Identification of HXKs in Yam and Validation of Proteins Interacting with DoHXK3

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

Abstract

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

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

Figures/Tables 7

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
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

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

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

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

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

Fig. 5 Subcellular localization ofDoHXK3GFP: Fluorescent channel. Merged: Merged channel

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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