Cloning Of Sheep Bone Morphogenetic Protein Receptor 1b Gene And Analysis Of Its Expression In Different Tissues Of The Sheep

Purpose: Hu sheep is an excellent breed of meat sheep in China,which has the characteristics of prolificacy,and the BMPR-1B gene(Fec B mutation)is the main gene for prolificacy Hu sheep.In order to further study the candidate gene BMPR-1B for prolificacy in Hu sheep,this study first screened the highly efficient breeding ewes carrying the homozygous Fec B Gene Mutation by detecting the RFLP polymorphism of Fec B Gene;using the mutation homozygous individuals as materials,the BMPR-1B coding sequence was cloned,and the BMPR-1B protein was prokaryotically expressed and purified,By Western blot technology,the expression profile of BMPR-1B protein in different tissues of the homozygous ewe was detected.This experiment provides a theoretical basis and experimental basis for the rapid detection method for detecting Fec B gene mutations,and also provides a factual basis for improving the efficiency of breeding new breeds of sheep for meat in production.Method: Hu sheep was used as the experimental material,and the Fec B genotype of Hu sheep was selected for verification.The Fec B gene was amplified using Hu sheep blood genomic DNA as a template,and the PCR products were analyzed by RFLP.Ava Ⅱ restriction endonuclease was used for PCR amplification.The amplified product was digested,and the Fec B genotype was determined based on the digestion results.In order to further study the BMPR-1B candidate gene for prolificacy Hu sheep,the ovarian c DNA of Hu sheep obtained by reverse transcription was used to clone the sequence of the prolificacy gene BMPR-1B,construct a prokaryotic expression vector,and express and purify the BMPR-1B protein in vitro.In order to detect the expression profile of BMPR-1B protein,the proteins from different tissues of Hu sheep’s ovary,ear,kidney,skeletal muscle and bone were extracted,and the expression level of BMPR-1B protein was analyzed by Western blot.In addition,the physicochemical properties,affinity/hydrophobicity,transmembrane domain,phosphorylation site,functional domain,tertiary structure and other aspects of Hu sheep BMPR-1B gene sequence were analyzed by bioinformatics.Results:(1)The genomic DNA extracted from the blood of Hu sheep was used as a template for amplification,and the PCR amplified product was analyzed by RFLP using Vpa K11 B Ⅰ(Ava Ⅱ)restriction endonuclease.The digested product was identified as two bands.It means that the Fec B gene of Hu sheep is mutated at 746,which proves that the Fec B genotype of Hu sheep was selected as homozygous mutant BB.(2)Using Hu sheep ovary as material,reverse transcription PCR to obtain BMPR-1B gene fragment,clone BMPR-1B gene,construct prokaryotic expression vector,express and purify recombinant protein in vitro,and obtain BMPR-1B recombinant protein concentration of 995 μg· m L-1.The successful expression of recombinant protein was verified by Western blot technology.Extract five kinds of whole proteins from Hu sheep’s ovary,ear,kidney,liver,and skeletal muscle.Western blot analysis of BMPR-1B protein expression profile revealed that BMPR-1B protein is expressed in ovary,ear,kidney,and skeletal muscle.It is highly expressed in the ovary but not expressed in the liver,indicating that BMPR-1B protein is expressed differently in different tissues.(3)Bioinformatics methods analyze the physical and chemical properties of BMPR-1B sequence.BMPR-1B is a fat-soluble transmembrane protein and a kinase membrane receptor protein.Protein network interaction analysis showed that BMPR-1B interacts with multiple bone morphogenetic proteins(BMPs),interacts with multiple SMAD family proteins,and mainly participates in the TGF-β signaling pathway.Conclusion:(1)The Fec B Gene of Hu sheep was mutated at 746 by RFLP polymorphism method,which is a high-efficiency breeding BB-type ewe flock.To lay the foundation for further establishment of Fec B gene homozygous and efficient breeding BB type ewes.(2)The recombinant expression vector p ET30a(+)-GST-BMPR-1B was successfully constructed,and the recombinant protein BMPR-1B was induced to express and purified in the host E.coli BL21(DE3),and the purified protein was verified as the target protein by Western blot technology.It provides a theoretical basis for the basic research of sheep BMPR-1B.(3)Western blot analysis of the protein expression profile of BMPR-1B revealed that the expression of BMPR-1B protein was specific in the ovary,ear,kidney,and skeletal muscle of Hu sheep,which laid the foundation for the study of BMPR-1B protein function.(4)Prediction and analysis of the structure and function of Hu sheep BMPR-1B protein by bioinformatics methods.BMPR-1B is a fat-soluble transmembrane protein,a kinase membrane receptor protein,and multiple bone morphogenetic proteins(BMPs)and The SMAD family proteins interact and mainly participate in the TGF-βsignaling pathway.