5‑AzaC缓解紫花苜蓿盐碱胁迫的生理效应及其对DNA甲基化酶基因表达的影响

doi:10.3864/j.issn.0578-1752.2025.21.017

摘要/Abstract

摘要：

【背景】土壤盐碱化是全球农业生产面临的主要生态问题之一，严重限制了作物的正常生长、产量形成及品质提升。紫花苜蓿（Medicago sativa）作为重要的多年生豆科牧草，生产力受到盐碱胁迫的严重制约，其应对盐碱胁迫的表观遗传学调控机制尚不清楚。DNA甲基化作为一种关键的表观遗传修饰，在植物适应非生物胁迫中发挥重要作用。【目的】在系统鉴定紫花苜蓿中DNA甲基化相关基因家族成员，解析其在盐碱胁迫下表达特征的基础上，通过施用DNA甲基化抑制剂5-氮杂胞苷（5-AzaC），探讨DNA甲基化在苜蓿耐盐碱性形成过程中的作用机制，以期为耐盐碱苜蓿种质改良提供理论依据。【方法】基于紫花苜蓿参考基因组，对DNA甲基转移酶和去甲基酶基因进行全基因组鉴定，并结合系统发育分析与保守结构域注释推测其功能。采用RT-qPCR检测这些基因在盐碱胁迫下的表达模式。以甘农3号为材料，在水培条件下设置不同浓度的5-AzaC预处理，筛选最佳浓度后，进一步测定植株的生长、生理和光合相关指标，以评价5-AzaC对紫花苜蓿耐盐碱性的调控作用。【结果】共鉴定出紫花苜蓿中13个DNA甲基转移酶基因和4个DNA去甲基酶基因，相关蛋白均定位于细胞核，且保守结构域完整。表达分析表明，MsCMT4、MsCMT6、MsCMT8和MsDML2在盐碱胁迫下显著上调，表明DNA甲基化与去甲基化过程均参与胁迫响应。生理测定结果显示，100 μmol·L^-1 5-AzaC显著缓解了盐碱胁迫造成的植株生长抑制，株高、鲜重和干重分别较对照提高12.62%、23.50%和18.67%。在光合色素代谢方面，5-AzaC有效抑制了叶绿素降解相关基因PAO、CAO和NYC的表达，减缓了色素降解。光合参数分析表明，5-AzaC处理显著提升了净光合速率（Pn）、气孔导度（Gs）、蒸腾速率（Tr），并增强了光系统II的量子效率（YII）和光化学猝灭系数（qP），表明其有助于维持光系统稳定性和高效性。在渗透调节方面，5-AzaC促进了可溶性糖的积累（增加37.21%），而对可溶性蛋白无显著影响。活性氧（ROS）分析显示，5-AzaC处理显著降低了H₂O₂和O₂^-·含量（分别下降22.8%和35.8%），同时超氧化物歧化酶（SOD）和过氧化氢酶（CAT）活性分别提高13.58%和21.82%，表明其能够通过增强抗氧化系统缓解氧化损伤。【结论】本研究系统解析了紫花苜蓿中DNA甲基化相关基因的家族组成及其在盐碱胁迫下的响应特征，揭示了DNA甲基化在紫花苜蓿耐盐碱性形成中的关键作用。外源施用5-AzaC可通过维持光合系统稳定、提升光合效率、促进渗透调节物质积累并增强ROS清除能力，从而有效改善紫花苜蓿的盐碱耐性。为阐释牧草应对非生物胁迫的表观遗传学机制提供了新的试验证据，并为耐盐碱苜蓿种质改良与利用提供了理论参考。

关键词: 盐碱胁迫, 紫花苜蓿, 表观遗传, 光合色素

Abstract:

【Background】Soil salinization is one of the major ecological challenges threatening global agricultural production, severely restricting crop growth, yield formation, and quality improvement. Alfalfa (Medicago sativa), as an important perennial legume forage, is particularly constrained by salt-alkali stress, while the epigenetic regulatory mechanisms underlying its response remain largely unknown. DNA methylation, as a key epigenetic modification, plays an essential role in plant adaptation to abiotic stresses. 【Objective】This study aimed to systematically identify DNA methylation-related gene families in alfalfa, characterize their expression patterns under salt-alkali stress, and further explore the role of DNA methylation in salt-alkali tolerance by applying the DNA methylation inhibitor 5-azacytidine (5-AzaC), thereby providing the theoretical insights for the genetic improvement of salt-alkali-tolerant alfalfa. 【Method】Based on the reference genome of alfalfa, DNA methyltransferase and demethylase genes were identified genome-wide, and their functions were inferred through phylogenetic analysis and conserved domain annotation. RT-qPCR was employed to analyze the expression patterns of these genes under salt-alkali stress. Using the cultivar Gannong No. 3 as plant material, a hydroponic salt-alkali stress system was established. Different concentrations of 5-AzaC were applied as pretreatments, and the optimal concentration was selected for subsequent assays. Plant growth, physiological, and photosynthetic parameters were then measured to evaluate the regulatory role of 5-AzaC in alfalfa salt-alkali tolerance. 【Result】A total of 13 DNA methyltransferase genes and 4 DNA demethylase genes were identified in alfalfa, all of which were localized to the nucleus with complete conserved domains. Expression analysis revealed that MsCMT4, MsCMT6, MsCMT8, and MsDML2 were significantly upregulated under salt-alkali stress, indicating the involvement of both methylation and demethylation processes in stress responses. Physiological analyses showed that 100 μmol·L^-1 5-AzaC significantly alleviated growth inhibition caused by salt-alkali stress, so plant height, fresh weight, and dry weight increased by 12.62%, 23.50%, and 18.67%, respectively, compared with the control. Regarding chlorophyll metabolism, 5-AzaC suppressed the expression of chlorophyll degradation-related genes (PAO, CAO, NYC), thereby delaying pigment degradation. Photosynthetic analysis indicated that 5-AzaC treatment markedly increased net photosynthetic rate (Pn), stomatal conductance (Gs), and transpiration rate (Tr), as well as the quantum efficiency of photosystem II (YII) and photochemical quenching (qP), suggesting enhanced stability and efficiency of the photosynthetic system. In terms of osmotic adjustment, 5-AzaC promoted soluble sugar accumulation (+37.21%) but had no significant effect on soluble protein. Reactive oxygen species (ROS) measurements showed that 5-AzaC reduced H₂O₂ and O₂^-·levels by 22.8% and 35.8%, respectively, while superoxide dismutase (SOD) and catalase (CAT) activities increased by 13.58% and 21.82%, respectively, indicating that 5-AzaC enhanced antioxidant capacity and alleviated oxidative damage. 【Conclusion】This study systematically characterized DNA methylation-related gene families in alfalfa and their responses to salt-alkali stress, revealing the pivotal role of DNA methylation in shaping salt-alkali tolerance. Exogenous application of 5-AzaC improved alfalfa tolerance by maintaining photosystem stability, enhancing photosynthetic efficiency, promoting osmolyte accumulation, and strengthening ROS scavenging capacity. These findings provided the new experimental evidence for understanding the epigenetic mechanisms of forage adaptation to abiotic stress and offer theoretical guidance for the improvement and utilization of salt-alkali-tolerant alfalfa germplasm.

Key words: salt-alkali stress, Medicago sativa, epigenetics, photosynthetic pigments

高荣, 李恒宇, 陈丽娟, 马晖玲. 5‑AzaC缓解紫花苜蓿盐碱胁迫的生理效应及其对DNA甲基化酶基因表达的影响[J]. 中国农业科学, 2025, 58(21): 4482-4496.

GAO Rong, LI HengYu, CHEN LiJuan, MA HuiLing. Physiological Effects of 5-AzaC on Alleviating Salt‑Alkali Stress in Alfalfa and Its Impact on the Expression of DNA Methylation Enzyme Genes[J]. Scientia Agricultura Sinica, 2025, 58(21): 4482-4496.

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基因名称 Gene name	正向引物 Forward primer（5′-3′）	反向引物 Reverse primer（5′-3′）
MsActin	GACAATGGAACTGGAATGG	CAATACCGTGCTCAATGG
MsCMT1	CGCCTGAGTTTGAGCGCA	TGGCATTTCGTCCCGAGT
MsCMT2	CCCCAGCGTTGTACCTGG	TCAGTATGAGGAGTGGTCCCA
MsCMT3	GCTGACTCTCAAAGTGCACC	GCACTCCCTTGAGGTCCC
MsCMT4	GACATACTGCCCAGCTGCA	GTCAAAGGAGGTCCGCCA
MsCMT5	AGGTTCTTCTCCACCCGGA	ACGGGAACAGCAACTGCA
MsCMT6	AGCTACGGGGCTTCTCCT	GCATTCCACTTGGTGCGC
MsCMT7	TGGCCTCAACGTTACGCA	TCCCTTCCTCGCCCTTGA
MsCMT8	GGACCTGGGCCCTTTGAA	GTCCACTGGGGTGAGTGC
MsCMT9	GCTGTTCCTGTGGCCCTT	CGGGCTAGACAACTTGGGT
MsDNMT1	TTCACCGAAGCTTGGCGT	CACTCGATCAACGGCGGA
MsMET1	GCTGCCGTCCGTAGTACC	CCATTGCCTACAGCTGGGA
MsMET2	CGTCCGCAGTACCGCAAA	CCATTGCCTACAGCTGGGA
MsMET3	CAGTCCGCAGTACCGCAA	CCATTGCCTACAGCTGGGA
MsDML1	TCTGGCAGAGATTTGTCCTGT	GCTGTTTGGCATTACTGTGGC
MsDML2	AACGAACGGTGCATGGGT	TGGTCTGCTTGCCAGTCTG
MsDML3	CAGGACTGGAGCAGAAGAACA	TCAGGGAGAGACAAGGGCA
MsDML4	TGGGATTGTACCTGCGGC	CCGAAGCCGCCTCTGTC
MsCAO	CCGGCATCTGGTCTGCAA	CCGGGCTTCGAAATCCCA
MsCLH	AGCTGCAATAACAAGTTGGCT	TTTCCGCCGCGACTATGG
MsNYC	GTTCCACGAATGCGAGTCG	GACCTCGCCGTACCCAAG
MsPAO	CCGAGAATGGCGGTGACA	CGTGTCGCCTCAGCCAAT

基因名称 Gene name	基因ID Gene ID	染色体 Chromosome	氨基酸数 Number of amino acids	分子量Molecular weigh	等电点 Point isoelectric	不稳定系数Instability index	亲水性 GRAVY value	亚细胞定位 Subcellular localization
MsMET1	MS.gene071054.t1	chr4.1	1653	185714.22	5.53	44.39	-0.498	Nuclear
MsMET2	MS.gene043208.t1	chr4.2	1506	169133.86	5.98	44.2	-0.492	Nuclear
MsMET3	MS.gene010135.t1	chr5.2	1311	146538.82	5.95	43.9	-0.381	Nuclear
MsCMT1	MS.gene056526.t1	chr8.3	1124	126873.89	5.78	42.68	-0.576	Nuclear
MsCMT2	MS.gene010674.t1	chr4.4	850	96315.79	5.22	43.52	-0.563	Nuclear
MsCMT3	MS.gene64852.t1	chr5.1	835	94196.68	5.36	41.38	-0.549	Nuclear
MsCMT4	MS.gene72088.t1	chr6.3	931	103979.18	5.67	44.82	-0.548	Nuclear
MsCMT5	MS.gene51465.t1	chr3.4	894	100820.99	6.28	36.94	-0.427	Nuclear
MsCMT6	MS.gene92341.t1	chr5.1	859	96724.01	7.93	38.79	-0.526	Nuclear
MsCMT7	MS.gene50806.t1	chr4.1	550	62072.73	4.67	37.23	-0.488	Nuclear
MsCMT8	MS.gene009335.t1	chr3.1	481	53450.93	5.99	36.33	-0.28	Nuclear
MsCMT9	MS.gene89792.t1	chr4.4	652	74165.94	5.85	42.92	-0.54	Nuclear
MsDNMT1	MS.gene002215.t1	chr2.3	427	48313.21	4.89	40.8	-0.397	Nuclear
MsDML1	MS.gene050207.t1	chr1.1	1497	168803.93	6.07	44.77	-0.732	Nuclear
MsDML2	MS.gene60556.t1	chr1.2	1795	202529.87	6.28	43.97	-0.776	Nuclear
MsDML3	MS.gene020552.t1	chr7.2	1798	201809.19	6.06	47.28	-0.747	Nuclear
MsDML4	MS.gene47350.t1	chr1.1	2232	247919.17	5.88	52.08	-0.776	Nuclear