Analysis of DNA Methylation and mRNA Expression Level of LEF1 Gene in Skin Tissues of Bashbay Sheep with Different Wool Colors

doi:10.6048/j.issn.1001-4330.2022.11.018

Abstract

Abstract:

【Objective】 The purpose of this study is to analyze the DNA methylation and mRNA expression levels of LEF1 gene in skin tissues of red brown and blue gray Bashbay sheep. 【Methods】 The methylation level and mRNA expression of LEF1 gene promoter region in different skin tissues of Bashbay sheep were detected by BSP (sulfite Modified Sequencing PCR) and RT-RCR (real-time fluorescent quantitative PCR). 【Results】 The methylation level of LEF1 gene promoter in the skin tissue of red brown Bashbay sheep was higher than that of blue gray, and the methylation CpG sites of LEF1 gene promoter were different. At the same time, SPSS software was used to analyze the correlation between the DNA methylation level of LEF1 gene promoter and the mRNA expression, and it was found that there was a significant negative correlation between them (P< 0.05). 【Conclusion】 The results show that DNA methylation level can regulate the formation of wool color in red brown and blue gray Bashbay sheep, and they can be used as a candidate genetic marker of Bashbay sheep wool color.

Key words: Bashbay sheep; LEF1gene; mRNA expression; DNA methylation

摘要：

【目的】分析LEF1基因在红棕色与青灰色巴什拜羊皮肤组织中DNA甲基化与mRNA的表达水平。【方法】运用BSP(亚硫酸盐修饰后测序PCR)与RT-RCR(实时荧光定量PCR),检测不同毛色巴什拜羊皮肤组织LEF1基因启动子区的甲基化水平与mRNA表达量。【结果】在红棕色巴什拜羊皮肤组织中的LEF1基因启动子区的甲基化水平高于青灰色的甲基化水平,且二者甲基化CpG位点不同,二者呈显著的负相关(P< 0.05)。【结论】DNA甲基化水平对红棕色与青灰色巴什拜羊的毛色形成具有调节作用,可作为一个候选的巴什拜羊毛色遗传标记。

关键词: 巴什拜羊, 毛色, LEF1基因, mRNA表达量, DNA甲基化

CLC Number:

S826.8

HOU Chenxi, HONG Wenjuan, HE Zonglong, Jueken Aniwashi. Analysis of DNA Methylation and mRNA Expression Level of LEF1 Gene in Skin Tissues of Bashbay Sheep with Different Wool Colors[J]. Xinjiang Agricultural Sciences, 2022, 59(11): 2742-2478.

侯晨曦, 洪文娟, 何宗龙, 决肯·阿尼瓦什. LEF1基因在巴什拜羊不同毛色皮肤组织中的DNA甲基化及mRNA表达水平分析[J]. 新疆农业科学, 2022, 59(11): 2742-2478.

Figures/Tables 9

References 22

[1]	杨广礼. 动物毛色形成的调控机制研究[J]. 黑龙江畜牧兽医, 2014,(5): 45-48.
	YANG Guangli. Study on the regulation mechanism of animal hair color formation[J]. Heilongjiang Animal Husbandry and Veterinary, 2014,(5): 45-48.
[2]	Gebreselassie G, Liang B, H Berihulay, et al. Genomic mapping identifies two genetic variants in the MC1R gene for coat colour variation in Chinese Tan sheep[J]. PLoS ONE, 2020, 15(8): e0235426. DOI URL
[3]	新疆家畜家禽品种志编写委员会. 新疆家畜家禽品种志[M]. 乌鲁木齐: 新疆人民出版社, 1985.
	Compilation Committee of Xinjiang Livestock and Poultry Breeds. Species of livestock and poultry in Xinjiang[M]. Urumqi: Xinjiang People's Publishing House, 1985.
[4]	Cieslak M, Reissmann M, Hofreiter M, et al. Colours of domestication[J]. Biological Reviews, 2011, 86(4): 885-899. DOI URL
[5]	徐伟, 封竣淇, 黄兰, 等. TYR基因研究进展[J]. 中国畜牧杂志, 2017, 53(4): 23-27.
	XU Wei, FENG Junqi, HUANG Lan, et al. Research progress of TYR gene[J]. Chinese Journal of Animal Husbandry, 2017, 53(4): 23-27.
[6]	Sethi J, Vidalpuig A. Wnt signalling and the control of cellular metabolism[J]. Biochemical Journal, 2010, 427(1): 1-17. DOI PMID
[7]	Van Genderen C, Okamura R M, Fariñas I, Quo R G, et al. Development of several organs that require inductive epithelial-mesenchymal interactions is impaired in LEF-1-deficient mice[J]. Genes Dev. 1994, 8(22): 2691-703. DOI URL
[8]	Ozren, Bogdanovi, Ryan, et al. DNA methylation and the preservation of cell identity-Science Direct[J]. Current Opinion in Genetics & Development, 2017, 46: 9-14.
[9]	Ito S, Wakamatsu K. Chemistry of mixed melanogenesis: pivotal roles of dopaquinone[J]. Photochemistry and Photobiology, 2008, 84(3): 582-592. DOI URL
[10]	Bonaventure J, Domingues M J, Larue L. Cellular and molecular mechanisms controlling the migration of melanocytes and melanoma cells[J]. Pigment Cell & Melanoma Research, 2013, 26(3):316-325.
[11]	Dasgupta R, Fuchs E. Multiple roles for activated LEF/TCF transcription complexes during hair follicle development and differentiation[J]. Development, 1999, 126(20): 4557-4568. DOI PMID
[12]	Guenther C.A, Tasic B, Luo L., et al. A molecular basis for classic blond hair color in Europeans[J]. Nature Genetics, 2014, 46(7): 748-752. DOI PMID
[13]	Stuckert A, Moore E, Coyle K P, et al. Variation in pigmentation gene expression is associated with distinct aposematic color morphs in the poison frog Dendrobates auratus[J]. BMC Evolutionary Biology, 2019, 19(1): 85. DOI PMID
[14]	Song X, Xu C, Liu Z, et al. Comparative Transcriptome Analysis of Mink (Neovison vison) Skin Reveals the Key Genes Involved in the Melanogenesis of Black and White Coat Color[J]. Scientific Reports, 2017, 7(1): 12461. DOI URL
[15]	杨磊, 王海东, 王祎, 等. Lef-1基因在棕色和白色羊驼皮肤差异表达[J]. 中国生物化学与分子生物学报, 2011, 27(11): 1061-1066.
	YANG Lei, WANG Haidong, WANG Yi, et al. Differential expression of LEF-1 gene in brown and white Alpaca Skin[J]. Chinese Journal of Biochemistry and Molecular Biology, 2011, 27(11): 1061-1066.
[16]	蒋雪, 臧艺玫. 表观遗传概述[J]. 生物学教学, 2015, 40(4): 2-4.
	JIANG Xue, ZANG Yimei. Overview of Epigenetics[J]. Biology Teaching, 2015, 40(4): 2-4.
[17]	钟焱, 徐慧, 彭凤兰. DNA甲基化在基因表达调控中的意义及研究进展[J]. 中国医药导报, 2019, 16(14): 33-36.
	ZHONG Yan, XU Hui, PENG Fenglan. Significance and research progress of DNA methylation in gene expression regulation[J]. China Medical Herald, 2019, 16(14): 33-36.
[18]	龙鑫. 乌珠穆沁羊与呼伦贝尔短尾羊尾椎形态、Brachyury(T-box)基因型特征及甲基化研究[D]. 呼和浩特: 内蒙古农业大学, 2019.
	LONG Xin. Study on morphology, Brachyury (T-box) genotype and methylation of Wuzhumuqin sheep and Hulunbeier sheep[D]. Hohhot: Inner Mongolia Agricultural University, 2019.
[19]	曼则热·朱尔丁, 姚力丹, 包敖敦格日乐, 等. 哈萨克羊MSTN基因的DNA甲基化分析[J]. 新疆农业科学, 2018, 55(7): 1326-1332. DOI
	Manzere Zhuerding, YAO Lidan, Baoaodun, Gerile, et al. DNA methylation analysis of MSTN gene in Kazakh sheep[J]. Xinjiang Agricultural Sciences, 2018, 55(7): 1326-1332.
[20]	刘亮, 朱正权, 阿满·哈迪尔别克, 等. DNA甲基化对miR-133a表达及胶质瘤细胞增殖及凋亡的影响[J]. 现代肿瘤医学, 2021, 29(8): 1296-1300.
	LIU Liang, ZHU Zhengquan, Aman Hadierbuick, et al. Effect of DNA methylation on mir-133a expression and proliferation and apoptosis of glioma cells[J]. Modern Oncology, 2021, 29(8): 1296-1300.
[21]	Chen C, Xue S, Zhang J, et al. DNA‐methylation‐mediated repression of miR‐766‐3p promotes cell proliferation via targeting SF2 expression in renal cell carcinoma[J]. International Journal of Cancer, 2017, 141(9): 1867-1878. DOI PMID
[22]	黄永震. 黄牛肌肉生长发育相关基因甲基化鉴定及IGF2和ZBED6基因的转录调控研究[D]. 杨凌: 西北农林科技大学, 2014.
	HUANG Yongzhen. Identification of methylation and transcriptional regulation of IGF2 and zbed6 genes in cattle muscle growth and development[D]. Yangling: Northwest University of agriculture and forestry science and technology, 2014.

引物名称 Gene name	引物序列 Primer sequence	退火温度 Annealing Temp	产物大小 Product size
β- actin	F: CCAACCGTGAGAAGATGACC R: CAGAGGCGTACAGGGACAG	58	97
LEF1	F:CTCTATCCAGGCTGGTCTGC R:GGCAGCTGTCATTCTTGGAC	58	108
LEF1 -BSP	F:TATTTAGGGAAGTTAAGGTGGAG R:CTAAACTAAACTACCAAAACGACC	58	160

引物名称 Gene name	引物序列 Primer sequence	退火温度 Annealing Temp	产物大小 Product size
β- actin	F: CCAACCGTGAGAAGATGACC R: CAGAGGCGTACAGGGACAG	58	97
LEF1	F:CTCTATCCAGGCTGGTCTGC R:GGCAGCTGTCATTCTTGGAC	58	108
LEF1 -BSP	F:TATTTAGGGAAGTTAAGGTGGAG R:CTAAACTAAACTACCAAAACGACC	58	160

试剂 Reagent	体积 Volume
DNA模板Template DNA	1 μL
PCR Forward Primer	0.5 μL
PCR Reverse Primer	0.5 μL
2×Taq PCR MasterMix	13 μL
ddH2O	10 μL
总体积Total volume	25 μL

试剂 Reagent	体积 Volume
DNA模板Template DNA	1 μL
PCR Forward Primer	0.5 μL
PCR Reverse Primer	0.5 μL
2×Taq PCR MasterMix	13 μL
ddH2O	10 μL
总体积Total volume	25 μL

程序 Program	温度 Temperature	时间 Time	循环数 Number of cycles
预变性 Pre-denaturation	94℃	5 min	-
变性 Denaturation	94℃	30s
退火 Anneal	62℃~52℃	30s	10cycle
延伸 Extension	72℃	1 min
变性 Denaturation	94℃	30s
退火 Anneal	最适温度	30s	30cycle
延伸 Extension	72℃	1 min
延伸 Extension	72℃	10 min	-
-	4℃	保存	-