Identification And Functional Verification Of EEF1D And PDE9A Associated With Milk Production Traits In Dairy Cattle

Our previous genome-wide association studies (GWAS) have identified38significant SNPs associated with one or multiple milk production traits in a Chinese Holstein population. The purpose of this study was to determine potential genes affecting milk yield and milk composition and function annotation based on such GWAS results and to validate there biological functions.(1) Based on the bovine genome sequence (Btau4.2), bioinformation and comparative analysis were conducted to determine potential genes and predict their functions. As a result, totally24genes corresponding to the aforementioned38SNPs were identified. Then,10out of such24genes were selected for further investigation. The mRNA expression patterns of10genes in8kinds of tissues of Holstein cows showed that both EEF1D and PDE9A genes were highly expressed in the mammary gland of lactating cows than in other7tiusses (cardiac muscle, uterus, kidney, liver, lung, ovary and small intestine). Combined with our previously GWAS results, EEF1D and PDE9A may be related to lactation capacity in Holstein cows. Determined the two genes as candidate genes for milk production traits of cows, and verified their functions in cellular level.(2) The mRNA sequences of both EEF1D and PDE9A genes were found to be not complete and kept changable in NCBI database, and alternative splicing of these genes exist in the mouse and human genes, so that variant splicing is likely to existed in cow. In the present study, we isolated the full-length mRNA sequences of EEF1D to determine if alternative splice variants existed. With rapid amplification of5’cDNA end (5’RACE) analysis, two novel alternative spliced transcript variants were observed in the mammary gland of lactating Holstein cow with length of1202bp and2195bp, named EEF1Da and EEF1Db. Such two variants contained the different first exon from each other (exon1a vs exon1b:294bp versus1287bp) with no overlap between the two types of exon1and the same remaining7exons. With quantitative real-time RT-PCR, the expression patterns of EEF1Da and EEF1Db were investigated across8kinds of tissues of lactating Chinese Holstein cows. It was found that the mRNA expression of EEF1Da was significantly higher than that of EEF1Db and similar to the overall mRNA level of EEF1D in the mammary gland. The PDE9A only exist1transcription in the breast tissue, connecting with the known sequence, get complete mRNA sequence.(3) Based on fresh mammary gland samples lactating Holstein cows from slaughterhouse, we separated the mammary epithelial cells and purified epithelial cells by enzyme digestion differences. Meanwhile, mammary epithelial cells can also be separated from the milk which was collected from Holstein cows in middle or late lactation. It was found that the celles have growth and proliferation ability and were able to express keratin18,some milk protein genes, DAGT1, GHR, EEF1D and PDE9A genes, so that the cell line can be used for gene function validation.(4) Based the complete mRNA sequence of EEF1D, we designed4siRNA fragments for RNAi and transfected them into bovine fibroblasts for12hours. Through real-time quantitative RT-PCR, EEF1D mRNA expression levels were detected. The result showed that3out of4siRNA can significantly reduced EEFID gene expression, of them2siRNA were used to construct shRNA vectors pGPU6-GFP-NEO-E1357and pGPU6-GFP-NEO-E1893, and also constructed the corresponding lentiviral vectors of shRNA. We did not conduct RNAi analysis for PDE9A due to its low expression level in such cell line. On the other hand, we synthesized the CDS sequences of both PDE9A and EEF1Da and connected them to the expression vectors pEGFP-Cl and pIRES-EGFP. Through vector transfection into293FT cells for48h, EEF1Da and PDE9A cDNAs were amplified with RT-PCR method. Result revealed that such vectors were available. In addition, the CDS sequences, we also had connected them to lentiviral vectors.Together, our results provided basis for further systematic function validation of EEF1Da and PDE9A genes and regulation on the formation of milk production traits.