| Buffalo is the second milk-producing livestock in the word,with strong resistance to disease and coarse feeding characteristics,adapt to hot and humid climate,very suitable for breeding in the south of China,is conducive to solving the plight of the relative lack of fresh milk in the south of China.Buffalo milk has much higher milk fat percentage and dry matter content than Holstein milk,so it enjoys the reputation of "gold in milk".However,the low milk yield of buffalo seriously restricts its development.Therefore,it is particularly important to screen and identify functional genes that regulate lactating traits in buffalo.In our previous study,genome-wide association analysis(GWAS)was conducted on lactating traits in buffalo,and it was found that MFSD14A gene was a candidate gene for regulating milk fat percentage and milk protein percentage in buffalo.However,the specific function and molecular mechanism of MFSD14A gene in regulating milk fat synthesis in buffalo is still unclear.MFSD14A gene is known to be a member of the major facilitator superfamily(MFS).MFS family proteins mainly play the function of membrane transport,and several MFS proteins have been found to be related to lactating traits of dairy cows.Therefore,this study took MFS protein as the research object.Firstly,gene family identification,evolutionary relationship analysis,expression pattern analysis and association analysis were carried out to screen out the gene with the greatest influence on milk fat content in buffalo milk.Then,in vitro cell experiments were used to explore the role and mechanism of this gene in milk fat synthesis.It provides theoretical basis for molecular breeding and genetic improvement of buffalo.This study consists of four parts:Trial 1: Function analysis of MFSD14A gene regulating milk fat traits in buffalo based on MFS superfamily theory.By whole-genome identification,36 MFS genes and 86 MFS protein sequences were obtained in this study.Subcellular localization showed that these proteins were located on cell membrane and organelle membrane.The motifs and domains of buffalo MFS protein were identified by MEME and NCBI-CCD search.A total of 15 motifs were obtained,which belong to MFS superfamily and have membrane transport function.Based on MUSCLE alignment,the MFS protein evolutionary trees of buffalo,cow,goat,sheep and horse were constructed and their evolutionary relationships were analyzed.Based on chromosome localization and collinearity analysis,the Ka/Ks of MFS protein between buffalo andcattle was less than 1,indicating that MFS protein played similar roles in buffalo and dairy cows.GO and KEGG enrichment analysis indicated that MFS genes mainly played membrane transport function in buffalo.SNP-trait association analysis showed that a large number of molecular markers in MFS gene were significantly associated with lactating traits of buffalo.SNP located in MFSD14A gene was significantly associated with milk fat percentage and milk protein percentage of buffalo,and there were significant differences among individuals with different genotypes.Therefore,it is speculated that MFSD14A regulates buffalo milk fat traits through membrane transport function.Trial 2: RNAi experiment of MFSD14A gene in mammary epithelial cells.We used RNAi to knock down the expression of MFSD14A gene in mammary epithelial cells of buffalo and dairy cows.The results showed that the cycle of mammary epithelial cells was significantly affected,as G0/G1 phase cells were significantly decreased,G2/M phase cells were significantly increased,and triglyceride content was significantly decreased.The expression levels of ACSL1,ACSS2,FABP3 and SCD,which regulate milk fat synthesis,were significantly decreased,but the intracellular milk protein content was not significantly changed.The fatty acid content of buffalo mammary epithelial cells was detected by gas chromatography after MFSD14A gene interference,and the contents of palmitic acid,stearic acid and oleic acid were significantly decreased by more than 30%.After palmitic acid,stearic acid and oleic acid were added to the medium,the intracellular triglyceride content increased significantly.The contents of palmitic acid,stearic acid and oleic acid in the supernatant of cell culture medium were significantly higher than those in the control group,indicating that the number of fatty acids entering cells was significantly reduced after MFSD14A gene interference.Trial 3: Effect of MFSD14A gene overexpression on mammary epithelial cells.In order to further identify the function of MFSD14A gene.The MFSD14A gene expression vector was constructed and transiently transfected.We found that overexpression of MFSD14A gene significantly affected the cell cycle of mammary epithelial cells,with a significant decrease in the number of cells in S phase and an increase in the number of cells in G2/M phase.When MFSD14A was overexpressed alone,the intracellular triglyceride content did not change significantly,and the expression levels of ACSL1,FABP3,SCD and other genes did not change significantly.However,the intracellular triglyceride content of palmitic acid and stearic acid was significantly higher than that of control group.The expression levels of genes regulating milk fat synthesis,such as FABP3 and ACSL1,were also significantly up-regulated.In other words,when MFSD14A gene was overexpressed,the addition of fatty acids as substrate could promote the synthesis of triglycerides in breast epithelial cells.Trial 4: The mechanism of MFSD14A gene regulating milk fat synthesis and cell cycle in buffalo mammary epithelial cells.We sequenced the m RNA of buffalo mammary epithelial cells after MFSD14A gene interference.642 down-regulated genes and 362 up-regulated genes were identified.The GO function and KEGG pathway enrichment analysis of differentially expressed genes showed that these genes were mainly enriched in DNA replication,cell cycle and PPAR signaling pathways.SLC27A6 was significantly upregulated in PPAR signaling pathway,while FABP3,FABP4,ACOX2 and other genes were significantly down-regulated.In conclusion,based on genome-wide family genes analysis,protein structure analysis and mark-trait association analysis,MFSD14A gene was screened as an important gene regulating milk fat traits in buffalo.In vitro cell experiments showed that the gene could regulate the transport and uptake of long chain fatty acids in mammary epithelial cells.Transcriptome sequencing revealed that the gene regulates milk fat synthesis in mammary epithelial cells through PPAR signaling pathway. |
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