| In this study,245 fresh milk samples(4:3:3 mixture)were collected from healthy first-trimester Holstein with similar weight and age in mid-to late-lactation(180-2 10 d),and those with somatic cell counts less than 200,000 cells mL-1 and extreme differences in milk fat percentage were selected after DHI measurement.Twenty-six samples were screened for extreme differences in milk fat percentage,with milk fat percentage greater than 4.4%in the high milk fat percentage group(HF group,13 samples)and milk fat percentage less than 3.0%in the low milk fat percentage group(LF group,13 samples).The samples were analyzed by metabolomics using ultra performance liquid chromatography-mass spectrometry(UHPLC-MS),and then the milk fat candidate genes corresponding to the significantly different metabolites of high milk fat were obtained by combined metabolism-transcriptomics analysis,and the key genes of milk fat metabolism were further identified by RT-qPCR.PDGFD gene overexpression and interference vectors were constructed,and the expression levels of triglyceride(TAG)content,lipid droplet secretion and genes related to milk fat synthesis were measured after transfection of dairy mammary epithelial cells(BMECs).The main findings were as follows.(1)In this study,metabolomic analysis of high and low milk fat rate milk samples yielded 877 metabolites;60 significantly different metabolites were obtained in positive and negative ion mode(P<0.05),25 up-regulated and 35 down-regulated;18 of these significantly different metabolites had significant ability to discriminate between high and low milk fat rates:DL-2-(acetylamino)-3phenylpropionic acid,PC(14:0e/9:0),1-methylguanine,glyceryl laurate,ethyl 1-(3-nitro-2thienyl)piperidine-4-carboxylate,N-phenylacetylglutamine,maltitol,5,6-dihydroxyindole-2-carboxylic acid,5-methyl-DL-tryptophan,Lysopa 16:0,epitestosterone,5-hydroxyindole-3-acetic acid,PA(16:0/18:3),delta-tocopherol,bile acid,L-histidine,cortisol,N-oleoylglycine.(2)PDGFD was screened as a key candidate gene for milk lipids by combined metabolic and transcriptomic analysis.mRNA expression of PDGFD gene was highest in mammary gland,which was highly significantly higher than intestine,liver,heart,kidney and ovary(P<0.01),and the expression level of PDGFD gene in mammary gland was highly significantly higher than other candidate differential genes BCAT1,HK2,TIAM1(P<0.01).(3)PDGFD gene overexpression vector was constructed to obtain pcDNA3.1-PDGFD recombinant plasmid.After successful transfection of BMECs,it was found that the content of TAG was significantly higher than that of the control group(P<0.05),and the content of secreted lipid droplets was also significantly higher than that of the control group;the expression levels of mRNA of milk lipid synthesis-related genes SREBP1,ACSL1,ACSS2,PLIN2,CD36 and XDH genes were significantly upregulated(P<0.05).This indicates that PDGFD genes can promote the synthesis of milk lipids in BMECs.(4)Interference with PDGFD gene revealed that the TAG content in BMECs was extremely significantly lower than that in the control group(P<0.01),and the lipid droplet content was also significantly lower than that in the control group;the mRNA expression levels of related lipogenic genes SREBP1,ACSL1,ACSS2,and PLIN2 were significantly down-regulated(P<0.05).It showed that interfering with PDGFD showed a significant inhibition of milk lipid synthesis in cows,and this inhibition effect was more obvious than the promotion effect of overexpressing PDGFD.In summary,the PDFGD gene plays a regulatory role in the metabolism of milk fat in Holstein cows,and the results of this study provide a valuable theoretical basis for milk fat synthesis in cows,as well as a reference for studying the lactation process of cows. |
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