MfERF053增强活性氧清除能力和抗逆相关基因表达提高拟南芥抗旱性

doi:10.11686/cyxb2025359

摘要/Abstract

摘要：

干旱胁迫制约植物生长与农业生产，挖掘抗旱基因并解析其机制对分子育种至关重要。黄花苜蓿MfERF053属于AP2/ERF家族，前期发现其在干旱下表达上调且过表达拟南芥具有抗旱表型，但机制未明。本研究以拟南芥Col-0野生型（Col）及MfERF053过表达株系（#19、#20、#33）为材料，经自然干旱胁迫处理后，测定植株表型、生理及分子指标，以明确其抗旱机制。结果显示：干旱胁迫下，过表达株系萎蔫延迟、复水恢复好；相对含水量显著高于Col（P<0.05），相对电导率与丙二醛含量显著低于Col（P<0.05）；光系统Ⅱ功能更稳定，最大光化学效率、电子传递速率显著高于Col（P<0.05）；过表达株系叶片氯化硝基四氮唑蓝（NBT）、3，3'-二氨基联苯胺（DAB）染色程度明显浅于Col，表明·O??、H?O?积累减少，且过氧化氢酶和过氧化物酶活性显著高于Col（P<0.05）。分子层面，干旱胁迫下MfERF053过表达株系中，活性氧（ROS）清除相关基因（AtCAT1、AtPOD3、AtSOD4）及干旱响应（AtRD22、AtRD29A）、低温/干旱交叉响应（AtCOR15A、AtCOR47）、钾离子转运（AtHAK5）、脱落酸（ABA）信号通路（AtABI5）等抗逆相关基因的表达量均显著上调（P<0.05），且多数基因表达量显著高于Col（P<0.05），仅AtRD29A和AtABI5在个别过表达株系中与Col无显著差异。综上，MfERF053可通过增强ROS清除能力、上调抗逆相关基因的表达、协同改善拟南芥干旱下的生理稳态与分子响应，最终提高其抗旱性，为耐旱作物/牧草分子育种提供重要的基因资源与理论依据。

关键词: MfERF053, 拟南芥, 抗旱性, 活性氧清除, 抗逆相关基因

Abstract:

Drought stress restricts plant growth and agricultural production， and mining drought-tolerannce genes and analyzing their mechanisms are crucial for molecular breeding. Medicago falcataMfERF053 belongs to the AP2/ERF transcription factor family. Previous studies have shown that its expression is upregulated under drought stress， and Arabidopsis thaliana overexpressing this gene exhibits a drought-tolerant phenotype. However， the underlying mechanism remains unclear. In this study， A. thaliana Col-0 wild type （Col） and MfERF053-overexpressing lines （#19， #20， #33） were used as materials. After natural drought stress treatment， plant phenotypic， physiological， and molecular indicators were determined to clarify the drought-tolerance mechanism of MfERF053. The results showed that under drought stress： the overexpressing lines exhibited delayed leaf wilting and better recovery ability after rehydration； their relative water content （RWC） was significantly higher than that of Col （P<0.05）， while their relative electrical conductivity （REC） and malondialdehyde （MDA） content were significantly lower than those of Col （P<0.05）； the function of photosystem Ⅱ （PSⅡ） was more stable， with the maximum photochemical efficiency （F_v/F_m） and electron transport rate （ETR） significantly higher than those of Col （P<0.05）； the degree of nitroblue tetrazolium （NBT）［for superoxide anion （·O??） localization］ and 3，3'-diaminobenzidine （DAB）［for hydrogen peroxide （H?O?） localization］ staining in leaves of the overexpressing lines was significantly lighter than that in Col， indicating reduced accumulation of ·O?? and H?O?， and the activities of catalase （CAT） and peroxidase （POD） were significantly higher than those in Col （P<0.05）. At the molecular level， under drought stress， the expression levels of stress-tolerance-related genes in MfERF053-overexpressing lines， including reactive oxygen species （ROS）-scavenging-related genes （AtCAT1， AtPOD3， AtSOD4）， drought-responsive genes （AtRD22， AtRD29A）， cold/drought cross-responsive genes （AtCOR15A， AtCOR47）， potassium ion transport gene （AtHAK5）， and abscisic acid （ABA） signaling pathway gene （AtABI5）， were all significantly up-regulated （P<0.05）. Most of these genes exhibited significantly higher expression levels in overexpressing lines than in Col （P<0.05）， except that AtRD29A and AtABI5 in individual overexpressing lines showed no significant difference compared with Col. In conclusion， MfERF053 was found to enhance ROS scavenging capacity， upregulate the expression of stress-tolerant-related genes， and synergistically improve the physiological homeostasis and molecular response of A. thaliana under drought， ultimately improving its drought tolerance. This study provides important genetic resources and a theoretical basis for the molecular breeding of drought-tolerant crops/forages.

Key words: MfERF053, Arabidopsis thaliana, drought resistance, reactive oxygen species scavenging, stress-resistant-related genes

李倩, 段伟, 张雪莉, 刘丽君, 王玉祥. MfERF053增强活性氧清除能力和抗逆相关基因表达提高拟南芥抗旱性[J]. 草业学报, 2026, 35(8): 157-169.

Qian LI, Wei DUAN, Xue-li ZHANG, Li-jun LIU, Yu-xiang WANG. MfERF053 enhances drought resistance of Arabidopsis thaliana by improving reactive oxygen species scavenging capacity and promoting expression of stress-resistance-related genes[J]. Acta Prataculturae Sinica, 2026, 35(8): 157-169.

图/表 7

表1 PCR引物

Table 1 PCR primers

引物 Primer	正向引物/反向引物 Forward primer/reverse primer （5′-3′）
qAtactin-F	CGCCATCCAAGCTGTTCTC
qAtactin-R	TCACGTCCAGCAAGGTCAAG
AtCAT1-F	GTCCTGGGATTCAGACAGGC
AtCAT1-R	GGCTCACGTTAAGACGAGT
AtPOD3-F	CCAATCCAGAAACGGAAGTG
AtPOD3-R	TCTGACGCTTCTGACGACAC
AtSOD4-F	GAAGAACCTTGCTCCTTCCAG
AtSOD4-R	GATTGGCAGTTGTGTCAACAAC
AtRD22-F	ACTTGGTAAATATCACGTCAGGCT
AtRD22-R	CTGAGGTGTTCTTGTGGCATACC
AtRD29A-F	GATATCGACAAGGATGTGCCG
AtRD29A-R	GTATCCAGGTCTTCCCTTCGC
AtCOR47-F	GCTTCGTCGTCGTTTCTCT
AtCOR47-R	CCAATGTATCTGCGGTTTGA
AtHAK5-F	TGCATCACTGGGACGGAGGCA
AtHAK5-R	GTACGCAGCCTGCCCGCAAT
AtABI5-F	AGAGGGATAGCGAACGAGTCTAGTC
AtABI5-R	GTTCGGTTTGGATTAGGTTTAGG

图1 自然干旱胁迫下野生型和过表达MfERF053拟南芥的表型Col：野生型Wild type； #19、#20、#33： MfERF053过表达的3个独立株系3 independent MfERF053-overexpressing lines；下同The same below.

Fig.1 Phenotypes of wild-type and MfERF053-overexpressing A. thaliana under natural drought stress

图2 干旱胁迫下野生型和过表达MfERF053拟南芥的相对含水量、相对电导率及丙二醛含量对比*表示同一株系在对照与干旱胁迫间差异显著（P<0.05），不同小写字母表示在对照组中各株系之间差异显著（P<0.05），不同大写字母表示在干旱胁迫组中各株系之间差异显著（P<0.05），下同。* indicate a significant difference （P<0.05） between the control and drought stress group for the same line. Different lowercase letters indicate significant differences （P<0.05） among different plant lines in the control group， while different uppercase letters indicate significant differences （P<0.05） among different plant lines in the drought stress group， the same below.

Fig.2 Comparison of relative water content， relative electrical conductivity， and malondialdehyde content between wild-type and MfERF053-overexpressing A. thaliana under drought stress

图3 干旱胁迫下野生型和过表达MfERF053拟南芥的叶绿素荧光对比Fv/Fm：最大光化学效率Maximum photochemical efficiency； NPQ：非光化学淬灭Non-photochemical quenching； ETR：电子传递效率Electron transport rate； Alpha：光能利用效率Light use efficiency.

Fig.3 Comparison of chlorophyll fluorescence between wild-type and MfERF053-overexpressing A. thaliana under drought stress

图4 干旱胁迫下野生型和过表达MfERF053拟南芥的活性氧含量及抗氧化酶活性对比A：氯化硝基四氮唑蓝染色［用于超氧阴离子（·O2-）定位检测］ Nitroblue tetrazolium chloride （NBT） staining for localization detection of superoxide anion （·O??）； B： 3，3'-二氨基联苯胺染色［用于过氧化氢（H2O2）定位检测］ 3，3'-diaminobenzidine （DAB） staining for localization detection of hydrogen peroxide （H?O?）； CAT：过氧化氢酶 Catalase； POD：过氧化物酶 Peroxidase； SOD：超氧化物歧化酶 Superoxide dismutase.

Fig.4 Comparison of reactive oxygen species content and antioxidant enzyme activities between wild-type and MfERF053-overexpressing A. thaliana under drought stress

图5 干旱胁迫下野生型和过表达MfERF053拟南芥的抗氧化酶相关基因表达对比AtCAT1：拟南芥过氧化氢酶1基因 A. thaliana catalase 1 gene； AtPOD3：拟南芥过氧化物酶3基因 A. thaliana peroxidase 3 gene； AtSOD4：拟南芥超氧化物歧化酶4基因 A. thaliana superoxide dismutase 4 gene.

Fig.5 Comparison of antioxidant enzyme-related gene expression between wild-type and MfERF053-overexpressing A. thaliana under drought stress

图6 干旱胁迫下野生型和过表达MfERF053拟南芥中抗逆相关基因的相对表达量AtRD22：拟南芥干旱响应蛋白22基因 A. thaliana responsive to dehydration 22 gene； AtRD29A：拟南芥干旱响应蛋白29A基因 A. thaliana responsive to dehydration 29A gene； AtCOR15A：拟南芥冷调节蛋白15A基因 A. thaliana cold-regulated 15A gene； AtCOR47：拟南芥冷调节蛋白47基因 A. thaliana cold-regulated 47 gene； AtHAK5：拟南芥高亲和钾转运蛋白5基因 A. thaliana high-affinity K? transporter 5 gene； AtABI5：拟南芥脱落酸不敏感5基因 A. thaliana abscisic acid insensitive 5 gene.

Fig.6 Relative expression of stress-resistant-related genes in wild-type and MfERF053-overexpressing A. thaliana under drought stress

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