The Effect Of ACAA2 Gene On Sheep Preadipocyte Differentiation And The Creation Of Related Breeding Materials

Adipose tissue is an endocrine organ with energy storage and secretion of hormones.If adipose tissue is overgrown and developed,that means excessive fat deposition it not only can cause human obesity symptoms,such as diabetes,fatty liver,gallbladder disease,high blood pressure,Endocrine disorders and other related diseases,but also can reduce the animal carcass lean meat rate in animal husbandry production which will have a certain impact on meat quality.Preadipocytes are specialized cells that can proliferate and differentiate into adipocytes.Body fat deposition is the result of the proliferation and differentiation of precursor adipocytes.Therefore,the mechanism of regulation of preadipocyte differentiation can provide theoretical basis for controlling body fat deposition and provide a reference for livestock breeding and animal production.ACAA2(acety1-Coenzyme A acyltransferase2)is a key enzyme in the fatty acid oxidation step,which catalyzes the oxidation of fatty acids.Studies have shown that ACAA2 gene is a key gene involved in the lipid metabolism pathway,occupied the node position in the pathway,and initially established the gene as the key gene affecting lipid metabolism.Previous studies were based on the study of the polymorphism of the ACAA2 gene at the molecular level.It is not clear that the mechanism of ACAA2 how to regulates the differentiation of preadipocyte into adipocytes.In order to explore its role in cell differentiation,ACAA2 gene was used to investigate the role in regulating the differentiation of sheep preadipocyte by us:ing CRISPR/Cas9-mediated gene knockout and gene over expression.Sheep primary preadipocyte was used as the media.The main research contents are as follows:1.The coding region of ACAA2 gene was amplified by PCR in vitro.The results of agarose gel electrophoresis showed that the band size was consistent with the expected result.The homology of NCB2 was 100%.The results of bioinformatics analysis showed that the ACAA2 gene was encoded by 486 amino acids,and the theoretical isoelectric point was 5.06.It was an acidic protein,which was a hydrophobic protein.The ACAA2 protein has no signal peptide,which contains four phosphorylation sites,four N-glycosylation sites,and 10 O-glycosylation sites.The protein secondary structure contains 15 alpha helix,27 Beta turn,18 Turn turns,and 16 Coil Coil Spiral.Phylogenetic tree analysis showed that ACAA2 protein was closely related to goat evolution.2.The activity validation and construction of ACAA2 gene knockout vector:The target gene was ligated with pcDNA3.1 over expressing vector and then digested with EcoR I and Kpn I to obtain the ACAA2 target band.The results of the sequencing of ACAA2-pcDNA3.1 suggesyed the over expression vector was constructed successfully.The results showed that the mutant genome was detected by the endogenous activity of the gRNA target.The bands of 740bp and 454bp were successfully cleaved by T7E1,which indicated that the target had both exogenous and endogenous activities,which met the requirements of later experiments.The highest activity of ACAA2-Cas9/gRNA knockout vector was transfected into sheep ear fibroblasts.When the mass ratio of plasmid to liposome was 1:1,1:2.5,1:3,the transfection efficiency was 30%,50%,75%,then determined the best transfection conditions was 1:3.3.ACAA2 gene knockout and over expression affect the differentiation of Preadipocyte:we obtained higher purity of preadipocyte from adipose tissue of the 3-month-old sheep tail and subcutaneous,whose growth curve was "S" type,basically in line with in vitro Growth and increment of fat cells.Preimplantation of adipose cells after INS + DEX + IBMX induced differentiation,the number of intracellular lipid droplets increased,and the emergence of the iconic "alicyclic." Induced differentiation of cells by oil red O stained orange,preadipocyte were successfully induced into fat cells.ACAA5-gRNA5 and ACAA2-pcDNA3.1 were transfected into sheep preadipocyte for 48h,and induced by differentiation medium.After oil red 0 staining,the number of lipid droplets was significantly lower in ACAA5-gRNA5 transfection group after 6d and 10d than the control group,induced 4d and 10d,the number of lipid droplets increased significantly in the ACAA2-pcDNA3.1 group after 6d and 10d inducted.The expression levels of PPARy in ACAA2-gRNA group and ACAA2-pcDNA3.1 group were respectively 1.00 ± 0.01,0.42 ± 0.12 and 1.5 ± 0.27.The expression level of LPL was respectively 1.00±0.10,0.37 ± 0.15,1.47 ± 0.16;AP2 The expression level of the knockout group was significantly lower than that of the control group,and the expression level of the over expression group was significantly up-regulated compared with the control group.Indicating that knockout ACAA2,inhibit lipid droplets formation,inhibit the differentiation of preadipocytes;over expression of ACAA2,promote lipid droplets formation,promote preadipocyte differentiation.