Polymorphism, Expression, and Structure Analysis of a Key Gene ARNT in Sheep (Ovis aries)

SIMPLE SUMMARY: Although the fact that ARNT may be related to sheep embryonic muscle growth and development has been clearly shown by our prior TMT proteomic study, its polymorphism, expression, and function remain unknown. Our goal was to thoroughly examine the association between the ARNT SNP loci and sheep growth traits, and elucidate the possible role of the ARNT gene in the formation and growth of sheep muscles. In this study, we evaluated the expression of the ARNT gene in the Hu, Chinese merino, and Gangba sheep, screened the polymorphism of the ARNT gene in the Hu and Ujimqin sheep populations, cloned the CDS sequence of the ARNT gene in the Hu sheep, and compared the CDS and protein sequences of the ARNT using bioinformatic analysis. The main results of our study showed an association between growth traits and sheep ARNT SNP loci, and a nonconservative missense point mutation G > C in the Hu and Ujimqin sheep, which is the primary factor that may regulate how the ARNT protein functions in the biology of sheep muscle growth and development. This work offers new and valuable insights into the mining of essential genes and identification of the molecular markers for sheep growth traits. ABSTRACT: Growth traits are influential factors that significantly affects the development of the sheep industry. A previous TMT proteomic analysis found that a key protein in the HIF signaling pathway, ARNT, may influence embryonic skeletal muscle growth and development in sheep. The purpose of this study was to better understand the association between the polymorphisms of ARNT and growth traits of sheep, and the potential function of ARNT. Real-time qPCR (qRT-PCR) of ARNT was carried out to compare its expression in different developmental stages of the muscle tissues and primary myoblasts in the Hu, Chinese merino, and Gangba sheep. The genetic variance of ARNT was detected using the Illumina Ovine SNP 50 K and 600 K BeadChip in the Hu and Ujimqin sheep populations, respectively. The CDS sequence of the ARNT gene was cloned in the Hu sheep using PCR technology. Finally, bioinformatic analytical methods were applied to characterize the genes and their hypothetical protein products. The qRT-PCR results showed that the ARNT gene was expressed significantly in the Chinese merino embryo after 85 gestation days (D85) (p < 0.05). Additionally, after the sheep were born, the expression of ARNT was significant at the weaning stage of the Hu sheep (p < 0.01). However, there was no difference in the Gangba sheep.In addition, six SNP loci were screened using 50 K and 600 K BeadChip. We found a significant association between rs413597480 A > G and the Hu sheep weight at weaning and backfat thickness in the 5-month-old sheep (p < 0.05), and four SNP loci (rs162298018 G > C, rs159644025 G > A, rs421351865 G > A, and rs401758103 A > G) were also associated with growth traits in the Ujimqin sheep (p < 0.05). Interestingly, we found that a G > C mutation at 1948 bp in the cloned ARNT CDS sequence of the Hu sheep was the same locus mutation as rs162298018 G > C identified using the 600 K BeadChip, which resulted in a nonconservative missense point mutation, leading to a change from proline to alanine and altering the number of DNA, protein-binding sites, and the α-helix of the ARNT protein. There was a strong linkage disequilibrium between rs162298018 G > C and rs159644025 G > A, and the ARNT protein was conserved among the goat, Hu sheep, and Texel sheep. And, we propose that a putative molecular marker for growth and development in sheep may be the G > C mutation at 1948 bp in the CDS region of the ARNT gene. Our study systematically analyzed the expression, structure, and function of the ARNT gene and its encoded proteins in sheep. This provides a basis for future studies of the regulatory mechanisms of the ARNT gene.