Cloning Of Dmrt1 And Expression Analysis During Sex Determination Of Mauremys Mutica

Gender is one of the major propositions of life sciences.Gender determination and its regulatory mechanisms have always been one of the frontiers and hotspots of genetics,developmental biology,and evolutionary biology.Two sex determination methods are found in vertebrates: genetic sex determination(GSD)and environmental sex determination(ESD).Dmrt1 is a member of the Dmrt family and is considered to be the first conserved gene involved in the sex differentiation of invertebrates into human.It is an important regulator of testis formation and development and plays a key and essential role in animal sex determination and sex differentiation.Mauremys mutica are incubated in high temperature conditions to produce female offspring,which are incubated at low temperatures to produce male offspring.This sex-determined type is temperature-dependent sex determination,ie,environmental sex determination.Relative to the extensive study of hereditary sex-determining genes,the gender-regulating mechanisms of many environmental sex-determining species are not well understood.The turtles such as the Mauremys mutica are in the transition during the evolution system of the animal and have a special gender-determining mechanism.Studies on the sex determination and regulation mechanisms of the turtles have been receiving much attention.Therefore,the study of gender-regulated gene expression patterns in Mauremys mutica with temperature-dependent sex determination is of great significance for revealing the environmental sex determination mechanism.In this thesis,the Dmrt1 gene of the Mauremys mutica was studied and its sex determination mechanism was studied.The specific contents of this study are as follows:1.Cloning and analysis of Dmrt1 gene of Mauremys muticaIn this study,the full-length cDNA of Dmrt1 was screened and cloned from the transcriptome of male and female gonads of Mauremys mutica.The RACE-PCR results showed that the full length of the sequence was 1811 bp,including the 3’ non-coding region of 572 bp,and the 5’ end was non-coding.The region is 129 bp,and the open reading frame is 1110 bp,encoding a total of 370 amino acids.The multiple alignment of amino acid sequence showed that the Dmrt1 gene of the Mauremys mutica had the highest homology with the Trachemys scripta,reaching 97.57%;the homology with Dmrt1 of Pelodiscus sinensis was 75.54%.The homology with Mus musculus Dmrt1 was 63.25%,and the lowest homology with Diprt1 of Acipenser sinensis was 21.62%.Phylogenetic tree analysis showed that the Mauremys mutica was closely related to the tortoise,red-eared tortoise and western tortoise,and gathered together as a small branch.2.Tissue specificity analysis of Dmrt1 gene of the Mauremys muticaQuantitative results showed that the expression of Dmrt1 in the heart,liver,spleen,kidney,eye,brain,muscle and other tissues was extremely low,and it was expressed in the ovary with a high expression level in the testis.Significantly higher than other organizations.3.The Expression and distribution of Dmrt1 in Mauremys muticaIn situ hybridization analysis showed that Dmrt1 mRNA was specifically expressed in testis germ cells.The expression of Dmrt1 of the Mauremys mutica is mainly concentrated in Sertoli’s cells,and is expressed in a small amount in Spermatogonia and primary spermatocytes,but is hardly expressed in sperm cells and sperm.4.Specific expression analysis of Dmrt1 gene in the gonads of the Mauremys mutica.The results showed that the Dmrt1 gene of the Mauremys mutica,which was incubated at the male temperature(25 ℃)and from 32 °C to 25 °C,began its male-specific expression in the 19 th stage before the initiation of gonadal differentiation.The expression of Dmrt1 in the 19 th to 22 nd stages of embryo development at 25 °C was gradually increased and significantly higher than that at 32 °C.The above results indicate that Dmrt1 gene may be involved in sex determination and male gonadal differentiation in Mauremys mutica,and participates in the development of male gonads.This study lays a foundation for further revealing the temperature-dependent sex regulation mechanism of Mauremys mutica..