敲降TYRP1基因对香猪表皮黑素细胞黑色素生成的影响  

Effect of Knocking down TYRP 1 Gene on Melanin Production in theXiang Pig Epidermal Melanocytes

作　　者：毕欢 覃海 袁巍 张雨丹 张依裕[1,2,3] 陈伟[1,2,3] BI Huan;QIN Hai;YUAN Wei;ZHANG Yudan;ZHANG Yiyu;CHEN Wei(Key Laboratory of Animal Genetics,Breeding and Breeding of Plateau Mountain Areas,Ministry of Education,Guiyang 550025,China;Key Laboratory of Animal Genetics and Breeding of Guizhou Province,Guiyang 550025,China;School of Animal Science,Guizhou University,Guiyang 550025,China)

机构地区：[1]高原山地动物遗传育种与繁殖教育部重点实验室,贵阳550025 [2]贵州省动物遗传育种与繁殖重点实验室,贵阳550025 [3]贵州大学动物科学学院,贵阳550025

出　　处：《畜牧兽医学报》2023年第10期4143-4153,共11页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：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(贵大人基合字(2019)34号);贵州省优秀青年科技人才培养计划黔科合平台人才([2021]5630号);贵州省生猪种业发展项目(黔农计财[2022]10号);贵州省生猪产业发展项目(2021;2022)。

摘　　要：旨在探究酪氨酸相关蛋白酶1(tyrosinase related protein 1,TYRP1)对香猪原代表皮黑素细胞黑色素生成的影响。本研究选用具有“两头乌”毛色特征的3月龄健康香猪3头,每头猪采取头部黑色皮肤组织和背部白色皮肤组织。通过HE染色观察不同颜色香猪皮肤的毛囊结构及黑素细胞的分布特征;体外培养香猪原代表皮黑素细胞并通过多巴(L-Dopa)染色、实时荧光定量PCR和Western Blot方法进行鉴定;根据TYRP1基因序列构建5条siRNA,采用实时荧光定量PCR筛选出干扰效率最高的siRNA进行转染。成功干扰黑素细胞内TYRP1的表达后,分别通过实时荧光定量PCR、Western Blot、黑色素含量检测方法对黑素细胞内TYR、TYRP1及TYRP2的mRNA与蛋白相对表达量、细胞内总黑色素含量进行检测。结果显示,在香猪黑色和白色皮肤中都观察到完整的毛囊结构,黑素细胞主要分布在香猪黑色被毛皮肤中的毛囊外根鞘部位以及表皮。在MelM培养基作用下,黑素细胞生长旺盛,细胞形态、生长曲线、多巴染色、实时荧光定量PCR及Western Blot结果均表明培养的黑素细胞维持正常的生物学特性。敲降TYRP1后下调香猪表皮黑素细胞中黑色素生成相关基因TYR、TYRP1和TYRP2的mRNA和蛋白的表达,同时对黑色素的形成具有抑制作用。TYRP1基因能够影响香猪表皮黑素细胞黑色素的生成,研究结果可为探索TYRP1基因对香猪黑色素沉积的分子机制提供试验参考和基础数据。The aim of this study was to explore the effect of tyrosine-related protease 1(tyrosinase related protein 1,TYRP 1)on melanin production in skin melanocytes of Xiang pigs.Three 3-month-old healthy pigs with the hair color characteristics of"two-head-black"animals were selected.Each pig took The black skin tissue on the head and white skin tissue on the back were collected for each pig.The structure of hair follicles and the distribution characteristics of melanocytes were observed by HE staining;The epidermal melanocytes were cultured in vitro and identified by Dopa(L-Dopa)staining,quantitative real-time PCR and Western Blot methods;Five siRNAs were constructed from TYRP 1 gene sequence,and the siRNA with the highest interference efficiency was transfected by quantitative real-time PCR.After successful interference of TYRP 1 in melanocytes,the mRNA and protein expression levels of TYRP,TYRP 1 and TYRP 2 were measured by quantitative real-time PCR and Western Blot,the total intracellular melanin content also been measured.The results showed that intact hair follicle structures were observed in both the black and white skin of Xiang pigs,and the melanocytes were mainly distributed in the outer root sheath of the skin hair follicles and epidermes in the black hairy skin of Xiang pig.Under the treatment of MelM medium,the melanocytes grew vigorously,and the results of cell morphology,growth curve,Dopa staining,quantitative real-time PCR and Western Blot all indicated that the cultured melanocytes maintained normal biological characteristics.Knocking down of TYRP 1 downregulated the mRNA and protein expression of melanogenesis-related genes TYR,TYRP 1 and TYRP 2 in Xiang pig epidermal melanocytes,and also had an inhibitory effect on melanin formation.The TYRP 1 gene was able to affect the melanin production in melanocyte epidermal cells,the results provide experimental reference and basic data to explore the molecular mechanism of TYRP 1 gene on melanin deposition in Xiang pigs.

关 键 词：TYRP1 黑素细胞 SIRNA HE染色 多巴染色 黑色素 

分 类 号：S828.2[农业科学—畜牧学]