The effect of the SNP g.18475 A>G in the 3′UTR of NCF4 on mastitis susceptibility in dairy cattle
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Neutrophil cytosolic factor 4 (NCF4) is a member of the nicotinamide adenine dinucleotide phosphate oxidase subunit. This protein functions as an essential factor in the host defense against the progression of bacterial infection. To explore the variability of the NCF4 gene and the susceptibility of cows to mastitis, NCF4 functional single nucleotide polymorphism (SNP) of the 3′ untranslated region (3′UTR) and its targeted microRNA (miRNA) were identified. One SNP g.18475 A>G in the 3′UTR of NCF4 was found within the binding seed region of bta-miR-2426. We constructed two recombinant pMIR-REPORT™ vectors with the A or G allele in the g.18475 locus and transiently co-transfected the vectors in human embryo kidney 293T (HEK 293T) cells, along with bta-miR-2426 mimics. A luciferase assay indicated that this SNP affects the binding of NCF4 and bta-miR-2426. In addition, the association analysis results showed that cows with the GG genotype in SNP g.18475 A>G had a relatively lower SCS value than cows with the AA genotype. Finally, quantitative real-time PCR (RT-qPCR) results showed that the cows with genotype GG had a relatively higher expression of NCF4 mRNA compared to the cows with genotype AA. NCF4 expression was regulated by the miRNA–mRNA interaction mechanism, and an important role for NCF4 in mastitis susceptibility in dairy cow was suggested.
KeywordsBovine NCF4 Mastitis SCS Bta-miR-2426 3′UTR SNP
This study was supported by grants from the National Natural Science Foundation of China (31371255, 31401050, 31672397, and 31671286), the Major Project of National Transgenics in China (2014ZX08007-001), the National Cow Industrial Technology System Program (CARS-37), the Cow Innovation Team of the Shandong Province Modern Agricultural Industry Technology System (SDAIT-12-011-02), and the Natural Science Foundation of Shandong Province (ZR2013CQ035).
Compliance with ethical standards
All experiments were carried out according to the Regulations for the Administration of Affairs Concerning Experimental Animals published by the Ministry of Science and Technology, China, in 2004 and approved by the Animal Care and Use Committee from the Dairy Cattle Research Center, Shandong Academy of Agricultural Sciences, Shandong, PR China.
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