蒙古冰草咖啡酸氧甲基转移酶基因AmCOMT1的鉴定及功能分析

doi:10.11983/CBB24013

植物学报 ›› 2024, Vol. 59 ›› Issue (3): 383-396.DOI: 10.11983/CBB24013

蒙古冰草咖啡酸氧甲基转移酶基因AmCOMT1的鉴定及功能分析

杜锦瑜¹^,²^,³^,⁴, 孙震²^,³^,⁴, 苏彦龙²^,³^,⁴, 王贺萍²^,³^,⁴, 刘亚玲⁵, 吴振映²^,³^,⁴, 何峰²^,³^,⁴^,^*(), 赵彦¹^,^*(), 付春祥²^,³^,⁴^,^*()

¹内蒙古农业大学草原与资源环境学院/农业农村部饲草栽培、加工与高效利用重点实验室/草地资源教育部重点实验室, 呼和浩特 010018
²中国科学院青岛生物能源与过程研究所, 青岛 266101
³山东能源研究院, 青岛 266101
⁴青岛新能源山东省实验室, 青岛 266101
⁵内蒙古草业技术创新中心有限公司, 呼和浩特 010018

收稿日期:2024-01-24 接受日期:2024-03-20 出版日期:2024-05-01 发布日期:2024-04-17
通讯作者: *E-mail: hefeng@qibebt.ac.cn; zhaoyannmg@163.com; fucx@qibebt.ac.cn
基金资助:
内蒙古自治区种业科技创新重大示范工程“揭榜挂帅”项目(2022JBGS0014);中央引导地方科技发展资金(2022ZY006);青岛新能源山东实验室“抓攻关”项目(QNESLKPP202302);国家林业和草原局重大应急科技“揭榜挂帅”项目(202201);呼和浩特市重大科技专项(2021-重-社-2)

Identification and Functional Analysis of an Agropyron mongolicum Caffeic Acid 3-O-methyltransferase Gene AmCOMT1

Jinyu Du¹^,²^,³^,⁴, Zhen Sun²^,³^,⁴, Yanlong Su²^,³^,⁴, Heping Wang²^,³^,⁴, Yaling Liu⁵, Zhenying Wu²^,³^,⁴, Feng He²^,³^,⁴^,^*(), Yan Zhao¹^,^*(), Chunxiang Fu²^,³^,⁴^,^*()

¹Key Laboratory of Grassland Resources and Education Ministry/Key Laboratory of Forage Cultivation, Processing and Efficient Utilization of Ministry of Agriculture and Rural Affairs/College of Grassland and Resources Environment of Inner Mongolia Agricultural University, Hohhot 010018, China
²Qingdao Institute of Bioenergy and Process, Chinese Academy of Sciences, Qingdao 266101, China
³Shandong energy Research Institute, Qingdao 266101, China
⁴Qingdao New Energy Shandong Laboratory, Qingdao 266101, China
⁵Inner Mongolia Grass Technology Innovation Center, Hohhot 010018, China

Received:2024-01-24 Accepted:2024-03-20 Online:2024-05-01 Published:2024-04-17
Contact: *E-mail: hefeng@qibebt.ac.cn; zhaoyannmg@163.com; fucx@qibebt.ac.cn

摘要/Abstract

摘要： 蒙古冰草(即沙芦草(Agropyron mongolicum))是我国北方代表性的多年生牧草之一, 具有较强的耐寒和耐旱能力。在植物中, 咖啡酸氧甲基转移酶基因(COMT)是参与木质素和褪黑素生物合成的关键基因, 在调节植物生长、品质和抗逆性中发挥重要作用。通过分析蒙古冰草全长转录组数据, 从蒙古冰草中克隆了COMT候选基因AmCOMT1。该基因在茎秆和根等木质素含量高的组织中高表达, 且其表达受多种非生物胁迫诱导。在拟南芥(Arabidopsis thaliana)野生型(Col-0)和突变体(omt1-2)中过表达AmCOMT1, 显著促进了转基因拟南芥的木质素合成, 使突变体的木质素单体和组分恢复至野生型水平, 同时Col-0/35S:AmCOMT1中木质素总量提高11%。此外, AmCOMT1过表达显著提高了Col-0/35S:AmCOMT1转基因拟南芥的褪黑素含量。在盐胁迫条件下, 该株系平均根长相比野生型拟南芥提高20.3%, 表现出更强的抗逆性。综上,蒙古冰草AmCOMT1基因在木质素和褪黑素合成中发挥关键作用, 可提高转基因拟南芥的抗逆性, 在蒙古冰草等单子叶牧草遗传改良方面具有重要应用潜力。

关键词: 蒙古冰草, 咖啡酸氧甲基转移酶, 木质素, 褪黑素, 盐胁迫

Abstract: Agropyron mongolicum is one of northern China’s most representative perennial forage grasses, showing strong tolerance to cold and drought. In plants, caffeic acid O-methyltransferase (COMT) is a key gene involved in the biosynthesis of lignin and melatonin, and plays an important role in regulating plant growth, biomass quality, and stress tolerance. In this study, through the analysis of the full-length transcriptome data of A. mongolicum, the COMT candidate gene AmCOMT1 was cloned. AmCOMT1 is highly expressed in tissues with high lignin content, such as stem and root, and its expression is induced by a variety of abiotic stresses, including drought and salt. Overexpression of AmCOMT1 in Arabidopsis wild type (Col-0) and mutant (omt1-2) significantly promoted the synthesis of lignin in transgenic Arabidopsis, restoring the lignin content and composition of the mutant to wild type level and the lignin content in Col-0/35S:AmCOMT1 was increased by 11%. In addition, overexpression of AmCOMT1 increased the melatonin content in Col-0/35S:AmCOMT1 transgenic Arabidopsis. Under salt stress conditions, the average root length of this transgenic line increased by 20.3% compared to the wild type, showing higher stress tolerance. In this study, we identified AmCOMT1 from A. mongolicum as a key gene regulating both lignin biosynthesis and melatonin biosynthesis, improving the stress tolerance of transgenic Arabidopsis. Our results highlighted the application potential of AmCOMT1 in genetic improvement of forage grasses through molecular breeding.

Key words: Agropyron mongolicum, caffeic acid 3-O-methyltransferase, lignin, melatonin, salt stress

杜锦瑜, 孙震, 苏彦龙, 王贺萍, 刘亚玲, 吴振映, 何峰, 赵彦, 付春祥. 蒙古冰草咖啡酸氧甲基转移酶基因AmCOMT1的鉴定及功能分析. 植物学报, 2024, 59(3): 383-396.

Jinyu Du, Zhen Sun, Yanlong Su, Heping Wang, Yaling Liu, Zhenying Wu, Feng He, Yan Zhao, Chunxiang Fu. Identification and Functional Analysis of an Agropyron mongolicum Caffeic Acid 3-O-methyltransferase Gene AmCOMT1. Chinese Bulletin of Botany, 2024, 59(3): 383-396.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB24013

https://www.chinbullbotany.com/CN/Y2024/V59/I3/383

图/表 7

表1 本研究使用的引物

Table 1 Primers used in this study

Purpose	Primer name	Sequence (5′-3′)
Gene clone	AmCOMT1-CDS-F	ATGGGCTCCATCGCCGCCGG
	AmCOMT1-CDS-R	CTACTTAGTGAACTCGATGGCCC
	AmCOMT1-pMDC32-F	CTAGAGGATCCCCGGATGGGCTCCATCGCCG
	AmCOMT1-pMDC32-R	GATCGGGGAAATTCGCTACTTAGTGAACTCGATGGCC
	AmCOMT1-pGWC-F	AGCAGGCTTTGACTTTATGGGCTCCATCGCCGCCGG
	AmCOMT1-pGWC-R	TGGGTCTAGAGACTTCTACTTAGTGAACTCGATGGCCC
Positive detection	M13F	ACTGGCCGTCGTTTTAC
	M13R	GTCATAGCTGTTTCCTG
	pMDC32-JC-F	CACTATCCTTCGCAAGACCCTTC
	pMDC32-JC-R	TTGAACGATCGGGGAAATTCGAG
	PGWC-JC-F	TAATACGACTCACTATAGGG
	PGWC-JC-R	ATTTAGGTGACACTATAG
qRT-PCR	AmCOMT1-qRT-F	GACGCTGCTCAAGAACTGCT
	AmCOMT1-qRT-R	CCATGCGTTGGCGTAGATGT
	18s-qRT-F	CAATGGGAAGCAAGGCTGTAA
	18s-qRT-R	AACAATCCGAACTGAGGCAATC
	AtACTIN-F	CATCAGGAAGGACTTGTACGG
	AtACTIN-R	GATGGACCTGACTCGTCATAC
Identification of Arabidopsis mutant	LP	TCCGGTTTGCAAGTATTTGAC
	BP	ATTTTGCCGATTTCGGAAC
	RP	CTAGGGTCAGTCCCGTGGTAC

图1 不同物种中COMT蛋白的系统进化树分析通过NCBI数据库获得不同物种来源的COMT蛋白和拟南芥CCoAOMT序列, 与本实验克隆到的AmCOMT1序列经MEGA软件进行比对后通过邻接法(Neighbor-Joining, NJ)构建系统进化树(Bootstrap=1 000)

Figure 1 Phylogenetic tree of COMT proteins in different species COMT protein sequences from different species and CCoAOMT sequence from Arabidopsis were obtained from NCBI database, and the phylogenetic tree (Bootstrap=1 000) was constructed by Neighbor-Joining (NJ) method with MEGA with AmCOMT1.

图2 蒙古冰草AmCOMT1与其它物种中COMT蛋白序列比对

Figure 2 Alignment of AmCOMT1 with COMT sequences from different species

图3 AmCOMT1在蒙古冰草不同组织及不同胁迫诱导下的表达情况 (A) 蒙古冰草不同组织中AmCOMT1的表达(R: 根; S: 茎; LB: 叶片; LS: 叶鞘; I: 幼穗); (B), (C) 50和150 mmol·L-1 NaCl处理下AmCOMT1的表达; (D), (E) 75和150 mmol·L-1甘露醇处理下AmCOMT1的表达。柱状图中的不同小写字母表示根据ANOVA分析结果(n=3)数据间存在显著性差异(P<0.05), NS表示差异不显著, ** P<0.01。

Figure 3 Expression of AmCOMT1 in different tissue of Agropyron mongolicum and under different treatments (A) The expression of AmCOMT1 in different tissues of A. mongolicum (R: Root; S: Stem; LB: Leaf blade; LS: Leaf sheath; I: Inflorescence); (B), (C) The expression of AmCOMT1 under 50 and 150 mmol·L-1 NaCl treatments; (D), (E) The expression of AmCOMT1 under 75 and 150 mmol·L-1 mannitol treatments. Different lowercase letters in the bar chart indicate significant differences (P<0.05) among the data according to the results of ANOVA analysis (n=3), NS indicate not significant, ** P<0.01.

图4 转基因拟南芥的表型和木质素染色分析 (A), (B) 转基因拟南芥中AmCOMT1的表达水平; (C) 转基因拟南芥在培养箱中生长8周后与Col-0及omt1-2的生长状态比较(bar=3.5 cm); (D), (E) 转基因拟南芥茎切片的间苯三酚染色(D)和Mäule染色结果(E) (bars=100 μm)。

Figure 4 Phenotypic and lignin staining analysis of transgenic Arabidopsis (A), (B) Expression level of AmCOMT1 in transgenic Arabidopsis; (C) 8-week-old transgenic Arabidopsis plants comparing with Col-0 and omt1-2; (D), (E) Lignin staining of stem cross section of different Arabidopsis lines with phloroglucinol staining method (D) and Mäule staining method (E) (bars=100 μm).

图5 过表达AmCOMT1对拟南芥Col-0和omt1-2突变体木质素含量(A)、组分(B)和S/G比例(C)的影响柱状图中的不同小写字母表示根据ANOVA分析结果(n=3)数据间存在显著性差异(P<0.05)。

Figure 5 Effects of AmCOMT1 overexpression on lignin content (A), composition (B), and S/G ratio (C) of Arabidopsis Col-0 and omt1-2 Different lowercase letters in the bar chart indicate significant differences (P<0.05) among the data according to the results of ANOVA analysis (n=3).

图6 过表达AmCOMT1对转基因拟南芥褪黑素合成和耐盐性的影响 (A) 拟南芥不同株系中的褪黑素含量; (B), (C) 盐胁迫处理后拟南芥不同株系的根长统计和生长表型(bars=1 cm)。柱状图中的不同小写字母表示根据ANOVA分析结果(n=3)数据间存在显著性差异(P<0.05)。

Figure 6 Overexpression of AmCOMT1 improved melatonin synthesis and salt tolerance in transgenic Arabidopsis (A) Melatonin content in different Arabidopsis lines; (B), (C) Root length and growth phenotype of different Arabidopsis lines growing on plates after NaCl treatment (bars=1 cm). Different lowercase letters in the bar chart indicate significant differences (P<0.05) among the data according to the results of ANOVA analysis (n=3).

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蒙古冰草咖啡酸氧甲基转移酶基因AmCOMT1的鉴定及功能分析

Identification and Functional Analysis of an Agropyron mongolicum Caffeic Acid 3-O-methyltransferase Gene AmCOMT1

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