Screening Of Function Genes Related To Milk Fat Mebabolism With MRNA-miRNA Analysis In Cattle And Function Verification For KLF6 Gene

As an important economic trait of dairy cows,milk fat percentage is one of the important factors affecting milk quality,and it is also an important subject in the field of cow genetics and breeding.With the application of new breeding methods such as genome-wide association analysis,dairy cow molecular breeding has developed very rapidly.However,the screening of functional genes related to important economic traits and the analysis of regulatory mechanisms of functional genes are still hot and difficult in the field of cow genetics and breeding.In this study,we studied bovine mammary epithelial cells（BMECs）using RNA-seq and mi RNA-seq conjiont analysis method to screen the functional genes related to dairy quality,and to verify the regulation mechanism of KLF6 and other genes on lipid metabolism,which provided candidate genes for the improvement of high-quality dairy cows,and a theoretical basis for understanding the regulation mechanism of lipid metabolism in bovine mammary epithelial cells and analysis of the regulation mechanism of important economic milk quality.1 Screening and identification of milk fat metabolism function gene in cowA total of 829 differentially expressed genes（DEGs）were screened by RNA-seq in bovine primary mammary epithelial cells from cows with high（4.85%）and low（3.41）milk fat percentages.Furthermore,a total of 190 DEGs（including 159up-regulated DEGs and 31 down-regulated DEGs）and 33 differentially expressed mi RNAs（DERs,including 11 up-regulated DERs and 22 down-regulated DERs）were identified by conjoint analysis of RNA-seq and mi RNA-seq data previously published by our laboratory.The m RNA-mi RNA regulatory network related to milk fat metabolism was constructed based on the target relationship between m RNA and mi RNAs.The results of luciferase reporter and q PCR experiments showed that there was a target relationship between mi R-148a and PDE4D gene,mi R-2382-5p and SOD3,NDRG2,and KLF6 genes,mi R-2425-5p and ADAMTS1 gene,indicating the accuracy of the target relationship prediction.Performing functional annotations on the 190 DEGs identified in the conjoint analysis,and analyzing 27 DEGs target regulated by 18 DERs significantly enriched in GO terms（p<0.05）related to lipid metabolism,to construct the m RNA-mi RNA regulatory network related to lipid metabolism,of which mi R-21-3p and mi R-148a could promote the synthesis of triglyceride in BMECs,and in contrast,mi R-2382-5p and mi R-2425-5p could reduce the content of triglyceride in BMECs.Furthermore,results showed that mi R-2382-5p could promote triglyceride decomposition by regulating lipid metabolism related genes.At the same time,the functional verification of two target genes NDRG2 and KLF6 of mi R-2382-5p at the cellular level showed that the expression levels of NDRG2 and KLF6 gene were positively correlated with triglyceride and cholesterol levels in BMECs.2 Regulation mechanism of KLF6 gene on milk fat metabolism at cellular levelThe study constructed KLF6 gene knockout BMECs(KLF6^-/-)using CRISPR/Cas9 technique.The results of PCR product sequencing showed that the KLF6 gene produced a large segment deletion of the DNA sequence,and the amino acid coding sequence was transcoded.The q PCR results showed that the m RNA of KLF6 gene was not expressed.At the same time,the triglyceride and cholesterol content in KLF6^-/-significantly decreased（p<0.05）,the content of monounsaturated fatty acids decreased significantly（p<0.05）,and the content of 10 fatty acids were lower（p<0.05）in KLF6^-/-than that in wild type BMECs（WT）,such as palmitoleic acid,oleic acid,andγ-linoleic acid.A total of 4936 up-regulated and 4596 down-regulated DEGs were obtained from RNA-seq data of KLF6^-/-and WT,including FASN,FABP3,VLDLR,NDRG2,and PPARγgenes,which related to lipid metabolism.Screening GO terms related to lipid metabolism（p<0.05）,including fatty acidβ-oxidation,adipocyte differentiation regulation,brown adipocyte differentiation regulation,very long chain fatty acid metabolism process,and fatty acid biosynthesis process to construct the regulatory network between lipid metabolism related GO terms and DEGs.KEGG pathway enrichment analysis of DEGs showed that 47 DEGs were significantly enriched in fatty acid metabolism pathway,14 DEGs were significantly enriched in fatty acid biosynthesis pathway,and 21 DEGs were significantly enriched in fatty acid extension pathway（p<0.05）.Metabolomics analysis of WT and KLF6^-/-were carried out,342 metabolit es were identified by positive ion mode and 294 metabolites were identified by negative ion mode.The highest proportion of metabolites was lipid and lipid-li ke molecules（29.874%）,followed by organic acids and derivatives（24.843%）,indicating that lipid metabolism is an important biological process in mammary epithelial cells.The analysis of metabolites in negative ion mode showed that t here were significant differences in metabolites content of many lipids and lipi d-like molecules（p<0.05）,including 1,2-distearoyl-sn-glycero-3-phospho-l-serin e,1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine,1,2-distearoyl-sn-glycero-3-phospho-l-serine,and 1-stearoyl-2-oleoyl-sn-glycero-3-phospho-（1’-sn-glycerol）.K EGG enrichment analysis of differential metabolites was performed,and the sig nificantly enriched pathways included oxidative phosphorylation,citric acid cycl e,metabolism of glycerophospholipid,insulin signaling,and glucagon signaling（p<0.05）.3 Regulation mechanism of KLF6 gene on milk lipid metabolism in miceBreast tissue-specific knockout mouse model of the KLF6 gene was constructed using the CRISPR/Cas9 technique.The results of PCR products showed that the lox P site of KLF6 gene was recognized by Cre gene in homozygous mice,q PCR and Western Blot showed that there was no expression of KLF6 m RNA and protein in the mammary gland tissue of homozygous mice.Furthermore,the histomorphological observation of mice showed that all organs（heart,liver,spleen,lung,kidney,muscle and fat）of mice were normal,indicating that knockout of KLF6 gene in breast tissue did not cause disease in mice.ELISA kit was used to detect triglyceride content in milk.The results showed that triglyceride content in milk of homozygous mice was significantly lower than that in heterozygous and wild mice（p<0.05）.The morphological analysis of breast tissue showed that the diameter of adipocytes in breast tissue of homozygous mice was smaller than that of wild mice,and the contents of triglyceride and cholesterol in breast tissue of homozygous mice were significantly reduced（p<0.05）.In addition,the expression levels of related genes regulating lipolysis（ATGL,PKA,HSL,and FABP4）were significantly increased in breast tissue in homozygoated mice.In conclusion,this study accurately selected candidate regulators for lipid metabolism such as KLF6 gene by m RNA-mi RNA analysis.In BMECs,KLF6 gene participates in fatty acid metabolism and metabolism related pathways by regulating milk fat metabolism related genes,affects the content of lipid and lipid-like molecular,and then acts on the lipid metabolic indicators such as triglyceride,cholesterol and fatty acid.In mouse breast tissue,KLF6 gene can regulating on the regulatory pathway of adipocytes,causing morphological changes in adipose cell in breast tissue and triglyceride and cholesterol contents,thus affecting the milk fat percentage.The regulation mechanism of KLF6 gene on milk lipid metabolism was clarified from the level of cells and laboratory animals,which providing candidate genes for improvement of dairy cows and theoretical basis for further understanding the mechanism of lipid metabolism in dairy cows.