山药AGPL基因家族鉴定及DoAGPL1互作蛋白筛选

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

摘要/Abstract

摘要：

目的鉴定并分析山药DoAGPL基因家族（DoAGPLs）成员，为阐明山药淀粉合成分子机制及抗逆代谢网络提供依据。方法采用生物信息学方法对DoAGPLs编码蛋白的理化性质、系统进化关系及基因结构等方面进行分析；利用RT-qPCR分析其在不同发育时期、组织及非生物胁迫下的表达模式；对DoAGPL1进行亚细胞定位，采用酵母双杂交技术（Y2H）和双分子荧光互补（BiFC）实验验证DoAGPL1与DoAGPS、DoMADS的互作关系。结果鉴定出6个山药DoAGPLs成员，编码275-561个氨基酸，系统发育树将其分为4支，含10个保守基序。RT-qPCR显示，DoAGPL1在块茎中高表达，在块茎膨大早期表达下调、盛期上调，DoAGPL1可能是调控块茎淀粉合成的一个核心基因。此外，DoAGPL1对低温和干旱胁迫有响应，且各基因成员在不同非生物胁迫中具有不同程度的表达。亚细胞定位表明DoAGPL1蛋白定位于细胞核和细胞膜，通过酵母双杂交筛选出DoAGPS、DoMADS为DoAGPL1的互作蛋白并验证它们之间存在互作关系。结论从山药转录组和蛋白组中共鉴定出6个DoAGPLs成员，其中DoAGPL1基因受低温和干旱胁迫上调，并在山药块茎中高表达，表明其在山药响应逆境及块茎膨大阶段的淀粉合成中具有潜在调控作用。

关键词: 山药, 淀粉合成, DoAGPL1基因, 表达分析, 互作蛋白

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

聂雨杉, 张艳芳, 袁舒琪, 李娜, 索宁宁, 谭桂莲, 霍秀文. 山药AGPL基因家族鉴定及DoAGPL1互作蛋白筛选[J]. 生物技术通报, 2026, 42(9): 316-326.

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.

图/表 12

表1 山药DoAGPLs蛋白理化性质

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

图1 DoAGPLs的多序列比对及系统进化分析A：多序列比对；B：DoAGPLs的系统进化树分析

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

图2 DoAGPLs保守基序分析A：基序序列徽标（每个字母的高度表示该位置氨基酸出现的频率）；B：保守基序

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

图3 DoAGPLs在山药不同发育时期及不同组织的表达模式分析A：山药转录组数据中DoAGPLs的表达分析；B：DoAGPLs在不同组织中的表达分析

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

图4 不同非生物胁迫处理下DoAGPLs的表达模式分析A：低温处理；B：干旱处理；C：盐处理；不同小写字母表示P<0.05水平差异显著

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

图5 DoAGPL1的亚细胞定位

Fig. 5 Subcellular localization of DoAGPL1

图6 诱饵载体 pBT3-SUC-DoAGPL1 的自激活检测

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

图7 阳性克隆菌落PCR检测M：DL2 000 DNA标记；1-16：菌液PCR结果

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

表2 互作蛋白Blast分析

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

图8 DoAGPL1与DoAGPS/MADSF互作验证

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

图9 AGPL、AGPS、MADS载体构建的质粒PCR和双酶切鉴定M1，M2：DL2 000 DNA标记；+：阳性对照；-：阴性对照；2：PCR扩增产物；3：重组质粒；4：阳性对照；5：酶切鉴定

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

图10 双分子荧光互补验证蛋白互作关系

Fig. 10 BiFC verifying the interaction of proteins

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