Cloning， Subcellular Localization and Expression Analysis of MtZHD4 Gene from Medicago truncatula

doi:10.13560/j.cnki.biotech.bull.1985.2024-1086

Abstract

Abstract:

Objective This study aims to clone and analyze the expression characteristics of the Medicago truncatulaMtZHD4 gene, in order to gain a deeper understanding of its function, and to provide a theoretical basis for its involvement in the growth and development of M. truncatula, as well as in the synthesis, regulation, and signaling of plant endogenous hormones. Method The M. truncatula cultivar 'R108' was used as the material. The MtZHD4 gene was cloned, and bioinformatics analysis and subcellular localization were performed. Real-time quantitative PCR (qPCR) was employed to analyze the expression pattern of MtZHD4 gene in different tissues, hormones, and stress treatments. Result The open reading frame (ORF) of the MtZHD4 gene was 1 092 bp, encoding a protein of 363 amino acids. Prediction of the conserved domains revealed that MtZHD4 contained two ZF-HD protein domains, which belonged to the ZF-HD dimmer and homeo ZF-HD superfamilies. Subcellular localization indicated that MtZHD4 was located in the nucleus and cytoplasm of the cell. Analysis of promoter cis-acting element showed that the promoter of MtZHD4 contained multiple cis-elements involved in plant growth and development, hormone response, and light response. The results of tissue-specific expression analysis revealed that the expressions of MtZHD4 varied significantly across different tissues, with the highest expression in the roots and lower levels in the leaves and pods. After treatment with different hormones and stress conditions, the expression of the MtZHD4 gene showed an upregulation trend under ABA induction, a downregulation trend under IAA induction, and a pattern of first increasing and then decreasing under 6-BA, MeJA, and SA induction. Under salt stress, the expression of the MtZHD4 gene increased, while under drought stress, the expression first increased and then decreased. Conclusion MtZHD4 gene responds to different hormones and abiotic stresses, and may regulate the growth and development of M. truncatula through related hormone signaling pathways, and play a positive regulatory role under salt and drought stress.

Key words: Medicago truncatula, ZF-HD transcription factor, bioinformatics analysis, subcellular localization, tissue differences, hormone treatment, salt stress, drought stress

YANG Chun, WANG Xiao-qian, WANG Hong-jun, CHAO Yue-hui. Cloning， Subcellular Localization and Expression Analysis of MtZHD4 Gene from Medicago truncatula[J]. Biotechnology Bulletin, 2025, 41(5): 244-254.

Figures/Tables 9

Table 1 Primer sequences

引物名称 Primer name	引物序列 Primer sequence （5′-3′）
MtZHD4-F	ATGGAGCTGAGCAGCAGCCAGGACG
MtZHD4-R	TTAGGGGGTGGGCAGGTGGTTGGCG
3302Y-F	TGACGCACAATCCCACTATCCT
3302Y-R	CCGTCCAGCTCGACCAGGAT
MtZHD4-3302Y-F	CGGGGGACTCTTGACCATGGATGGAGCTGAGCAGCAGC
MtZHD4-3302Y-R	ACTAGTCAGATCTACCATGGTTAGGGGGTGGGCAGGTG
MtActin-F	TGATCTGGCTGGTCGTGACCTTA
MtActin-R	ATGCCTGCTGCTTCCATTCCTAT
MtZHD4-RT-F	CGGCGAGAAGGAGTGGTA
MtZHD4-RT-R	GCTTGATGGGCTTGTTGTT

Fig. 1 Cloning of MtZHD4 gene and PCR detection of constructed expression vector as well as the sequence analysis of homologous proteinA: Cloning of MtZHD4 gene in Medicago truncatula. B: PCR detection of expression vector 3302Y-MtZHD4. C: Evolutionary tree of MtZHD4 homologous protein sequence

Fig. 2 Multiple sequence alignment of MtZHD4 homologous proteinsA: Conserved motif analysis of MtZHD4 protein in M. truncatula (Mt: M. truncatula; Tp: Trifolium pratense; Ps: Pisum sativum; Vv: Vicia villosa; Ca: Cicer arietinum;Ap: Abrus precatorius; Ss: Spatholobus suberectus; Vu: Vigna unguiculata;Cc: Cajanus cajan; Pa: Populus alba; Tc: Theobroma cacao; Va: Vigna angularis; Pn: Populus nigra; Pt: Populus trichocarpa). B: LOGO diagram of the conserved domain of MtZHD4 protein in M. truncatula. C: Amino acid sequence alignment of MtZHD4 homologous protein in M. truncatula

Table 2 Conserved motifs of MtZHD4 homologous proteins

序号 No.	基序序列 Sequence of motif	基序长度 Width （aa）	基序位点 Sites （bp）	最大似然比 LLR	E值 E-value
1	VKVRYREKLKNEAAEKGGNAADGVGEFMPKGEEGVVZAL	39	28	2 468	2.2e-720
2	CQEIGVKRRVLKVWMHNNKHNLAKKNPPT	29	14	1 112	2.0e-300
3	NCSACHCHRNFHRKEVEGEPTSCD	24	19	942	6.4e-207
4	HNHIISSSAPALPSE	15	42	804	1.6e-110
5	MELSSQEGEIPIPJN	15	13	451	5.7e-082
6	SDEQEDGGGVV	11	15	352	9.2e-051

Table 3 Physicochemical properties of M. truncatula MtZHD4 protein

一级结构特征 Characteristics of primary structure	预测结果 Predicted results
氨基酸数量 Number of amino acids	363
分子量 Molecular weight （Da）	40 848.88
分子式 Molecular formula	C₁₇₆₄H₂₇₀₀N₅₅₆O₅₃₆S₁₈
带正电荷残基总数 Total number of positively charged residues	30
带负电荷残基总数 Total number of negatively charged residues	31
不稳定指数 Instability coefficient	60.41
脂溶系数 Aliphatic index	56.45
总平均亲水性 Average hydrophobicity	-0.9

Fig. 3 Analysis of MtZHD4 protein structure and functionA: Protein sequence analysis. B: Secondary structure of protein (blue indicates α-helix, purple indicates β-sheet, and yellow indicates random coil). C: Tertiary structure of protein (the yellow ribbon indicates the protein backbone, and blue indicates the ZF-HD domain). D: Analysis of transmembrane structure analysis. E: Prediction of signal peptide. F: Prediction of hydrophobicity. G: Prediction of protein domain

Fig. 4 Analysis of cis-acting elements in the MtZHD4 gene promoterA: The black line indicates the promoter length of MtZHD4 gene, and the boxes with different colors indicate cis-acting elements with different functions. B: The different color boxes at the bottom left indicate the number of elements related to plant growth and development, hormone response, metabolism and stress response, and the length bars of different colors on the bottom right indicate the proportion of cis-acting elements

Fig. 5 Subcellular localization of MtZHD4

Fig. 6 Expression pattern analysis of MtZHD4Different letters indicate significant differences at P<0.05. The values are in means ±SD (n=4)

References 37

1	Glazebrook J. Genes controlling expression of defense responses in Arabidopsis—2001 status [J]. Curr Opin Plant Biol, 2001, 4(4): 301-308.
2	Singh K, Foley RC, Oñate-Sánchez L. Transcription factors in plant defense and stress responses [J]. Curr Opin Plant Biol, 2002, 5(5): 430-436.
3	Wang WL, Wu P, Li Y, et al. Genome-wide analysis and expression patterns of ZF-HD transcription factors under different developmental tissues and abiotic stresses in Chinese cabbage [J]. Mol Genet Genomics, 2016, 291(3): 1451-1464.
4	Hu W, DePamphilis CW, Ma H. Phylogenetic analysis of the plant-specific zinc finger-homeobox and mini zinc finger gene families [J]. J Integr Plant Biol, 2008, 50(8): 1031-1045.
5	Windhövel A, Hein I, Dabrowa R, et al. Characterization of a novel class of plant homeodomain proteins that bind to the C4 phosphoenolpyruvate carboxylase gene of Flaveria trinervia [J]. Plant Mol Biol, 2001, 45(2): 201-214.
6	Mukherjee K, Brocchieri L, Bürglin TR. A comprehensive classification and evolutionary analysis of plant homeobox genes [J]. Mol Biol Evol, 2009, 26(12): 2775-2794.
7	Ariel FD, Manavella PA, Dezar CA, et al. The true story of the HD-zip family [J]. Trends Plant Sci, 2007, 12(9): 419-426.
8	Tan QKG, Irish VF. The Arabidopsis zinc finger-homeodomain genes encode proteins with unique biochemical properties that are coordinately expressed during floral development [J]. Plant Physiol, 2006, 140(3): 1095-1108.
9	Lai W, Zhu CX, Hu ZY, et al. Identification and transcriptional analysis of zinc finger-homeodomain (ZF-HD) family genes in cucumber [J]. Biochem Genet, 2021, 59(4): 884-901.
10	Wang H, Yin XJ, Li XQ, et al. Genome-wide identification, evolution and expression analysis of the grape (Vitis vinifera L.) zinc finger-homeodomain gene family [J]. Int J Mol Sci, 2014, 15(4): 5730-5748.
11	Niu HL, Xia PL, Hu YF, et al. Genome-wide identification of ZF-HD gene family in Triticum aestivum: molecular evolution mechanism and function analysis [J]. PLoS One, 2021, 16(9): e0256579.
12	Xu Y, Wang YH, Long QZ, et al. Overexpression of OsZHD1, a zinc finger homeodomain class homeobox transcription factor, induces abaxially curled and drooping leaf in rice [J]. Planta, 2014, 239(4): 803-816.
13	Khatun K, Nath UK, Robin AHK, et al. Genome-wide analysis and expression profiling of zinc finger homeodomain (ZHD) family genes reveal likely roles in organ development and stress responses in tomato [J]. BMC Genomics, 2017, 18(1): 695.
14	Liu MY, Wang XX, Sun WJ, et al. Genome-wide investigation of the ZF-HD gene family in Tartary buckwheat (Fagopyrum tataricum) [J]. BMC Plant Biol, 2019, 19(1): 248.
15	Park HC, Kim ML, Lee SM, et al. Pathogen-induced binding of the soybean zinc finger homeodomain proteins GmZF-HD1 and GmZF-HD2 to two repeats of ATTA homeodomain binding site in the calmodulin isoform 4 (GmCaM4) promoter [J]. Nucleic Acids Res, 2007, 35(11): 3612-3623.
16	Perrella G, Davidson MLH, O'Donnell L, et al. ZINC-FINGER interactions mediate transcriptional regulation of hypocotyl growth in Arabidopsis [J]. Proc Natl Acad Sci USA, 2018, 115(19): E4503-E4511.
17	Tran LS P, Nakashima K, Sakuma Y, et al. Co-expression of the stress-inducible zinc finger homeodomain ZFHD1 and NAC transcription factors enhances expression of the ERD1 gene in Arabidopsis [J]. Plant J, 2007, 49(1): 46-63.
18	Ye QY, Zhou CE, Lin H, et al. Medicago2035: genomes, functional genomics and molecular breeding [J]. Mol Plant, 2024: S1674-2052(24)00399-X.
19	Nandety RS, Wen JQ, Mysore KS. Medicago truncatula resources to study legume biology and symbiotic nitrogen fixation [J]. Fundam Res, 2023, 3(2): 219-224.
20	Figueiredo DD, Barros PM, Cordeiro AM, et al. Seven zinc-finger transcription factors are novel regulators of the stress responsive gene OsDREB1B [J]. J Exp Bot, 2012, 63(10): 3643-3656.
21	Chen CJ, Wu Y, Li JW, et al. TBtools-II: a "one for all, all for one" bioinformatics platform for biological big-data mining [J]. Mol Plant, 2023, 16(11): 1733-1742.
22	谢宏, 李舒文, 董迪, 等. 蒺藜苜蓿MtDWF1基因克隆、亚细胞定位及表达特征分析 [J]. 草地学报, 2023, 31(4): 984-991.
	Xie H, Li SW, Dong D, et al. Cloning, subcellular localization and expression pattern of MtDWF1 gene in Medicago truncatula [J]. Acta Agrestia Sin, 2023, 31(4): 984-991.
23	Liu MD, Liu H, Liu WY, et al. Systematic analysis of zinc finger-homeodomain transcription factors (ZF-HDs) in barley (Hordeum vulgare L.) [J]. Genes, 2024, 15(5): 578.
24	Bueso E, Muñoz-Bertomeu J, Campos F, et al. ARABIDOPSIS THALIANA HOMEOBOX25 uncovers a role for Gibberellins in seed longevity [J]. Plant Physiol, 2014, 164(2): 999-1010.
25	雷艳芳. 蒺藜苜蓿遗传图谱的构建及种子产量相关性状的遗传分析 [D]. 兰州: 甘肃农业大学, 2009.
	Lei YF. Construction of genetic map of Medicago truncatula and genetic analysis of seed yield-related traits [D]. Lanzhou: Gansu Agricultural University, 2009.
26	庞丹丹, 刘玉飞, 田易萍, 等. 茶树ZF-HD转录因子基因家族的鉴定及表达分析 [J]. 南方农业学报, 2021, 52(3): 632-640.
	Pang DD, Liu YF, Tian YP, et al. Identification and expression analysis of ZF-HD transcription factor gene family in Camellia sinensis [J]. J South Agric, 2021, 52(3): 632-640.
27	Sun JH, Xie MM, Li XX, et al. Systematic investigations of the ZF-HD gene family in tobacco reveal their multiple roles in abiotic stresses [J]. Agronomy, 2021, 11(3): 406.
28	Hong SY, Kim OK, Kim SG, et al. Nuclear import and DNA binding of the ZHD5 transcription factor is modulated by a competitive peptide inhibitor in Arabidopsis [J]. J Biol Chem, 2011, 286(2): 1659-1668.
29	时丕彪, 王德领, 蒋润枝, 等. 藜麦ZF-HD转录因子的全基因组鉴定及其对盐胁迫的响应分析 [J]. 江苏农业学报, 2022, 38(2): 304-312.
	Shi PB, Wang DL, Jiang RZ, et al. Genome-wide identification of ZF-HD transcription factors and expression analysis of response to salt stress in quinoa [J]. Jiangsu J Agric Sci, 2022, 38(2): 304-312.
30	Guo YQ, Zhu C, Zhao SS, et al. De novo transcriptome and phytochemical analyses reveal differentially expressed genes and characteristic secondary metabolites in the original oolong tea (Camellia sinensis) cultivar ‘Tieguanyin’ compared with cultivar ‘Benshan’ [J]. BMC Genomics, 2019, 20(1): 265.
31	张晋玉, 晁毛妮, 杜弘杨, 等. 大豆ZF-HD转录因子GmZHD1的克隆及表达分析 [J]. 华北农学报, 2017, 32(2): 1-7.
	Zhang JY, Chao MN, Du HY, et al. Cloning and expression analysis of ZF-HD transcription factor GmZHD1 in Glycine max [J]. Acta Agric Boreali Sin, 2017, 32(2): 1-7.
32	吴国江, 周伟, 李艳肖, 等. 高粱ZF-HD基因家族鉴定与盐碱胁迫下的表达分析 [J]. 浙江农业学报, 2024, 36(6): 1217-1231.
	Wu GJ, Zhou W, Li YX, et al. Identification and expression analysis under saline-alkali stress of ZF-HD gene family in sorghum [J]. Acta Agric Zhejiangensis, 2024, 36(6): 1217-1231.
33	付鸿博, 李杰, 杨永超, 等. 石榴ZF-HD基因家族鉴定与分析 [J]. 河南农业科学, 2022, 51(6): 119-125.
	Fu HB, Li J, Yang YC, et al. Identification and analysis of the ZF-HD gene family in Punica granatum [J]. J Henan Agric Sci, 2022, 51(6): 119-125.
34	He K, Li CX, Zhang ZY, et al. Genome-wide investigation of the ZF-HD gene family in two varieties of alfalfa (Medicago sativa L.) and its expression pattern under alkaline stress [J]. BMC Genomics, 2022, 23(1): 150.
35	Zhou CZ, Zhu C, Xie SY, et al. Genome-wide analysis of zinc finger motif-associated homeodomain (ZF-HD) family genes and their expression profiles under abiotic stresses and phytohormones stimuli in tea plants (Camellia sinensis) [J]. Sci Hortic, 2021, 281: 109976.
36	Shan XY, Yan JB, Xie DX. Comparison of phytohormone signaling mechanisms [J]. Curr Opin Plant Biol, 2012, 15(1): 84-91.
37	Xu XR, Zhou H, Yang QH, et al. ZF-HD gene family in rapeseed (Brassica napus L.): genome-wide identification, phylogeny, evolutionary expansion and expression analyses [J]. BMC Genomics, 2024, 25(1): 1181.

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