Isolation And Identification Of Buffalo ADFP Gene And Its Function In Milk Fat Synthesis

ADFP gene encodes lipid droplet coated protein 2,which plays an important role in the formation of intracellular lipid droplets,regulation of lipid storage and metabolism,oxidation of fatty acids and other physiological functions.It has been reported that ADFP can promote the production of fat droplets and the secretion of milk fat globules in mammary epithelial cells of mice and dairy goats,but the specific function of ADFP in the milk fat synthesis process of buffalo is still unclear.The functional diversity of ADFP gene may be closely related to the occurrence of alternative splicing isomer.There is no relevant study on the identification of the alternative splicing isomer of buffalo ADFP gene.Using buffalo mammary tissue cDNA as template,the buffalo ADFP gene was isolated and identified by RT-PCR to determine whether there were alternative splicing isomer in the gene;The bioinformatics analysis of the obtained code product was carried out,and its basic functions were preliminarily studied;qPCR technique was used to detect the selective spliceosome that play a major role in buffalo mammary tissue.Further,the function of ADFP in the process of buffalo milk fat synthesis was verified by in vitro cellular function experiments.The relative m RNA expression of buffalo ADFP gene in 9 tissues of buffalo was detected,and the tissue expression specificity of buffalo m RNA gene was analyzed.The following results are obtained:(1)Four alternative splicing isomer of the buffalo ADFP gene were obtained,named ADFP＿X1,ADFP＿X3,ADFP＿X4 and ADFP＿X6,which are 1425 bp,1353bp,1344 bp and1218bp,respectively,encoding 475,450,447 and 405 amino acids.Compared with common cattle,goats,sheep and other bovidae species,the sequence consistency of buffalo was96%～99.8%,and the sequence difference was small.ADFP＿X1,ADFP＿X3 and ADFP＿X4were completely consistent with the alternative splicing isomer by NCBI database.ADFP＿X6 was a newly discovered isomer in this study,and the sequence consistency with ADFP＿X1,ADFP＿X3 and ADFP＿X4 was 97.8%,98% and 91.5%,respectively.Except for ADFP＿X6,the other three alternative splicing isomer have complete functional domains,but ADFP＿X6 has 45 amino acids missing and does not have complete functional domains.(2)Four alternative splicing isomer of ADFP gene were expressed in both lactation and non-lactation breast tissues,and the expression levels of ADFP＿X3,ADFP＿X4 and ADFP＿X6 were significantly higher than those of ADFP＿X1(P<0.001).At the same time,the expression levels of ADFP＿X3,ADFP＿X4 and ADFP＿X6 were significantly higher in lactation than in non-lactation(P<0.001),and the expression abundance of ADFP＿X3,ADFP＿X4 and ADFP＿X6 were different in different lactation periods,suggesting that ADFP gene may be involved in the process of milk production in buffalo.(3)After overexpression of ADFP＿X3 in mammary epithelial cells,the expressions of milk fat synthesis genes PPARG,LPL,SCD,FASN and SREBP-1c were significantly increased,while the triglyceride content and lipid droplet accumulation in mammary epithelial cells were significantly increased(P<0.01).After overexpression of ADFP＿X4,the content of triglycerides in mammary epithelial cells increased significantly(P<0.05),but the expression of milk fat synthesis related genes and lipid droplet changes were not significant.The inability to interfere with a particular variant spliceosome interferes with the entire transcription process of the ADFP gene.After interference with ADFP＿X3 in mammary epithelial cells,the expressions of milk fat related genes PPARG,LPL,SCD,FASN and SREGBP-1c were significantly decreased,while the number and volume of lipid droplets in mammary epithelial cells were significantly reduced.The results indicated that ADFP＿X3,one of the four variable spliceosomes of ADFP gene,plays an important role in the synthesis of buffalo milk fat.(4)The relative expression level of ADFP＿X3 in 9 tissues of buffalo was detected by real-time quantitative fluorescent PCR(qPCR).The results showed that ADFP＿X3 was expressed in all tissues of buffalo in lactation period,with a high level of expression in mammary tissue(P<0.01),followed by heart and muscle.It is expressed at relatively low levels in the kidneys,lungs and brain.The results of this study showed that ADFP plays a more important role in lipid synthesis in buffalo mammary epithelial cells,and its main role is to regulate the secretion and size of lipid droplets.Among the four splicing isomers obtained from mammary glands,ADFP＿X3 plays a major role in the synthesis of buffalo milk fat.In addition,this study also revealed that ADFP gene can participate in milk fat synthesis in mammary epithelial cells through PPAR signaling pathway.The results of this study can provide a basis for the analysis of the milk fat synthesis mechanism of milk buffalo,which has important scientific value,and can also provide a reference for the molecular breeding of water buffalo.