miR-2366靶向DCT基因调控香猪黑色素细胞黑色素生成的分子机制研究
The molecular mechanism of miR-2366 targeting DCT gene in Xiang pig melanocytes melanogenesis
毕欢 1袁巍 1张雨丹 1陈伟1
作者信息
- 1. 贵州大学动物科学学院/高原山地动物遗传育种与繁殖教育部重点实验室/贵州省动物遗传育种与繁殖重点实验室,贵州贵阳 550025
- 折叠
摘要
[目的]明确miR-2366通过靶向作用多巴色素异构酶基因(DCT)对黑色素细胞中黑色素生成的影响,为解析剑白香猪"两头乌"毛色的形成机制打下理论基础.[方法]以剑白香猪为研究对象,通过酶解法测定不同毛色皮肤组织中总黑色素含量,利用碱溶法测定不同颜色毛发中不同种类黑色素含量,以实时荧光定量PCR检测miR-2366和DCT、TYR和TYRP1基因在不同毛色皮肤组织中的表达情况;根据miR-2366与DCT基因的靶向关系,人工合成双荧光素酶报告载体(pmirGL0-DCT-3'-UTR-wt和pmirGL0-DCT-3'-UTR-mut)及miRNA-2366过表达载体mimic、抑制载体inhibitor和阴性对照NC,分别转染剑白香猪黑素细胞后,通过实时荧光定量PCR和Western blotting检测DCT、TYR和TYRP1基因的相对表达量及其蛋白表达水平,并以碱溶法检测转染黑色素细胞中的总黑色素含量.[结果]在剑白香猪黑色皮肤组织中,总黑色素含量极显著高于白色皮肤组织(P＜0.01,下同);在黑色毛发中,总黑色素、棕黑色素和真黑色素含量也极显著高于白色毛发;在黑色皮肤组织中,miR-2366的相对表达量极显著低于白色皮肤组织,而DCT、TYR和TYRP1基因的相对表达量极显著高于白色皮肤组织.在转染试验中,miR-2366 mimic能极显著下调DCT、TYR和TYRP1基因表达,显著降低DCT、TYR和TYRP1蛋白表达;导致黑色素细胞中黑色素含量极显著降低36.0%;而miR-2366 inhibitor极显著显著上调DCT、TYR和TYRP1基因及其蛋白的表达,促使黑色素细胞中黑色素含量极显著上升高20.0%.[结论]miR-2366通过靶向负调控DCT基因表达而调节黑色素细胞中的黑色素含量,进而调控剑白香猪毛色的形成,提示miR-2366对黑色素的生成具有调控作用.
Abstract
[Objective]To clarify the effect of miR-2366 on melanin production in melanocytes by targeting DCT gene,and to lay a theoretical foundation for analyzing the formation mechanism of"two-black-head"coat color in Jianbai Xiang pigs.[Method]With the Jianbai Xiang pig,the amount of total melanin in skin tissue and the amount of hair in different colors was determined by enzymatic hydrolysis;The content of different types of melanin in hair of different col-ors were determined by alkaline dissolution method;The expression levels of miR-2366 and DCT and TYR,TYRP1 were analyzed in different coat color skin tissues by quantitative real-time PCR technology.Based on the targeting relationship of miR-2366 and the DCT gene,Artificial dual luciferase reporter vector(pmirGL0-DCT-3'-UTR-wt and pmirGL0-DCT-3'-UTR-mut)and miRNA-2366 overexpression vector mimic,inhibitory vector inhibitor and negative control NC,After the transfection of the white pig melanocytes,respectively,The relative expression levels of DCT,TYR and TYRP1 genes and their protein expression levels were determined by quantitative real-time PCR and Western blotting,And the total melanin content in the transfected melanocytes was determined by alkaline lysis.[Result]The melanin content was signifi-cantly higher in the Jianbai Xiang pig black skin tissue than the content in the white skin tissue(P<0.01,the same below).In the black hair,the total melanin,phaeomelanin and eumelanin content were significantly higher than those in the white hair.miR-2366 was significantly higher in white skin,and DCT,TYR,TYRP1 were opposite to miR-2366-the expressions were significantly higher than white skin.In the transfection assay,miR-2366 mimic significantly downregulated the ex-pression of DCT,TYR and TYRP 1 protein,TYR and TYRP1;especially in melanocytes;miR-2366 inhibitor signifi-cantly increased the expression of DCT,TYR and TYRP1 protein,increasing the content of melanin in melanocytes by 20.0%.[Conclusion]The miR-2366 regulates the melanin content in melanocytes by targeting and negatively regulating DCT gene expression,and then regulates the formation of pig hair color,suggesting that miR-2366 regulates the produc-tion of melanin.
关键词
剑白香猪/miR-2366/多巴色素异构酶基因(DCT)/黑色素Key words
Jianbai Xiang pigs/miR-2366/DCT gene/melanin引用本文复制引用
基金项目
贵州省科技计划项目(黔科合支撑[2022]一般087)
贵州大学引进人才科研项目(QKHZC[2022]General 087)
出版年
2023
国家科技期刊平台
NSTL
万方数据