Analysis Of Promoter Activity Of CGI-58 Gene In Qinchuan Beef Cattle

CGI-58 is an accelerating factor of adipose triglyceride lipase(ATGL),which plays a key role in triglyceride metabolism.In this study,the blood,tissue and cell samples were collected from Qinchuan beef cattle.The transcriptional regulation mechanism of CGI-58 gene and its association with beef quality were investigated from the level of molecular level,cell and DNA-protein interaction.In this study,the role of CGI-58 gene in lipid metabolism was studied at the transcriptional level,and its relationship with meat quality was discussed.It was helpful for the improvement of meat quality.This study has the following results:1.The tissue expression profiles of CGI-58 Qinchuan beef calf and adult cattle were constructed by qRT-PCR using the cDNA of each tissue at different growth stages.In this study,the expression of CGI-58 gene and ATGL gene were detected in muscle and adipose tissue of 6 months and 18 months Qinchuan beef cattle.It was found that CGI-58 gene and ATGL gene showed an approximate trend with the change of age.2.The relative expression of CGI-58 and ATGL genes was detected and identified in Qinchuan beef cattle pradipocytes.It was found that they were expressed during days 0~10 after inducing.3.The transcription initiation site of CGI-58 gene was determined by 5'rapid-amplification of cDNA ends(5'RACE).The fragment of the 5-'regulatory region of CGI-58 gene was amplified by PCR.The bioinformatics software predicted the presence of a CpG island and 11 important transcription factors in the amplified fragment.The recombinant plasmids of CGI-58 were transfected into preadipocyte.The key transcriptional regulatory regions of CGI-58 were determined and the effect of transcription factors could be determined.4.The mutations of ZEB1 transcription factor binding site in CGI-58 gene can significantly improve the promoter activity of CGI-58 gene in Qinchuan beef cattle by site-directed mutagenesis(P<0.05),and proved by gel shift assays.The ZEB1 transcription factor can specifically bind to the CGI-58 gene DNA sequence.5.A novel single nucleotide polymorphism(SNP)g.14451079A> C in the 5'-regulatory region of CGI-58 gene of Qinchuan beef cattle was detected by direct sequencing.The results showed that in the Qinchuan beef cattle population,the SNP g.14451079A> C site was associated with the meat quality(P<0.01)and had a significant effect on the promoter activity(P<0.05).This study shows that the important transcription factor ZEB1 in the lipid-regulating network has transcriptional regulation of CGI-58 gene in Qinchuan beef cattl.Moreover,the single nucleotide polymorphism of the CGI-58 gene promoter region g.14451079A>C site was associated with the meat quality and had a significant effect on the promoter activity.