The Effects Of GH And GHR Genes On The Growth Performance Of Guizhou Local Cattle

Guanling cattle,Sinan cattle and Weining cattle are excellent local breeds in Guizhou Province.Some studies show that they all contain 8 kinds of amino acids necessary for human body.They are a kind of dietary meat with high protein content and low fat content,which are popular among people.However,they also have problems such as small physique and slow growth speed,which to some extent affect their development.In this study,Guizhou local cattle(Guanling cattle,Sinan cattle and Weining cattle)were selected as the main research objects,and growth hormone(GH)and growth hormone receptor(GHR)were selected as candidate genes.The polymorphism of GH and GHR genes was detected.By the detection of polymorphism of GH and GHR genes and the correlation between SNPs of the two genes and growth traits of Guizhou local cattle,coupleed with analysis of their expression,we have the expression level of growth related factors and cell proliferation rate were detected from the cell level.Also,The CDS region of GH and GHR gene was cloned.The eukaryotic expression vector of GH and GHR gene was constructed and transfected into the primary skeletal muscle cells of Guanling cattle.It aims to explore the mechanism of the influence of GH and GHR genes on the growth and development of Guizhou local cattle,and provide a scientific basis for the breeding and utilization of Guizhou local cattle.The research results are as follows:1.Two SNPs were detected in exon 5 of GH gene of Guizhou local cattle(Guanling cattle,Sinan cattle and Weining cattle),namely Exon5-G1570 C and Exon5-A1720 C.Eight SNPs were detected in GHR gene,which were Exon9-C162 T,Exon9-C201 T,Exon9-G243 C,Exon9-G383 A,Exon9-A495 T,Exon9-C622 T,Exon9-C642 T and Exon9-A650 C,respectively.Sinan cattle had no Exon9-G243 C.Further correlation analysis showed that there was no significant difference in Exon5-G1570 C of GH gene in Guanling cattle(P>0.05),but in Exon5-A1720 C genotype CC,there was significant difference in body height,cross height,chest circumference and abdominal circumference for AC and AA(P<0.05),and in oblique length and ischial end width for AC or AA(P<0.05);in Sinan cattle,there was significant difference in body height for GC and CC in Exon5-G1570 C genotype GG(P<0.05),while the CC of Exon5-A1720 C genotype had significant effect on AA(P<0.05).In Weining cattle,the results showed that the CC of Exon5-G1570 C genotype had significant effect on GG,while the CC of Exon5-A1720 C genotype had significant effect on AA(P<0.05),while the CC of AC genotype had significant effect on AA(P<0.05).There was no significant difference between Guanling cattle and Sinan cattle in GHR gene mutation sites,but in Weining cattle,GC of Exon9-G243 C genotype was significant to GG and CC in chest circumference(P<0.05),AA of Exon9-A650 C genotype was significant to CC in body oblique length and ischial end width(P<0.05),AA of chest depth was significant to AC and CC(P<0.05),and the other six SNPs were not significant.The results showed Exon5-G1570 C,Exon5-A1720 C,Exon9-G243 C and Exon9-A650 C these four SNPs had significant effects on the growth characteristics of local cattle in Guizhou,suggesting that they could be used as candidate molecular markers for the growth and development of local cattles in Guizhou.The genetic analysis showed that except Exon5-A1720 C in three cattle breeds and Exon9-G243 C in Weining cattle,the other SNPs did not deviate from hardy-Weinberg equilibrium,which may be caused by genetic drift,mutation or breeding measures.Bioinformatics analysis showed that the relative molecular weight of GH and GHR protein was 27385.45 or 70872.71?respectively,and the theoretical isoelectric point was 6.90 or 4.60?both of them were transmembrane proteins?with high hydrophilicity and stability.Except the secondary structure of Exon9-G383 A m RNA,there were changes happend in other SNPs before and after the mutation.2.The m RNA of GH and GHR genes were detected in the related skeletal muscle tissues(psoas muscle and dorsal longest muscle)of Guizhou local cattle,and the expression trend tended to be the same.GH and GHR genes of the three cattle breeds were highly expressed in psoas muscle.which indicated that GH and GHR genes were related to the growth and development of Guizhou local cattle psoas muscle.3.The CDS regions of GH and GHR genes of Guanling cattle were cloned successfully,and the eukaryotic expression vectors of p EGFP-N3-GH and p EGFP-N3-GHR were constructed.by caomparison of the NCBI and to the sequencing results,it was clear that the eukaryotic expression vectors of p EGFP-N3-GH and p EGFP-N3-GHR were fully constructed,which provides experimental basis for cell transfection.4.The primary skeletal muscle cells of Guanling cattle were successfully isolated and cultured and the recombinant plasmids p EGFP-N3-GH and p EGFP-N3-GHR were transfected.The growth factors IGF-1 and IGF-2 were detected by q RT-PCR and CCK-8 respectively before and after transfection.The results showed that over expression of GH and GHR genes could significantly increase the m RNA expression of IGF-1 and IGF-2,in four periods(6h,12 h,24h,48h)The proliferation rate of the overexpression group was significantly higher than that of the blank group(P<0.01).It is suggested that GH and GHR genes can promote the proliferation and differentiation of the skeletal muscle cells of Guanling cattle,and have a positive regulatory effect on them.