Expression and Regulation Analysis of LEF1, YWHAZ and WNT2 Genes in Bashibai Sheep Skins with Different Coat Colors

doi:10.6048/j.issn.1001-4330.2022.11.019

Abstract

Abstract:

【Objective】 To explore the relationship between LEF1, YWHAZ and WNT2 gene expression and coat color and lay the foundation for further analysis of the molecular mechanisms of LEF1, YWHAZ and WNT2 genes in the Wnt/β-actenin signaling pathway. 【Methods】 In the experiment, 8 black and white coats of Bashibai sheep were randomly selected, skin tissues were collected, and the quantitative reverse transcription polymerase chain reaction (qRT-PCR) method was used to determine the amount of LEF1, WNT2 and YWHAZ genes expression in sheep skin which different colors of Bashibai sheep. And it was verified in two-way with the FPKM value of LEF1, YWHAZ and WNT2 gene transcriptome sequencing results. 【Results】 The results showed that the relative expression level of LEF1 gene in black Bashibai sheep skin tissue was (4.66±0.59), and that in white Bashibai sheep skin tissue was (0.43±0.15); the relative expression level of WNT2 gene in black Bashibai sheep tissue was (7.35±0.77), (0.36±0.11) in white Bashibai sheep skin tissue; the relative expression level of YWHAZ gene in black Bashibai sheep tissue was (4.44±0.57), and (1.02±0.23) in white Bashibai sheep skin tissue. The FPKM values of the transcriptome data of LEF1, YWHAZ, and WNT2 genes were consistent with the trend of qRT-PCR results. 【Conclusion】 Explanation: Firstly, the experimental data is authentic and reliable, and secondly, the experimental results prove that LEF1, WNT2, and YWHAZ genes are of different colors in Bashibai sheep, which is expressed in sheep skin tissues. Moreover, the expression levels of LEF1, WNT2 and YWHAZ genes in black sheep skin are higher than those in white sheep skin, indicating that LEF1, WNT2 and YWHAZ are involved in the formation of coat color, which can be used as the correlation between sheep coat color potential gene research and coat color.

Key words: Bashibai sheep; coat color; lymphatic enhancement factor(LEF1); WNT2; tyrosine3-monooxygenase/tryptophan5-monooxygenase activation protein zeta(YWHAZ); quantification reverse transcription Polymerase Chain Reaction

摘要：

【目的】研究LEF1、YWHAZ及WNT2基因表达量与毛色之间的关系,为分析LEF1、YWHAZ及WNT2基因在Wnt/β-actenin信号通路中的分子机制奠定基础。【方法】采用随机选取黑色和白色被毛的巴什拜羊各8只,采集皮肤组织,通过定量反转录聚合酶连锁反应(qRT-PCR)方法测定LEF1、WNT2及YWHAZ基因在不同毛色巴什拜羊皮肤中的表达量,并与LEF1、YWHAZ及WNT2基因转录组测序结果的FPKM值进行双向验证。【结果】LEF1基因在黑色巴什拜羊皮肤组织相对表达量是(4.66±0.59),在白色巴什拜羊皮肤组织是 (0.43±0.15);WNT2基因在黑色巴什拜羊组织相对表达量是(7.35±0.77),在白色巴什拜羊皮肤组织是(0.36±0.11);YWHAZ基因在黑色巴什拜羊组织相对表达量是 (4.44±0.57),在白色巴什拜羊皮肤组织是(1.02±0.23)。LEF1、YWHAZ及WNT2基因的转录组数据的FPKM值与qRT-PCR结果趋势一致。【结论】LEF1、WNT2及YWHAZ基因在不同毛色的巴什拜羊皮肤组织中均有表达。且LEF1、WNT2及YWHAZ基因在黑色绵羊皮肤中的表达量极显著高于白色绵羊皮肤(P<0.01),LEF1、WNT2及YWHAZ参与毛色的形成过程,可作为绵羊毛色潜在基因研究与毛色之间的相关性。

关键词: 绵羊, 毛色, LEF1, WNT2, YWHAZ, 定量反转录聚合酶连锁反应

CLC Number:

S862.8

HONG Wenjuan, HOU Chenxi, HE Zonglong, Jueken Aniwashi. Expression and Regulation Analysis of LEF1, YWHAZ and WNT2 Genes in Bashibai Sheep Skins with Different Coat Colors[J]. Xinjiang Agricultural Sciences, 2022, 59(11): 2749-2757.

洪文娟, 侯晨曦, 何宗龙, 决肯·阿尼瓦什. 不同毛色巴什拜羊皮肤组织中LEF1、YWHAZ及WNT2基因差异表达[J]. 新疆农业科学, 2022, 59(11): 2749-2757.

Figures/Tables 8

Fig.1 Total RNA quality detection

Table 1 qRT-PCR results of LEF1 gene in the skin of black and white Bumbashibai sheep

毛色 Coat colour	基因 Gene	平均CT值	△CT	△△CT	2^-△△CT
黑色Black	LEF1 β-actin	29.66 26.72	3.46±0.50	-2.14±0.11	4.66^A±0.59
白色White	LEF1 β-actin	29.40 23.68	5.40±0.64	1.94±0.247	0.43^B±0.15

Fig.2 Agarose gel electrophoresis of qRT-PCR products Note:M:DL2000 DNA Marker; 1-3:β-actin gene; 4-8:YWHAZ gene; 9-11:WNT2 gene; 12-14:LEF1 gene

Fig.3 Melting and amplification curves of the three target genes and internal reference gene

Table 2 qRT-PCR results of WNT2 gene in the skin of black and white bashibai sheep

毛色 Coat colour	基因 Gene	平均CT值	△CT	△△CT	2^-△△CT
黑色Black	WNT2 β-actin	31.67 26.72	6.03±0.39	-2.38±0.35	7.35^A±0.77
白色White	WNT2 β-actin	31.37 23.68	7.42±0.76	2.39±0.66	0.36^B±0.11

Table 3 qRT-PCR results of YWHAZ gene in the skin of black and white Bashibai sheep

毛色 Coat colour	基因 Gene	平均CT值	△CT	△△CT	2^-△△CT
黑色Black	YWHAZ β-actin	25.87 26.72	1.47±0.14	-1.81±0.21	4.44^A±0.57
白色White	YWHAZ β-actin	26.62 23.68	3.27±0.40	0.81±0.15	1.02^B±0.23

Fig.5 Relative expression levels of LEF1, WNT2 and YWHAZ mRNA in Bashibai sheep skin with different coat color Note: ＊＊ shoulder mark indicated extremely significant difference (P < 0.01)

Fig.6 FPKM values of LEF1, Wnt2 and YWHAZ genes in transcriptome

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