Screening And Functional Analysis Of Polymorphisms Of GH And IGF-1 Gene In Miniature Pigs

Miniature pigs have become the most widely used non-rodent large-scale experimental animals in the biomedical field because of their short stature,high inbreeding coefficient,and ease of handling.They are also considered as human disease research model because of their physiological characteristics,anatomical features,and disease progression are similar to human.In recent years,the view that miniature pigs are the most suitable donor for xenotransplantation has become a consensus in the international transplantation field.Therefore,the studies on the development and utilization of miniature pigs have received widespread attention from the biomedical community and clarifying the formation mechanism of miniature pig dwarfism will provide theoretical support for its better utilization.The growth and development regulation of animals is affected by many factors,such as genetic factors,environmental factors,nutrition and hormones.Among them,the growth axis of GH-IGF-1 plays a crucial role in the growth of organisms,and a large number of heritable mutations have been accumulated between large pigs and miniature pigs under the long-term influence of these factors.Therefore,it is of great significance to elucidate the mechanism of GH and IGF-1 gene mutations.In this study,the Chinese abundant miniature pig and local large pig resources were fully utilized.Firstly,the gene expression differences were detected among pigs with different bodysize and the SNPs in the coding region of GH and IGF-1 gene among them were screened and analyzed,expecting find out the SNPs that have potential functions.The results showed that the m RNA and protein expression levels of IGF-1 in large pigs were significantly higher than that in miniature pigs(P<0.05)at the same stage of growth(1 week after birth).A total of 17 SNPs were detected in pig GH gene,among which 6 have significant correlation with pig bodysize(P<0.001),they were g.237 bp A>G in intron 1,g.283 bp T>C,g.309 bp A>G and g.318 bp A>G in exon 2,g.540 bp A>G and g.544 bp A>G in intron 2,of which the 3 SNPs in exon 2 are missense mutations and encode 9th(p.Val9Ala),22th(p.Gln22Arg)and 25th(p.Asp25Gly)amino acids in the signal peptide regions of GH gene,respectively.In addition,based on the bodysize,the 3 missense SNPs formed a strong linkage,the CGG was the advantage haplotype of the large pigs,while the TAA is the dominant haplotype of miniature pigs,and the haplotype frequency is significantly different between large pigs and miniature pigs(P<0.001);In the same time,only one synonymous mutation(c.258A>G)of IGF-1 gene was screened between Large White pigs and Bama Xiang pigs,of which G is the dominant allele of large pigs,A is the dominant allele of minipigs and the SNP is located in the critical site for the binding of IGF-1 and IGF-1R.The synonymous mutation changed the m RNA secondary structure of the IGF-1 gene according to the prediction of the online bioinformatics software.In addition,statistical analysis of the codon usage frequency revealed that the frequency of codons was higher in large pigs,whereas the of codon usage frequency in miniature pigs was lower.Based on the 3 missense mutations in the coding sequence of the signal peptide of porcine GH gene,eight signal peptides were composed and the coding sequence of which were inserted into the p EGFP-N1 vector and fused with the EGFP gene to form recombination vectors p EGFP-N1-sp1-8.48 h after the p EGFP-N1-sp1-8 and p EGFP-N1 transfected into GH3 cells,the expression level of EGFP in the cells and supernatant were detected by western blotting.Results showed that compared to Ala,the Val at 9thamino acid of the signal peptide promoted protein synthesis,the Gln in22 th amino acid and the Gly in 25 th amino acid of the signal peptide promoted EGFP secretion.In order to elucidate the molecular mechanism of different signal peptides affecting protein synthesis,the stability of the m RNA was further tested and the results confirmed there were differences in the m RNA stability of the 8 signal peptide groups,which were consistent with both the secondary structure and the minimum free energy of the m RNA of the 8 signal peptides.The above results indicated that the missense mutations in the porcine signal peptide of GH gene significantly affect the protein synthesis and secretion,which is presumably caused by the changes in the secondary structure of the m RNA and consequently altered m RNA stability.Since only one synonymous mutation(c.258A>G)was screened in the exons of pig IGF-1 gene and the mutation altered the m RNA secondary structure of the gene,the IGF-1 expression vectors with G allele and A allele were constructed and transfected into MC-3T3 cells.Results showed that the expression level of IGF-1 with allele G was significantly higher than that with allele A at both transcriptional and translational levels(P<0.05).Then the effect of the synonymous mutation on m RNA and protein stabilities were further detected through the Act D and CHX interference,the results confirmed that the stabilities of IGF-1 with G allele is significantly lower than the IGF-1 with allele A,which were consistent with the secondary structure and minimum free energy of the m RNA that encoding the C region of the IGF-1 gene.Since this synonymous SNP is located in the key region of IGF-1 binding to IGF-1R,the binding affinity of IGF-1 and IGF-1R in pig fetal fibroblasts with GG and AA genotypes were examined by co-immunoprecipitation and immunofluorescence techniques and confirmed the IGF-1 with GG genotype binded more IGF-1R than IGF-1 with AA genotype compared to total IGF-1R.To reveal why this mutation affects the binding affinity of IGF-1 to IGF-1R,the differential immunodetection methods were used to detect the effect of this synonymous mutation on the conformation of the protein by a conformation-sensitive IGF-1 antibody.The results showed there was a difference in conformation of IGF-1 protein between the two genotypes,suggesting the synonymous mutation c.258A>G changed the conformation of IGF-1 and thus affected its binding affinity to IGF-1R.The results in this study resolved the function of the SNPs in GH and IGF-1 gene,which are the key genes of GH-IGF-1 axis that involved in the formation of miniature pigs,the results may provide theoretical data for the exploitation and application of miniature pigs.