College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China.
Department of Animal Sciences, Purdue University, West Lafayette, IN 47907-2054, USA.
J Anim Sci. 2022 May 1;100(5). doi: 10.1093/jas/skac131.
Variation in genes of the serotonergic system influences aggressive behavior by affecting serotonin levels in the central and cortical nervous system. SLC6A4 (serotonin transporter) is a master regulator of 5-HT signaling and involved in the regulation of aggressive behavior in humans and rodents. To identify potential functional single nucleotide polymorphisms (SNPs) for the porcine SLC6A4 gene associated with aggressive behavior, a total of 500 pigs (268 barrows and 232 gilts) were selected and mixed in 51 pens. Their behavior was recorded and observed for 72 h after mixing. Based on a composite aggressive score (CAS), the most aggressive and the least aggressive pigs within each pen were selected separately (a total of 204 pigs). Ear tissue was sampled to extract genomic DNA. Eight SNPs in the 5'-flanking region, coding region, and 3'-untranslated region (3'-UTR) of SLC6A4 were genotyped, of which 6 SNPs had significant differences (P < 0.05) in allele frequency between the most aggressive and least aggressive pigs. Luciferase activity was greater in plasmids of genotype GG than plasmids of genotype CC of rs345058216 (P < 0.01). Computational analysis nominated MAZ as putative transcription factor (TF) with higher probability to bind the SLC6A4 promoter at the SNP (rs345058216) site. Also, we demonstrated that MAZ overexpression modulates SLC6A4 promoter activity in allele-specific manner with an in vitro assay. In addition, we demonstrated that SLC6A4 was a direct target of miR-671-5p. The dual luciferase reporter gene assay and cell transfection were performed to examine the role of miR-671-5p in regulating SLC6A4 expression. The luciferase assays revealed that the SNP rs332335871 affects regulation of miR-671-5p in SLC6A4 expression. After overexpression of miR-671-5p in porcine primary neural cells, the SLC6A4 mRNA levels can be significantly reduced. In conclusion, we here found that miR-671-5p and MAZ mediated porcine SLC6A4 expression level, which provides the possible molecular mechanism of aggressive behavior.
5-羟色胺能系统基因的变异通过影响中枢和皮质神经系统中的 5-羟色胺水平来影响攻击行为。SLC6A4（血清素转运体）是 5-HT 信号的主要调节因子，参与人类和啮齿动物攻击行为的调节。为了鉴定与攻击行为相关的猪 SLC6A4 基因的潜在功能单核苷酸多态性（SNP），总共选择了 500 头猪（268 头公猪和 232 头母猪），并将它们混合在 51 个围栏中。记录它们的行为，并在混合后 72 小时进行观察。基于综合攻击得分（CAS），每个围栏中最具攻击性和最不具攻击性的猪分别被单独选择（总共 204 头猪）。采集耳组织提取基因组 DNA。在 SLC6A4 的 5'-侧翼区、编码区和 3'-非翻译区（3'-UTR）中对 8 个 SNP 进行了基因分型，其中 6 个 SNP 在最具攻击性和最不具攻击性的猪之间等位基因频率有显著差异（P<0.05）。rs345058216 处基因型 GG 的质粒的荧光素酶活性大于基因型 CC 的质粒（P<0.01）。计算分析提名 MAZ 作为具有更高概率结合 SNP（rs345058216）位点 SLC6A4 启动子的假定转录因子（TF）。此外，我们通过体外实验证明 MAZ 过表达以等位基因特异性方式调节 SLC6A4 启动子活性。此外，我们还证明 SLC6A4 是 miR-671-5p 的直接靶标。进行双荧光素酶报告基因检测和细胞转染以检查 miR-671-5p 在调节 SLC6A4 表达中的作用。荧光素酶测定表明 SNP rs332335871 影响 miR-671-5p 对 SLC6A4 表达的调节。在猪原代神经细胞中过表达 miR-671-5p 后，SLC6A4 mRNA 水平可显著降低。总之，我们发现 miR-671-5p 和 MAZ 介导猪 SLC6A4 的表达水平，这为攻击行为提供了可能的分子机制。
