Stydy On Polymorphism Of Five Genes Of Yanbian Yellow Cattle And Regulation Of UCP2 Gene On Differentiation Of Bovine Preadipocytes

This experiment using the method of candidate genes for combining modern genotyping technology,aims to explore the yanbian cattle fibroblast growth factor(FGF14)14,decoupling associated protein 2(UCP2),decoupling associated protein 3(UCP3),myosin heavy chain 3(MYH3),myosin heavy chain 8(MYH8)gene polymorphism and its effect on the growth characteristics,in order to yanbian cattle breeding and germplasm resources protection to provide scientific basis.A total of120 24-month-old Yanbian yellow cattle were selected as the research objects.DNA mixed pool sequencing and high-resolution melting curve(HRM)typing were used to investigate whether there was any correlation between different genotypes of the five candidate genes and growth traits of Yanbian yellow cattle,and genetic statistical analysis was conducted.The results showed that there was one mutation in each of the five candidate genes detected in Yanbian yellow cattle.There was A>G mutation in the third exon of FGF14 gene at site 631947,and the AG genotype was significantly correlated with height at withers,body weight,height at hip cross and rump length traits(P<0.05).There was a C>G mutation at position 53412568 in the 4th exon of UCP2 gene,and the CG genotype was significantly correlation to the height at back trait(P<0.05),and there was a significant correlation between CC genotype and body weight and abdominal perimeter traits(P<0.05).There was a C>T mutation at53443536 in the 5th exon of the UCP3 gene and the CC genotype is significantly correlated with chest circumference traits(P<0.05).There was a T>G mutation at29602748 in the 41 th exon of the MYH3 gene and the GG genotype was height at hip cross and hip width traits are significantly correlated(P<0.05).There was a C>T mutation at 29406042 in the 36 th exon of MYH8 gene,and there was a significant correlation between TT genotype and CT genotype in body weight traits(P<0.05)),at the same time,TT genotype was significantly correlated with chest circumference traits(P>0.05).UCP2 is a member of the decouple protein family,and is one of the key genes involved in the body energy balance and the regulation of lipid metabolism in the form of fatty acids.In this study,UCP2 was used as a candidate gene to explore the molecular genetic markers of Yanbian yellow cattle and the function of UCP2 gene in the differentiation process of Yanbian yellow cattle adipocytes.Firstly,induced differentiation and identification of Yanbian yellow cattle precursor adipocytes were performed to detect the expression of UCP2.Secondly,si RNA interference and overexpression vectors of UCP2 gene were constructed by molecular techniques and transfected into adipocytes.The effects of UCP2 gene on the growth and development of Yanbian yellow cattle precursor adipocytes were investigated by oil red O staining,triglyceride determination,RT-q PCR,CCK-8 and flow cytometry.The results showed:Yanbian cattle fat precursor cells morphological observation showed a long spindle,growing towards into fibrous form,oil red O staining showed that can be observed by dyed orange lipid drops into ring shape,into fat genes PPAR gamma,C/EBP alpha,LPL and FASN expression patterns show successful build yanbian cattle precursor cells differentiation model,The content of triglyceride was gradually increased,and the expression of UCP2 gene was firstly increased and then decreased,and the expression level reached the highest on the fourth day.The expression of UCP2 in UCP2 overexpression group was significantly higher than that in control group(P<0.001),and the expression of lipid-related genes PPARγ,C/EBPα,LPL and FASN were all increased,which played a positive role in lipid accumulation and triglyceride content.UCP2 interference significantly increased the expression of proliferation marker genes PCNA and Cyclin D1(P<0.001),and significantly increased the proliferation activity of cells(P<0.05).The UCP2 overexpression group significantly increased the expression of pro-apoptotic gene Bax(P<0.001),and significantly inhibited the expression of anti-apoptotic gene Bcl2(P<0.01).Similarly,the apoptosis detection results showed that the UCP2 overexpression group significantly promoted the apoptosis of cells(P<0.05).Finally,the expression of key genes in the JNK signaling pathway was detected,and the expression levels of both JNK1 and JNK2 in the UCP2 overexpression group were significantly increased on the 4th day(P<0.001),with no significant effect later(P>0.05).In conclusion,the five candidate genes selected in this study all have certain effects on the growth and development of Yanbian yellow cattle.The exploration of the candidate gene UCP2 in the differentiation process of Yanbian yellow cattle adipocytes provides basic reference materials for the function of UCP2 gene and meat quality gene screening.