The Analysis Of Core Promoter And Expression Regulation Of Porcine IGF1 Gene

Insulin-like growth factor system is composed of insulin-like growth factor-?(IGF-?)and insulin-like growth factor-?(IGF-?), receptor and insulin-like growth factor binding proteins(IGFBPs) and insulin-like growth factor combining protease,etc. IGF is mainly secreted by the liver cells in the systemic circulation,it is controlled by growth hormone(GH) that secreted from pituitary. Located in the center of the growth axis,growth hormone is the main regulating animal growth hormones. Many studies have shown that the GH for animal growth regulation after birth is mediated by insulin-like growth factor 1(IGF1). Evidences also proved that IGF1 promote tissue growth and participate in metabolic activity of the organization in vivo and in vitro. IGF1 is an important control factor of the body's growth, development and metabolism, as well as mediates GH to play its role on growing activity. At present, according to the IGF1 gene of livestock and poultry on the effects of growth and carcass traits was carried out very quickly, the results of the study will help to further understand the mechanism of the IGF1 and make its application in animal genetics and breeding.More previous studies focus on the level of genome DNA polymorphism and the relation between growth and meat traits, and they found some polymorphism loci associated with production performance. However, the difference of individual character, gene expression level of the research may find that the main cause of character differences rather than the expression the differences of protein.Given the IGF1 play an important role in the process of animal life activities, identifying its mechanism will help human to understand and adjust life process is of great significance. This study used the technology of analysis of promoter activity, Ch IP, the overexpression, q RT-PCR et al.The results confirmed that the IGF1 gene in the porcine fetal fibroblast cells can be transcriptionally regulated through poly(d A:d T) tract, C/EBP? signaling pathways. The results will help to further understand the molecular regulation mechanism of IGF1, help tounderstand the mechanism of pig growth and development, and then to find the molecular basis of improving the efficiency of pig growth.The main results were as follows:1.Got the IGF1 gene cloned from +1(the first base of m RNA sequence published online as+1) backward to 2775 bp of the 5' regulatory region.2.Successfully built the porcine IGF1 gene promoter, and built six reporter gene recombinants and transfected with the porcine fetal fibroblast, the results showed that the region of-381/+2 had the highest activity and it was speculated to present the cis-acting element.3. Successfully constructed IGF1 core promoter containing different genotypes poly(d A: d T)tract, the reporter gene recombinants were transfected in the porcine fetal fibroblast, the results showed that the activity of IGF1 core promoter containing 9-T?10-T?11-T genotype poly(d A: d T) tract increased in turn, while the activity of IGF1 core promoter containing12-T genotype poly(d A: d T) tract decreased significantly and was much less than that the activity of IGF1 core promoter containing 11-T genotype poly(d A: d T) tract.4.By transfected with pc DNA3.1-C/EBP? and IGF1 core promoter containing different genotype poly(d A: d T) tract, the result showed that the activity of IGF1 core promoter co-transfected with C/EBP? was prominently lower than that the activity of each the same genotype IGF1 core promoter without co-transfected with C/EBP?, which revealed that the transcription factor C/EBP? could repress the expression of IGF1 core promoter containing different genotype poly(d A:d T) tract. In addiction, the activity of IGF1 core promoter co-transfected with C/EBP? was similar, which revealed that different genotype poly(d A:d T) tract might affect the regulation of C/EBP? on the expression of IGF1, and the activity of IGF1 containing 11-T genotype poly(d A:d T) tract reduced the most prominent,and the 10-T?9-T reduced less, the 12-T reduced least, the level of the change was similar to the effect of different genotype poly(d A:d T) tract acting on the expression of IGF1.5.The result of Ch IP showed that the transcription factor C/EBP? could bind to the site-286 of the upstream of porcine IGF1,which made the expression of IGF1 repressed in the porcine fetal fibroblast,while the result of analysis of IGF1 promoter revealed that theregion of-381/-100 was the core promoter which upregulated the expression of IGF1.Thereby, the transcription factor C/EBP? was a repressor of the expression of IGF1.