Variation in genes of the serotonergic system influence 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.

中文翻译：

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猪 SLC6A4 基因功能性单核苷酸多态性的鉴定与混合后断奶仔猪的攻击行为相关

血清素能系统基因的变异通过影响中枢和皮质神经系统中的血清素水平来影响攻击行为。SLC6A4（5-羟色胺转运蛋白）是 5-HT 信号转导的主要调节因子，参与调节人类和啮齿动物的攻击行为。为鉴定与攻击行为相关的猪 SLC6A4 基因的潜在功能性单核苷酸多态性 (SNP)，总共选择了 500 头猪（268 头公猪和 232 头母猪）并在 51 个猪栏中混合。混合后记录并观察他们的行为 72 小时。根据综合攻击性评分 (CAS)，分别选择每个围栏内攻击性最强和攻击性最低的猪（总共 204 头猪）。对耳组织取样以提取基因组 DNA。5'-侧翼区、编码区中的八个 SNP，对 SLC6A4 的 3' 和 3'-非翻译区 (3'-UTR) 进行了基因分型，其中 6 个 SNP 在攻击性最强和攻击性最小的猪之间的等位基因频率有显着差异 (P < 0.05)。rs345058216 基因型 GG 质粒中的荧光素酶活性高于基因型 CC 质粒 (P < 0.01)。计算分析指定 MAZ 作为推定的转录因子 (TF)，在 SNP (rs345058216) 位点结合 SLC6A4 启动子的可能性更高。此外，我们通过体外试验证明 MAZ 过表达以等位基因特异性方式调节 SLC6A4 启动子活性。此外，我们证明 SLC6A4 是 miR-671-5p 的直接靶标。进行双荧光素酶报告基因测定和细胞转染以检查 miR-671-5p 在调节 SLC6A4 表达中的作用。荧光素酶测定显示 SNP rs332335871 影响 SLC6A4 表达中 miR-671-5p 的调节。在猪原代神经细胞中过表达 miR-671-5p 后，SLC6A4 mRNA 水平可显着降低。总之，我们在这里发现 miR-671-5p 和 MAZ 介导猪 SLC6A4 表达水平，这提供了攻击行为的可能分子机制。