过表达桑树MnERF2增强拟南芥抗旱性

doi:10.13560/j.cnki.biotech.bull.1985.2025-0448

摘要/Abstract

摘要：

目的 AP2/ERF家族转录因子是植物特有的调控因子，在植物响应非生物胁迫（如低温、干旱、水涝及高盐）中发挥关键作用。解析桑树ERF转录因子基因MnERF2在干旱胁迫中的分子功能，为桑树抗逆育种提供理论基础和基因资源。方法通过花序浸染法获得MnERF2过表达拟南芥株系，分别对转基因幼苗及成苗进行干旱胁迫处理，观测其生长表型，并测定生理生化指标。结果过表达MnERF2拟南芥株系失水率比野生型显著降低。干旱胁迫下，过表达幼苗根系发育优于野生型，成苗的生物量积累和叶绿素含量显著提高。此外，干旱胁迫下过表达株系中超氧化物歧化酶（SOD）、过氧化物酶（POD）、过氧化氢酶（CAT）和谷胱甘肽S-转移酶（GST）活性显著增强，抗坏血酸（ASA）、谷胱甘肽（GSH）和脯氨酸含量显著增加，同时显著降低相对电导率及丙二醛（MDA）和活性氧（ROS；O₂^•-、H₂O₂、•OH）含量。结论 MnERF2具有正向调控干旱胁迫响应的功能，过表达MnERF2通过提高植株水分保持能力和生物量，促进脯氨酸生物合成、增加抗氧化酶活性和抗氧化物质含量以提高转基因拟南芥抗旱性。

关键词: 拟南芥, 过表达, 桑树, MnERF2, 抗旱性

Abstract:

Objective AP2/ERF transcription factors are plant-specific regulators that play a critical role in plant responses to abiotic stresses, such as low temperature, drought, waterlogging, and high salinity. Elucidating the molecular function of the mulberry ERF transcription factor MnERF2 in drought stress response may provide a theoretical foundation and genetic resources for stress-resistant mulberry breeding. Method MnERF2-overexpressing Arabidopsis thaliana lines were obtained via the floral dip method. Both transgenic seedlings and mature plants were subjected to drought stress, followed by phenotypic observation and physiological/biochemical analysis. Result The water loss rate of MnERF2-overexpressing A. thaliana lines is significantly lower than that of the wild-type. The overexpressingseedlings presented superior root development and mature transgenic plants showed significant increase in biomass accumulation and chlorophyll content under drought stress. The overexpressing lines demonstrated enhanced activities of antioxidant enzymes, including superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and glutathione S-transferase (GST). Levels of ascorbic acid (ASA), glutathione (GSH), and proline significantly elevated, meanwhile the relative electrical conductivity, malondialdehyde (MDA) content, and the levels of reactive oxygen species (ROS; O₂^•-, H₂O₂, •OH) markedly reduced under drought stress. Conclusion MnERF2 positively regulates the responses to drought stress. Overexpressing MnERF2 increase the tolerance to drought in transgenic A. thaliana by improving water retention and biomass accumulation, promoting proline biosynthesis, boosting antioxidant enzyme activity, and increasing antioxidant content.

Key words: Arabidopsis thaliana, overexpression, mulberry, MnERF2, resistance to drought

董亚茹, 聂玉霞, 朱红, 王照红, 刘惠芬, 郭光. 过表达桑树MnERF2增强拟南芥抗旱性[J]. 生物技术通报, 2026, 42(4): 182-189.

DONG Ya-ru, NIE Yu-xia, ZHU Hong, WANG Zhao-hong, LIU Hui-fen, GUO Guang. Overexpression of Mulberry MnERF2 Enhances Resistance to Drought in Arabidopsis thaliana[J]. Biotechnology Bulletin, 2026, 42(4): 182-189.

图/表 8

表1 本研究所用引物

Table 1 Primers used in this study

用途 Application	引物名称 Primer name	序列 Sequence （5′‒3′）
PCR验证 PCR verification	pROKⅡ-F	CTATCCTTCGCAAGACCCTTCCTC
PCR验证 PCR verification	pROKⅡ-R	GACGTTGTAAAACGACGGCCAGTG
实时荧光定量 PCR Quantitative real-time PCR	MnERF2-F	TCCCGTTGGAAGCCGGGA
	MnERF2-R	CGACAGAGGCGGGACGTT
	TUB2-F	GCCAATCCGGTGCTGGTAACA
	TUB2-R	CATACCAGATCCAGTTCCTCCTCCC
	AtACTIN-F	CCAGCCATCGCTCATCGGAATG
	AtACTIN-R	CAGACACTGTATTTTCTCTCTG

图1 过表达MnERF2拟南芥抗性筛选（A）、PCR验证（B）及表达分析（C）M：Marker 2 000；WT：野生型拟南芥；‒：阴性对照；L1‒L5：转化株系。不同小写字母表示差异显著（P<0.05）。下同

Fig. 1 Resistance screening(A), PCR verification(B), and expression analysis (C) of A. thaliana overexpressing MnERF2M: Marker 2 000. WT: Wild-type Arabidopsis thaliana. ‒: Negative control. L1‒L5: Transgenic lines. Different lowercase letters indicate significant differences (P<0.05). The same below

图2 过表达MnERF2拟南芥幼苗的抗旱性评价A：转基因拟南芥和WT植株生长情况（比例尺=1 cm）；B：转基因拟南芥和WT植株初生根长；C：转基因拟南芥和WT植株在正常生长条件下失水率

Fig. 2 Evaluation of drought resistance in A. thaliana seedlings overexpressing MnERF2A: The growth of transgenic A. thaliana and WT plants (Scale bar=1 cm). B: Primary root length of transgenic A. thaliana and WT plants. C: Water loss rate of transgenic A. thaliana and WT plants under normal growth conditions

图3 干旱胁迫下过表达MnERF2拟南芥成苗生长特性A：表型（比例尺=1 cm）；B：鲜重；C：总叶绿素含量

Fig. 3 Growth characteristics of MnERF2-overexpressing A. thaliana seedlings under drought stressA: Phenotypes (Scale bar=1 cm). B: Fresh weight. C: Total chlorophyll content

图4 干旱胁迫下过表达MnERF2拟南芥相对电导率及MDA含量分析

Fig. 4 Analysis of relative electrolyte leakage and MDA content in MnERF2-overexpressing A. thaliana under drought stress

图5 干旱胁迫下过表达MnERF2拟南芥O2•-、H2O2、•OH含量分析

Fig. 5 Content analysis of O2•-, H2O2, •OH in MnERF2-overexpressing A. thaliana under drought stress

图6 干旱胁迫下过表达MnERF2拟南芥SOD、POD、CAT和GST活性分析

Fig. 6 Activity analysis of SOD, POD, CAT and GST in MnERF2-overexpressing A. thaliana under drought stress

图7 干旱胁迫下过表达MnERF2拟南芥AsA、GSH和脯氨酸含量分析

Fig. 7 Content analysis of AsA, GSH and proline in MnERF2-overexpressing A. thaliana under drought stress

参考文献 40

[1]	Sarkar T, Ravindra KN, Sidhu GK, et al. Overexpression of phosphoenol pyruvate carboxylase gene of Flaveria trinervia in transgenic mulberry (Morus spp.) leads to improved photosynthesis rate and tolerance to drought and salinity stresses [J]. Plant Cell Tissue Organ Cult, 2023, 156(1): 26.
[2]	Wang Y, Jiang W, Cheng JS, et al. Physiological and proteomic analysis of seed germination under salt stress in mulberry [J]. Front Biosci, 2023, 28(3): 49.
[3]	Wei H, Wang X, Wang KT, et al. Transcription factors as molecular switches regulating plant responses to drought stress [J]. Physiol Plant, 2024, 176(3): e14366.
[4]	Han DG, Han JX, Xu TL, et al. Isolation and preliminary functional characterization of MxWRKY64, a new WRKY transcription factor gene from Malus xiaojinensis Cheng et Jiang [J]. Vitro Cell Dev Biol Plant, 2021, 57(2): 202-213.
[5]	Sarkar T, Thankappan R, Kumar A, et al. Stress inducible expression of AtDREB1A transcription factor in transgenic peanut (Arachis hypogaea L.) conferred tolerance to soil-moisture deficit stress [J]. Front Plant Sci, 2016, 7: 935.
[6]	Dong XM, Han BC, Yin XY, et al. Genome-wide identification of the GRAS transcription factor family in autotetraploid cultivated alfalfa (Medicago sativa L.) and expression analysis under drought stress [J]. Ind Crops Prod, 2023, 194: 116379.
[7]	Abiri R, Shaharuddin NA, Maziah M, et al. Role of ethylene and the APETALA 2/ethylene response factor superfamily in rice under various abiotic and biotic stress conditions [J]. Environ Exp Bot, 2017, 134: 33-44.
[8]	Ma ZM, Hu LJ, Jiang WZ. Understanding AP2/ERF transcription factor responses and tolerance to various abiotic stresses in plants: a comprehensive review [J]. Int J Mol Sci, 2024, 25(2): 893.
[9]	Feng K, Hou XL, Xing GM, et al. Advances in AP2/ERF super-family transcription factors in plant [J]. Crit Rev Biotechnol, 2020, 40(6): 750-776.
[10]	Nakano T, Suzuki K, Fujimura T, et al. Genome-wide analysis of the ERF gene family in Arabidopsis and rice [J]. Plant Physiol, 2006, 140(2): 411-432.
[11]	Jofuku KD, den Boer BG, Montagu MV, et al. Control of Arabidopsis flower and seed development by the homeotic gene APETALA2 [J]. Plant Cell, 1994, 6(9): 1211-1225.
[12]	Zhang GY, Chen M, Chen XP, et al. Phylogeny, gene structures, and expression patterns of the ERF gene family in soybean (Glycine max L.) [J]. J Exp Bot, 2008, 59(15): 4095-4107.
[13]	Zhuang J, Deng DX, Yao QH, et al. Discovery, phylogeny and expression patterns of AP2-like genes in maize [J]. Plant Growth Regul, 2010, 62(1): 51-58.
[14]	Nie SM, Wang D. AP2/ERF transcription factors for tolerance to both biotic and abiotic stress factors in plants [J]. Trop Plant Biol, 2023, 16(3): 105-112.
[15]	He YR, Jia RR, Qi JJ, et al. Functional analysis of citrus AP2 transcription factors identified CsAP2-09 involved in citrus canker disease response and tolerance [J]. Gene, 2019, 707: 178-188.
[16]	Yu Y, Yu M, Zhang SX, et al. Transcriptomic identification of wheat AP2/ERF transcription factors and functional characterization of TaERF-6-3A in response to drought and salinity stresses [J]. Int J Mol Sci, 2022, 23(6): 3272.
[17]	Kong LF, Song Q, Wei HB, et al. The AP2/ERF transcription factor PtoERF15 confers drought tolerance via JA-mediated signaling in Populus [J]. New Phytol, 2023, 240(5): 1848-1867.
[18]	Huang SZ, Ma ZM, Hu LJ, et al. Involvement of rice transcription factor OsERF19 in response to ABA and salt stress responses [J]. Plant Physiol Biochem, 2021, 167: 22-30.
[19]	Lv KW, Li J, Zhao K, et al. Overexpression of an AP2/ERF family gene, BpERF13, in birch enhances cold tolerance through upregulating CBF genes and mitigating reactive oxygen species [J]. Plant Sci, 2020, 292: 110375.
[20]	Huang JY, Zhao XB, Bürger M, et al. Two interacting ethylene response factors regulate heat stress response [J]. Plant Cell, 2021, 33(2): 338-357.
[21]	董亚茹, 赵东晓, 耿兵, 等. 桑树MnERF2的表达分析[J]. 生物技术通报, 2022, 38(11): 112-121.
	Dong YR, Zhao DX, Geng B, et al. Expression analysis of MnERF2 in mulberry [J]. Biotechnol Bull, 2022, 38(11): 112-121.
[22]	王学奎. 植物生理生化实验原理和技术 [M]. 2版. 北京: 高等教育出版社, 2006.
	Wang XK. Principles and techniques of plant physiological biochemical experiment [M]. 2nd ed. Beijing: Higher Education Press, 2006.
[23]	Nie XG, Wang L. Plant species compositions alleviate toxicological effects of bisphenol A by enhancing growth, antioxidant defense system, and detoxification [J]. Environ Sci Pollut Res Int, 2022, 29(43): 65755-65770.
[24]	Satish Chandra K, Gurjar D. Plant transcription factors: an overview and its significance in abiotic stress [J]. J Adv Biol Biotechnol, 2024, 27(7): 853-867.
[25]	悦曼芳, 张春, 吴忠义. 植物转录因子AP2/ERF家族蛋白结构和功能的研究进展 [J]. 生物技术通报, 2022, 38(12): 11-26.
	Yue MF, Zhang C, Wu ZY. Research progress in the structural and functional analysis of plant transcription factor AP2/ERF protein family [J]. Biotechnol Bull, 2022, 38(12): 11-26.
[26]	Li C, Cui J, Shen YR, et al. BrERF109 positively regulates the tolerances of drought and salt stress in Chinese cabbage [J]. Environ Exp Bot, 2024, 223: 105794.
[27]	魏娜, 敬文茂, 许尔文, 等. 白花草木樨MaERF058基因耐旱功能验证 [J]. 草业学报, 2024, 33(8): 159-169.
	Wei N, Jing WM, Xu EW, et al. Functional analysis of the MaERF058 gene in response to drought stress in Melilotus albus [J]. Acta Pratac Sin, 2024, 33(8): 159-169.
[28]	Cheng MC, Liao PM, Kuo WW, et al. The Arabidopsis ETHYLENE RESPONSE FACTOR1 regulates abiotic stress-responsive gene expression by binding to different cis-acting elements in response to different stress signals [J]. Plant Physiol, 2013, 162(3): 1566-1582.
[29]	Shi K, Liu J, Liang H, et al. An alfalfa MYB-like transcriptional factor MsMYBH positively regulates alfalfa seedling drought resistance and undergoes MsWAV3-mediated degradation [J]. J Integr Plant Biol, 2024, 66(4): 683-699.
[30]	Miller G, Suzuki N, Ciftci-Yilmaz S, et al. Reactive oxygen species homeostasis and signalling during drought and salinity stresses [J]. Plant Cell Environ, 2010, 33(4): 453-467.
[31]	de Carvalho MHC. Drought stress and reactive oxygen species: Production, scavenging and signaling [J]. Plant Signal Behav, 2008, 3(3): 156-165.
[32]	Zhou JM, Dang Z, Cai MF, et al. Soil heavy metal pollution around the Dabaoshan Mine, Guangdong province, China [J]. Pedosphere, 2007, 17(5): 588-594.
[33]	Gill SS, Tuteja N. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants [J]. Plant Physiol Biochem, 2010, 48(12): 909-930.
[34]	Liang YQ, Li XS, Yang RR, et al. BaDBL1, a unique DREB gene from desiccation tolerant moss Bryum argenteum, confers osmotic and salt stress tolerances in transgenic Arabidopsis [J]. Plant Sci, 2021, 313: 111047.
[35]	Wu LJ, Zhang ZJ, Zhang HW, et al. Transcriptional modulation of ethylene response factor protein JERF3 in the oxidative stress response enhances tolerance of tobacco seedlings to salt, drought, and freezing [J]. Plant Physiol, 2008, 148(4): 1953-1963.
[36]	马福林, 马玉花. 干旱胁迫对植物的影响及植物的响应机制 [J]. 宁夏大学学报: 自然科学版, 2022, 43(4): 391-399.
	Ma FL, Ma YH. Effect of drought stress on plants and their response mechanism [J]. J Ningxia Univ Nat Sci Ed, 2022, 43(4): 391-399.
[37]	Ashraf M, Foolad MR. Roles of glycine betaine and proline in improving plant abiotic stress resistance [J]. Environ Exp Bot, 2007, 59(2): 206-216.
[38]	Sun SH, Hu CG, Qi XJ, et al. The AaCBF4-AaBAM3.1 module enhances freezing tolerance of kiwifruit (Actinidia arguta) [J]. Hortic Res, 2021, 8(1): 97.
[39]	Li BB, Wang XH, Wang XF, et al. An AP2/ERF transcription factor VvERF63 positively regulates cold tolerance in Arabidopsis and grape leaves [J]. Environ Exp Bot, 2023, 205: 105124.
[40]	罗巧玉, 谢惠春, 马永贵, 等. 水分胁迫对发草抗坏血酸‒谷胱甘肽循环的影响 [J]. 草业科学, 2025, 42(6): 1477-1485.
	Luo QY, Xie HC, Ma YG, et al. Effects of drought and waterlogging stress on the ascorbate-glutathione cycle of hairgrass [J]. Pratacult Sci, 2025, 42(6): 1477-1485.