary structure prediction is 4yj0.1.A,and has a clear zinc finger structure.(3)The pairwise amino acid distance between multiple species is greater than the pairwise nucleotide distance,indicating a possible adaptive selection.The positive selective detection of Dmrt1 gene in amphibian was detected by the codeml subroutine of PAML software,only one amino acid site was detected in many models that would be positive selection.Although the positive selection detected was weak,it presumed to be affected by gene duplication because of the high homology in the conserved sequence.In addtion,it had an impact on the evolution of Dmrt1 gene by amphibious sex chromosomes recombined during the long evolutionary process.(4)The expression of Dmrt1 in testis,ovary,liver,heart,kidney and spleen tissues of Vibrissaphora leishanensis adults were detected by fluorescence quantitative PCR.The expression of Dmrt1 gene in male testis was significantly higher than other tissues,suggesting that Dmrt1 gene may be related to the development and differentiation of male gonad in Vibrissaphora leishanensis.It needed further exploration on the role of specific sex determination.Our results suggested that,the Dmrt gene family was a class of highly conserved genes during the evolution of amphibians.Recombination of homomorphic sex chromosomes and gene duplication would have affected on the positive selection of the Dmrt1 gene.Meanwhile,it has significant male specificity on the expression of the Dmrt1 gene which was a good candidate gene for amphibian sex determination research. |
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