| Cis-9,trans-11-conjugated linoleic acid(c9,t11-CLA)is an important nutritional component in milk.Stearoyl coenzyme A desaturase(SCD1)is an important desaturase expressed in the mammary gland of dairy cows.Most of the c9,t11-CLA is derived from SCD1 catalyzed trans 11-octadecenoic acid(TVA)in mammary epithelial cells.In this study,immortalized dairy cow mammary epithelial cell line MAC-T was selected as the research object.CAY group(SCD1 dysfunction model),TVA group and Control group were set up,with3 replicates in each group.The relative m RNA expression of SCD1,TVA accumulation and c9,t11-CLA synthesis were determined by real-time PCR and gas chromatography,respectively.The transcriptomics and proteomics were used to analyze the transcription differences among the groups,and finally SCD1 related genes were screened out.The results are as follows:Experiment 1: MAC-T cells were treated with 10 n M CAY((SCD1 inhibitor))for 12 h and50μM TVA for 2.5 h.The results showed that the relative expression of SCD1 m RNA in CAY group was significantly lower than that in Control group and TVA group(P < 0.01);the addition of TVA significantly increased the expression of SCD1 m RNA compared with Control group(P< 0.01).The cumulative amount of TVA in CAY group was significantly higher than TVA group(P < 0.01),while the synthesis of c9,t11-CLA in CAY group was significantly lower than TVA group(P < 0.01).Experiment 2: 651 differential expressed genes were obtained by transcriptome analysis,CAY/Control had 456,CAY/TVA had 468,and TVA/Control had 25.The identified differentially expressed genes were analyzed by functional annotation and pathway analysis.The pathways related to energy metabolism and fatty acid metabolism included glycosphingolipid biosynthesis,fat digestion and absorption,linoleic acid metabolism,carbohydrate digestion and absorption.About 50 different genes such as PLPP3,CYP2S1,PCK2,CYP,EHHADH were involved.Experiment 3: Through TMT-labeled proteomics analysis,1326 differential proteins were found;CAY/Control had 1256,CAY/TVA had 625,and TVA/Control had 68.The pathways related to energy metabolism and fatty acid metabolism are glycolysis,pentose phosphate pathway,amino acid and nucleotide glycometabolism pathway,fatty acid coenzyme a biological process,fatty acid extender activity and fatty acid synthase activity functional pathway,lipoprotein catabolism and sphingolipid catabolism,involving the main differential proteins TPI1、PGAM1、GALT、PGM1、PFKL、PKM,etc.In this study,we successfully constructed the MAC-T cell model of SCD1 dysfunction,which proved that SCD1 deletion could inhibit the synthesis of c9,t11-CLA in cow mammary gland.In addition,about 50 SCD1-related genes were screened in transcriptome and 61SCD1-related proteins were screened in proteomics,which are mainly involved in the pathways related to energy metabolism and fat metabolism. |
