| Our previous study used RNA-seq to generate the bovine transcriptome from the mammary glands of four lactating Holstein cows with extremely high and low phenotypic values of milk protein and fat percentage. Among them,31differentially expressed genes were revealed. Gene ontology and pathway Integrated analysis of differential gene expression, previously reported quantitative trait loci, and genome-wide association studies indicated that TRIB3, SAA, VEGFA and PTHLH were the most promising candidate genes affecting milk protein and fat percentage. This study was designed to detect association between these differentially expressed genes with milk production traits in Chinese Holstein and find the key cause of mutations in key genes for further functional verification.In the present study, we performed a replication study on an independent dairy cattle population to evaluate whether such gene is associated with milk yield and milk composition traits. Through sequencing the entire coding region and-2000bp upstream regions of candidate genes with pooled DNA of21Chinese Holstein bulls, totally29SNPs were identified. A total of717Chinese Holstein individuals were genotyped for these identified SNPs using SNaPshot assay, MALDI-TOF-MS, Capillary electrophoresis and PCR-RFLP. The association analysis revealed that these identified SNPs were significantly associated with more than one trait. Besides, haplotype analysis revealed the same results. In addition, we also found that the mRNA level of SAA3.2gene was greatly higher in mammary gland than that in other7tissues. Meanwhile, the mRNA level of TRIB3gene was greatly higher in liver than that in other7tissues.For purpose of detecting the significant genetic effects for5’ regulatory region sites, we used the online prediction software TFSEARCH to predict the transcription factor binding sites, and the promoter activity assay, the gel shift assay (EMSA) and super gel mobility shift assay (super-shift EMSA) to verify the function of these causual mutations. The results showed that the transcription factor binding sites of SAA1gene locus c.-1788C>T and c.-963C>A were changed and the dual-luciferase activity also verified the prediction. The c.114G>A locus of SAA2gene changed the secondary structure of mRNA and affect the promoter activity by combination different transcription factors; SAA3.2gene locus c-1500A>G luciferase activity is significantly changed, and detected the C/EBPb transcription factor utilizing the super-shift EMSA. Additionally, we validated a length of52bp enhancer sequence of TRIB3genes by the promoter activity analysis.The genes TRIB3and SAA3.2were expressed in mammary epithelial cell lines (MAC-T) cells, so it is suitable for functional verification of these candidate genes. In transient transfection for MAC-T cells, the transfection efficiency of SAA3.2gene was70～80%while the efficiency of TRIB3gene was40～50%. After transient transfection of TRIB3and SAA3.2, we identified mRNA expression levels of related genes by real-time PCR. The results showed that the expression levels of these genes participated in fatty acid transportion (for example, LPL, APOA1and D36) change extremely significant (P<0.01), the changes of the other genes remained unobvious (P>0.05). We speculated that TRIB3and SAA3.2genes affect milk fat by influencing the expression of related genes in dairy cattle. Accordingly, bovine TRIB3and SAA3.2were considered to be functional genes for milk production traits, these results provided powerful evidence for further pathway analysis. |
