Construction Prokaryotic Expression Vector And Expression Of Chinese Holstein Cows SRY Gene HMG Box

It becomes more and more important to control the sex of cows,because the value of cows is higher than most of other animals and sex determination of cows will lead to a rapid increase in the economy.Chinese Holstein cow is a local species in China,who will be regards as a more significative object of study.It has more practical significance to an investigation in sex-determination mechanisms,which will help people to know the theory and practical of cows' sex-determination,and also,it is possible to propose the new method of the sex identity.The sex Control of cows is a biotechnology technique that can remarkably improve the economic returns of stockbreeding.Further more,it has a very important significance for stock breeding,breeding,production,the prevention and cure of genetic disease.Since this century,the investigation of sex formation mechanism has developed from morphology level to molecular level.The sex determination of mammalian depend on the sex-determining region of Y gene(SRY)which makes the primordial sex gland develop to be the testicle.Numerous investigations indicated that SRY genic mutation could cause the genitical gland of people or the others mammalian impaired development.In addition,through some investigation in the cases of sex reversal,sex determination is a multi-gene cascade connection regulatory process.The method of sex-controlled has developed from individual level,cellular level to molecular level,and it provided the theory guide and advanced technology instrument for the subsequent study.In the male sex determination is switched by a single gene on the short arm of the Y chromosome,named as SRY.SRY gene encoding proteinum which has a length contain 70 amino acid residue possess the character of DNA-binding protein,it belongs to a family of High Mobility Group(HMG),encodes a Protein with an HMG-like DNA-binding domain,which Probably acts as a local organizer of chromatin structure.The High Mobility Group is a convseve region of SRY gene,it is combined with 80 amino acid,reside in many DNA binding protein.as yet,all the SRY gene mutation point that cause sex reversal located in the sequence of High Mobility Group.It explain that SRY gene is the nucleus gene of sex-determination,can derivn spermary growth,and has critical effect for testicle development.This paper used genetic engineering technology to clone and express the core sequence of SRY gene High Mobility Group box.It must be very meaningful to control sex of cows on molecular level.In this paper,we cloned of bovine SRY gene and identified of protein in vitro and get know further about the mechanism of action of SRY gene.All of those establish substantial foundation for the clarification of sex-determination mechanism.In this paper,the main contents of study as follows:(1)To draw blood of male Holstein cow,extract RNA reference molecular cloning technique,amplification cDNA sequence of SRY gene with RT-PCR technique,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 pMD18-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 99.5%.(2)According sequences of bovine SRY gene and vector pET-28a(+),primers that contain restricted inner enzyme EcoRâ… ,Xhoâ… sites were designed.The vector pMD18-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 pMD18-T/SRY were digested by restricted inner enzyme EcoRâ… ,Xhoâ… and products digested were linked by T4 DNA ligase emzyme to construct vector pET-28a/SRY.The vector pET-28a/SRY was tansformed to E.coli BL21(DE3) and analysis of the vector sequence was done by bioloyical company.The result of analysis suggested the vector pET-28a/SRY was obtained sucessfully.E.coli BL21(DE3)containing pET-28a/SRY was induced by IPTG to express SRY protein of bovine,while the procuct expressed was analysized by SDS-PAGE and the 28 kDa band was observed in PAGE.These studies implement prokaryotic expression of SRY gene with modern molecular organism technique,procured complete SRY proteinum,depuration and identification.Through above reseach,offered very important valuable data for eukaryotic expression,resistance analysis and triturate diagnostic reagent,to establish important hylic groundwork for the constitution and function study of SRY gene in future.