蒺藜苜蓿MtZHD4基因克隆、亚细胞定位及表达分析

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

摘要/Abstract

摘要：

目的对蒺藜苜蓿（Medicago truncatula）MtZHD4基因进行克隆和表达特征分析，以深入了解其功能，为蒺藜苜蓿生长发育和植物内源激素合成、调控及信号传导等方面提供理论基础。方法以蒺藜苜蓿‘R108’为材料，克隆得到MtZHD4基因，对其进行生物信息学分析和亚细胞定位，利用实时荧光定量PCR技术对MtZHD4基因在不同组织、不同激素和胁迫处理下的表达模式进行分析。结果 MtZHD4基因开放阅读框为1 092 bp，编码363个氨基酸，蛋白保守结构域预测发现MtZHD4含有2个ZF-HD蛋白结构域，分别属于ZF-HD dimmer和homeo ZF-HD超家族。亚细胞定位结果显示，MtZHD4定位于细胞核和细胞质。启动子顺式作用元件分析表明，MtZHD4基因启动子中含有参与植物生长发育、激素响应、光响应等多个顺式作用元件。组织特异性表达分析结果显示，MtZHD4基因在根中表达量最高，在叶和荚果中较低。经不同激素和胁迫处理后，MtZHD4基因的表达量在ABA诱导下出现上调趋势，IAA诱导下呈现下调趋势，在6-BA、MeJA和SA诱导下表现为先上升后下降；在盐胁迫下，MtZHD4基因的表达量呈上升趋势，而在干旱胁迫下，表达量则先上升后下降。结论 MtZHD4基因对不同激素和非生物胁迫具有响应，可能通过相关激素信号转导调控蒺藜苜蓿的生长发育，并在盐和干旱胁迫下起正向调控作用。

关键词: 蒺藜苜蓿, ZF-HD转录因子, 生物信息学分析, 亚细胞定位, 组织差异, 激素处理, 盐胁迫, 干旱胁迫

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

杨春, 王晓倩, 王红军, 晁跃辉. 蒺藜苜蓿MtZHD4基因克隆、亚细胞定位及表达分析[J]. 生物技术通报, 2025, 41(5): 244-254.

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.

图/表 9

表1 引物序列

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

图1 MtZHD4基因克隆和构建的表达载体PCR检测以及同源蛋白序列分析A：蒺藜苜蓿MtZHD4基因的克隆；B：表达载体3302Y-MtZHD4的PCR检测；C：MtZHD4同源蛋白序列进化树

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

图2 MtZHD4同源蛋白多序列比对A：蒺藜苜蓿MtZHD4蛋白的保守基序分析（Mt：蒺藜苜蓿；Tp：红车轴草；Ps：豌豆；Vv：毛苕子；Ca：鹰嘴豆；Ap：相思子：Ss：密花豆；Vu：豇豆；Cc：木豆；Pa：银白杨；Tc：可可；Va：赤豆；Pn：黑杨；Pt：毛果杨）；B：蒺藜苜蓿MtZHD4蛋白保守结构域的LOGO图；C：蒺藜苜蓿MtZHD4同源蛋白氨基酸序列比对

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

表2 MtZHD4同源蛋白的保守基序

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

表3 蒺藜苜蓿MtZHD4蛋白理化性质

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

图3 MtZHD4蛋白结构与功能分析A：蛋白序列分析；B：蛋白二级结构（蓝色表示α-螺旋，紫色表示β-折叠，黄色表示无规则卷曲）；C：蛋白三级结构（黄色带状代表蛋白主链，蓝色为ZF-HD结构域）；D：跨膜结构分析；E：信号肽预测；F：亲疏水性预测；G：蛋白结构域预测

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

图4 MtZHD4基因启动子顺式作用元件分析A：黑线表示MtZHD4基因的启动子长度，线条上的方块表示启动子区的顺式作用元件，图下方不同颜色的方框代表具有不同功能的顺式作用元件；B：左下方不同颜色方框代表与植物生长发育、激素响应、代谢和胁迫相关元件的数量，右下方不同颜色的长度条表示该基因作用元件所占比例

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

图5 MtZHD4亚细胞定位

Fig. 5 Subcellular localization of MtZHD4

图6 MtZHD4表达模式分析不同字母代表P<0.05时的显著差异，数值为平均值±SD（n=4）

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

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