Genetic Effects Analysis And Functional Verification Of DDIT3 Associated With Milk Production Traits

As known,it is a key step for improving milk quality to detect and identify the key genes affecting milk production traits in dairy cattle.Based on transcriptome sequencing,DNA damage-induced transcription factor 3(DDIT3)(p=4.01E-05)was identified as an important candidate gene for milk production traits in dairy cattle.Therefore,in this study,the genetic effect of DDIT3 was analyzed and its function was further verified by DDIT3 interference.The results were as follows:(1)Genetic effect analysis: The polymorphisms of DDIT3 were detected by pooled sequencing,and two SNPs sites were found in the 5’ promoter region.Furthermore,the genetic effects of SNP loci and haplotypes were determined by association analysis with five milk production traits(milk yield,milk fat yield,milk fat percentage,milk protein yield and milk protein percentage)of Chinese Holstein cattle.It was shown that the two SNPs of DDIT3 gene were significantly associated with milk fat trait(p=0.0001;p=0.0006)and milk yield(p=0.0063),and the two loci were completely linked.Therefore,DDIT3 gene was identified as an important candidate gene for milk production traits in dairy cattle.(2)Screening optimal si RNA interference sequence of DDIT3: Firstly,the p GBCas9-2A-puro-2A-GFP vector was successfully constructed by CRISPR-Cas9 and transfected into bovine mammary epithelial cells by lipofectamine,but the transfection efficiency was very low,so lentivirus interference technique was used to interfere with DDIT3 gene in further experiment.Based on the DDIT3 sequence of bovine,the sh RNA was designed and synthesized and a sh RNA recombinant lentivirus vectorwas constructed.The recombinant Lentivirus was packaged by transfecting the 293 T cell with the recombinant vector packaging plasmids(p Gag/Pol,p Rev and p VSV-G).The DDIT3 lentivirus sh RNA interference vector was used to infect bovine mammary epithelial cells(MAC-T)and DDIT3-LV3-D287 was successfully obtained with 60% interference efficiency.(3)Functional verification of DDIT3 gene: After the interference fragments were infected with MAC-T cells,the total RNA was extracted and detected,and the database was constructed and sequenced by RNA-seq.3838 differential genes in the group compared with the blank group were screened,including 1989 up-regulated genes and 1949 down-regulated genes.The screened differential genes were analyzed by GO and Pathway enrichment analysis.The enrichment results showed that DDIT3 gene mainly affected the pathways related to cell proliferation,cell senescence,apoptosis and immune inflammation.