山羊AGRP基因克隆及在黑色素细胞中的表达研究  

Cloning of goat AGRP gene and its expression in melanocytes

作　　者：李杰[1,2] 孙晓燕 蒋婧[1,2] 任航行 王高富[1,2] 陈灿灿 LI Jie;SUN Xiaoyan;JIANG Jing;REN Hangxing;WANG Gaofu;CHEN Cancan(Chongqing Academy of Animal Sciences,Chongqing 402460,China;Chongqing Engineering Technology Center for Goat Research,Chongqing 402460,China)

机构地区：[1]重庆市畜牧科学院,重庆402460 [2]重庆市山羊工程技术研究中心,重庆402460

出　　处：《黑龙江畜牧兽医》2024年第15期49-54,共6页Heilongjiang Animal Science And veterinary Medicine

基　　金：重庆市自然科学基金面上项目(cstc2020jcyj-msxmX0114);重庆市科研院所绩效激励引导专项(cstc2021jxj180018);重庆市畜牧科学院财政专项(22544C-23)。

摘　　要：为了研究刺鼠相关蛋白(agouti-related protein,AGRP)基因在动物皮肤黑色素沉积中的作用,试验以酉州乌羊为研究对象,利用TRIzol法提取皮肤组织RNA,通过RT-PCR扩增AGRP基因编码区(coding region,CDS)序列,采用生物信息学软件预测AGRP蛋白质结构和功能,构建系统进化树分析山羊与其他哺乳动物AGRP基因的保守性;体外培养黑色素细胞,利用实时荧光定量PCR检测AGRP基因在细胞增殖和分化阶段不同时间点的相对表达量。结果表明:酉州乌羊AGRP基因CDS序列长度为405 bp,编码135个氨基酸,编码蛋白质分子量为14871.36 u,理论等电点为5.70;AGRP蛋白二级结构由3个α-螺旋和1个β-折叠以αααβ的顺序组成;AGRP蛋白的氨基酸序列在不同物种间高度相似;在构建的系统进化树中,酉州乌羊与绵羊和山羊的亲缘关系最近,与小鼠和人的亲缘关系最远。AGRP基因在黑色素细胞增殖和分化阶段的不同时间点均有表达,随着细胞培养时间的延长,AGRP基因的相对表达量在增殖阶段先逐渐降低再升高,在分化阶段逐渐升高,与0小时比较均差异极显著(P<0.01)。说明AGRP基因在黑色素细胞中具有时空特异性,在黑色素沉积过程中发挥重要作用。In order to study the role of agouti-related protein(AGRP)gene in the skin melanin deposition of animals,Youzhou dark goat(Capra hircus)was selected as the research object.RNA was extracted from skin tissue by Trizol;the coding region(CDS)of AGRP was amplified by RT-PCR,and its structure and function were analyzed by bioinformatics.Phylogenetic tree was constructed to analyze the conservation of AGRP in goats and other mammals.Melanocytes were cultured in vitro,the relative expression of AGRP gene at different time points of cell proliferation and differentiation were detected by real-time quantitative PCR.This result showed that the CDS sequence length of AGRP gene in Youzhou dark goat was 405 bp,encoding 135 amino acids.The molecular weight of the encoded protein was 14871.36 u,and its theoretical isoelectric point was 5.70.The secondary structure of AGRP protein contained 3α-helices and 1β-folded in the order ofαααβ.The amino acid sequence of AGRP protein was highly similar among different species.In the phylogenetic tree,Youzhou dark goat has the closest relationship with Ovis aries and Capricus,but the relationship between Mus culus and Homo sapiens was the furthest.AGRP gene was expressed at different time points in the proliferation and differentiation stages of melanocytes.In the proliferation stage,the AGRP gene expression decreased gradually and then increased with the extension of cell culture time.And in the differentiation stage,the gene expression increased gradually with the extension of cell culture time,and the difference was extremely significant compared with 0 hour(P<0.01).The results indicated that AGRP gene had spatio-temporal specificity in melanocytes,which played an important role in the process of melanin deposition.

关 键 词：酉州乌羊 AGRP 生物信息学 黑色素细胞 基因表达 

分 类 号：S827[农业科学—畜牧学] Q785[农业科学—畜牧兽医]