Bovine SOX9 Cloning And Analysis Of Sex-related Gene Expres Sion In Early Fetus

Dairy cattle is an important economical animal in agriculture and the proportion of the male and female offspring is 1:1.However only the female can product milk and the male is not wanted.because of their bad quality beef and low feed-reture rate.So,the sex control of dairy cattle is important to the agricultural development and the mechanism of the sex determination becames the focus in agricultural animal reproduction.Previous study shows that SRY is at the top of the cascade of genetic activity lead-ing to male development in mammals. Sex determining region Y-box 9 (SOX9) which acts as the capital regulatory signal downstream SRY play a pivotal role in the genital ridge differentiation, gonadogenesis, Sertoli cell differentiation and testis cords development.In the present study, SOX9 containing complete ORF was amplified from Holstein dairy cattle us-ing RACE technique. Then the SOX9 homology,chromosomal localization,SOX9 amino acid se-quence,SOX9 conservative domain,SOX9 phosphorylation and methylation site,and signal peptide were analyzed by bioinformatics. Meanwhile,four sex related genes(SRY,SOX9,DAX1 and FOXL2) was detected by real-time qPCR, and the standard curves of these genes were drawn. The main contents are as follows:The cloning of complete SOX9 cDNA. According to the partial sequence of dairy cattle SOX9(AF278703), we have amplified the complete coding region(2846bp)of bovine SOX9 by RACE technique,construct the prokaryotic expression vector and eukaryotic expression vector of the SOX9 ORF.Structure and functional prediction of SOX9. BLAST in GenBank/Blast indicates that SOX9 con-tainning a 1575bp complete coding region is a novel gene.The deduced protein of SOX9 is composed of 524 amino acids with a calculated molecular weight of approximately 57.17334KD. SOX9 is located at chromosome 19.Four sex related genes(SRY,SOX9,DAX1 and FOXL2) and a intemalcontrol gene GAPDH were cloned into T-A cloning vector, respectively, and then standard curve of real-time qPCR was established.The result of real-time qPCR showed that the sex determining time of male and female is 35-39dpc and 39-41dpc respectively, and sex differentiation ended at 41-43dpc.SRY is the male sex determining gene, while FOXL2 may play a role of a female sex determining gene.