红麻DNA甲基化响应镉胁迫及甲基化差异基因的表达分析

doi:10.3724/SP.J.1006.2021.04269

Abstract

Abstract:

DNA methylation is one of the important epigenetic modifications in plants and plays an important role in response to stress. However, there are few reports of the changes of DNA methylation levels in plants under cadmium stress. The seedlings of kenaf P3A were treated with 300 μmol L^-1 CdCl₂ by hydroponics, the agronomic traits and cadmium contents were investigated. The changes of DNA methylation level of root under cadmium stress were analyzed by using methylation sensitive amplified polymorphism (MSAP) method. In addition, the differential methylation fragments were recovered, cloned, and sequenced. The relative expression levels of differential methylated genes revealed that Cd stress significantly inhibited the plant height, stem diameter, root length, root surface area, and total fresh weight of kenaf seedlings. The whole DNA methylation ratio in root was 68.23% and 62.78% under cadmium and control treatments, respectively. Among them the total methylation ratio was 37.50% and 36.36%, and the semi-methylation ratio was 25.28% and 31.87%, respectively. These results indicated that cadmium stress significantly increased the DNA methylation level of roots DNA. Seven differential methylated genes which involved in stress resistance were characterized differentially expressed under cadmium stress, suggesting that the change of DNA methylation played an important role in response to cadmium stress. This study provides a theoretical basis for further exploring the potential mechanism of DNA methylation in response to cadmium stress in plants.

Key words: kenaf, cadmium stresses, DNA methylation, methylation-sensitive amplified polymorphism (MSAP), real-time fluorescent quantitative PCR (qRT-PCR)

LU Hai, LI Zeng-Qiang, TANG Mei-Qiong, LUO Deng-Jie, CAO Shan, YUE Jiao, HU Ya-Li, HUANG Zhen, CHEN Tao, CHEN Peng. DNA methylation in response to cadmium stress and expression of different methylated genes in kenaf[J].Acta Agronomica Sinica, 2021, 47(12): 2324-2334.

Figures/Tables 9

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Fig. 1

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Table 6

Comparisons of differentially methylated sequences"

序列编号 Sequence number	甲基化差异片段比对结果 Comparisons of differentially methylated sequences	功能注释 Functional annotation
1	Leucine-rich repeat receptor-like kinases, (LRR-RLK) At5g15730, Gossypium	充当胞外信号的受体, 参与各种环境及发育信号的感知和传递^[22]。 As the receptor of extracellular signal, it participates in the perception and transmission of various environmental and developmental signals^[22].
2	Zinc finger protein XlCGF8.2DB-like (XlCGF8.2DB), Bemisia tabaci gastrula	参与一些重要的调控过程, 如; 形态建成、花粉发育、胚发育、胁迫反应等^[23]。 Widely involved in regulatory of important processes, such as morphogenesis, pollen development, embryo development, stress response, and so on^[23].
3	Ankyrin-3-like, Gossypium arboreum	尚未见报道。 Has not been reported.
4	Trehalose-phosphate phosphatase D (GaTPPD), Gossypium arboreum	海藻糖磷酸磷酸酶在植物生长和胁迫反应中起重要作用^[24]。 Trehalose phosphatase plays an important role in plant growth and stress response^[24].
5	Serine/threonine-protein kinase, Gossypium raimondii	丝氨酸/苏氨酸蛋白激酶, 催化多种功能蛋白的磷酸化, 调控植物非生物胁迫^[25]。 Serine/threonine protein kinases, catalyze the phosphorylation of various functional proteins and regulate abiotic stress in plants^[25].
6	Peroxidasin (Pxdn) transcript variant X6, Castor canadensis	过氧化物酶, 植物抗逆过程中的关键酶之一^[26]。 Peroxidase is one of the key enzymes in plant stress resistance^[26].
7	Protein NRT1/ PTR FAMILY 2.7 (NPF2.7), Gossypium hirsutum	NRT1/PTR家族蛋白参与转运植物激素及次生代谢物合成过程。 NRT1/PTR family proteins is involved in the transport of plant hormones and the synthesis of secondary metabolites.
8	60S ribosomal protein L16(RPL16), Gossypium hirsutum	参与细胞中蛋白质合成及调控基因表达^[27]。 It is involved in protein synthesis and gene expression regulation^[27].
9	Receptor-like protein kinase At2g46850, Durio zibethinus	类受体激酶通过接收和传递胞外信号调控细胞的生理反应, 参与植物生长发育过程。 Receptor like kinases are involved in plant growth and development by receiving and transmitting extracellular signals to regulate cell physiological responses.
10	Transcript variant X2, Gossypium raimondii	转录变异体X2, 参与细胞增殖及细胞周期活动的调节。 Transcription variant X2 is involved in the regulation of cell proliferation and cell cycle activity.
11	3-ketoacyl-CoA synthase 1 (GrKCS1), Gossypium raimondii	3-酮脂酰辅酶A合成酶, 在抵抗干旱和盐害等非生物胁迫过程中起着重要的作用。 3-ketoacyl coenzyme A synthetase plays an important role in abiotic stresses such as drought and salt stress.
12	L-ascorbate oxidase homolog (L-AAOh), Gossypium hirsutum	抗坏血酸氧化酶, 在调节植物发育过程和应激反应方面发挥重要作用^[21]。 L-Ascorbic acid oxidase plays an important role in regulating plant development and stress response^[21].
13	Indole-3-acetic acid-amido synthetase (GH3.1), Camellia sinensis	吲哚-3醋酸-酰胺合成酶, 通过调节植物体内激素的动态平衡, 参与调控植物的生长发育过程^[18]。 Indole-3 acetate amide synthetase is involved in the regulation of plant growth and development by regulating the dynamic balance of hormones in plants^[18].
14	Acyl-CoA-binding domain-containing protein 4 (ACBD4)	酰基辅酶A结合域蛋白, 在植物的生长发育、生物和非生物胁迫应答中起重要的作用。 Acyl-CoA-binding domain proteins play an important role in plant growth and development, biological and abiotic stress response.
序列编号 Sequence number	甲基化差异片段比对结果 Comparisons of differentially methylated sequences	功能注释 Functional annotation
15	ABC transporter G family member 26 (ABCG26), Gossypium arboreum	ABC 转运蛋白G家族参与植物信号转导、次生代谢物运输和非生物胁迫响应等过程^[20]。 ABC transporter G family are involved in plant signal transduction, secondary metabolite transport and abiotic stress response^[20].
16	NADP-dependent malic enzyme (NADP-ME) At1G65930, Gossypium hirsutum	NADP依赖苹果酸酶, 通过苹果酸代谢来平衡细胞内的pH来防御生物和非生物胁迫^[19]。 NADP relies on malic acid enzyme to balance intracellular pH through malate metabolism to protect against biotic and abiotic stresses^[19].
17	NAC domain-containing protein 71 (GrNAC71)	NAC转录因子, 在逆境胁迫信号转导过程中发挥重要作用^[28]。 NAC transcription factor plays an important role in stress signal transduction^[28].
18	F-box protein At3g07870, Gossypium raimondii	参与调控胞内蛋白降解、受体识别和信号传导^[29]。 It is involved in the regulation of intracellular protein degradation, receptor recognition and signal transduction^[29].
19	Pentatricopeptide repeat-containing protein (PPRC) At5g55740, Gossypium arboreum	从RNA剪切、编辑、降解和翻译等多个方面影响细胞器中RNA的新陈代谢^[30]。 RNA metabolism in organelles is affected by RNA splicing, editing, degradation, and translation^[30].
20	Ethylene-responsive transcription factor 2 (ERF2)	乙烯转录因子(AP2/ERFs)调控激素响应非生物胁迫^[31]。 Ethylene transcription factors (AP2/ERFs), widely involved in plant hormone regulation and thus response to abiotic stress^[31].

Table 6

Fig. 3

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引物类型 Primer type	引物名称及序列 Primer name and sequence (5′-3′)
引物类型 Primer type	EcoR I (E)			Hpa II/Msp I (HM)
接头 Adapter	EA1	CTCGTAGACTGCGTACC	HMA1	GACGATGAGTCTAGAA
接头 Adapter	EA2	AATTGGTACGCAGTC	HMA2	CGTTCTAGACTCATC
预扩增	E0	GACTGCGTACCAATTCA	HM0	GATGAGTCTAGAACGGT
Pre-amplification	E1	GACTGCGTACCAATTCAAC	HM1	GATGAGTCTAGAACGGTAG
	E2	GACTGCGTACCAATTCAAG	HM2	GATGAGTCTAGAACGGTAC
	E3	GACTGCGTACCAATTCACA	HM3	GATGAGTCTAGAACGGTTG
选择性扩增	E4	GACTGCGTACCAATTCACT	HM4	GATGAGTCTAGAACGGTTC
Selective amplification	E5	GACTGCGTACCAATTCACC	HM5	GATGAGTCTAGAACGGTGT
	E6	GACTGCGTACCAATTCACG	HM6	GATGAGTCTAGAACGGTGC
	E7	GACTGCGTACCAATTCAGC	HM7	GATGAGTCTAGAACGGTCT
	E8	GACTGCGTACCAATTCAGG	HM8	GATGAGTCTAGAACGGTCG

引物名称 Primer name	正向引物 Forward sequence (5′-3′)	反向引物 Reverse sequence (5′-3′)
GH3.1	GACCGCCGTCTGTAACTCG	GGTTCCACAACTCCGCCC
GrKCS1	GCCGCCAGTGTGATGGATAT	CGGTGCTCGGTTCTCCATT
NADP-ME	TTCATCTTCCCTTCGCCTT	CATCCCAACAACACCTTCCT
ABCG26	ATTGGTACGCAGTCACGATG	TTCACCTTCATTTGACACGG
L-AAOh	AAGCCTTTCCGATTGTTCTGC	GGAGTTAGATTTTTGTTGTTGGGAG
LRR-RLK	GCATATACACGATAAGAAACCGC	CATGAAGATATTCCAAGCCCC
GaTPPD	CCTTCCTCCCTTGGGTTAGT	TTCTGTTCGCCTGTGGTTTT
XlCGF8.2DB	GACTGCGTACCAATTCAAGGC	AATTGTTGCCATATTTCTCCCAT
His3 (reference gene)	GTGGAGTCAAGAAGCCTCACAG	ATGGCTCTGGAAACGCAAA

CdCl₂浓度 Concentration of CdCl₂(μmol L^-1)	株高 Plant height (cm)	茎粗 Stem diameter (mm)	全鲜重 Fresh weight (g)	根鲜重 Root fresh weight (g)	根长 Root length (cm)	根表面积 Root surface area (cm²)
0	29.24 a	2.49 a	21.19 a	9.27 a	275.32 a	23.68 a
300	19.94 b	1.70 b	9.54 b	5.50 b	186.74 b	8.98 b

CdCl₂浓度 Concentration of CdCl₂(μmol L^-1)	株高 Plant height	茎粗 Stem diameter	全鲜重 Fresh weight	根鲜重 Root fresh weight	根长 Root length	根表面积 Root surface area
0	0	0	0	0	0	0
300	21.52	21.07	54.93	40.67	64.27	49.66

甲基化类型 Methylation type	各类型的条带数和比率 Band numbers and ratio of each type		变化比率(上升↑, 下降↓) Exchange rate (up↑, down↓)
甲基化类型 Methylation type	0 μmol L^-1	300 μmol L^-1	变化比率(上升↑, 下降↓) Exchange rate (up↑, down↓)
类型I (无甲基化) Type I (Unmethylation)	356	304	↓ 14.61
类型II (半甲基化) Type II (Hemi-methylation)	242	305	↑ 26.03
类型III, IV (全甲基化) Type III and IV (Full methylation)	359	348	↓ 3.06
半甲基化率Hemi-methylated ratio (%)	25.28	31.87	↑ 26.07
全甲基化率Fully methylated ratio (%)	37.50	36.36	↓ 3.04
甲基化率/MSAP Total methylated ratio/MSAP (%)	62.78	68.23	↑ 8.68

DNA methylation in response to cadmium stress and expression of different methylated genes in kenaf

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