铅胁迫下红麻生理特性及DNA甲基化分析

doi:10.3724/SP.J.1006.2021.04104

Abstract

Abstract:

DNA methylation plays an important role in response to plant biotic and abiotic stresses, but there are few reports on the changes of plant DNA methylation level under lead stress. In this study, kenaf (Hibiscus cannabinus L.) P3A was used as the material, the seedlings were cultured in Hoagland solution, and treated with at different concentrations (0, 200, 400, and 600 μmol L ^-1) of PbCl₂. The changes of agronomic traits, ROS content and antioxidant enzyme activity of root were investigated. The changes of DNA methylation level in roots under 600 μmol L ^-1 lead stress were determined by methylation-sensitive amplification polymorphism (MSAP). The differentially methylated genes (DMGs) were cloned, sequenced, and functionally annotated. In addition, the expression levels of DMGs were investigated by qRT-PCR. The results showed that the stem diameter, root length and root surface area of seedlings were significantly inhibited by different concentrations of PbCl₂ stress. And the plant height and total fresh weight of kenaf seedlings were significantly reduced under 400 μmol L ^-1 concentration or more of lead stress. The content of lead, O₂ ^? and MDA, and activities of SOD were increased significantly in kenaf seedlings roots, CAT activity increased first and then decreased with the increase of lead concentration, the POD activity showed a trend of decreasing first and then increasing. MSAP analysis of roots treated with 0 μmol L ^-1 and 600 μmol L ^-1 PbCl₂ showed that DNA methylation rates were 71.13%, 62.20%, fully methylated ratio was 50.52%, 37.80%, and hemi-methylated ratio were 20.62%, 24.40%, respectively. In other words, lead stress significantly reduced DNA methylation rate and total methylation rate, whereas increased the hemi-methylation rate of roots of kenaf seedlings. qRT-PCR analysis showed that there were also differences in gene expression of seven DMGs closely related to resistance. It suggested that the change of DNA methylation level played an important role in kenaf response to lead stress in kenaf. This study provides a theoretical basis for further exploring the potential mechanism of DNA methylation in response to plant abiotic stress, and improving soil lead pollution in kenaf production.

Key words: kenaf, lead stress, DNA methylation, MSAP, qRT-PCR, antioxidant enzyme system

LI Zeng-Qiang, DING Xin-Chao, LU Hai, HU Ya-Li, YUE Jiao, HUANG Zhen, MO Liang-Yu, CHEN Li, CHEN Tao, CHEN Peng. Physiological characteristics and DNA methylation analysis under lead stress in kenaf (Hibiscus cannabinus L.)[J].Acta Agronomica Sinica, 2021, 47(6): 1031-1042.

Figures/Tables 9

Table 1

Table 2

Table 3

Fig. 1

Fig. 2

Fig. 3

Table 4

Table 5

Comparisons analysis of differentially methylated sequences"

基因名 Gene name	片段大小 Fragment length (bp)	基因代号或ID Gene symbol or ID	基因全称 Gene full name	功能注释 Functional annotation
7-dlgase	255	Hca.05G0009940	Hca. flavonol-3-O-glycoside-7-O-glucosyltransferase 1	将UDP-糖基供体转移到花色素的C7羟基(羧基)^[18]。 Transfer UDP glycosyl donor to C7 hydroxy (carboxy) of anthocyanins.
AHL23	110	Hca.02G0040470	Hca. AT hook motif domain containing protein	AT-hook家族蛋白在植物生长发育、激素信号转导和逆境胁迫应答中发挥重要作用^[19]。 At-hook family proteins play an important role in plant growth and development, plant hormone signal transduction and stress response.
CesA2	140	Hca.03G0045600	Hca. CESA1-cellulose synthase	CesA影响初生壁和次生壁的生物合成,响应植物的抗病性^[20]。 CesA affects cell wall biosynthesis and responds to plant disease resistance.
chD12	136	CP023742	Gossypium hirsutum cultivar TM1 chromosome D12	尚未见报道。 Has not been reported.
EF1B/S6	131	Hca.09G0002430	Hca. eukaryotic translation initiation factor 3 subunit D	尚未见报道。 Has not been reported.
ETO1	178	Hca.13G0028050	Hca. ethylene-responsive protein related	乙烯调控种子萌发、器官衰老、生物和非生物胁迫等过程。 Ethylene regulates seed germination, organ senescence, biotic and abiotic stress.
GTPase	361	Hca.01G0050250	Hca. RAN GTPase-activating protein 1	参与调控细胞周期中各个时期的细胞生命活动。 It is involved in the regulation of cell activities at various stages of the cell cycle.
Kinase	165	Hca.02G0009450	Hca. protein kinase family protein	尚未见报道。 Has not been reported.
mit-gene	247	KR736346	Gossypium trilobum mitochondrion, complete genome	尚未见报道。 Has not been reported.
NPF5.4	269	LOC108487536	Gossypium arboreum protein NRT1/ PTR FAMILY 5.4-like	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 are involved in the transport of plant hormones and the synthesis of secondary metabolites.
Phosphatase	248	Hca.13G0013080	Hca. phosphatidic acid phosphatase-related	参与磷酸基团转移、代谢等生理过程。 It is involved in the transfer and metabolism of phosphate groups.
PLK	265	Hca.15G0014780	Hca. receptor-like protein kinase	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 protein kinase regulates cellular physiological responses by receiving and transmitting extracellular signals and are involved in plant growth and development.
PME7	117	Hca.01G0006960	Hca. pectinesterase	果胶酯酶抑制剂在植物生长发育和响应逆境胁迫中发挥重要作用^[21]。 Pectinesterase inhibitor plays an important role in plant growth and response to stress.
PMT24	171	Hca.15G0021740	Hca. methyltransferase	催化腐胺向N-甲基腐胺的转化。 Catalyze the conversion of putrescine to N-methylputrescine.
RaBa1f	170	LOC105766406	Gossypium raimondii ras-related protein RABA1f	RABA家族蛋白在调控根毛扩张?细胞壁组分、响应生物胁迫等方面发挥重要作用。 RABA family protein plays an important role in regulating root hair expansion, cell wall components and responding to biological stress.
SAM-MET	114	Hca.18G0000220	Hca. S-adenosyl-L-methionine-dependent methyltransferases	催化的甲基化修饰对植物信号传导、染色体表达和基因沉默等起重要的调节作用。 Catalytic methylation plays an important role in regulating signal transduction, chromosome expression and gene silencing in plants.
SGT1	300	Hca.02G0014470	Hca. SGT1 protein	SGT1基因与植物与植物响应生物和非生物胁迫密切相关。 SGT1 gene is closely related to plant response to biotic and abiotic stress.
TIM21	257	Hca.12G0027090	Hca. mitochondrial import inner membrane translocase subunit Tim	负责线粒体内膜的转运与装配^[22]。 Responsible for the transport and assembly of mitochondrial intima.
TPR	353	Hca.17G0008870	Hca. tetratricopeptide repeat domain containing protein	介导与蛋白质的相互作用。 Mediates interactions with proteins.
VPS13F	102	Hca.05G0005060	Hca. vacuolar protein sorting-associated protein 16	液泡分选相关蛋白参与调控主根的发育和植株生长素响应过程。 Vacuolar protein sorting-associated protein are involved in the regulation of taproot development and auxin response.
WD40	140	Hca.01G0001440	Hca. katanin p80 WD40 repeat-containing subunit B1 homolog 1	WD40是拟南芥生长发育和胁迫信号传递的关键调控因子^[23]。 WD40 is a key regulator of Arabidopsis growth and stress signal transmission.
β-man6	188	Hca.02G0010790	Hca. alpha-mannosidase 2	α-甘露聚糖酶引导蛋白的跨膜运输。 alpha-mannosidase guides the transmembrane transport of proteins.

Table 5

Fig. 4

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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 Pre-amplification	E0	GACTGCGTACCAATTCA	HM0	GATGAGTCTAGAACGGT
预扩增 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′)
AHL23	TCCTCCATCCGAACCAGTGTG	CAAGGGAATCCAGAGAGACCATC
CesA2	CCTAAAAATGCCGAACTCTACGC	TAGAAAATCTGGACTCTCCTGGTGC
ETO1	ATCTTTTCCGAGTTTGGTGTATCCT	CGCTCCCTCCTTTCTTCGTC
NPF5.4	TGAAGTTAGCAGCATCGGTTG	TTCCAACACTTCCAAGAGGGT
PLK	TTGCGGCACTACGAGGTTGTT	CCCCAATCATTCAATCTCCATACC
PME7	CGCTGGACAAGAAAGATACCG	GACACGCCGAGTGACATCATAGA
RABA1f	ACAAGGCTCACCATTTCCACAT	GGGTCCCAGATACGAGGTTTTC
SGT1	TCATTGCCAACACTAAAGCACACAT	CCTAAGGCTACTCTGGAATCTGGGT
VPS13F	TTGAGCCATCGTGGTGAAGGG	GCAGCAGGATTTGCGGTGTT
WD40	ATGCCCTCCTTCAATGCGT	CCCAATACCGATGAACAGCC
β-man6	GATGATTGGTGGAGGAATGAGCA	GTCGTCTGCTGCTTTAGAGGTCAC
His3 (reference gene)	GTGGAGTCAAGAAGCCTCACAG	ATGGCTCTGGAAACGCAAA

PbCl₂浓度 Concentration of PbCl₂(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	18.12±0.42 a	2.01±0.02 a	14.33±0.61 a	2.71±0.17 a	105.79±12.13 a	16.70±1.38 a
200	18.18±0.20 a	1.78±0.01 b	13.58±0.50 a	2.48±0.15 a	76.99±4.53 b	13.53±0.61 b
400	14.90±0.05 b	1.64±0.06 c	8.91±0.16 b	1.27±0.01 b	34.60±1.09 c	6.51±0.32 c
600	12.18±0.20 c	1.52±0.03 c	7.27±0.27 b	1.00±0.04 b	28.79±1.84 c	5.12±0.44 c

甲基化类型 Methylation type	PbCl₂浓度PbCl₂ concentration		变化比率(上升↑, 下降↓) Exchange rate (up↑, down↓)
甲基化类型 Methylation type	0 μmol L^-1	600 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)	83	110	32.53↑
类型II (半甲基化) Type II (hemi-methylation)	61	71	16.39↑
类型III, IV (全甲基化) Type III and IV (full methylation)	147	110	25.17↓
半甲基化率Hemi-methylated ratio (%)	20.62	24.40	18.33↑
全甲基化率Fully methylated ratio (%)	50.52	37.80	34.20↓
甲基化率/MSAP Total methylated ratio/MSAP (%)	71.13	62.20	12.55↓

Physiological characteristics and DNA methylation analysis under lead stress in kenaf (Hibiscus cannabinus L.)

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