铁甲秋海棠DNA甲基转移酶全基因组鉴定及表达分析(长英文摘要）

doi:10.11983/CBB24010

植物学报 ›› 2024, Vol. 59 ›› Issue (5): 726-737.DOI: 10.11983/CBB24010 cstr: 32102.14.CBB24010

铁甲秋海棠DNA甲基转移酶全基因组鉴定及表达分析(长英文摘要）

陈婷欣¹, 符敏³, 李娜², 杨蕾蕾², 李凌飞²^,^*(), 钟春梅¹^,^*()

¹华南农业大学生物质工程研究院, 农业农村部能源植物资源与利用重点实验室, 广东省农林生物质工程技术研究中心, 广州 510642
²深圳市中国科学院仙湖植物园, 深圳市南亚热带植物多样性重点实验室, 深圳 518004
³中山市农业科技推广中心, 中山 528400

收稿日期:2024-01-22 接受日期:2024-05-07 出版日期:2024-09-10 发布日期:2024-08-19
通讯作者: *李凌飞, 深圳市中国科学院仙湖植物园副研究员, 硕士生导师, 中国野生植物保护协会秋海棠专业委员会副主任委员, 广东省植物学会理事, 《广东农业科学》青年编委。主要从事园艺植物种质资源收集与利用、植物基因组学及园艺品质形成分子生物学等研究。主持国家自然科学基金等项目6项, 以第一或通讯作者(含共同)在Nature Plants、New Phytologist和Journal of Experimental Botany等期刊发表论文30余篇, 其中SCI论文13篇。授权国家发明专利6件, 获登录或评定植物新品种6个。E-mail: lingfei_li@szbg.ac.cn; 钟春梅, 华南农业大学生物质工程研究院副教授, 硕士生导师, 农业农村部能源植物资源与利用重点实验室副主任兼秘书, 中国植物生理与分子生物学学会能源植物专业委员会委员。主要从事薯蓣属药食两用植物的品质调控与品种培育等研究。主持国家自然科学基金等项目4项, 以第一或通讯作者(含共同)在Plant Physiology、Horticulture Research和Journal of Experimental Botany等期刊上发表论文20余篇, 其中SCI论文14篇。授权国家发明专利5件。E-mail: zhongchunmei@scau.edu.cn
基金资助:
广东省自然科学基金(2021A1515011315);深圳市城管科研项目(202408);深圳市城管科研项目(202205)

Identification and Expression Analysis of DNA Methyltransferase in Begonia masoniana

Chen Tingxin¹, Fu Min³, Li Na², Yang Leilei², Li Lingfei²^,^*(), Zhong Chunmei¹^,^*()

¹Guangdong Engineering Technology Research Center of Agricultural and Forestry Biomass, Key Laboratory of Energy Plants Resource and Utilization, Ministry of Agriculture and Rural Affairs, Institute of Biomass Engineering, South China Agricultural University, Guangzhou 510642, China
²Key Laboratory of Southern Subtropical Plant Diversity, Fairy Lake Botanical Garden, Shenzhen & Chinese Academy of Sciences, Shenzhen 518004, China
³Zhongshan Agricultural Science and Technology Extension Center, Zhongshan 528400, China

Received:2024-01-22 Accepted:2024-05-07 Online:2024-09-10 Published:2024-08-19
Contact: *E-mail: lingfei_li@szbg.ac.cn; zhongchunmei@scau.edu.cn

摘要/Abstract

摘要： DNA甲基化是重要的表观遗传修饰之一, 参与调控植物基因组稳定性、发育及胁迫响应等过程。DNA甲基转移酶是DNA甲基化的关键酶。为了解铁甲秋海棠(Begonia masoniana) DNA甲基转移酶的功能, 采用生物信息学方法从铁甲秋海棠基因组中鉴定出5个编码DNA甲基转移酶的基因。根据序列特征将其分为CMT、MET和DRM三类。不同类别成员的基因序列长度和内含子数量存在明显差异, 但同类成员的基因结构和保守结构域具有高度保守性。这些蛋白均定位于细胞核, 且基因启动子含有大量的光响应、MYB结合及植物激素响应等元件。激素响应模式分析表明, CMT3类在GA、SA和NAA处理下基因表达显著降低, CMT2类在MeJA和NAA处理下基因表达显著降低, 而MET类和DRM类分别在GA和ABA处理下基因表达显著升高。此外, 组织特异性分析发现, 叶片中BmaCMT2-5和BmaDRM2-2的表达量明显高于其它组织器官, 且这2个酶的编码基因与BmaMET1-15在叶片红色部分的表达高于绿叶部分, 推测这3个DNA甲基转移酶可能在叶斑形成过程中发挥重要作用。

关键词: 铁甲秋海棠, 叶斑, DNA甲基转移酶, 植物激素

Abstract: INTRODUCTION: DNA methylation is one of the important epigenetic modifications involved in the regulation of plant genome stability, development and stress responses. DNA methylation introduces methylation groups into DNA molecules, thereby altering the activity of DNA segments. DNA methylation is catalyzed by DNA methyltransferase, a process by which methyl groups formed from S-adenosyl-L-methionine are transferred via covalent links to specific locations in the DNA sequence to form N4-methylcytosine, 5-methylcytosine, N6-methyladenine, or 7-methylguanine. However, there are few reports about the effects of DNA methyltransferase on leaf variegation formation and stress response of Begonia.
RATIONALE: Studies have shown that DNA methylation is involved in regulating the formation of leaf color, flower color and leaf variegation, as well as responses to stresses and hormones. As an endemic species of Begonia, Begonia masoniana has unique and beautiful leaf markings, pink, dark green and light green in different developmental stages. It has high ornamental value and is an excellent foliage plant. Therefore, based on the genomic data, this study conducted genome-wide identification and expression pattern analysis of DNA methyltransferase genes, aiming to explore the genetic resources that regulate the formation of leaf variegation.
RESULTS: To investigate whether DNA methyltransferase is involved in the regulation of leaf variegation formation and stress response in B. masoniana, bioinformatics analysis was used to identify the genes encoding DNA methyltransferase. Five genes were obtained from the genome of B. masoniana. According to the protein structural characteristics, their encoded proteins were divided into three categories including CMT, MET and DRM. The sequence length and intron number of these genes were significantly categorized into different subgroups, but their structure and conserved domains in the same subgroup were highly conserved. In addition, all the encoded proteins were predicted to locate in the nucleus. The promoters of these genes contain a large number of cis-acting elements such as light response, MYB binding, and plant hormone response elements. Analysis of hormone response patterns showed that the gene expression of CMT3 was significantly decreased under GA, SA and NAA, and the gene expression of CMT2 was significantly decreased under MeJA and NAA, while MET-type and DRM-type genes displayed significantly increased expression under GA and ABA treatments. In addition, tissue specific analysis showed that the expression levels of BmaCMT2-5 and BmaDRM2-2 in leaves were significantly higher than those of other tissues, while the expressions of these two genes and BmaMET1-15 in red part of leaves were significantly higher than that of green part, implying that these three genes may be involved in regulating the formation of leaf variegation.
CONCLUSION: The structure and function of DNA methyltransferase genes vary significantly across different categories in B. masoniana. However, within each category, members display high conservation in gene structure, conserved domains, motifs, and evolutionary patterns. These genes are likely to play crucial roles in the growth and development of diverse tissues and organs, as well as in responding to various biological and abiotic stresses. Moreover, based on the differential expression patterns of BmaCMT2-5, BmaMET1-15, and BmaDRM2-2 genes between leaf variegation and non-variegation areas, coupled with the abundance of MYB regulatory elements related to anthocyanin synthesis in their promoters, it is hypothesized that these genes may contribute to the formation of leaf variegation. As the current understanding of the functional roles of these methyltransferase genes is largely speculative, future research should focus on their functional validation, which will involve utilizing reverse genetics techniques coupled with phenotypic observations to determine their involvement in specific biological processes. Additionally, physiological, biochemical, and molecular biological methods should be employed to elucidate the precise mechanisms of their actions.

Relative expression levels of DNA methyltransferase genes in different tissues and organs of Begonia masoniana

Key words: Begonia masoniana, leaf variegation, DNA methyltransferase, phytohormones

陈婷欣, 符敏, 李娜, 杨蕾蕾, 李凌飞, 钟春梅. 铁甲秋海棠DNA甲基转移酶全基因组鉴定及表达分析(长英文摘要）. 植物学报, 2024, 59(5): 726-737.

Chen Tingxin, Fu Min, Li Na, Yang Leilei, Li Lingfei, Zhong Chunmei. Identification and Expression Analysis of DNA Methyltransferase in Begonia masoniana. Chinese Bulletin of Botany, 2024, 59(5): 726-737.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB24010

https://www.chinbullbotany.com/CN/Y2024/V59/I5/726

图/表 7

表1 铁甲秋海棠DNA甲基转移酶qRT-PCR引物

Table 1 DNA methyltransferase qRT-PCR primers for Begonia masoniana

Gene name	Gene ID	Primer sequence (5′-3′)
BmaCMT2-5	Bma021768.1	F: CTGCTGGCTGCTATGGTCTT	R: ACATCATGTGTGGGTAGCGG
BmaCMT3-11	Bma005730.1	F: CCAACAGTAGTTACACGCGCGG	R: CCGGAAAACCTTGCAGTCTCGC
BmaCMT3-14	Bma011172.1	F: TGCCGAGCAAACGTCAAGCG	R: ACTTCAGGCGCCGAAGAAGC
BmaMET1-15	Bma013045.1	F: CGGCTAACGCTTGCATCGCT	R: AGGAGCAGCCGCCCATATGA
BmaDRM2-2	Bma014831.1	F: CTGCCGCAAGAAAGAGAGGT	R: GAAGGCCACCACTTCCTTGT

图1 拟南芥(At)、甘蓝型油菜(Bna)、水稻(Os)和铁甲秋海棠(Bma) DNA甲基转移酶系统发育树(A)与蛋白结构域分布图(B) 采用MEGA7, 邻接法。Bootstrap值为1 000。数字表示自展值。CMT: 染色质甲基化酶; MET: 甲基转移酶; DNMT: 从头DNA甲基转移酶; DRM: 结构域重排甲基化酶; aa: 氨基酸

Figure 1 DNA methyltransferase phylogenetic tree (A) and protein domain map (B) of Arabidopsis thaliana (At), Brassica napus (Bna), Oryza sativa (Os) and Begonia masoniana (Bma) Using the Neighbor-joining method in MEGA 7 software. The value of bootstrap is 1 000. Numbers indicate bootstrap values. CMT: Chromomethylases; MET: Methyltransferase; DNMT: de novo DNA methyltransferase; DRM: Domains rearranged methylase; aa: Amino acid

表2 铁甲秋海棠DNA甲基转移酶基本理化性质分析

Table 2 Analysis on the physical and chemical properties of DNA methyltransferase in Begonia masoniana

Gene name	CDS (bp)	Protein			Chr.	N-glycosyl-sites	Subcellular localization
Gene name	CDS (bp)	Amino acid (aa)	pI	Molecular weight (Da)	Chr.	N-glycosyl-sites	Subcellular localization
BmaCMT2-5	2547	848	6.3013	97206.68	Chr.5	4	Nucleus
BmaCMT3-11	2892	963	6.0700	109079.09	Chr.11	4	Nucleus
BmaCMT3-14	2511	836	4.7815	94618.65	Chr.14	4	Nucleus
BmaMET1-15	3645	1214	6.3066	136797.74	Chr.15	6	Nucleus
BmaDRM2-2	1773	590	4.8922	66506.45	Chr.2	2	Nucleus

图2 铁甲秋海棠(Bma) DNA甲基转移酶基因结构(A)和保守基序分布图(B) CDS同表2。

Figure 2 Gene structure of DNA methyltransferase (A) and distribution map of conserved motif (B) of Begonia masoniana CDS is the same as shown in Table 2.

图3 铁甲秋海棠DNA甲基转移酶基因启动子序列顺式元件数量(A)和分布(B) MeJA: 茉莉酸甲酯

Figure 3 Number (A) and distribution (B) of cis-elements in DNA methyltransferase gene promoter sequence of Begonia masoniana MeJA: Methyl jasmonate

图4 铁甲秋海棠DNA甲基转移酶基因在不同组织器官中的相对表达量 ACT7为内参基因, 基因相对表达量为平均值±标准差。DNA甲基转移酶在不同组织中的最小表达量视为“1”。不同小写字母表示各处理间差异显著(P<0.05)。

Figure 4 Relative expression levels of DNA methyltransferase genes in different tissues and organs of Begonia masoniana ACT7 was the internal reference gene and the relative gene expression was means±SD. The minimum expression of DNA methyltransferase in different tissues is regarded as “1”. Different lowercase letters indicate significant differences among different treatments (P<0.05).

图5 铁甲秋海棠DNA甲基转移酶基因在GA、ABA、SA、MeJA和NAA处理(6小时)下的相对表达量将对照组的相对表达量视为“1”。GA: 赤霉素; ABA: 脱落酸; SA: 水杨酸; MeJA: 茉莉酸甲酯; NAA: 萘乙酸。*表示处理组与对照组差异显著(P<0.05); **表示处理组与对照组差异极显著(P<0.01)。

Figure 5 Relative expression levels of DNA methyltransferase genes in Begonia masoniana treated with GA, ABA, SA, MeJA and NAA (6 h) The relative expression of control is regarded as “1”. GA: Gibberellin; ABA: Abscisic acid; SA: Salicylic acid; MeJA: Methyl jasmonate; NAA: 1-naphthalene acetic acid. * indicate that the difference between treatment group and control group is significant (P<0.05); ** indicate that the difference between treatment group and control group is extremely significant (P<0.01).

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铁甲秋海棠DNA甲基转移酶全基因组鉴定及表达分析(长英文摘要）

Identification and Expression Analysis of DNA Methyltransferase in Begonia masoniana

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