College of Animal Science and Technology, Northwest A&F University, Yangling, 712100, Shaanxi, People's Republic of China.
Shaanxi Beef Cattle Engineering Research Center, Yangling, 712100, Shaanxi, People's Republic of China.
Sci Rep. 2017 Aug 11;7(1):7867. doi: 10.1038/s41598-017-08384-1.
The SIX1 homeobox gene belongs to the six homeodomain family and is widely thought to play a principal role in mediating of skeletal muscle development. In the present study, we determined that the bovine SIX1 gene was highly expressed in the longissimus thoracis and physiologically immature individuals. DNA sequencing of 428 individual Qinchuan cattle identified nine single nucleotide polymorphisms (SNPs) in the promoter region of the SIX1 gene. Using a series of 5' deletion promoter plasmid luciferase reporter assays and 5'-rapid amplification of cDNA end analysis (RACE), two of these SNPs were found to be located in the proximal minimal promoter region -216/-28 relative to the transcriptional start site (TSS). Correlation analysis showed the combined haplotypes H-H (-GG-GA-) was significantly greater in the body measurement traits (BMTs) than the others, which was consistent with the results showing that the transcriptional activity of Hap2 was higher than the others in Qinchuan cattle myoblast cells. Furthermore, the electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation assay (ChIP) demonstrated that NRF1 and ZSCAN10 binding occurred in the promoter region of diplotypes H-H to regulate SIX1 transcriptional activity. This information may be useful for molecular marker-assisted selection (MAS) in cattle breeding.
SIX1 同源盒基因属于六同源结构域家族，被广泛认为在介导骨骼肌发育中发挥主要作用。本研究表明，牛 SIX1 基因在胸最长肌和生理上未成熟的个体中高度表达。对 428 头秦川牛个体的 DNA 测序，在 SIX1 基因的启动子区域鉴定出 9 个单核苷酸多态性（SNP）。通过一系列 5'缺失启动子质粒荧光素酶报告基因检测和 5'-快速扩增 cDNA 末端分析（RACE），发现其中两个 SNP 位于转录起始位点（TSS）前 -216/-28 的近端最小启动子区域。相关性分析表明，与其他组合单倍型相比，组合单倍型 H-H（-GG-GA-）在体尺性状（BMTs）中显著更大，这与在秦川牛成肌细胞中 Hap2 的转录活性高于其他单倍型的结果一致。此外，电泳迁移率变动分析（EMSA）和染色质免疫沉淀分析（ChIP）表明，NRF1 和 ZSCAN10 在二倍体型 H-H 的启动子区域结合，以调节 SIX1 的转录活性。这些信息可能对牛的分子标记辅助选择（MAS）有用。
