花生转录因子AhHDZ70的生物信息学分析及耐盐耐旱性研究

doi:10.13560/j.cnki.biotech.bull.1985.2025-0901

摘要/Abstract

摘要：

目的干旱和盐胁迫是严重影响我国花生产量及品质的环境因素。探究AhHDZ70（Ah12g074600.1）基因结构和功能，揭示其在花生耐旱和耐盐性方面的作用，为花生抗盐和抗旱育种提供重要的基因资源。方法利用生物信息学方法对AhHDZ70进行理化性质、基因结构、系统发育、启动子顺式元件、蛋白互作分析；采用荧光定量PCR技术分析AhHDZ70基因在不同组织器官和盐、干旱、高温、低温胁迫下的表达量；利用烟草瞬时转化技术进行AhHDZ70基因的亚细胞定位分析；并采用转基因技术在拟南芥中进行遗传转化，通过盐和干旱胁迫处理进一步验证该基因的生物学功能。结果 AhHDZ70编码326个氨基酸，是不稳定的亲水蛋白，无信号肽，其二级和三级结构以无规则卷曲为主。结构域分析表明，AhHDZ70包含4个外显子和3个内含子。AhHDZ70启动子区包含2个干旱诱导功能元件（MBS基序）、1个逆境胁迫响应元件（TC-rich repeats基序）和1个低温响应元件（LTR基序）。转录组结合RT-qPCR分析表明，AhHDZ70在种子中的表达量最高，正向响应盐、干旱、高温和低温胁迫。亚定位实验结果显示，AhHDZ70定位于细胞核。AhHDZ70异源过表达拟南芥获得纯合的转基因植株，通过盐、干旱胁迫处理发现，AhHDZ70过表达转基因植株的发芽率均显著高于WT，且根长极显著长于WT。结论 AhHDZ70属于HD-ZIP Ⅱ转录因子，定位于细胞核。AhHDZ70可能参与对花生多个组织生长发育的调控，并可能正向调控花生对盐和干旱胁迫的响应。

关键词: 花生, 转录因子, AhHDZ70, 生物信息学分析, 表达模式, 耐盐, 耐旱, 基因功能

Abstract:

Objective Drought and salinity stress are critical environmental factors that severely affect the yield and quality of peanuts (Arachis hypogaea L.) in China. Investigating the structure and function of AhHDZ70 (Ah12g074600.1) gene will reveal its function in tolerance of peanut to drought and salt, and provide essential gene resources for peanut salt resistance and drought resistance breeding. Method Bioinformatics method were applied to analyze the physical and chemical properties, gene structure, phylogeny, cis-element of promoter, protein interaction of AhHDZ70. Fluorescence quantitative PCR technology was used to identify the expressions of AhHDZ70 gene in different tissues and organs, and under salt, drought, high temperature and low temperature stresses. Subcellular localization analysis was performed using tobacco transient transformation technology. Genetic transformation was carried out in Arabidopsis and biological function of AhHDZ70 was further verified by salt and drought stress treatment. Result AhHDZ70 encoded 326 amino acids, which was an unstable hydrophilic protein with no signal peptide, its secondary and tertiary structures were mainly characterized by irregular curling. Domain analysis showed that AhHDZ70 contained 4 exons and 3 introns. The AhHDZ70 promoter region contained two drought-induced functional elements (MBS motif), one adversity stress response element (TC-richrepeats motif), and one cryogenic response element (LTR motif). The main form of its secondary structure and tertiary structure was irregular curl. Transcriptome combined with RT-qPCR analysis showed that AhHDZ70 had the highest expression in seeds and responded positively to salt, drought, high temperature and low temperature stress. The results of subcellular localization showed that AhHDZ70 was located in the nucleus. AhHDZ70 heterologously overexpressed Arabidopsis to obtain homozygous transgenic plants. Under salt and drought stress, the germination rate of AhHDZ70-overexpressed transgenic lines was significantly higher than that of WT, and the root length was significantly longer than WT. Conclusion AhHDZ70 belongs to the HD-ZIP II transcription factor and is located in the nucleus. AhHDZ70 might play a regulatory role in the growth and development of various tissues in peanuts. Additionally, AhHDZ70 is likely involved in positively regulating peanut's response to salt and drought stress.

Key words: peanut, transcription factor, AhHDZ70, bioinformatics analysis, expression pattern, tolerance to salt, tolerance to drought, gene function

吴翠翠, 陈登科, 兰刚, 夏芝, 李朋波. 花生转录因子AhHDZ70的生物信息学分析及耐盐耐旱性研究[J]. 生物技术通报, 2026, 42(1): 198-207.

WU Cui-cui, CHEN Deng-ke, LAN Gang, XIA Zhi, LI Peng-bo. Bioinformatics Analysis of Peanut Transcription Factor AhHDZ70 and Its Tolerances to Salt and Drought[J]. Biotechnology Bulletin, 2026, 42(1): 198-207.

图/表 8

表1 所用相关引物

Table 1 Primers used in the study

基因名称 Gene name	正向引物 Forward primer （5′‒3′）	反向引物 Reverse primer （5′‒3′）	实验类型 Type of experiment
AhHDZ70	TTGGAGAGAACACGGGGGACGAATTCATGGAATATGCAACATATTCATCAGC	GCCTGCAGGTCGACTCTAGAGGATCCGGACCAAAAGTCCCACCATTG	过表达实验、亚定位实验、过表达PCR鉴定
β-Actin	CAGGATTTGCCGGTGATGATG	TCTGTTGGCCTTCGGGTTGAG	内参
AhHDZ70	AGATGAGTTTGGGTTTGAGT	GAGGTAGAGGCTTCTGGAT	RT-qPCR

图1 AhHDZ70与其他物种同源蛋白的进化树分析Ah：四倍体花生；Ad：二倍体花生；Ca：鹰嘴豆；Cc：木豆；Gm：栽培大豆；Gs：野生大豆；La：窄叶羽扇豆；Mt：苜蓿；Ps：豌豆；Pv：菜豆

Fig. 1 Phylogenetic tree analysis of homologous proteins between AhHDZ70 and other speciesAh: Arachis hypogaea; Ad: Arachis duranensis; Ca: Cicer arietinum; Cc: Cajanus cajan; Gm: Glycine max; Gs: Glycine soja; La: Lupinus angustifolius; Mt: Medicago truncatula; Ps: Pisum sativum; Pv: Phaseolus vulgaris

图2 AhHDZ70启动子分析（A）及互作蛋白预测（B）

Fig. 2 Analysis of AhHDZ70 promoter (A) and prediction of interaction protein (B)

图3 AhHDZ70组织器官和非生物胁迫的表达模式分析A：AhHDZ70在22个组织器官的表达模式；B：AhHDZ70及其同源基因AhHDZ5在非生物胁迫下的组织表达模式。Vegetative shoot tip：主茎营养茎尖；Reproductive shoot tip：第一侧生殖芽尖；Root：根；Nodule：根瘤；Flowers：花；Pistil：雌蕊；Stamen：雄蕊；Aerial peg：果针；Subterranean peg：入土果针；Main stem leaf：主根；Lateral stem leaf：侧枝叶；Leaf：叶；Pod pattee 3：荚果发育期3；Pericarp pattee 5：果皮发育期5；Pericarp pattee 6：果皮发育期6；Seed pattee 6：种子发育期6；Seed pattee 7：种子发育期7；Seed pattee 8：种子发育期8；Seed pattee 10：种子发育期10；Seed pattee5：种子发育期5；Expansion pod：膨大期果针；Gynophore stalk：雌蕊柄。L：叶片；R：根

Fig. 3 Analysis of AhHDZ70 expression patterns in tissues, organs, and abiotic stressA: Expression patterns of AhHDZ70 in 22 tissues and organs. B: Expression patterns of AhHDZ70 under abiotic stress. L: Leaf. R: Root

图4 AhHDZ70在花生不同组织器官及非生物胁迫下的RT-qPCR鉴定A：AhHDZ70在花生不同组织器官的RT-qPCR分析；B-E：AhHDZ70在盐、干旱、低温和高温胁迫下的RT-qPCR分析。通过双尾t检验分析差异显著性，误差线代表平均值±SD，*P<0.05， **P<0.01，下同

Fig. 4 RT-qPCR identification of AhHDZ70 in different tissues and organs of peanut under abiotic stressA: RT-qPCR analysis of AhHDZ70 in different tissues and organs of peanuts. B-E: RT-qPCR analysis of AhHDZ70 under salt, drought, low temperature and high temperature stress. The difference significance was analyzed by two-tailed t-test. Error bars indicate mean ±SD, *P<0.05, **P<0.01,the same below

图5 AhHDZ70蛋白在烟草细胞中的亚细胞定位

Fig. 5 Subcellular localizations of AhHDZ70 proteins in tobacco leaves (bar=50 μm)

图6 AhHDZ70过表达株系的盐和干旱胁迫下发芽率鉴定

Fig. 6 Identification of the germination rate in AhHDZ70 overexpression lines under salt and drought stress

图7 AhHDZ70过表达株系的盐胁迫和干旱胁迫根长实验

Fig. 7 Root length experiments of salt stress and drought stress in AhHDZ70 overexpressed strain

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