5-氮胞苷对体细胞胚胎诱导水曲柳DNA甲基化的影响

doi:10.3969/j.issn.1004-1524.20240997

浙江农业学报 ›› 2025, Vol. 37 ›› Issue (8): 1648-1657.DOI: 10.3969/j.issn.1004-1524.20240997

5-氮胞苷对体细胞胚胎诱导水曲柳DNA甲基化的影响

丛建民¹(), 黄威剑¹, 尹欣幸¹, 刘金平¹, 赵永²^,^*()

1.台州科技职业学院农业与生物工程学院,作物研究所,浙江台州 318020
2.白城师范学院生命科学学院,吉林白城 137000

收稿日期:2024-11-19 出版日期:2025-08-25 发布日期:2025-09-03
作者简介:丛建民(1974—),男,吉林大安人,博士,教授,研究方向为生物化学与分子生物学。E-mail:congjianmin@126.com
通讯作者: *赵永,E-mail:13500835885@139.com
基金资助:
国家自然科学基金(30872008)

Effect of 5-azacytosine on somatic embryo-induced DNA methylation in Fraxinus mandschurica

CONG Jianmin¹(), HUANG Weijian¹, YIN Xinxing¹, LIU Jinping¹, ZHAO Yong²^,^*()

1. Crop Research Institute, School of Agricultural and Biological Engineering, Taizhou Vocational College of Science and Technology, Taizhou 318020, Zhejiang, China
2. College of Life Sciences, Baicheng Normal University, Baicheng 137000, Jilin, China

Received:2024-11-19 Online:2025-08-25 Published:2025-09-03
Contact: ZHAO Yong

摘要/Abstract

摘要： 为研究5-氮胞苷(5-azacytidine, 5-AzaC)在水曲柳体细胞胚胎发生中的表观遗传调控作用,通过在培养基中添加20.0 μmol·L^-1 5-AzaC诱导水曲柳体细胞胚胎发生,以未添加5-AzaC为对照,采用甲基化敏感扩增多态性(methylation sensitive amplification polymorphism, MSAP)法分析外植体基因组DNA的胞嘧啶甲基化水平和模式,以及变异对体细胞胚胎发生的影响。结果表明:5-AzaC可以降低水曲柳外植体的褐化率,引起外植体基因组DNA甲基化状态发生变化,具体表现为全甲基化比例升高,半甲基化比例降低。5-AzaC抑制外植体基因组DNA的胞嘧啶甲基化,诱导基因组DNA去甲基化变异,从而降低体细胞胚胎的诱导率。研究结果表明,表观遗传参与调控树木体细胞胚胎发生,为树木体细胞胚胎发生提供了理论指导。

关键词: 水曲柳, 体细胞胚胎, DNA甲基化, 5-氮胞苷, 表观遗传

Abstract:

To investigate the role of 5-azacytidine (5-AzaC) in the epigenetic regulation of genomic DNA methylation during somatic embryogenesis in Fraxinus mandsharica Rupr., somatic embryos were induced by adding 20.0 μmol·L^-1 5-azacytidine to the medium, with untreated samples as the control, methylation-sensitive amplification polymorphism (MSAP) was used to analyze the effects of cytosine methylation levels, patterns, and variations in the genomic DNA of explants on somatic embryogenesis. The results showed that, the browning rate of the explants was reduced by 5-AzaC treatment, which led to alterations in the genomic DNA methylation status, specifically an increase in fully methylated cytosines and a decrease in hemimethylated cytosines. 5-AzaC inhibited cytosine methylation and induced DNA demethylation variation in explant genomic DNA, thereby reducing the induction rate of somatic embryos. These findings demonstrated that epigenetic mechanisms participate in regulating somatic embryogenesis in trees, which provided theoretical guidance for the study of tree somatic embryogenesis.

Key words: Fraxinus mandsharica, somatic embryo, DNA methylation, 5-azacytidine, epigenetics

中图分类号:

S718.3

丛建民, 黄威剑, 尹欣幸, 刘金平, 赵永. 5-氮胞苷对体细胞胚胎诱导水曲柳DNA甲基化的影响[J]. 浙江农业学报, 2025, 37(8): 1648-1657.

CONG Jianmin, HUANG Weijian, YIN Xinxing, LIU Jinping, ZHAO Yong. Effect of 5-azacytosine on somatic embryo-induced DNA methylation in Fraxinus mandschurica[J]. Acta Agriculturae Zhejiangensis, 2025, 37(8): 1648-1657.

图/表 8

参考文献 22

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外植体类型 Explant type	总位点 Total sites	未甲基化位点数(占比) Non-methylation sites (ratio/%)	甲基化的CCGG位点数(占比/%) Methylation sites of CCGG(ratio/%)
外植体类型 Explant type	总位点 Total sites	未甲基化位点数(占比) Non-methylation sites (ratio/%)	甲基化总位点 Methylation total sites	全甲基化位点 Holomethylation sites	半甲基化位点 Semimethylation site
HT	381	268(70.34)	113(29.66)	68(17.85)	45(11.79)
HY	390	276(70.77)	114(29.23)	68(17.44)	46(11.79)
HD	390	276(70.77)	114(29.23)	71(18.21)	43(11.02)
HW	388	272(70.10)	116(29.90)	75(19.33)	41(10.57)
WD	389	274(70.44)	115(29.56)	75(19.28)	40(10.28)
WW	390	276(70.77)	115(29.23)	69(17.69)	45(11.54)
5-HT	388	272(70.10)	116(29.90)	68(17.53)	48(12.37)
5-HY	389	274(70.44)	115(29.56)	63(16.20)	52(13.36)
5-HD	389	274(70.44)	115(29.56)	67(17.22)	48(12.34)
5-HW	389	274(70.44)	115(29.56)	64(16.45)	51(13.11)
5-WD	389	274(70.44)	115(29.56)	67(17.22)	48(12.34)
5-WW	389	274(70.44)	115(29.56)	68(17.48)	47(12.08)

外植体类型 Explant type	总位点 Total sites	未甲基化位点数(占比) Non-methylation sites (ratio/%)	甲基化的CCGG位点数(占比/%) Methylation sites of CCGG(ratio/%)
外植体类型 Explant type	总位点 Total sites	未甲基化位点数(占比) Non-methylation sites (ratio/%)	甲基化总位点 Methylation total sites	全甲基化位点 Holomethylation sites	半甲基化位点 Semimethylation site
HT	381	268(70.34)	113(29.66)	68(17.85)	45(11.79)
HY	390	276(70.77)	114(29.23)	68(17.44)	46(11.79)
HD	390	276(70.77)	114(29.23)	71(18.21)	43(11.02)
HW	388	272(70.10)	116(29.90)	75(19.33)	41(10.57)
WD	389	274(70.44)	115(29.56)	75(19.28)	40(10.28)
WW	390	276(70.77)	115(29.23)	69(17.69)	45(11.54)
5-HT	388	272(70.10)	116(29.90)	68(17.53)	48(12.37)
5-HY	389	274(70.44)	115(29.56)	63(16.20)	52(13.36)
5-HD	389	274(70.44)	115(29.56)	67(17.22)	48(12.34)
5-HW	389	274(70.44)	115(29.56)	64(16.45)	51(13.11)
5-WD	389	274(70.44)	115(29.56)	67(17.22)	48(12.34)
5-WW	389	274(70.44)	115(29.56)	68(17.48)	47(12.08)

模式类型 Type of Patterns	位点类型Type of sites				HT	HY	HD	HW	WD	5-HT	5-HY	5-HD	5-HW	5-WD	5-WW
	未褐化未长大WW		其他类型 Other types
	H	M	H	M
1	-	-	-	-	23	21	15	21	24	21	17	18	16	19	19
2	+	-	-	-	1	2	1	0	0	3	7	6	7	5	5
3	-	+	-	-	0	1	8	3	0	0	0	0	1	0	0
4	-	-	+	-	0	0	2	3	3	0	0	3	1	2	3
5	+	-	+	-	44	44	42	41	40	45	45	42	44	43	42
6	+	+	+	-	1	1	1	1	2	0	0	0	0	0	0
7	-	-	-	+	1	0	0	0	0	0	1	1	1	1	1
8	-	+	-	+	43	44	44	45	45	45	44	43	43	43	43
9	+	+	-	+	1	1	1	0	0	0	0	1	1	1	1
10	+	-	+	+	1	2	2	3	3	2	1	2	2	2	2
11	-	+	+	+	1	2	2	3	3	2	1	2	2	2	2
12	+	+	+	+	274	272	272	270	270	272	274	272	272	272	272

模式类型 Type of Patterns	位点类型Type of sites				HT	HY	HD	HW	WD	5-HT	5-HY	5-HD	5-HW	5-WD	5-WW
	未褐化未长大WW		其他类型 Other types
	H	M	H	M
1	-	-	-	-	23	21	15	21	24	21	17	18	16	19	19
2	+	-	-	-	1	2	1	0	0	3	7	6	7	5	5
3	-	+	-	-	0	1	8	3	0	0	0	0	1	0	0
4	-	-	+	-	0	0	2	3	3	0	0	3	1	2	3
5	+	-	+	-	44	44	42	41	40	45	45	42	44	43	42
6	+	+	+	-	1	1	1	1	2	0	0	0	0	0	0
7	-	-	-	+	1	0	0	0	0	0	1	1	1	1	1
8	-	+	-	+	43	44	44	45	45	45	44	43	43	43	43
9	+	+	-	+	1	1	1	0	0	0	0	1	1	1	1
10	+	-	+	+	1	2	2	3	3	2	1	2	2	2	2
11	-	+	+	+	1	2	2	3	3	2	1	2	2	2	2
12	+	+	+	+	274	272	272	270	270	272	274	272	272	272	272

变异类型 Mutation types		带型Types				数量Number
		未褐化未长大WW		其他类型 Other types		HT	HY	HD	HW	WD	5-HT	5-HY	5-HD	5-HW	5-WD	5-WW
		H	M	H	M	HT	HY	HD	HW	WD	5-HT	5-HY	5-HD	5-HW	5-WD	5-WW
A	A1	+	+	+	-	1	1	1	1	2	0	0	0	0	0	0
	A2	+	+	-	+	1	1	1	0	0	0	0	1	1	1	1
B	B1	+	-	+	+	1	2	2	3	3	2	1	2	2	2	2
	B2	+	-	-	-	1	2	1	0	0	3	7	6	7	5	5
C	C1	-	+	+	+	1	2	2	3	3	2	1	2	2	2	2
	C2	-	+	-	-	0	1	8	3	0	0	0	0	1	0	0
D	D1	-	-	+	-	0	0	2	3	3	0	0	3	1	2	3
	D2	-	-	-	+	1	0	0	0	0	0	1	1	1	1	1
总计Total						6	9	17	13	11	7	10	15	15	13	14

5-氮胞苷对体细胞胚胎诱导水曲柳DNA甲基化的影响

Effect of 5-azacytosine on somatic embryo-induced DNA methylation in Fraxinus mandschurica

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参考文献 22

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模式类型 Pattern types	位点类型Site types				数量Number
	对照Control		其他类型Other types		5-HT	5-HY	5-HD	5-HW	5-WD	5-WW
	H	M	H	M	5-HT	5-HY	5-HD	5-HW	5-WD	5-WW
1	-	-	-	-	21	16	11	16	21	19
2	+	-	-	-	2	5	6	7	6	5
3	-	+	-	-	1	0	0	1	0	0
4	-	-	+	-	0	0	2	0	0	3
5	+	-	+	-	45	46	41	41	40	42
6	-	-	-	+	0	2	9	2	1	1
7	+	-	-	+	0	0	0	2	0	0
8	-	+	-	+	44	44	44	45	45	43
9	+	+	-	+	0	1	1	2	2	1
10	+	-	+	+	1	1	1	1	2	0
11	-	+	+	+	2	1	1	0	0	2
12	+	+	+	+	272	272	272	270	270	272

甲基化变异类型 Methylation mutation types		数量Number
甲基化变异类型 Methylation mutation types		5-HT	5-HY	5-HD	5-HW	5-WD	5-WW
甲基化上升位点	CNG位点升高CNG increase sites	0	0	0	0	0	0
Methylation increase site	CG位点升高CG increase sites	0	1	1	2	2	1
	CG/CNG位点升高CG/CNG increase sites	0	0	0	0	0	0
甲基化位点上升总数(比例)Total methylation increase sites(rate)		0(0)	1(20%)	1(7.15%)	2(40%)	2(40%)	1(40%)
甲基化下降的位点	CNG位点下降CNG decreased site	1	3	12	3	3	1
Decreased methylation site	CG位点下降CG decreased site	2	1	1	0	0	2
	CG/CNG下降位点CG/CNG decreased site	0	0	0	0	0	0
甲基化位点下降总数(比例)Total methylation decreased sites(rate)		3(100%)	4(80%)	13(92.85%)	3(60%)	3(60%)	3(75%)
甲基化总变异位点Total methylation mutation sites		3	5	14	5	5	4

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