Expression Of Bovine SRY Gene And Identification Of Its Protein Binding

It becomes increasingly important to contol the sex of bovine, because the value of cow is higher than most of other animals and sex determination of bovine will stimulate increasing of economy. The centre of controlling bovine sex is understanding mechism of sex forming. SRY(Sex-determining Region on the Y Chromosome) has been proposed to be the master gene regulating the cascade of testis determination. And, MIS(Mullerian Inhibiting Substance) is necessary for sex determination of mammals. MIS inhibits the forming of mullian duct that constructs the gonad structure of female. Previous studies demonstated the MIS gene (also known as AMH) as a potential target of SRY.There are seldom studies about bovine sex determination. It's still unknown that wether the bovine SRY can bind promoter of bovine MIS gene and research of bovine SRY expression in prokaryotes wasn't reported.The purpose of this study is identifing the binding of SRY to promoter of MIS gene. The SRY gene was expressed in prokaryotes, the promoters of MIS genes were cloned, and the binding between SRY protein and promoters of MIS genes was studied. These studies must be very meaningful for controlling sex of bovine on molecular level. In this paper, we cloned of bovine SRY gene and identified of protein in vitro.(1) The genomic DNA of bovine was extracted, and primers by which the ORF of SRY gene can be amplificated were sythesized(SRY gene contains single extron, so its ORF can be obtained through genomic DNA as template). Products amplificated were linked to vector pGEM-T and analysis of product linked was done by bioloyical company. The result suggested that SRY gene ORF of bovine was obtained and the homogeneity between this ORF and sequence bovine SRY gene in GeneBank is 100%.(2) According sequences of bovine SRY gene and vector pET-28a(+), primers that contain restricted inner enzyme EcoRâ… ,Salâ… sites were designed. The vector pGEM-T/SRY being used as template, the ORF of SRY gene was amplificated in order to get enzyme sites in its sequence. The product of PCR and the vector pGEM-T/SRY were digested by restricted inner enzyme EcoRâ… ,Salâ… and products digested were linked by T4 DNA ligant emzyme to...