Yuan Zehu, Ge Ling, Su Pengwei, Gu Yifei, Chen Weihao, Cao Xiukai, Wang Shanhe, Lv Xiaoyang, Getachew Tesfaye, Mwacharo Joram M, Haile Aynalem, Sun Wei
Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China, Yangzhou University, Yangzhou 225009, China.
International Joint Research Laboratory in Universities of Jiangsu Province of China for Domestic Animal Germplasm Resources and Genetic Improvement, Yangzhou University, Yangzhou 225009, China.
Animals (Basel). 2023 Oct 11;13(20):3173. doi: 10.3390/ani13203173.
Previously, was identified as a candidate gene associated with sheep growth traits. This study aimed to investigate the direct role of in regulating myogenesis in embryonic myoblast cells and to investigate the association between single-nucleotide polymorphisms (SNPs) in its promoter region and sheep growth traits. The function of in myoblast proliferation and differentiation was detected after small interfering RNAs (siRNAs) knocked down the expression of . Cell proliferation was detected using CCK-8 assay, EdU proliferation assay, and flow cytometry cell cycle analysis. Cell differentiation was detected via cell immunofluorescence and the quantification of myogenic regulatory factors (MRFs). SNPs in the promoter region were detected using Sanger sequencing and genotyped using the improved multiplex ligation detection reaction (iMLDR) technique. As a result, a notable decrease ( < 0.01) in the percentage of EdU-positive cells in the siRNA-694-treated group was observed. A significant decrease ( < 0.01) in cell viability after treatment with siRNA-694 for 48 h and 72 h was detected using the CCK-8 method. The quantity of S-phase cells in the siRNA-694 treatment group was significantly decreased ( < 0.01). After interfering with in myoblasts during induced differentiation, the relative expression levels of MRFs were markedly ( < 0.05 or < 0.01) reduced compared with the control group on days 5-7. The myoblast differentiation in the siRNA-694 treatment group was obviously suppressed compared with the control group. SNP1, SNP2, SNP3, and SNP4 were significantly ( < 0.05) associated with all traits except body weight measured at birth and one month of age. SNP5 was significantly ( < 0.05) associated with body weight, body height, and body length in six-month-old sheep. In conclusion, interfering with can inhibit the proliferation and differentiation of ovine embryonic myoblasts. SNPs in its promoter region can serve as potential useful markers for selecting sheep growth traits.
此前， 被鉴定为与绵羊生长性状相关的候选基因。本研究旨在探讨 在调节胚胎成肌细胞肌生成中的直接作用，并研究其启动子区域单核苷酸多态性（SNP）与绵羊生长性状之间的关联。在小干扰RNA（siRNA）敲低 的表达后，检测其在成肌细胞增殖和分化中的功能。使用CCK-8法、EdU增殖法和流式细胞术细胞周期分析法检测细胞增殖。通过细胞免疫荧光和肌源性调节因子（MRF）定量检测细胞分化。使用桑格测序法检测启动子区域的SNP，并使用改进的多重连接检测反应（iMLDR）技术进行基因分型。结果显示，在siRNA-694处理组中观察到EdU阳性细胞百分比显著降低（ < 0.01）。使用CCK-8法检测发现，用siRNA-694处理48小时和72小时后细胞活力显著降低（ < 0.01）。siRNA-694处理组中S期细胞数量显著减少（ < 0.01）。在诱导分化过程中干扰成肌细胞中的 后，与对照组相比，在第5 - 7天MRF的相对表达水平显著降低（ < 0.05或 < 0.01）。与对照组相比，siRNA-694处理组的成肌细胞分化明显受到抑制。SNP1、SNP2、SNP3和SNP4与除出生时和1月龄时测量的体重外的所有性状显著相关（ < 0.05）。SNP5与6月龄绵羊的体重、体高和体长显著相关（ < 0.05）。总之，干扰 可抑制绵羊胚胎成肌细胞的增殖和分化。其启动子区域的SNP可作为选择绵羊生长性状的潜在有用标记。
