Molecular Identification,Tissue Expression And Polymorphism Analysis Of Hu Sheep Growth-related Genes ATF4,RAC1 And RAN

The breeding and production of Hu sheep plays an important role in the production of meat sheep in China.The ATF4,RAC1,and RAN genes are important genes related to sheep growth.This study selected Hu sheep as the experimental material and cloned the ATF4,RAC1,and RAN genes of Hu sheep through c DNA end rapid amplification technology.Next,the DNA and protein sequence characteristics of the three genes were analyzed using bioinformatics tools.Finally,real-time fluorescence quantitative PCR method was used to analyze the expression of these three genes in different tissue parts of Hu sheep.The main experimental results of this study are as follows:Activating transcription factor 4(ATF4)belongs to the activating transcription factor/ cyclic AMP response element-binding protein family of basic region-Leucine zipper(Bzip)transcription factors.In this study,the full-length c DNA of ATF4 gene of Hu sheep was obtained by RACE-PCR.The full-length of ATF4 gene was 1219-bp,encoding 353 amino acids.In this study,the c DNA end rapid amplification method was used to obtain the full length of the c DNA sequence of the ATF4 gene of Hu sheep.The total length is 1219-bp,which encodes 353 amino acids.The protein encoded by this gene has high homology of 99.83%,97.87%,96.14%,and 94.95%with the ATF4 protein of goat,antelope,Canadian deer,and buffalo,respectively.This protein has a Basic leucine zipper(b ZIP)domain of b ZIP transcription factors: a DNA-binding and dimerization domain.Computer-aided analysis showed that the genome sequence of the gene contains 2 exons and 1 intron,a total of 1154-bp.Through further calculation and analysis,it was found that there were 7 mutation sites in the coding region of Hu sheep ATF4 gene.Analysis of tissue expression profile revealed that the ATF4 gene in Hu sheep strongly expressed in muscle and fat and moderately expressed insmall intestine,fat,and spleen,and moderately expressed in lungs,kidneys,small intestine,liver,heart,and spleen.The Ras-related C 3botulinum toxin substrate 1(RAC1)gene belongs to the RAC subfamily,and its promoter region is rich in GC bases and has the characteristics of housekeeping gene.In this study,the c DNA sequence of Hu sheep RAC1 gene was obtained by rapid amplification of c DNA ends.In full length,the c DNA of this gene is 904-bp in length and encodes 192 amino acids.The protein encoded by this gene has 100.00%,98.44%,95.26%,91.15%,90.62%,89.58%,88.36% homology with the RAC1 protein of Japanese sea cucumber,fruit fly,zebrafish,bony fish,Pacific white shrimp,human,and mosquito butterfly,respectively.This protein has a domain of Ploop＿NTPase domain superfamily.Through further calculation and analysis,it is found that the genome sequence of Hu sheep RAC1 gene contains 6 exons and 5introns,a total of 2555-bp.Further comparison and analysis of the sheep gene database revealed that there were 10 mutation sites in the gene coding region of the Hu sheep RAC1 gene.The tissue expression profile analysis showed that the RAC1 gene of Hu sheep is highly expressed in the spleen,fat,and kidneys,moderately expressed in the lungs,and weakly expressed in muscles,liver,heart,and small intestine.Ras.Related nuclear protein(RAN)gene is the most abundant small G protein in eukaryotic cells,which contains 216 amino acids and molecular weight of 24 kda.In this study,the RACE-PCR method was used to obtain the full-length c DNA sequence of the Hu sheep RAN gene.The full-length c DNA of the gene is 1041-bp and encodes216 amino acids.The Hu sheep RAN protein has high homology of 87.04%,93.98%,91.59%,100%,89.35%,90.45%,and 89.50% with that of human,house mice,bony fish,salmon lice,mussels,yellow croaker and fruit fly,respectively.This protein has a domain of P-loop＿NTPase domain superfamily.Through further calculation and analysis,it is found that the genome sequence of the gene contains 4 exons and 3introns,a total of 16454-bp.Further comparison and analysis in the sheep gene database found that there were 8 mutation sites in the coding region of Hu sheep RAN gene.Analysis of tissue expression profile revealed that the RAN gene of Hu sheep is highly expressed in muscles,liver,it is strongly expressed with muscle and fat and moderately expressed kidneys,lungs,and heart.These experimental results lay a molecular foundation for further improving the growth traits of Hu sheep using ATF4,RAC1 and RAN genes in the future.