The Relationship Between Genes Related To Energy Metabolism And Cis9,Trans11-conjugated Linoleic Acid Synthesis In Mammary Gland Of Dairy Cows

Cis9,trans11 conjugated linoleic acid(c9,t11 CLA)is an important nutritional component of milk.In the mammary gland of cows,Stearoyl Co A desaturase 1(SCD1)catalyzes the synthesis of c9,t11-CLA from trans 11 octadecanoic acid(TVA).The team’s previous research has shown that after the SCD1 gene is suppressed,the synthesis of c9,t11-CLA in bovine mammary epithelial cells(MAC-T cells)decreases,and energy metabolism related genes such as PGLS,PFKL,ALDOA,TPI1,GAPDH,PGK1,PGAM1,ENO1,PKM,LDHA,and LDHB are upregulated.Therefore,this experiment took SCD1 as the entry point,through cell test and animal verification test,using RNA interference(RNAi),real-time polymerase chain reaction(Real time PCR),parallel reaction monitoring(PRM)and other technologies to study the relationship between the above 11 energy metabolism related genes and the synthesis of c9,t11-CLA in cow mammary gland.The test results are as follows:Experiment 1: Design specific small interfering RNA(siRNA: siRNA-1,siRNA-2,siRNA-3)based on the mRNA sequences of 11 candidate genes(PGLS,PFKL,ALDOA,TPI1,GAPDH,PGK1,PGAM1,ENO1,PKM,LDHA,and LDHB).When the coverage rate of MAC-T cells in cows reaches 80%,use Lipofectamine (?)RNAi MAX transfection reagents were used to transport siRNA or scaffold into cells,and 11 candidate gene silenced MAC-T cell models were established.The results showed that the specific siRNAs designed in this study significantly reduced the relative mRNA expression of corresponding genes(P<0.01),when PGLS(siRNA-1,siRNA-2,siRNA-3),PFKL(siRNA-1,siRNA-2,siRNA-3),ALDOA(siRNA-1,siRNA-2,siRNA-3),TPI1(siRNA-1,siRNA-2,siRNA-3),GAPDH(siRNA-1,siRNA-2,siRNA-3),PGK1(siRNA-1,siRNA-2,siRNA-3),PGAMA 1(siRNA-1),ENO1(siRNA-1,siRNA-2,siRNA-3),After PKM(siRNA-2,siRNA-3),LDHA(siRNA-1,siRNA-2,siRNA-3),and LDHB(siRNA-3)were silenced,the relative mRNA expression of SCD1 significantly increased(P<0.01).When LDHB(siRNA-2)was silenced,the relative mRNA expression of SCD1 significantly increased(P<0.05).Experiment 2: Milk epithelial cells(MECs)of dairy cows were isolated from milk.First,the relative expression of LSP1,HBA and HBB genes in MAC-T cells,MECs and Milk somatic cell(MSCs)was measured by Real time PCR.The results showed that there was no significant difference in the relative expression of LSP1,HBA and HBB genes between MECs and MAC-T cells,indicating that the isolated MECs had high purity,It can be used for subsequent Real time PCR and PRM tests.Then,based on the content of c9 and t11-CLA in milk,MECs were divided into c9,t11-CLA high yielding dairy cows(HIGH group)and c9,t11-CLA low yielding dairy cows(LOW group).Real time PCR was used to compare and analyze the expression differences of 11 candidate genes between the two groups of MECs.The results showed that the relative mRNA expression levels of PFKL and PGAM1 were significantly lower in c9,t11-CLA high yielding dairy group(HIGH group)than in c9,and t11-CLA low yielding dairy group(LOW group)(P<0.01);The relative mRNA expression levels of ALDOA,GAPDH,and PGK1 were significantly lower in the HIGH group than in the LOW group(P<0.05);The relative mRNA expression levels of PGLS,TPI1,ENO1,PKM,LDHA,and LDHB were not significantly different between the HIGH and LOW groups(P>0.05).The PRM results showed a negative correlation between ALDOA,GADPH,PGAM1,and PKM and SCD1.This experiment validated the relationship between 11 energy metabolism related genes and SCD1 through cell experiments.Animal validation experiments were used to determine the negative correlation between ALDOA,GADPH,PGAM1 and SCD1,which in turn participated in the synthesis of c9,t11-CLA in the mammary gland of cows.