A post-GWAS confirming the genetic effects and functional polymorphisms of AGPAT3 gene on milk fatty acids in dairy cattle

Background: People are paying more attention to the healthy and balanced diet with the improvement of their living standards. Milk fatty acids(FAs) have been reported that they were related to some atherosclerosis and coronary heart diseases in human. In our previous genome-wide association study(GWAS) on milk FAs in dairy cattle, 83 genomewide significant single nucleotide polymorphisms(SNPs) were detected. Among them, two SNPs, ARS-BFGL-NGS-109493 and BTA-56389-no-rs associated with C18 index(P = 0.0459), were located in the upstream of 1-acylglycerol-3-phosphate O-acyltransferase 3(AGPAT3) gene. AGPAT3 is involved in glycerol-lipid, glycerol-phospholipid metabolism and phospholipase D signaling pathways. Hence, it was inferred as a candidate gene for milk FAs. The aim of this study was to further confirm the genetic effects of the AGPAT3 gene on milk FA traits in dairy cattle.Results: Through re-sequencing the complete coding region, and 3000 bp of 5′ and 3′ regulatory regions of the AGPA T3 gene, a total of 17 SNPs were identified, including four in 5′ regulatory region, one in 5′ untranslated region(UTR),three in introns, one in 3′ UTR, and eight in 3′ regulatory region. By the linkage disequilibrium(LD) analysis with Haploview4.1 software, two haplotype blocks were observed that were formed by four and 12 identified SNPs,respectively. Using SAS9.2, we performed single locus-based and haplotype-based association analysis on 24 milk FAs in 1065 Chinese Holstein cows, and discovered that all the SNPs and the haplotype blocks were significantly associated with C6:0, C8:0 and C10:0(P < 0.0001–0.0384). Further, with Genomatix, we predicted that four SNPs in 5′ regulatory region(g.146702957 G > A, g.146704373 A > G, g.146704618 A > G and g.146704699 G > A) changed the transcription factor binding sites(TFBSs) for transcription factors SMARCA3, REX1, VMYB, BRACH, NKX26, ZBED4, SP1, USF1, ARNT and FOXA1. Out of them, two SNPs were validated to impact transcriptional activity by performing luciferase assay that the alleles A of both SNPs, g.146704373 A > G and g.146704618 A > G, increased the transcriptional activities of AGPAT3 promoter compared with alleles G(P = 0.0004).Conclusions: In conclusion, our findings first demonstrated the significant genetic associations of the AGPAT3 gene with milk FAs in dairy cattle, and two potential causal mutations were detected.

Double⁃Shear Experiments of Cold⁃Formed Steel Stud⁃to⁃Sheathing Connections at Elevated Temperatures

The behavior of cold⁃formed steel(CFS)stud⁃to⁃sheathing connections at elevated temperatures is an important parameter for the fire resistance design and modeling of mid⁃rise CFS structures.In this paper,three kinds of sheathings,namely,medium⁃and low⁃density calcium⁃silicate boards and oriented strand board,were selected for double⁃shear experiments on the mechanical properties of 253 screw connections at ambient and elevated temperatures.The effects of the shear direction,screw edge distance and the number of screws on the behavior of the connections were studied.The results showed that the shear direction and the screw edge distance more significantly influenced the peak deformation,while their impacts on the peak load varied with the type of sheathings.Compared with the single⁃screw connections,the peak loads of the specimens with double⁃screw connections obviously increased but did not double.Finally,a simplified load⁃displacement curve model of stud⁃to⁃sheathing connections at elevated temperature was generated first by establishing the prediction formula for characteristic parameters,such as the peak load,the peak deformation and the elastic stiffness,and then by considering whether the curves corresponded to stiffness increase phenomena.The present investigation provides basic data for future studies on the numerical modeling of CFS structures under fire conditions.