Exonic Snps Detection Of Pig IGF1R Gene And The Influence Of IGF1R Intracellular Domain SNPs On Its Expression Levels

Bama minipig is the unique miniature pig breed in our country.As a dwarf breed,there are many characteristic genetic markers in its nurturing process,such as the microsomatia of morphological markers,which means that all parts of the body size are reduced in Bama minipig and are proportioned when compared with the Large pig.The microsomatia of the Bama minipig may be caused by the lack of growth hormone,and be regulated by the GHRH-growth hormone-IGF1 axis.IGF1 R is one of the cell membrane receptors in this axis and plays an important role in the signaling transmission and cascade.Only when IGF1 combines with IGF1 R,the extracellular signal could transduce to intracellular effectors and plays a central role in growth and development.Many studies have shown that the polymorphisms of IGF1 R can affect the growth traits,at the same time,it was reported that IGF1 R is the potential candidate gene for regulating pig growth and carcass traits after birth.Firstly,this study screened exonic SNPs which located in IGF1 R gene in Bama minipigs and Large pigs,analysed whether these SNPs influenced the m RNA transcription and protein translation.Between the Bama minipigs and Large pigs(Large White pig,Landrace and Duroc),there were 9 synonymous SNPs located in the IGF1 R gene exons,and they were g.267380 G>C,g.286723 T>G,g.288540 C>T,g.296721 T>C,g.310130 C>T,g.310196 C>T,g.312691 C>T,g.312757 C>T,g.329372 C>T,respectively.These 9 SNPs were all homozygous genotypes in these pig breeds.Through the linkage disequilibrium and haplotype analysis,these 9 SNPs formed a complete LD and two haplotypes,GTCTCCCCC haplotype was the unique haplotype in Bama minipigs and CGTCTTTTT was the unique haplotype in Large pigs.Through constructing two haplotype expression vectors of the pig IGF1 R intracellular domain(the haplotype of Bama minipig was CCCCC and named as pc DNA3.1(+)-My JKCF-BM,the haplotype of Large pig was TTTTT and named as pc DNA3.1(+)-My JKCF-LP).At 48 h post transient transfection,the My JKCF-LP m RNA expression in pc DNA3.1(+)-My JKCF-LP was significantly higher than the My JKCF-BM m RNA expression in pc DNA3.1(+)-My JKCF-BM.Using the actinomycin D to interfere with the RNA synthesis at 24 h post-transfection,the My JKCF-BM m RNA stability was more stabilized than My JKCF-LP.Through m RNA secondary structure prediction,the m RNA minimum free energy of the haplotype of IGF1 R intracellular domain in Bama minipig and Large pig were-497.30 kcal/mol and-495.20 kcal/mol,respectively.Therefore,the haplotype in Bama minipig was more stabilized than the haplotype in Large pig.Simultaneously,the m RNA secondary structure of IGF1 R intracellular domain has difference in these two haplotypes.By western blot and gray level analysis,at 48 h post transient transfection,the My JKCF-LP protein expression level of pc DNA3.1(+)-My JKCF-LP was significantly higher than the My JKCF-BM protein expression level of pc DNA3.1(+)-My JKCF-BM.Via codon preference prediction,Bland-Altman consistency analysis and Fisher exact test,we analyzed using-frequency and distribution characteristic of 29 codons which were influenced by 9 SNPs located in IGF1 R gene between Bama minipig and Large pig.The results showed that the codon usage bias of these 29 codons have different distributions between Bama minipig and Large pig,the low frequency codes were more likely appeard in Large pig,but on the contrary,the high frequency codes were more inclined to appear in Bama minipig.The above results indicate that exonic SNPs of intracellular domain of IGF-1R in Bama minipig and Large pig affect m RNA transcription and protein translation.Therefore,these SNPs regulate the expression of IGF1 R in different pig breeds.