Identification of the AGPL Gene Family in Yam and Screening of DoAGPL1 Interacting Proteins

doi:10.13560/j.cnki.biotech.bull.1985.2025-1247

Abstract

Abstract:

Objective This study aimed to identify and analyze the members of the DoAGPL gene family in yam (Dioscorea opposita), providing a foundation for elucidating the molecular mechanisms of starch synthesis and the stress-responsive metabolic network in yam. Method We characterized the DoAGPL family using bioinformatics to assess the physicochemical properties, phylogeny, and gene structures of its members. RT-qPCR was employed to evaluate their expression patterns in different developmental stages, tissues, and abiotic stress conditions. Additionally, we determined the subcellular localization of DoAGPL1 and demonstrated its interactions with DoAGPS and DoMADS through yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays. Result Six DoAGPL members were identified in yam, encoding proteins of 275–561 amino acids. Phylogenetic analysis classified them into four clades, and ten conserved motifs were identified. RT-qPCR analysis revealed that DoAGPL1 was highly expressed in tubers. Its expression was down-regulated during the early stage of tuber expansion and up-regulated during the peak expansion stage, suggesting that DoAGPL1 may serve as a core regulator of starch synthesis in tubers. Furthermore, DoAGPL1 responded to low-temperature and drought stresses, and the expression of DoAGPL members varied in different abiotic stress conditions. Subcellular localization analysis revealed that the DoAGPL1 protein was localized to the nucleus and the cell membrane. Yeast two-hybrid screening identified DoAGPS and DoMADS as DoAGPL1-interacting proteins, and the interactions were further confirmed. Conclusion Six DoAGPL members were identified from yam transcriptomic and proteomic data. DoAGPL1 was upregulated by cold and drought stresses and highly expressed in tubers, indicating a potential role in regulating stress responses and starch synthesis during tuber expansion.

Key words: Dioscorea opposita Thunb., starch synthesis, DoAGPL1 gene, expression analysis, interacting protein

NIE Yu-shan, ZHANG Yan-fang, YUAN Shu-qi, LI Na, SUO Ning-ning, TAN Gui-lian, HUO Xiu-wen. Identification of the AGPL Gene Family in Yam and Screening of DoAGPL1 Interacting Proteins[J]. Biotechnology Bulletin, 2026, 42(9): 316-326.

Figures/Tables 12

Table 1 Physicochemical properties of DoAGPLs protein

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
DoAGPL1	533	59 161.22	7.05	35.403	-0.116	82.48	细胞膜/叶绿体
DoAGPL2	533	59 182.18	6.53	34.883	-0.114	82.48	细胞质
DoAGPL3	550	60 270.60	6.84	44.561	-0.229	87.45	叶绿体
DoAGPL4	561	62 279.13	9.25	37.753	-0.243	85.92	叶绿体
DoAGPL5	307	34 795.22	6.02	37.229	-0.181	88.93	细胞质
DoAGPL6	275	31 206.12	7.72	35.696	-0.193	90.07	细胞质

Fig. 1 Multiple sequence alignment and phylogeneic tree analysis of DoAGPLsA: Multiple sequence alignment. B: Phylogeneic tree analysis of DoAGPLs

Fig. 2 Conserved motifs analysis of the DoAGPLsA: Motif sequence logo (the height of each letter indicates the frequency of amino acid occurrence at that position); B: conserved motifs

Fig. 3 Analysis of expression patterns of the DoAGPLs in different developmental stages and tissues of the yamA: Expression analysis of DoAGPLs in yam transcriptome data. B: Expression analysis of DoAGPLs in different tissues of yam

Fig. 4 Expression pattern analysis of DoAGPLs under different abiotic stressA: Low-temperature treatment. B: Drought treatment. C: Salt treatment. Different lowercase letters indicate significant difference at P<0.05 level

Fig. 5 Subcellular localization of DoAGPL1

Fig. 6 Self-activation detection of the bait vector pBT3-SUC-DoAGPL1

Fig. 7 Positive clonal colonies were detected by PCRM: DL2 000 DNA marker; 1-16: PCR results of 1-16 bacterial solution

Table 2 Blast analysis of interacted proteins

编号 No.	功能注释 Function annotation	序列号 Serial number
1	葡萄糖-1-磷酸腺苷基转移酶小亚基	XM_039260986.1
2	SPX结构域含蛋白4	XM_039273168.1
3	NAC结构域的蛋白74样	XM_039268517.1
4	MADS-box蛋白	XM_039259047.1

Fig. 8 Verification of interaction between DoAGPL1 and DoAGPS/MADS

Fig. 9 AGPL, AGPS, MADS plasmid PCR and double enzyme digestion identification of vector constructionM1, M2: DL2 000 DNA marker; +: positive control; -: negative control; 2: PCR amplification product; 3: recombinant plasmid; 4: positive control; 5: enzyme digestion identification

Fig. 10 BiFC verifying the interaction of proteins

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