MC1R c.676A>G与山羊皮肤色素沉积的关系及其对黑色素合成的影响

doi:10.3969/j.issn.1004-1524.2022.12.06

浙江农业学报 ›› 2022, Vol. 34 ›› Issue (12): 2629-2639.DOI: 10.3969/j.issn.1004-1524.2022.12.06

MC1R c.676A>G与山羊皮肤色素沉积的关系及其对黑色素合成的影响

蒋婧¹^,²(), 任航行¹^,², 周鹏¹^,², 孙晓燕¹^,², 李杰¹^,², 付琳¹^,², 张丽¹^,², 刘良佳¹^,², 王高富¹^,²^,^*()

1.重庆市畜牧科学院,重庆荣昌 402460
2.重庆市山羊工程技术研究中心,重庆荣昌 402460

收稿日期:2022-01-12 出版日期:2022-12-25 发布日期:2022-12-26
通讯作者: 王高富
作者简介:*王高富,E-mail:wanggaofs20031216@163.com
蒋婧(1986—),女,重庆忠县人,硕士,副研究员,主要从事分子遗传与羊育种研究。E-mail:jiangjing19@163.com
基金资助:
重庆市基础研究与前沿探索专项(重庆市自然科学基金)(cstc2018jcyjAX-0042);重庆市畜牧科学院市级财政科研项目(19504);重庆市畜牧科学院市级财政科研项目(020208)

Relationship between MC1R c.676A>G and goat skin pigmentation and its effect on melanin synthesis

JIANG Jing¹^,²(), REN Hangxing¹^,², ZHOU Peng¹^,², SUN Xiaoyan¹^,², LI Jie¹^,², FU Lin¹^,², ZHANG Li¹^,², LIU Liangjia¹^,², WANG Gaofu¹^,²^,^*()

1. Chongqing Academy of Animal Sciences, Rongchang 402460, Chongqing, China
2. Chongqing Engineering Research Center for Goat, Rongchang 402460, Chongqing, China

Received:2022-01-12 Online:2022-12-25 Published:2022-12-26
Contact: WANG Gaofu

摘要/Abstract

摘要：

为探讨山羊MC1R基因c.676A>G突变与山羊皮肤色素沉积的关系,及其对黑色素合成和cAMP信号通路下游色素相关基因表达的影响:一方面采用直接测序法检测酉州乌羊及其杂交后代群体(120个个体)MC1R基因c.676A>G的突变情况,用色差仪测量不同基因型个体的腹部皮肤色度值,分析c.676A>G突变与皮肤色素沉积的关系;另一方面,构建野生型(MC1R c.676A)和突变型(MC1R c.676G)真核表达载体,采用脂质体介导法转染到小鼠皮肤黑色素瘤细胞(B16-F10)中进行过表达,利用酶标仪检测各组细胞黑色素含量的差异,并通过实时荧光定量PCR方法检测各组细胞中cAMP信号通路下游色素相关基因(MITF、TYR、TYRP1、DCT)的表达差异性。结果显示:酉州乌羊及其杂交后代群体中,c.676A>G位点均以GG基因型占优势,不同基因型群体的皮肤色度值差异不显著(P>0.05)。MC1R突变组(MC1R c.676G)细胞中的黑色素含量极显著(P<0.05)高于MC1R野生组(MC1R c.676A)。MITF、TYRP1和DCT基因在MC1R突变组中的相对表达量极显著(P<0.01)高于野生组,TYR基因在MC1R突变组细胞中的相对表达量显著(P<0.05)高于野生组。综上,MC1R基因c.676A>G不直接影响山羊皮肤色素沉积,但MC1R c.676G能促进B16-F10细胞中黑色素的合成,也能促进cAMP信号通路下游MITF、TYR、TYRP1和DCT等基因的表达,推测MC1R基因c.676A>G与黑色素合成密切相关。

关键词: 山羊, MC1R基因, 突变, 黑色素合成, cAMP信号通路

Abstract:

In order to reveal the relationship between goat MC1R gene c.676A>G mutation and goat skin pigmentation, and the effect of mutation on melanin synthesis and the expression of pigment-related genes in the downstream of the cAMP signaling pathway: on the one hand, the direct sequencing was used to detect the MC1R c.676A>G mutation of Youzhou dark-skin goats and the hybrid offsprings (120 individuals), and the abdominal skin color of individuals of different genotypes was determined with the colorimeter, and the relationship between c.676A>G mutation and skin pigmentation was analyzed; on the other hand, the eukaryotic expression vectors of wild type (MC1R c.676A) and mutant type (MC1R c.676G) were constructed and transfected into B16-F10 cells for overexpression by liposome-mediated method, and the differences of melanin in each group were detected by the microplate reader and the expression differences of pigment-related genes in the downstream of the cAMP signaling pathway (MITF, TYR, TYRP1, DCT) in cells were detected by quantitative real-time PCR. The results showed that the c.676A>G site of Youzhou dark-skin goats and their hybrid offsprings was dominated by the GG genotype, and the skin color values of different genotype populations did not show significant difference (P>0.05). The melanin in mutant group cells (MC1R c.676G) showed significantly (P<0.01) higher value than that of the wild group cells (MC1R c.676A). The relative expression of MITF, TYRP1 and DCT in the mutant group cells was significantly (P<0.01) higher than that of the wild group, and the relative expression of TYR in the mutant group was significantly (P<0.05) higher than that of the wild group. In summary, MC1R c.676A>G did not directly affect goat skin pigmentation. However, MC1R c.676G could promote the synthesis of melanin in B16-F10 cells and could increase the expression of MITF, TYR, TYRP1 and DCT genes in the downstream of cAMP signaling pathway, indicating that MC1R c.676A>G was closely related to melanin synthesis.

Key words: goats, MC1R gene, mutation, melanin synthesis, cAMP signaling pathway

中图分类号:

S827

蒋婧, 任航行, 周鹏, 孙晓燕, 李杰, 付琳, 张丽, 刘良佳, 王高富. MC1R c.676A>G与山羊皮肤色素沉积的关系及其对黑色素合成的影响[J]. 浙江农业学报, 2022, 34(12): 2629-2639.

JIANG Jing, REN Hangxing, ZHOU Peng, SUN Xiaoyan, LI Jie, FU Lin, ZHANG Li, LIU Liangjia, WANG Gaofu. Relationship between MC1R c.676A>G and goat skin pigmentation and its effect on melanin synthesis[J]. Acta Agriculturae Zhejiangensis, 2022, 34(12): 2629-2639.

图/表 8

表1 实验用引物的基本参数

Table 1 Parameters of primers used in experiment

引物名称 Primer name	正向引物序列 Forward primer sequences(5'→3')	反向引物序列 Reverse primer sequences(5'→3')	产物大小 Product size/bp
Goat-K-MC1R	TGAGAGCAAGCACCCTTTCCT	GGTCTAGCGATCCTCTTTGTCA	1 062
Goat-MC1R	CTCCATCTTCTACGCCCTGC	GGACGACCGTGTGGTTGTAG	134
Mouse-ACTB	TCCAGCCTTCCTTCTTGGGTAT	GTTGGCATAGAGGTCTTTACGG	104
Mouse-MITF	CAACGGGAACAGCAACGAGC	TTCACCAGATCAGGCGAGCA	164
Mouse-TYR	AATCCTAACTTACTCAGCCCAGC	TAATAGTGGTCCCTCAGGTGTTC	120
Mouse-TYRP1	ACTGATGCGGTCTTTGACGAATG	GCCTGTTATGTCCAATAGGTGCG	91
Mouse-DCT	TCTTCCTAACCGCAGAGCAACTT	GATTCCAATGACCACTGAGAGAG	101

表2 酉州乌羊及其杂交后代MC1R基因c.676A>G位点的基因型(等位基因)频率与遗传特性

Table 2 Genotype (allele) frequency and genetic characteristics of MC1R gene c.676A>G locus in Youzhou dark-skin goats and its hybrid progeny

群体 Group									基因型及其频率 Genotype and frequency
酉州乌羊Youzhou dark-skin goats	0.709	0.291	1.410	0.248	0.617	0.432	A	0.188	AA	0.059(1)
									AG	0.235(4)
							G	0.812	GG	0.706(12)
杂交后代Hybrid offsprings	0.751	0.248	1.331	0.217	4.971	0.025	A	0.145	AA	0.049(5)
									AG	0.194(20)
							G	0.854	GG	0.757(78)

表3 MC1R基因c.676A>G位点多态性下酉州乌羊及其杂交后代的皮肤色度值

Table 3 Skin color of Youzhou dark-skin goats and their hybrid offsprings under different MC1R gene c.676A>G locus polymorphism

基因型 Genotype	酉州乌羊 Youzhou dark-skin goats	杂交后代 Hybrid offsprings
AA	70.07±12.51	31.48±5.59
AG	79.67±6.25	29.92±2.79
GG	75.68±3.61	27.14±1.41

图1 不同时间各组细胞的形态 A、B、C、D分别对应于空白对照组(BC)、阴性对照组(NC)、MC1R野生组(MC1R-A)和突变组(MC1R-G)细胞。1、2、3分别代表转染前、转染24 h和分化72 h。

Fig.1 Cell morphology of each group at different time A, B, C, D represent BC group, NC group, MC1R-A group and MC1R-G group, respectively. 1, 2 and 3 represent pre-transfection, 24 h after transfection and 72 h after differentiation, respectively.

图2 不同时间各组细胞的转染结果 A、B、C、D分别对应于空白对照组(BC)、阴性对照组(NC)、MC1R野生组(MC1R-A)和突变组(MC1R-G)细胞。1、2分别表示转染24 h和分化72 h。

Fig.2 Results of cell transfection in each group at different time A, B, C, D represent BC group, NC group, MC1R-A group and MC1R-G group, respectively. 1 and 2 represent 24 h after transfection and 72 h after differentiation, respectively.

图3 分化72 h时各组细胞山羊MC1R基因的相对表达量柱上无相同大写字母的表示差异极显著(P<0.01)。下同。

Fig.3 Relative expression level of MC1R gene in each group at 72 h of differentiation Bars marked without the same uppercase letters indicate significant difference at P<0.01. The same as below.

图4 各组细胞的黑色素合成率柱上无相同小写字母的表示差异显著(P<0.05)。下同。

Fig.4 Percentage of melanogenesis in each group Bars marked without the same lowercase letters indicate significant difference at P<0.05. The same as below.

图5 各组细胞色素相关基因的表达水平

Fig.5 Expression level of cytochrome related genes in each group

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MC1R c.676A>G与山羊皮肤色素沉积的关系及其对黑色素合成的影响

Relationship between MC1R c.676A>G and goat skin pigmentation and its effect on melanin synthesis

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