山药HXKs鉴定及DoHXK3互作蛋白验证

doi:10.13560/j.cnki.biotech.bull.1985.2025-1246

摘要/Abstract

摘要：

目的己糖激酶（hexokinase, HXK）催化植物蔗糖代谢，其编码基因HXK是调控糖代谢的关键基因。对山药DoHXK家族成员进行鉴定、表达模式分析及互作蛋白筛选，解析该家族在糖代谢过程中的核心作用，为探究山药蔗糖代谢促进淀粉合成调控机制及抗逆代谢提供依据。方法基于山药不同发育阶段转录组和蛋白组联合分析筛选山药HXK家族，对其理化性质、系统进化关系、亲疏水性、亚细胞定位等进行分析，通过RT-qPCR检测DoHXKs在不同发育时期、不同组织，以及低温、干旱、盐胁迫下的表达模式。对DoHXK3进行亚细胞定位分析并筛选其互作蛋白。结果共鉴定到4个DoHXKs成员，平均氨基酸个数为602，平均分子量为58.45 kD，平均等电点为6.56。与几内亚山药的亲缘关系最近。DoHXK家族成员表现出明显的时空表达特异性：在组织水平上，多在块茎中高表达；在发育水平上，以块茎膨大前期的表达水平最为显著。DoHXKs均能响应3种非生物胁迫（低温、干旱、盐），其中，DoHXK2和DoHXK3受低温显著诱导，DoHXK2-4受盐胁迫强烈诱导，DoHXK1和DoHXK3表达量在干旱胁迫后显著上调。DoHXK3可能是调控山药生长及应对非生物胁迫的关键因子。亚细胞定位结果显示，DoHXK3定位于细胞核和细胞膜。利用酵母双杂交系统筛选并验证了DoHXK3的2个互作蛋白——DoA2C和DoERF3A，分别在植物抵御逆境胁迫和调控生长发育中发挥重要功能。结论山药的4个DoHXKs家族成员共同参与对低温、干旱及盐等胁迫的响应。特别是DoHXK3，作为调控生长发育与抗逆的关键基因，在山药生长前期占主导地位并受低温、盐胁迫强烈诱导。蛋白互作研究表明，DoHXK3及其互作蛋白协同调控山药发育并在逆境响应中起核心调控作用。

关键词: 山药, HXKs, 表达分析, 亚细胞定位, 酵母双杂交, 互作蛋白筛选及验证, 非生物胁迫

Abstract:

Objective Hexokinase (HXK) catalyzes sucrose metabolism in plants, and the HXK gene is a key regulator of sucrose metabolism. Identification of DoHXK family members in yam, analysis of their expression patterns, and screening of interacting proteins aim to elucidate the core role of this family in sucrose metabolism, providing a basis for exploring the regulatory mechanism of starch synthesis promoted by sucrose metabolism and the metabolic response to abiotic stress in yam. Method The HXK gene family of yam was identified through an integrated analysis of transcriptomic and proteomic data from different developmental stages of yam. Physicochemical properties, phylogenetic relationships, hydrophilicity/hydrophobicity, and subcellular localization of the DoHXKs were then analyzed. The expression patterns of DoHXKs in different developmental stages, tissues, and under low-temperature, drought, and salt stress conditions were detected via RT-qPCR. Additionally, subcellular localization analysis of DoHXK3 was conducted, and its interacting proteins were screened. Result A total of four DoHXKs were identified, with an average of 602 amino acids, an average molecular weight of 58.45 kD, and an average isoelectric point of 6.56. They were most closely related to those of Dioscorea alata. The DoHXK family members showed obvious spatiotemporal expression specificity: at the tissue level, they were mostly highly expressed in tubers; at the developmental level, the expression level was most significant in the early stage of tuber expansion. DoHXKs could respond to three abiotic stresses (low temperature, drought, and salt), among which DoHXK2 and DoHXK3 were significantly induced by low temperature, DoHXK2-4 were strongly induced by salt stress, and the expression levels of DoHXK1 and DoHXK3 were significantly upregulated after drought stress. DoHXK3 might be a key factor in regulating the growth of yam and responding to abiotic stress. Subcellular localization results showed that DoHXK3 was located in the nucleus and cell membrane. Two interacting proteins of DoHXK3, DoA2C and DoERF3A, were screened and verified by the yeast two-hybrid system, which play important roles in plant resistance to abiotic stress and regulation of growth and development, respectively. Conclusion The four DoHXK family members in yam jointly participate in the response to low temperature, drought, and salt stress. Particularly, DoHXK3, as a key gene regulating growth and development and stress resistance, plays a dominant role in the early growth stage of yam and is strongly induced by low temperature and salt stress. Protein interaction studies indicated that DoHXK3 and its interacting proteins cooperatively regulate the development ofyamand play a core regulatory role in abiotic stress response.

Key words: Dioscorea opposita Thunb, HXKs, expression analysis, subcellular localization, yeast two-hybrid, screening and validation of interacting proteins, abiotic stress

袁舒琪, 张艳芳, 聂雨杉, 李娜, 索宁宁, 谭桂莲, 霍秀文. 山药HXKs鉴定及DoHXK3互作蛋白验证[J]. 生物技术通报, 2026, 42(9): 306-315.

YUAN Shu-qi, ZHANG Yan-fang, NIE Yu-shan, LI Na, SUO Ning-ning, TAN Gui-lian, HUO Xiu-wen. Identification of HXKs in Yam and Validation of Proteins Interacting with DoHXK3[J]. Biotechnology Bulletin, 2026, 42(9): 306-315.

图/表 7

表1 山药DoHXKs理化性质

Table 1 Physicochemical properties of DoHXKs in D. opposita

基因名称 Gene name	蛋白长度 Protein length （aa）	相对分子量 Molecular weight （Da）	理论等电点 Isoelectric point	不稳定指数 Instability index	亲水性 Grand average of hydropathicity	脂肪系数 Aliphatic index	亚细胞定位预测 Subcellular localization prediction
DoHXK1	535	58 350.27	6.67	36.698	-0.013	99.51	叶绿体
DoHXK2	518	56 139.20	5.43	43.944	-0.093	88.32	叶绿体、细胞核
DoHXK3	538	57 964.62	6.75	39.814	0.085	96.25	叶绿体、细胞膜
DoHXK4	820	90 279.70	7.38	52.810	-0.066	98.9	叶绿体

图1 DoHXKs的多序列比对及系统发育树分析A：蛋白多序列比对；红色区域：完全保守；黄色区域：相似性较高；白色区域：相似性较低；黑框所选区域相似性较高；B：蛋白系统进化树

Fig. 1 Multiple sequence alignment and phylogenetic tree analysis of DoHXKsA: Protein multiple sequence alignment; red regions: completely conserved; yellow regions: high similarity; white regions: low similarity; the black-framed region has high similarity. B: Protein phylogenetic tree

图2 DoHXKs保守基序分析A：不同基序在每个DoHXK序列中的位置；B：不同基序

Fig. 2 Conserved motif analysis of DoHXKsA: Positions of different motifs in each DoHXK sequence. B: Different motifs

图3 DoHXKs不同发育时期及不同组织基因表达量A：DoHXKs在山药不同发育阶段的基因表达量；B：DoHXKs在山药不同组织的基因表达量。不同小写字母表示差异达到显著水平（P<0.05）。下同

Fig. 3 Expression of DoHXKs in different developmental stages and tissuesA: Gene expression levels of DoHXKs at different developmental stages of yam. B: Gene expression levels of DoHXKs in different tissues of yam. Different lowercase letters indicate significant differences (P<0.05). The same below

图4 DoHXKs对低温（A）、干旱（B）和盐（C）处理的RT-qPCR验证

Fig. 4 RT-qPCR validation of expression of DoHXKs in the response to cold (A), drought (B), and salt (C) treatment

图5 DoHXK3的亚细胞定位

Fig. 5 Subcellular localization ofDoHXK3

图6 蛋白质互作关系验证A：自激活检测结果；B：加3-AT抑制结果；C：酵母双杂交互作验证；D、E：DoHXK3与DoERF3A（D）、DoA2C（E）相互作用的双分子荧光互补验证

Fig. 6 Verification of protein interaction relationshipsA: Self-activation assay results. B: Result of 3-AT inhibition. C: Y2H assay for protein interaction verification. D, E: Verification of the interaction between DoHXK3, DoERF3A (D) and DoA2C (E) by bimolecular fluorescence complementation

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