鸡VVD候选基因<em>ST3GAL4</em>甲基化水平与ALP活性和基因表达的关系

doi:10.3969/j.issn.1674-7968.2023.02.014

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摘要肢体内外翻畸形(valgus-varus deformity, VVD)是肉鸡(Gallus gallus)养殖业中最常见的腿病。为了探究VVD相关基因ST3 β-半乳糖苷α-2,3-唾液酸转移酶4 (ST3 β-galactoside α-2,3-sialyltransferase 4, ST3GAL4)启动子区甲基化水平与血清碱性磷酸酶(alkaline phosphatase, ALP)活性和基因表达的关系,选用VVD和正常肉鸡检测其血清ALP活性,采用qPCR技术检测ST3GAL4基因在不同组织中的差异表达,筛选差异表达组织,进一步利用亚硫酸氢盐测序法(bisulfite sequencing PCR, BSP)检测ST3GAL4基因在血液和差异表达组织中的甲基化水平。结果表明,VVD组ALP活性显著低于正常组(P<0.05),腿肌ST3GAL4基因的表达量显著高于正常组(P<0.05)。甲基化水平检测结果表明：在血液中,VVD组ST3GAL4启动子区总体甲基化水平高于正常组(P=0.051),CpG25甲基化差异显著(P<0.05);在腿肌组织中,VVD组ST3GAL4启动子区总体甲基化水平极显著低于正常组(P<0.01),CpG11、CpG14、CpG15甲基化差异达到显著水平(P<0.05)。ST3GAL4启动子区转录因子预测结果显示：关键位点CpG15可能与转录因子CRE结合蛋白1 (cAMP response element binding protein 1, CRE-BP1)、cAMP应答元件结合蛋白(cAMP response element binding protein, CREB)和细胞质多聚腺苷酸化元件结合蛋白(cytoplasmic polyadenylation element binding protein, CPE bind)相结合,关键位点CpG25可能与转录因子特异蛋白1 (specificity protein 1, Sp1)和激活蛋白-2α (activator proein-2α, AP-2α)相结合。相关性分析结果表明,血液ST3GAL4启动子区关键位点CpG25甲基化水平和血清ALP活性呈负相关,腿肌组织ST3GAL4启动子区关键位点CpG15甲基化水平和基因表达量也呈负相关。本研究揭示了VVD肉鸡ST3GAL4基因启动子区甲基化水平与血清ALP活性、基因表达水平存在一定联系,为肉鸡抗病育种工作提供参考数据。

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	马岩超
	李建增
	张露洁
	蔡春霞
	蒋瑞瑞
	张彦华
	田亚东
	韩露
	吉进卿
	韩瑞丽

关键词 ：鸡, 肢体内外翻畸形(VVD), ST3 β-半乳糖苷α-2,3-唾液酸转移酶4 (ST3GAL4), 甲基化, 碱性磷酸酶(ALP), 基因表达

Abstract：Valgus-varus Deformity (VVD) is one of the most common leg diseases in broiler (Gallus gallus) breeding industry. To explore the relationship between the methylation level of the promoter region of the VVD related gene ST3 β-galactoside α-2,3-sialyltransferase 4 (ST3GAL4) and the activity and gene expression of alkaline phosphatase (ALP), the differential expression of ST3GAL4 in different tissues was detected by qPCR, and the differential expression tissues were screened. Further, bisulfite-sequencing PCR (BSP) were carried out to detect the methylation level of ST3GAL4 in blood and differentially expressed tissues. The results showed that the ALP activity of broilers in VVD group was significantly lower than that in normal group (P<0.05); The expression level of ST3GAL4 gene in leg muscle of broilers in VVD group was significantly higher than that in normal group (P<0.05). The results of methylation level showed that the overall methylation level of blood ST3GAL4 promoter region in VVD group was higher than that in normal group (P=0.051), and the methylation level of CpG25 was significantly different (P<0.05). In leg muscle tissue, the overall methylation level of ST3GAL4 promoter region of broilers in VVD group was significantly lower than that in normal group (P<0.01), and the methylation levels of CpG11, CpG14 and CpG15 were significantly different (P<0.05). The prediction results of transcription factors in ST3GAL4 promoter region showed that the key site CpG15 might bind to the transcription factors cAMP response element binding protein 1 (CRE-BP1), cAMP response element binding protein (CREB) and cytoplasmic polyadenylation element binding protein (CPE bind), and the key site CpG25 might bind to the transcription factors specific protein 1 (Sp1) and activator protein-2α (AP-2α). Correlation analysis showed that the methylation level of CpG25 in the ST3GAL4 promoter region in blood was negatively correlated with serum ALP activity, and the methylation level of CpG15 in the ST3GAL4 promoter region in leg muscle was also negatively correlated with gene expression. This study reveals that the methylation level of the promoter region of ST3GAL4 gene in VVD broilers is correlated with gene expression level and serum ALP activity, which provides reference data for disease resistance breeding of broilers.

Key words： Chicken Valgus-varus deformity (VVD) ST3 β-galactoside α-2,3-sialyltransferase 4 (ST3GAL4) Methylation Alkaline phosphatase (ALP) Gene expression

收稿日期: 2022-04-25

ZTFLH:

S831.2

基金资助:河南省自然科学基金(222300420458); 中原学者首席科学家工作室(30601985)

通讯作者: *rlhan@126.com

引用本文:

马岩超, 李建增, 张露洁, 蔡春霞, 蒋瑞瑞, 张彦华, 田亚东, 韩露, 吉进卿, 韩瑞丽. 鸡VVD候选基因ST3GAL4甲基化水平与ALP活性和基因表达的关系[J]. 农业生物技术学报, 2023, 31(2): 361-369.
MA Yan-Chao, LI Jian-Zeng, ZHANG Lu-Jie, CAI Chun-Xia, JIANG Rui-Rui, ZHANG Yan-Hua, TIAN Ya-Dong, HAN Lu, JI Jin-Qing, HAN Rui-Li. Relationship Between the Methylation Level of VVD Candidate Gene ST3GAL4 and ALP Activity and Gene Expression in Chicken (Gallus gallus). 农业生物技术学报, 2023, 31(2): 361-369.

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http://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2023.02.014 或 http://journal05.magtech.org.cn/Jwk_ny/CN/Y2023/V31/I2/361

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