| In mammals, male sex determination and initiating testis development are under the control of the Sry (sex-determining region of the Y chromosome). In 1991, Koopman reported that Sry gene can direct male development in an XX transgenic mouse. Sry protein contains a DNA binding domain known as the HMG-box. The HMG box is the singular part of Sry conserved between species, and point mutations in the corresponding region of the SRY gene cause sex reversal in humans. In human, SRY protein was detected in the nuclei of Sertoli cells, and was speculated that it worked as a transcriptional factor.Although the testis-inducing function of Sry has been clearly demonstrated, its direct target gene or genes still await identification. As one likely Sry target, the Sry-related gene Sox9 has emerged. Most other genes which are involved in this complex process and play important roles in male sex differentiation have been identified, including Sf1 (Steroidogenic fator-1), Gata4 (GATA binding protein 4), Wt1 (Wilms'tumor gene), Sox9 (Sry-related HMG box-9), Amh (Anti Mullerian Hormone), and Dax1 (Dosage sensitive sex reversal locus-1). At present, almost all the researches about mammals are focus on human and mouse, and there are fewer researches on other animals.Cattle is thought to be an important economic animal, it is great valuable in animal husbandry. The male calves are valueless. At present, the sex control technologies being used in cow production is separation of X and Y sperms. Its disadvantages are expensive, low efficiency, reduction of fertility rate, and so on. Researches on molecular regulation mechanism about sex determination, finding the method to interfere the regulation net of sex determination can provide a new means for cow sex control. The researches among different species could also help us to further understand the mechanisms of sex determination and the evolutionary rules in mammalian sex determination.In this research, we cloned the 1838bp Sry sequences, including the 5'flanking and ORF. Bovine genital ridge cells and ovary granulosa cells were isolated and cultured in vitro. We identified the sex and characteristic of the genital ridge cells. The information of Sry 5'flanking and ORF were analyzed by bioinformatics method. The potential transcriptional start site and transcriptional factor binding sites in Sry 5'flanking 1066bp were predicted. To analyzing the evolution of bovine Sry, we compared Sry protein sequences among 10 species. We also predicted and compared the three-dimensional structure SRY/Sry between human and bovine. To find the core promoter of bovine Sry, We constructed 13 deletion recombinants with different length of Sry 5' flanking, promoter activity analysis was performed in bovine genital ridge cells by the dual-luciferase reproter assay system. Sry gene was overexpressed by pcDNA3.1-Sry in genital ridge cells and granulosa cells, after that six genes which were identified as important genes in sex determination in mammals, including Sf1, Gata4, Wt1, Sox9, Amh and Dax1 were examined by semi-quantitative RT-PCR to observe their expression change influenced by Sry. Subcellular localization was also performed to analyze the disposition of Sry protein in genital ridge cells and granulosa cells. We got the conclusion below:1. The 1838bp Sry sequences, including the 5'flanking and ORF, were cloned and secquenced. Blast anslysis indicated that the sequences were consistent with bovine Sry sequences in GenBank(No. AB039748.1).2. Bovine genital ridge cells were isolated and cultured in vitro, and it were identified that it came from a male genital of sex differentiation period; they could grow normally in vitro, and be passaged stablely.3. Bovine ovary granulosa cells .were isolated cultured in vitro, they could grow normally in vitro, and be passaged stablely.4. The bovine Sry 5'flanking sequences of 1066bp were analyzed by bioinformatics method, it were indicated that there were 3 potential transcriptional start sites at -93,-419,-722(ATG, A was 0 point), and the transcriptional factor binding sites (TFBS) prediction indicated that there were copious potential TFBS in the fragment, which was consistent with the special complicated regulation mechanism of Sry.5. Comparison of 10 species SRY/Sry sequences showed that bos taurus was more homologous to capra hircus(93.5%); bos taurus was more divergent from mus musculus (60.4%).6. Evolution analysis of SRY/Sry sequences of 10 species showed bos taurus and capra hicus were more relative, and belonged to a group, they are reach to the distal end of the cladogram, indicated that artiodactylous animals has greater variation than others by means of natural selection.7. Comparison of SRY/Sry primary structure of human and bovine, the HMG-box was the singular conserved part, but they were similar in the three-dimensional structure. They both can recognize the 7bp conserved binding site sequences. It indicated that although the Sry primary structure has greater variation among different species, the three-dimensional structure and mode of action were similar.8. We constructed 13 deletion recombinants with luciferase reporeter, the promoter acativity of bovine Sry 5'flanking 1056bp was analyzed by dual-luciferase reproter assay system in genital ridge cells, the core promoter of bovine Sry was located at the -599~-565bp upstream of Sry translation start site ATG (A is 0 site.), a fragment of 35bp. 9. Sry was overexpressed in genital ridge cells by pcDNA3.1-Sry, after that six genes which were identified as important genes in sex determination in mammals, including Sf1, Gata4, Wt1, Sox9, Amh and Dax1 were examined by semi-quantitative RT-PCR to observe their expression change influenced by Sry. The results indicated that Sry can up-regulate the Sox9 expression.10. Subcellular localization was performed to analyze the disposition of Sry protein in genital ridge cells and granulosa cells. Bovine Sry protein localizated in the cell nuclei, it confirmed that bovine Sry working as transcription factor.
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