Multi-omics Study Of Milk Production Traits In Dairy Cattle And Functional Validation Of Candidate Gene RPL8

Milking performance is the most important economic traits of dairy cows.Detection and identification key genes on milking performance has been a research hotspot for dairy cattle.The rapid development of high-throughputomics technology have provided new opportunities for the detection and identification of key genes for dairy cows' milk traits.To identify key genes and IncRNAs affecting milk traits,we performed longitudinal GWAS combined transcriptome and proteome studies,thus providing evidence for cow' molecular breeding.The study consisted of four parts:Part ?:Based on a 9,615 Chinese Holstein cattle population,we performed longitudinal GWAS by using the test-day records of the first,second,and third parity as the response variable.Milk yield,milk fat percentage,and milk protein percentage were used for longitudinal GWAS.In total,84 SNPs were observed to be genome-wise significantly associated with one or more milk traits.The most significant SNPs associated with three milk traits were within DGAT1 and GHR genes.In addition,we proposed a novel QTL that significantly affected milk fat percentage and it is worthy to further study.The present study not only proved the previous findings well,but also provided resources for the functional study of milk traits.Part ?:To further detection candidate genes and IncRNAs for milk production traits at the transcriptome level,four Holstein cows' mammary tissues at peak and late lactation stages were examined by biopsy.The transcriptome of mammary gland tissue was profiled using RNA sequencing technology.In total,we found 254 mRNAs and 117 IncRNAs were differentially expressed at two lactation stages respectively.Combining the GO,KEGG and QTL,we proposed 7 genes?CDKN1A,PIM1,SOCS3,DDIT4,FADD,SLC27A and RPL8?and 12 IncRNAs?XLOC₀00178,XLOC₁69978,XLOC₁98815,XLOC₂26575,XLOC₂74111,XLOC₃06924,XLOC₃98297,XLOC₄12271,XLOC₅18578,XLOC₀00752,XLOC₆26085 and XLOC₂50904?that might affect milk production traits.Part ?:In current study,we adopted tandem mass tags?TMT?label-based quantitative analysis technique to investigate proteome changes occurring in bovine mammary gland from peak to late lactation stages genes and identify candidate genes for milk traits at protein level.The mammary tissues were the samples used in Part ?.A total of 3,753 proteins and 179 differentially expressed proteins were identified.We observed five new DEPs?AACS,DHCR7,GSTM3,SFRP1 and SFRP4?and nine functional well-studies known proteins?PLIN2,LPIN1,PLIN3,GSN,CD74,MMP2,SOD1,SOD3 and GPX3?related to milk traits and mammary morphology.Part ?:Integrating the results of GWAS,transcriptome and proteome as well as considering the previous findings in our team,we selected the RPL8 gene as a candidate gene for further validation.First we explored the function of RPL8 gene in milk fat metabolism by RNAi.The results indicated expression levels of 11 genes involved in milk fat metabolism changed after RPL8 silencing.Based on the results of RNAi and the interaction of RPL8 with the genes related to lipid metabolism,we speculated that RPL8 may play a role in denovo synthesis of fatty acids.Previous findings in our team showed that the mutation at P18 resulted in the change of transcription factor PAX6 binding.Further we detected the role of PAX6 on RPL8 using RNAi and co-immunoprecipitation.The results demonstrated the PAX6 contributed to positive regulation of RPL8 expression.In summary,all findings herein indicated that the complex genetic mechanism of milkingperformance of dairy cows was affected by numerous protein-encoding genes as well as many regulatory pathways.We analyzed the genetic basis of milk traits by multi-omics approach,which will provide multi-omics molecular support and valuable gene resources for molecular breeding of dairy cows more effectively.