菊花和菊花脑DNA甲基化相关酶基因鉴定及表达分析

doi:10.16420/j.issn.0513-353x.2020-1069

摘要/Abstract

摘要：

基于传统菊花（Chrysanthemum × morifolium Ramat.）品种‘金背大红’转录组数据和菊花脑（C. nankingense）的基因组数据,通过生物信息学方法鉴定了二者的DNA甲基化相关酶基因,分析其编码蛋白结构域并构建系统发育树,还分析了基因的表达情况。从‘金背大红’中鉴定出了13个DNA甲基转移酶基因和11个去甲基化酶基因,从菊花脑中鉴定出10个DNA甲基转移酶基因和5个去甲基化酶基因。MET1同源氨基酸序列系统进化分析结果表明,菊花‘紫精灵’‘金背大红’和‘禾城星火’与甘菊[C. lavandulifolium（Fisch. ex Trautv.）Ling et Shih]处于较近的分支中,这表明他们可能有更近的演化关系,而菊花脑和菊花‘泉乡水长’与甘菊的进化关系可能较远。DNA甲基化相关酶基因在不同组织及不同菊花品种中的表达水平存在较大差异。

关键词: 菊花, DNA甲基化, DNA甲基转移酶, DNA去甲基化酶, 基因鉴定, 表达

Abstract:

In this study,based on the transcriptome data of the traditional chrysanthemum （Chrysanthemum × morifolium Ramat.）species‘Jinbeidahong’and the genome data of Chrysanthemum nankingense,the genes encoding DNA methylation-related enzyme were identified through bioinformatics from the‘Jinbeidahong’and C. nankingense,phylogenetic trees and gene expression were analyzed. Totally 13 DNA methyltransferase genes and 11 demethylase genes were identified from‘Jinbeidahong’,and 10 DNA methyltransferase genes and five demethylase genes were identified from C. nankingense. The evolutionary relationship demonstrated that C. lavandulifolium（Fisch. ex Trautv.）Ling et Shih were more closely associated with chrysanthemum‘Zijingling’‘Jinbeidahong’‘Hechengxinghuo’than C. nankingense and‘Quanxiangshuichang’. The transcripts levels of DNA methylation-related enzyme genes in multiple tissues and chrysanthemum varieties were quite different.

Key words: chrysanthemum, DNA methylation, DNA methyltransferase, DNA demethylase, gene identification, gene expression

中图分类号:

S682.11

王莹, 艾鹏慧, 李帅磊, 康冬茹, 李忠爱, 王子成. 菊花和菊花脑DNA甲基化相关酶基因鉴定及表达分析[J]. 园艺学报, 2022, 49(4): 827-840.

WANG Ying, AI Penghui, LI Shuailei, KANG Dongru, LI Zhongai, WANG Zicheng. Identification and Expression Analysis of Genes Related to DNA Methylation in Chrysanthemum × morifolium and C. nankingense[J]. Acta Horticulturae Sinica, 2022, 49(4): 827-840.

图/表 9

表1 荧光定量引物序列

Table 1 Appendix a sequences for fluorescent quantitative

基因 Gene	上游引物序列（5′-3′） Forward primer sequence	下游引物序列（5′-3′） Reverse primer sequence
Cm-methylase1	AACTGGTCACTATACAACTG	ATCATCTGCCGTCATTAC
Cm-methylase2	GACGCTCTCCAAGATTAG	ACCACCATCATTAGATTCAA
Cm-methylase10	TGACTCCGATAGGCTACTT	GGTATCCGAATCAATATCAACATT
Cm-demethylase3	TGGAGAATGATGGAAGTG	GCCTGTTACCTTGGATAA
Cn-methylase1	TGTCAAAATCTGCTGGAGG	TTGTCATTATTGTTGGGGG
Cn-methylase2	AAAAAAGAAACGAAAGGGTC	ATAAAGAAAGCGAGAAATGG
Cn-methylase3	TACAATTCATGGTGCCTTGC	ATTTTGCGCTTCCATATGCT
Cn-methylase4	GCTTCTTCCACTTCCTCCCC	CTTAAACCCCCACCTCTCGT
Cn-methylase5	TTGCCTCAGTTTCCTTTACC	AGCACCTCCATACTCCATTT
Cn-methylase6	AATCACTTTGCTATCTCTCT	TTACTTACTTGTGTCTCTGG
Cn-methylase7	TCTTTTGCTCTTGCTCACAC	CACACACAGCCCACACATTT
Cn-methylase8	CCAAGCTAATGTGTGGCTTC	GCTGTCCATTAAACTTTGGG
Cn-methylase9	TTTTGGAAGTGAATACAGGG	ATGGTTGACATAGTGGAGAA
Cn-methylase10	ATGACAGTGAAGGACAGCGA	GAAATGAGGGGTATGAAAGG
Cn-demethylase1	CAAACCTAAGAAACCGCCAA	CAACATCTCCAAAGCCACAA
Cn-demethylase2	CTGAAGAAACTGGTAGACAA	CAAAATCAAGAACCTAATGG
Cn-demethylase3	ACAGATAAGGACCACGAATG	GGACTGAATAACCAGCAAAC
Cn-demethylase4	CCACCAGAAGGAAAATGTCG	CTCACCAGGATTGTCCCACG
Cn-demethylase5	AAACCGAAGCCAATACTGAG	TTCTAAGAGTGGGTGGGAGT
Actin	AGCTTGCATATGTTGCTCTTGA	TTACCGTAAAGGTCCTTCCTGA

图1 ‘金背大红’菊（Cm）和菊花脑（Cn）DNA甲基转移酶（A）和去甲基化酶（B）的系统发育树和保守结构域采用MEGA7,邻接聚类方法,Bootstrap的值为1 000。数字表示自展值。 At:拟南芥;Sl:番茄;Qs:欧洲栓皮栎;Fv:森林草莓;Bna:甘蓝型油菜。

Fig. 1 Phylogenetic tree and conserved domains of DNA methylases（A）and demethylases（B）of Chrysanthemum ‘Jinbeidahong’（Cm）and Chrysanthemum nankingense（Cn） Using the Neighbour-joining method in MEGA 7 software. The value of bootstrap is 1 000. Numbers indicate bootstrap values. At:Arabidopsis thaliana;Sl:Solanum lycopersicum;Qs:Quercus suber;Fv:Fragaria vesca;Bna:Brassica napus.

图2 不同物种中MET1的系统发育树和保守结构域采用MEGA7,邻接聚类方法,Bootstrap的值为1 000。数字表示自展值。 Cl:甘菊;Cn:菊花脑;Aa:青蒿;Cc:刺苞菜蓟;Ls:莴苣;Ha:向日葵;Os:水稻;Zm:玉米;At:拟南芥。

Fig. 2 Phylogenetic tree and conserved domains of MET1 family in different species Using the Neighbour-joining method in MEGA 7 software. The value of bootstrap is 1 000. Numbers indicate bootstrap values. Cl:C. lavandulifolium;Cn:C. nankingense;Aa:Artemisia annua;Cc:Cynara cardunculus;Ls:Lactuca saliva;Ha:Helianthus annuus;Os:Oryza sativa;Zm:Zea mays;At:Arabidopsis thaliana.

表2 ‘金背大红’菊的DNA甲基转移酶基因和去甲基化酶基因的结构分析

Table 2 Structural analysis of DNA methyltransferases and demethylase genes of Chrysanthemum‘Jinbeidahong’

酶 Enzyme	基因 Gene name	CDS长度/bp CDS length	蛋白质 Protein			类型 Type
酶 Enzyme	基因 Gene name	CDS长度/bp CDS length	长度/aa Length	分子量/Da Mw	等电点 pI	类型 Type
甲基转移酶	Cm-methylase1	4 800	1 599	178 739.29	5.75	MET1
Methyltransferase	Cm-methylase2	2 788	925	103 843.57	6.14	CMT
	Cm-methylase3	2 595	864	97 299.75	4.91	CMT
	Cm-methylase4	3 093	1 030	116 027.35	5.92	CMT
	Cm-methylase5	2 511	836	94 321.79	5.95	DNMT2
	Cm-methylase6	1 317	438	49 870.56	5.24	DNMT2
	Cm-methylase7	1 053	350	39 764.17	5.13	DNMT2
	Cm-methylase8	921	306	34 963.24	4.91	DNMT2
	Cm-methylase9	1 788	595	66 491.85	4.89	DRM
	Cm-methylase10	1 833	610	68 071.51	4.96	DRM
	Cm-methylase11	654	217	24 671.57	8.97	DRM
	Cm-methylase12	1 122	373	40 971.16	4.34	DRM
	Cm-methylase13	1 041	346	39 705.42	5.83	DNMT2
去甲基化酶	Cm-demethylase1	4 638	1 545	173 065.09	8.26	DME
Demethylase	Cm-demethylase2	4 503	1 500	170 278.85	6.35	DML3
	Cm-demethylase3	3 822	1 273	144 744.28	8.93	DML3
	Cm-demethylase4	1 281	426	47 988.14	5.92	ROS1
	Cm-demethylase5	2 928	975	110 623.30	5.53	ROS1
	Cm-demethylase6	1 584	527	60 170.57	5.78	ROS1
	Cm-demethylase7	3 027	1 008	115 750.42	5.78	ROS1
	Cm-demethylase8	2 751	916	103 280.83	8.51	DME
	Cm-demethylase9	3 672	1 223	137 284.38	6.74	DME
	Cm-demethylase10	4 503	1 500	169 026.50	6.05	ROS1
	Cm-demethylase11	864	287	32 569.63	4.37	DML3

表3 菊花脑的DNA甲基转移酶基因和去甲基化酶基因的结构分析

Table 3 Structural analysis of DNA methyltransferases and demethylase genes of Chrysanthemum nankingense

酶 Enzyme	基因 Gene Name	CDS长度/bp CDS length	外显子数 Number of exons	蛋白质Protein			类型 Type
酶 Enzyme	基因 Gene Name	CDS长度/bp CDS length	外显子数 Number of exons	长度/aa Length	分子量/Da Mw	等电点 pI	类型 Type
甲基转移酶	Cn-methylase1	4 803	3	1 600	178 962.60	5.78	MET1
Methyltransferase	Cn-methylase2	1 029	4	342	39 472.94	9.68	DRM
	Cn-methylase3	2 019	12	672	75 038.30	5.01	DRM
	Cn-methylase4	1 470	9	489	54 992.14	5.19	DRM
	Cn-methylase5	879	10	292	32 439.25	7.63	CMT
	Cn-methylase6	2 730	20	909	101 888.25	6.20	CMT
	Cn-methylase7	1 887	18	628	71 372.50	4.99	CMT
	Cn-methylase8	543	4	181	20 947.95	7.57	DNMT2
	Cn-methylase9	1 155	9	384	44 105.80	6.36	DNMT2
	Cn-methylase10	1 038	9	345	39 700.18	5.27	DNMT2
去甲基化酶	Cn-demethylase1	4 254	18	1 417	160 835.45	7.46	DML3
Demethylase	Cn-demethylase2	2 805	14	934	106 836.11	8.79	DML3
	Cn-demethylase3	2 613	15	870	99 036.82	9.20	DML3
	Cn-demethylase4	5 244	20	1 747	194 573.45	6.65	DME
	Cn-demethylase5	4 707	18	1 568	175 882.89	5.96	ROS1

图3 ‘金背大红’菊幼苗期部分DNA甲基转移酶基因和去甲基化酶基因在根、茎和叶的表达分析将根的表达水平设为1。*表示与其差异显著（P < 0.05）,**表示与其差异极显著（P < 0.01）。下同。

Fig. 3 Relative expression of DNA methylases and demethylases gene in root,stem and leaf of Chrysanthemum‘Jinbeidahong’seedling stage Expression leves in root were arbitrarily set to 1. Use * to indicate that the difference is significant（P < 0.05）,** to indicate that the difference is significant extremely（P < 0.01）. The same below

图4 ‘金背大红’菊开花期DNA甲基转移酶基因和去甲基化酶基因在根、茎、叶和花的表达分析

Fig. 4 Relative expression of DNA methylases and demethylases gene in root,stem,leaf and flower of Chrysanthemum‘Jinbeidahong’during flowering stage

图5 不同品种菊花叶片组织中DNA甲基转移酶基因和去甲基化酶基因的表达分析 1:‘金背大红’;2:‘千丝万缕’;3:‘银龙分水’;4:‘国庆红’;5:‘南农红袖’;6:‘紫精灵’;7:‘杭白菊’;8:‘紫蝶翻飞’;9:‘天女的神秘’;10:‘玉台一号’。将‘金背大红’的表达水平设为1,*表示与其差异显著（P < 0.05）,**表示与其差异极显著（P < 0.01）。

Fig. 5 Relative expression of DNA methylases and demethylases in different chrysanthemum cultivars 1:‘Jinbeidahong’;2:‘Qiansiwanlü’;3:‘Yinlongfenshui’;4:‘Guoqinghong’;5:‘Nannong Hongxiu’;6:‘Zijingling’;7:‘Hangbaiju’;8:‘Zidie Fanfei’;9:‘Tiannvdeshenmi’;10:‘Yutaiyihao’. Expression leves of‘Jinbeidahong’were arbitrarily set to 1, use * to indicate that the difference is significant（P < 0.05）,** to indicate that the difference is significant extremely（P < 0.01）.

图6 菊花脑幼苗期DNA甲基转移酶（A）和去甲基化酶（B）在根、茎和叶的表达分析

Fig. 6 Expression analysis of DNA methyltransferase（A）and demethylase（B）in root,stem and leaf of Chrysanthemum nankingense seedling stage

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