Expression Regulation Of TBC1D7 And GRB10 Genes And Correlation Between Two Genes And Growth Traits Of Guanling Cattle

Guanling cattle is an excellent local beef cattle breed resource in Guizhou Province.Studies have shown that its muscle protein contains a complete variety of amino acids,which are rich in essential amino acids and umami amino acids.The meat is tender and delicious,and it is a source of high quality protein,which is loved by consumers.However,problems such as slow growth rate and poor meat performance of Guanling cattle have affected its industrial development to a certain extent.Target protein of rapamycin(m TOR)signaling pathway is an important pathway involved in physiological functions such as metabolism,protein synthesis and cell growth.The TBC1 domain family member 7(TBC1D7)and growth factor receptor-binding protein 10(GRB10)are important members in the upstream and downstream of m TOR signaling pathway,respectively,which have been shown to be related to growth and development.In this study,Guizhou Guanling cattle were used as experimental subjects,TBC1D7 and GRB10 genes were selected as candidate genes,and single nucleotide polymorphisms(SNPs)in TBC1D7 and GRB10 genes were detected by direct sequencing method and bioinformatics analysis was conducted;Each mutation site was typed,8 growth traits were determined,and the correlation between the SNPs genotypes of the two genes and the growth traits of Guanling cattle was analyzed;Gene m RNA expression levels in 9 tissues of Guanling cattle were measured and analyzed in different periods;The eukaryotic expression vectors of TBC1D7 and GRB10 genes were constructed and transfected into Guanling bovine myoblasts to detect the expression levels of major genes involved in growth and development.By exploring the effects of TBC1D7 and GRB10 genes on the growth and development of cattle,it provides theoretical reference for the scientific selection and breeding of Guanling cattle.The main research results are as follows:1、A total of 5 SNPs were detected in Guanling bovine TBC1D7 gene,which were:g.12972T>C and g.12984A>G on exon 5;g.20538C>T and g.20577T>C on exon 6 and g.24807G>A on exon 8.A total of 5 SNPs were detected in GRB10gene.g.193768C>G and g.194018G>A on intron 11,g.194184G>A on exon 12,and g.207578G>A and g.207620G>C on intron 15,respectively;Bioinformatics analysis showed that g.12972T>C,g.12984A>G,g.20538C>T,g.20577T>C and g.194184G>A sites changed the m RNA secondary structure free energy;The relative molecular weights of Guanling bovis TBC1D7 and GRB10 proteins were 33904.44k Da and59857.50k Da,respectively.Both of them were hydrophilic and did not have signal peptides.Both of them had potential N-glycosylation sites,and the two proteins were significantly different in spatial structure.2、Each of the 10 SNP sites in the two genes had three genotypes,The number of effective alleles was higher,and the polymorphism information content was moderate polymorphism(0.25<PIC<0.5).The genotype distribution of all loci deviated significantly from Hardy-Weinberg equilibrium(P<0.05)byχ~2test;TBC1D7 gene g.24807G>A variant locus heterozygous(GA)individuals have significantly greater body steep length,chest circumference,and Posterior buttock circumference than mutant(AA)individuals(P<0.05).GRB10 gene g.194018G>A locus heterozygous(GA)individuals have significantly greater body steep length,chest circumference,and posterior buttock circumference than mutant(AA)individuals(P<0.05),g.194184G>A variant locus heterozygous The bust and posterior buttock circumference of GA individuals were significantly larger than those of wild type(GG)and mutant individuals(AA)(P<0.05).3、TBC1D7 gene was expressed in heart,liver,lung,kidney,brain,spinal cord,small intestine,longissimus dorsi and hindmuscle of Guanling bovine.The expression levels in liver and spinal cord were significantly higher than those in other tissues(P<0.01).The expression level in the lungs was the next highest.It’s also expressed to some extent in the brain,kidneys and small intestine.Expression of heart,dorsal longest muscle and hind leg muscle was significantly lower than that of other tissues(P<0.01);GRB10 gene was expressed in all 9 tissues of Guanling cattle.The expression level in liver was significantly higher than that in other tissues(P<0.01),followed by lung,and to some extent in kidney,small intestine,heart and spinal cord.The expression levels in longissimus dorsi muscle,hind muscle and brain were significantly lower than those in other tissues(P<0.01).4、The CDS regions of TBC1D7 and GRB10 genes were successfully cloned,and pEGFP-N3-TBC1D7 and pEGFP-N3-GRB10 eukaryotic expression vectors were constructed.After overexpression of TBC1D7 gene in bovine myoblasts,it was found that GRB10 gene expression increased significantly,IGF1 gene expression decreased significantly,and IGF2 gene expression decreased slightly;After overexpression of GRB10 gene,it was found that the expression of TBC1D7 gene increased significantly,while the expression of IGF1 and IGF2 genes did not change significantly.