紫花苜蓿MsCZF29基因克隆及耐冻功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2026-0186

摘要/Abstract

摘要：

目的 CZF锌指蛋白（Zinc finger proteins, ZFPs）在植物响应生物及非生物胁迫中起到重要的调控作用。克隆紫花苜蓿MsCZF29基因并分析其耐冻功能，为揭示该基因调控拟南芥耐冻机制奠定基础。方法以拟南芥为材料，根据前期紫花苜蓿抗冻转录组分析，筛选得到1个冷冻胁迫响应基因，将其克隆出来并命名为MsCZF29，并进行生物信息学分析和表型分析；构建过表达载体通过农杆菌转化法获得转基因植株（OE2-1、OE2-2和OE6-2）。观察冷冻胁迫下T₃代存活率表型、测定生理生化指标和相关冷响应基因的表达量；进行转录自激活活性验证。结果 MsCZF29编码区序列（CDS）全长为1 554 bp，编码517个氨基酸，是不稳定亲水蛋白。亚细胞定位发现该蛋白定位于细胞核，酵母自激活实验发现其具有转录激活活性。异源过表达MsCZF29显著增加了冷冻胁迫下拟南芥株系的过氧化氢酶（catalase, CAT）、超氧化物歧化酶（superoxide dismutase, SOD）和过氧化物酶（peroxidase, POD）活性，降低了相对电导率（REL）和丙二醛（MDA）含量。冷冻胁迫下过表达株系的存活率及生长状态明显优于野生型。结论成功克隆MsCZF29基因，其过表达在冷冻胁迫过程中发挥正向调控作用，可提高植物的耐冻性。

关键词: 紫花苜蓿, MsCZF29, 冷冻胁迫, 耐冻性, 生理指标

Abstract:

Objective CZF zinc finger proteins (ZFPs) play important regulatory roles in plant responses to biotic and abiotic stresses. Cloning the MsCZF29 gene from alfalfa and analyzing its freezing tolerance function lays a foundation for revealing the regulatory mechanism of this gene in freezing tolerance of Arabidopsis. Method Using Arabidopsis thaliana as the experimental model, we identified the freezing stress-responsive gene MsCZF29 based on previous transcriptomic analysis of cold tolerance in Medicago sativa. The gene was cloned and designated as MsCZF29, and bioinformatics analysis and phenotypic characterization were conducted on it. An overexpression vector was constructed and introduced into plants via Agrobacterium-mediated transformation to generate transgenic lines (OE2-1, OE2-2, and OE6-2). Under freezing stress conditions, we evaluated T₃ generation survival rates, measured key physiological and biochemical parameters, and quantified expression levels of relevant cold-responsive genes. Transcriptional autoactivation activity was verified using yeast assays. Result The MsCZF29 coding sequence (CDS) spans 1 554 bp, encoding 517 amino acids that form a protein which is both unstable and hydrophilic. Subcellular localization confirmed that the protein is localized in the nucleus, while yeast autoactivation assays demonstrated its transcriptional activation capability. Heterologous overexpression of MsCZF29 significantly enhanced the activities of catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) in Arabidopsis lines under freezing stress, while simultaneously reducing relative electrical conductivity (REL) and malondialdehyde (MDA) content. Transgenic lines exhibited markedly higher survival rates and superior growth status compared to wild-type plants under freezing stress conditions. Conclusion We successfully cloned MsCZF29 gene, demonstrating that its overexpression exerts a positive regulatory effect during freezing stress responses, thereby enhancing plant freezing tolerance.

Key words: alfalfa, MsCZF29 gene, freezing stress, freezing resistance, physiological indicators

范雨馨, 任乃芃, 轩一芙, 臧辉, 刘香萍. 紫花苜蓿MsCZF29基因克隆及耐冻功能分析[J]. 生物技术通报, 2026, 42(11): 154-163.

FAN Yu-xin, REN Nai-peng, XUAN Yi-fu, ZANG Hui, LIU Xiang-ping. Cloning and Freezing Tolerance Analysis of the MsCZF29 Gene in Alfalfa[J]. Biotechnology Bulletin, 2026, 42(11): 154-163.

图/表 6

图1 MsCZF29在本氏烟草叶片中的亚细胞定位

Fig. 1 Subcellular localization of MsCZF29 in tobacco leaves

图2 pGBKT7-MsCZF29在转化Y2H酵母菌株中的活性验证

Fig. 2 Activity validation of pGBKT7-MsCZF29 in transformed Y2H yeast strains

图4 野生型与转MsCZF29基因拟南芥在冷冻胁迫下的表型分析A：未处理下野生型与转基因拟南芥幼苗的表型；B：冷冻处理下野生型与转基因拟南芥幼苗的表型；C：冷冻处理前后拟南芥盆栽表型变化；D：冷冻后野生型与转基因型拟南芥的存活率

Fig. 4 Phenotypic analysis of wild-type and MsCZF29-transgenic A. thaliana under freezing stressA: Phenotypes of wild-type and transgenic A. thaliana seedlings under untreated conditions. B: Phenotypes of wild-type and transgenic A. thaliana seedlings under freezing treatment. C: Changes in the phenotypes of A. thaliana potted plants before and after freezing treatment. D: Survival rates of wild-type and transgenic A. thaliana after freezing

图5 野生型与转MsCZF29基因拟南芥在冷冻胁迫下的细胞膜透性分析

Fig. 5 Cell membrane permeability analysis of wild-type and MsCZF29-transgenic A. thaliana under freezing stress

图6 野生型与转MsCZF29基因拟南芥在冷冻胁迫下的抗氧化酶活性分析

Fig. 6 Analysis of antioxidant enzyme activity in wild-type and MsCZF29-transgenic A. thaliana under freezing stress

图7 拟南芥低温响应基因在MsCZF29转基因拟南芥中的表达量分析

Fig. 7 Expression analysis of the cold-responsive genes in MsCZF29 overexpressing A. thaliana

参考文献 36

[1]	轩一芙, 韩子坤, 贠新龙, 等. 8个紫花苜蓿品种在黑龙江省大庆地区生产性能综合评价 [J]. 草原与草坪, 2025, 45(4): 179-187.
	Xuan YF, Han ZK, Yun XL, et al. Comprehensive evaluation of production performance of eight alfalfa varieties in Daqing area, Heilongjiang Province [J]. Grassland Turf, 2025, 45(4): 179-187.
[2]	王园, 王晶, 李淑霞. 紫花苜蓿MsBBX24基因的克隆及耐盐性分析 [J]. 草业学报, 2023, 32(3): 107-117.
	Wang Y, Wang J, Li SX. Cloning of MsBBX24 from alfalfa (Medicago sativa) and determination of its role in salt tolerance [J]. Acta Prataculturae Sin, 2023, 32(3): 107-117.
[3]	杨青川, 康俊梅, 张铁军, 等. 苜蓿种质资源的分布、育种与利用 [J]. 科学通报, 2016, 61(2): 261-270.
	Yang QC, Kang JM, Zhang TJ, et al. Distribution, breeding and utilization of alfalfa germplasm resources [J]. Chin Sci Bull, 2016, 61(2): 261-270.
[4]	任卫波, 陈立波, 郭慧琴, 等. 紫花苜蓿耐寒越冬性研究进展 [J]. 中国草地学报, 2008, 30(2): 104-108.
	Ren WB, Chen LB, Guo HQ, et al. Progress in winter hardiness and survival of alfalfa [J]. Chin J Grassland, 2008, 30(2): 104-108.
[5]	Guo XY, Liu DF, Chong K. Cold signaling in plants: Insights into mechanisms and regulation [J]. J Integr Plant Biol, 2018, 60(9): 745-756.
[6]	Ruelland E, Vaultier MN, Zachowski A, et al. Chapter 2 cold signalling and cold acclimation in plants [M]//Advances in Botanical Research. Amsterdam: Elsevier 2009: 35-150.
[7]	杨梅. 紫花苜蓿MsVDAC基因克隆及抗寒耐旱功能分析 [D]. 哈尔滨: 东北农业大学, 2022.
	Yang M. Isolation of MsVDAC from alfalfa (Medicago sativa L.) and cold tolerance and drought resistance analysis [D]. Harbin: Northeast Agricultural University, 2022.
[8]	Riechmann JL, Heard J, Martin G, et al. Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes [J]. Science, 2000, 290(5499): 2105-2110.
[9]	赵欣羽, 张文颖, 张楚熠, 等. 艾基因组中MYC转录因子基因家族鉴定及表达分析 [J]. 中草药, 2025, 56(23): 8713-8722.
	Zhao XY, Zhang WY, Zhang CY, et al. Genome-wide identification and expression analysis of MYC transcription factor gene family in Artemisia argyi [J]. Chin Tradit Herb Drugs, 2025, 56(23): 8713-8722.
[10]	Laity JH, Lee BM, Wright PE. Zinc finger proteins: new insights into structural and functional diversity [J]. Curr Opin Struct Biol, 2001, 11(1): 39-46.
[11]	Liu K, Hou QD, Yu RR, et al. Genome-wide analysis of C2H2 zinc finger family and their response to abiotic stresses in apple [J]. Gene, 2024, 904: 148164.
[12]	Shuai Y, Feng GY, Yang ZF, et al. Genome-wide identification of C2H2-type zinc finger gene family members and their expression during abiotic stress responses in orchardgrass (Dactylis glomerata) [J]. Genome, 2022, 65(4): 189-203.
[13]	Sun SJ, Guo SQ, Yang X, et al. Functional analysis of a novel Cys2/His2-type zinc finger protein involved in salt tolerance in rice [J]. J Exp Bot, 2010, 61(10): 2807-2818.
[14]	Zhang H, Liu YP, Wen F, et al. A novel rice C2H2-type zinc finger protein, ZFP36, is a key player involved in abscisic acid-induced antioxidant defence and oxidative stress tolerance in rice [J]. J Exp Bot, 2014, 65(20): 5795-5809.
[15]	Joseph MP, Papdi C, Kozma-Bognár L, et al. The Arabidopsis zinc FINGER PROTEIN3 interferes with abscisic acid and light signaling in seed germination and plant development[J]. Plant Physiol, 2014, 165(3): 1203-1220.
[16]	Zhang X, Guo XP, Lei CL, et al. Overexpression of SlCZFP1, a novel TFIIIA-type zinc finger protein from tomato, confers enhanced cold tolerance in transgenic Arabidopsis and rice [J]. Plant Mol Biol Rep, 2011, 29(1): 185-196.
[17]	Han YC, Fu CC. Cold-inducible MaC2H2s are associated with cold stress response of banana fruit via regulating MaICE1 [J]. Plant Cell Rep, 2019, 38(5): 673-680.
[18]	Zhang HL, Sun Z, Feng S, et al. The C2H2-type zinc finger protein PhZFP1 regulates cold stress tolerance by modulating galactinol synthesis in Petunia hybrida [J]. J Exp Bot, 2022, 73(18): 6434-6448.
[19]	Berg JM, Shi YG. The galvanization of biology: a growing appreciation for the roles of zinc [J]. Science, 1996, 271(5252): 1081-1085.
[20]	Brown RS. Zinc finger proteins: getting a grip on RNA [J]. Curr Opin Struct Biol, 2005, 15(1): 94-98.
[21]	Kim DH, Yamaguchi S, Lim S, et al. SOMNUS, a CCCH-type zinc finger protein in Arabidopsis, negatively regulates light-dependent seed germination downstream of PIL5 [J]. Plant Cell, 2008, 20(5): 1260-1277.
[22]	Han GL, Qiao ZQ, Li YX, et al. The roles of CCCH zinc-finger proteins in plant abiotic stress tolerance [J]. Int J Mol Sci, 2021, 22(15): 8327.
[23]	Lin PC, Pomeranz MC, Jikumaru Y, et al. The Arabidopsis tandem zinc finger protein AtTZF1 affects ABA- and GA-mediated growth, stress and gene expression responses [J]. Plant J, 2011, 65(2): 253-268.
[24]	Wang WY, Liu BH, Xu MY, et al. ABA-induced CCCH tandem zinc finger protein OsC3H47 decreases ABA sensitivity and promotes drought tolerance in Oryza sativa [J]. Biochem Biophys Res Commun, 2015, 464(1): 33-37.
[25]	Ren NP, Liu JL, Wang HB, et al. Combined transcriptomic and proteomic analysis reveals the response mechanisms of alfalfa to freezing stress [J]. Front Plant Sci, 2026, 16: 1682825.
[26]	孙群, 胡景江. 植物生理学研究技术 [M]. 杨凌: 西北农林科技大学出版社, 2006.
	Sun Q, Hu JJ. Research technology of plant physiology [M]. Yangling: Northwest A&F University Press, 2006.
[27]	Li XG, Liang XQ, Li WH, et al. Isolation and functional analysis of MbCBF2, a Malus baccata (L.) borkh CBF transcription factor gene, with functions in tolerance to cold and salt stress in transgenic Arabidopsis thaliana [J]. Int J Mol Sci, 2022, 23(17): 9827.
[28]	Adnan M, Morton G, Hadi S. Analysis of rpoS and bolA gene expression under various stress-induced environments in planktonic and biofilm phase using 2^-ΔΔCT method [J]. Mol Cell Biochem, 2011, 357(1/2): 275-282.
[29]	Sun JQ, Jiang HL, Xu YX, et al. The CCCH-type zinc finger proteins AtSZF1 and AtSZF2 regulate salt stress responses in Arabidopsis [J]. Plant Cell Physiol, 2007, 48(8): 1148-1158.
[30]	Bai HR, Lin P, Li X, et al. DgC3H1 a CCCH zinc finger protein gene, confers cold tolerance in transgenic Chrysanthemum [J]. Sci Hortic, 2021, 281: 109901.
[31]	Xu LA, Xiong XP, Liu TT, et al. Heterologous expression of two Brassica campestris CCCH zinc-finger proteins in Arabidopsis induces cytoplasmic foci and causes pollen abortion [J]. Int J Mol Sci, 2023, 24(23): 16862.
[32]	Liu HM, Xiao SQ, Sui SZ, et al. A tandem CCCH type zinc finger protein gene CpC3H3 from Chimonanthus praecox promotes flowering and enhances drought tolerance in Arabidopsis [J]. BMC Plant Biol, 2022, 22: 506.
[33]	王砺寒. 水稻蛋白激酶OsMAPK6通过OsLIC和OsCATA调控抗病的机制研究 [D]. 武汉: 华中农业大学, 2022.
	Wang LH. Study on the mechanism of rice protein kinase OsMAPK6 regulating disease resistance through OsLIC and OsCATA [D]. Wuhan: Huazhong Agricultural University, 2022.
[34]	Kim WC, Kim JY, Ko JH, et al. AtC3H14, a plant-specific tandem CCCH zinc-finger protein, binds to its target mRNAs in a sequence-specific manner and affects cell elongation in Arabidopsis thaliana [J]. Plant J, 2014, 80(5): 772-784.
[35]	安飞飞, 李庚虎, 陈霆, 等. 植物耐寒生理及蛋白质组学研究进展 [J]. 中国农学通报, 2015, 31(14): 96-101.
	An FF, Li GH, Chen T, et al. Research progress on physiology and proteomics for cold tolerance in plants [J]. Chin Agric Sci Bull, 2015, 31(14): 96-101.
[36]	吴玥. 枳锌指蛋白PtrTZF1抗寒功能鉴定及机制解析 [D]. 赣州: 赣南师范大学, 2023.
	Wu Y. Functional characterization and mechanisms of cold stress tolerance conferred by a zinc finger protein PtrTZF1 in Poncirus trifoliata [D]. Ganzhou: Gannan Normal University, 2023.