| With the rapid development of animal husbandry and the improvement of human living standards,consumers' demand for milk quality is gradually increasing,and the requirements for milk quality are also higher and higher.Milk quality traits are complex economic traits,which are regulated by multiple genes.With the advent of the post genomics era,screening and mining candidate functional genes related to milk fat traits and with significant genetic effects has become the main challenge for dairy cattle genetic and breeding researchers.Glycerol-3-phosphate acyltransferase mitochondrial(GPAM)gene,belonging to the GPAT gene family,was located on bovine chromosome 26q22.Studies have shown that GPAM gene catalyzes the first step of phospholipid and triglyceride(TG)biosynthesis.Based on the results of previous omics analysis,GPAM gene may play an important role in regulating milk fat metabolism.In this study,GPAM was used as a candidate gene to verify the function of GPAM and bovine lipid metabolism at the cell level.Meanwhile,the correlation between GPAM and milk fat metabolism was analyzed on the basis of animal model.Using crispr-cas9 Methods: Taking bovine mammary epithelial cells as the research object,the GPAM gene knockout cell line was constructed,and the GPAM gene overexpression and interference vector were constructed.The m RNA and protein expression of the target gene after overexpression,interference and knock-out were detected respectively,and the contents of triglycerides,cholesterol and fatty acids in the cells were measured To verify the effect of GPAM gene on milk fat metabolism related genes at protein and protein levels.The experimental results show that: 1 After GPAM gene knockout,the expression of m RNA and protein was decreased;the content of triglyceride and cholesterol in the knockout cells decreased;the content of triglyceride and cholesterol decreased after GPAM gene knockout;the detection results of fatty acid content in cells showed that the increase of GPAM expression could reduce the content of butyrate,and the decrease of GPAM expression could make butyrate and octanoic acid contain The results showed that GPAM gene knockout could decrease the expression of ACSL5,FABP3,HSL,PRSS2 and agpat4.In this study,bovine GPAM gene knockout mammary epithelial cell line was used as the research object,and transcriptome sequencing was performed to further verify the function of candidate genes and analyze the relationship between the candidate genes and milk fat metabolism.A total of 360 up-regulated genes and 570 down regulated genes were found in transcriptome sequencing;352 genes were enriched in 3196 GO terms,613 of which were significantly enriched,including 402 biological process GO terms,72 cell components GO terms and 139 molecular function GO terms After GPAM gene knockout,the down regulated gene ACAT2 can form a complex with the up-regulated gene hmgcs1,and the down regulated gene acs2 may inhibit the expression of acaca and ACAT2.The up regulation of fatty acid metabolic pathway leads to the up regulation of pyruvate and pyruvate metabolic pathways,and the fatty acid metabolic pathway is affected by metabolic pathways.At the same time,the transcriptome sequencing results showed that GPAM gene was also related to the regulation of cell ATP response and proton transport ATPase activity,suggesting that GPAM gene may be involved in mitochondrial energy metabolism and ATP function.The m RNA and protein expression levels of some genes were verified by fluorescence quantitative PCR and Western blot,and the results were consistent with the transcriptome sequencing results.In order to further study the function of GPAM in cells,we studied the metabolism of mitochondrial energy of candidate gene.The experimental results showed that: in GPAM gene knockout cell lines,the respiratory capacity of mitochondria in cells was decreased,and the basic glycolysis capacity of cells was decreased;meanwhile,the transport of Na + / K + in cells was affected;meanwhile,the content of mitochondria in cells was decreased after GPAM gene knockout Less.At the same time,GPAM knockout resulted in the decrease of the activities of important regulatory enzymes required for mitochondrial glycolysis,which eventually led to significant inhibition of mitochondrial glycolysis.At the same time,the decrease of GPAM content in knockout cells significantly inhibited the oxidative phosphorylation of mitochondria and the tricarboxylic acid cycle.These results indicate that GPAM knockout can affect mitochondrial function and energy metabolism of breast epithelial cells.Using crispr-cas9 gene editing technology,the GPAM gene knockout mouse model was established to verify the regulation of gene expression at the model animal level.The results showed that there were no significant changes in the growth,development and reproduction of GPAM knockout mice.The results of serological test showed that the serum triglyceride content of GPAM knockout mice was significantly lower than that of wild-type mice(P < 0.05),but the content of cholesterol in serum was not significantly changed.The results of serum triglyceride and cholesterol in GPAM gene knockout mice were significantly lower than those in control group(P < 0.05)The contents of triglyceride and cholesterol in mammary gland and adipose tissue of knockout mice were significantly lower than those of wild type mice in control group The results of enzyme-linked immunosorbent assay showed that the content of triglycerides in the milk of GPAM knockout mice was significantly lower than that of wild mice(P < 0.05);the fatty acid content of tissues in mice mammary gland tissues was detected,and it was found that the contents of palmitic acid,linoleic acid and arachidonate of knockout mice were significantly lower than that of wild mice These results indicate that GPAM gene plays an important role in the regulation of milk fat metabolism and fatty acid metabolism in mice.Based on the previous transcriptome analysis,this study used GPAM as a candidate gene to verify the effect of GPAM gene on the content of fatty acids,triglycerides and cholesterol in cells,and the regulation of genes related to milk fat metabolism at the cell level;and the GPAM gene knockout mouse model was constructed to further verify the regulatory effect of GPAM gene on milk fat metabolism at the animal level.Histological observation showed that the diameter of adipocytes in the adipose tissue of knockout mice was slightly smaller than that of wild-type mice,the content of intramuscular fat was significantly less than that of wild-type mice,the diameter of adipocytes in breast tissue of wild-type mice was slightly larger than that of knockout mice,and no obvious morphological changes were observed in other tissues.At the same time,the mitochondrial activity and energy metabolism of mammary epithelial cells were detected.It provides high-quality gene resources and theoretical basis for the selection of milk quality traits and the cultivation of new varieties.Based on the previous transcriptome analysis,this study used GPAM as a candidate gene to verify the effect of GPAM gene on the content of fatty acids,triglycerides and cholesterol in cells at the cell level.On the basis of transcriptome analysis,we understood the regulatory effect of GPAM gene knockout on the genes related to milk fat metabolism;and constructed a GPAM gene knockout mouse model,which was further developed at the animal level To verify the regulation of GPAM gene on milk fat metabolism.At the same time,the mitochondrial activity and energy metabolism of mammary epithelial cells were detected.It provides high-quality gene resources and theoretical basis for the selection of milk quality traits and the cultivation of new varieties. |
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