| Miniature pigs have been developed and utilized as a experimental model due totheir small body length, following the mice, dogs and other animals in the researchfield of biomedical research or human disease controlling. Miniature pigs and largepigs exist mutations on the way of genetic breeding under different growingconditions and different economic conditions cultivation, To clarify the mechanism ofdwarf traits, the research on the growth and development characteristics of miniaturepigs and producing standardized mini-pigs has become the main target research areas.And in a variety of complex interactions, the growth is mainly regulated through theneuroendocrine axis of growth hormone and insulin like factors (GH-IGF axis). Atpresent, there were more research about IGF-1. As a result, it should be highlyconcerned that the structure and function of IGF-1R gene in the growth anddevelopment of the metabolic process.In this study, Xizang Tibetan pigs, Bama Xiang pigs are treated as subjects, JumuNo.1White pigs, Large White pigs and Northeast wild boars are regarded as controls,IGF-1R gene is choosen as a candidate gene. In order to analyze differences in laws,Western blotting and Immunohistochemistry approaches were used to analyze theexpression of IGF-1R. To investigate the reason for the difference of proteinexpression in pigs with different body length, Allele-specific PCR methods were usedto analyze the promoter,3’UTR and partial coding region of the IGF-1R gene in pigswith different body length. The transcription factor binding sites of the promoter andmiRNAs binding sites of3’UTR were predicted and analyzed by online software. Forthe SNP sites screened in the3’UTR, dual luciferase reporter gene vector thatcarriying different miRNA binding sites were constructed and then transfected them toPK cells, the luciferase activity was detected.There are significant difference of the IGF-1R protein expression in differentdevelopmental stages and different varieties of pigs. The IGF-1R gene can express in different tissues (liver, muscle, bone)of large pigs and miniature pigs in differentdevelopmental stages (born stage, weaning stage, adult stage). The expression ofIGF-1R in large pigs was significantly higher than that of miniature pigs (p<0.05).The details are as follows: In liver, Xizang Tibetan pigs IGF-1R protein contentincrese to maximun first then decreased, Jumu No.1White pig was as the same. While,Bama xiang pig was gradually increased; In muscle, the content distribution had thesame rules, both showed a gradual increase with age decline; The distribution of bonetissue is consistent between pigs, protein content reaches a maximum at birth stage,then showed a recovery trend after the first fall.Transcription factors of Sp1, MZF1, CdxA, AMl-1a, Nkx-2were detected in thepromoter region. Two polymorphisms in the promoter region of the gene, T-1165Cmutation and C-1440G mutation. For the two SNPs of the3’UTR region of IGF-1Rgene, TC is the dominant haplotype of miniature pigs while that for large pigs is CT,IGF-1R gene coding region was also detected, A C/T mutation was detected in theintron19of the gene. Results show that: different genotypes in pigs, more SNP locican affect pigs as long trait genetic markers.A large-scale pig and miniature pigs dual luciferase reporter gene vectors in the3’UTR of IGF-1R gene were constructed, indicating that the two SNP sites had lessaffection for post-transcriptional regulator.Through this study, the SNPs detected between pigs with different body lengthresulted in the expression difference of IGF-1R gene, provding genetical resourcesand theoretical basis for in-depth study of the growth and development of theminiature pigs, mechanism development and applications of molecular biologycharacteristics. |
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