南方农业学报2023，Vol.54Issue(12)：3674-3682,9.DOI:10.3969/j.issn.2095-1191.2023.12.022
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
摘要
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分类
农业科技引用本文复制引用
毕欢,袁巍,张雨丹,陈伟..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[J].南方农业学报,2023,54(12):3674-3682,9.基金项目
贵州省科技计划项目(黔科合支撑[2022]一般087) （黔科合支撑[2022]一般087）
贵州大学引进人才科研项目(贵大人基合字(2019)34号) Guizhou Province Science and Technology Plan Project(QKHZC[2022]General 087) （贵大人基合字(2019）
Gui-zhou University Introduced Talent Research Project(GDRJHZ(2019)34) （GDRJHZ(2019）
