Functional Validation Of GPIHBP1for Milk Production Traits And Detection Of Candidate Genes Based On RNA Sequencing

Our previous genome-wide association study (GWAS) in Chinese Holstein popμlation revealed Glycosylphosphatidylinositol-anchored HDL binding protein1(GPIHBP1) is a potential functional candidate gene for milk production traits. This study aimed at functional validation of GPIHBP1and detection of candidate genes based on RNA sequencing.We did overexpression and RNAi experiments on GPIHBP1in the bovine primary mammary epithelial cell and then identified mRNA expression patterns of related genes by real-time PCR. After transient transfection of GPIHBPl, the transcription levels of casein genes (CSN1S1, CSN1S2, CSN2and CSN3) and LTF reduced while the expression level of LGB increased. Meanwhile, the expression levels of LPL, CD36, VLDLR, ACACA and FASN increased with the overexpression of GPIHBPl. There were significant changes in the mRNA levels of CSN3, LTF, VLDLR, ACACA and FASN. In the RNAi experiment of GPIHBP1, the expression levels of casein genes (CSN1S1, CSN1S2, CSN2and CSN3) and LTF increased, while those of LGB, LPL, CD36, VLDLR, ACACA and FASN reduced. There were significant changes in the mRNA levels of LGB, LTF, ACACA and FASN. Accordingly, bovine GPIHBP1is considered to be a functional gene for milk production traits.In order to detect the cause mutation of GPIHBP1, we constructed promoter recombinant vectors of GPIHBP1and measured the promoter activity of different segments by dual-luciferase reporter system. Results revealed that the promoter activity was significantly different between wild type and mutation type (P<0.001). In addition, we predicted transcription factor binding sites, and based on the results, we did the gel retardation assays for G/A (Chr14:2553998) and C/A (Chrl4:2553653). We found that the SNPs had an effect on binding sites for transcription factors. Our results provide insights into the transcriptional regμlation of GPIHBP1. Furthermore, we detected the expression levels of GPIHBPl in15Chinese Holstein cows, and the results showed that the expression level of wild type were higher than that of mutation type (P<). Association analysis also revealed that mutation-type individuals outperformed wild-type ones in milk yield, milk protein yield, milk fat yield, milk protein percentage and milk fat percentage.Based the results of RNA-seq of bovine blood,1408different expression genes was tested, and53different expression genes were located in QTLs about milk production traits. Based the results of RNA-seq of milk,1459different expression genes was tested, and98different expression genes were located in QTLs about milk production traits. The results provided important information for investigation of transcriptome and detection of candidate genes of milk production traits.