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

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
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

References 37

[1]	Shan N, Wang PT, Zhu QL, et al. Comprehensive characterization of yam Tuber nutrition and medicinal quality of Dioscorea opposita and D. alata from different geographic groups in China [J]. J Integr Agric, 2020, 19(11): 2839-2848.
[2]	李月仙, 黄东益, 黄小龙, 等. 山药的研究进展 [J]. 中国农学通报, 2009, 25(9): 91-96.
	Li YX, Huang DY, Huang XL, et al. Progress of study on Dioscorea opposita thunb [J]. Chin Agric Sci Bull, 2009, 25(9): 91-96.
[3]	覃维治, 韦本辉, 甘秀芹, 等. 不同淮山药种质(品种)的粘度比较与聚类分析 [J]. 南方农业学报, 2012, 43(2): 176-179.
	Qin WZ, Wei BH, Gan XQ, et al. Comparative analysis of different yam (Dioscorea opposita Thunb.) germplasm resources and their clustering on the basis of viscosity characteristics [J]. J South Agric, 2012, 43(2): 176-179.
[4]	孙怡青. 山药(Dioscorea opposita thunb.)BAM1和BAM3基因克隆及BAM1功能鉴定与互作蛋白筛选 [D]. 呼和浩特: 内蒙古农业大学, 2025.
	Sun YQ. Cloning of BAM1 and BAM3 genes and functional identification of BAM1 with interaction protein screening in Chinese yam (Dioscorea opposita thunb.) [D]. Hohhot: Inner Mongolia Agricultural University, 2025.
[5]	Tuncel A, Okita TW. Improving starch yield in cereals by over-expression of ADPglucose pyrophosphorylase: Expectations and unanticipated outcomes [J]. Plant Sci, 2013, 211: 52-60.
[6]	Espada J. Enzymic synthesis of adenosine diphosphate glucose from glucose 1-phosphate and adenosine triphosphate [J]. J Biol Chem, 1962, 237(12): 3577-3581.
[7]	周彩琴, 王丽娟. 腺苷二磷酸葡萄糖焦磷酸化酶研究进展 [J]. 农业科学研究, 2011, 32(2): 56-59.
	Zhou CQ, Wang LJ. Advances of research on ADP-glucose pyrophosphorylase [J]. J Agric Sci, 2011, 32(2): 56-59.
[8]	Georgelis N, Braun EL, Shaw JR, et al. The two AGPase subunits evolve at different rates in angiosperms, yet they are equally sensitive to activity-altering amino acid changes when expressed in bacteria [J]. Plant Cell, 2007, 19(5): 1458-1472.
[9]	Crevillén P, Ventriglia T, Pinto F, et al. Differential pattern of expression and sugar regulation of Arabidopsis thaliana ADP-glucose pyrophosphorylase-encoding genes [J]. J Biol Chem, 2005, 280(9): 8143-8149.
[10]	Lu FH, Park YJ. Sequence variations in OsAGPase significantly associated with amylose content and viscosity properties in rice (Oryza sativa L.) [J]. Genet Res, 2012, 94(4): 179-189.
[11]	Tang XJ, Peng C, Zhang J, et al. ADP-glucose pyrophosphorylase large subunit 2 is essential for storage substance accumulation and subunit interactions in rice endosperm [J]. Plant Sci, 2016, 249: 70-83.
[12]	Han SX, Lin YH, Meng YY, et al. Genome-wide identification and analysis of SUS and AGPase family members in sweet potato: response to excessive nitrogen stress during storage root formation [J]. Int J Mol Sci, 2024, 25(15): 8236.
[13]	王明, 肖鑫辉, 应东山, 等. 木薯AGPase基因家族的生物信息学及其表达分析 [J]. 南方农业学报, 2019, 50(2): 222-229.
	Wang M, Xiao XH, Ying DS, et al. Bioinformatics and expression of AGPase gene family in cassava [J]. J South Agric, 2019, 50(2): 222-229.
[14]	Mistry J, Chuguransky S, Williams L, et al. Pfam: The protein families database in 2021 [J]. Nucleic Acids Res, 2021, 49(D1): D412-D419.
[15]	Artimo P, Jonnalagedda M, Arnold K, et al. ExPASy: SIB bioinformatics resource portal [J]. Nucleic Acids Res, 2012, 40(W1): W597-W603.
[16]	Horton P, Park KJ, Obayashi T, et al. WoLF PSORT: protein localization predictor [J]. Nucleic Acids Res, 2007, 35(): W585-W587.
[17]	Kumar S, Nei M, Dudley J, et al. MEGA: a biologist-centric software for evolutionary analysis of DNA and protein sequences [J]. Brief Bioinform, 2008, 9(4): 299-306.
[18]	Li N, Zhang YF, Huo XW, et al. Genome-wide identification and expression profiling of plasma membrane-localized SWEET gene family associated with sugar transport during yam Tuber Development [J]. Int J Mol Sci, 2025, 26(12): 5847.
[19]	Xing LN, Zhang YF, Xing YP, et al. Reference genes selection and validation in yam by real-time quantitative polymerase chain reaction [J]. Sci Rep, 2025, 15: 19947.
[20]	Qu JZ, Xu ST, Zhang ZQ, et al. Evolutionary, structural and expression analysis of core genes involved in starch synthesis [J]. Sci Rep, 2018, 8: 12736.
[21]	赵步洪, 张文杰, 常二华, 等. 水稻灌浆期籽粒中淀粉合成关键酶的活性变化及其与灌浆速率和蒸煮品质的关系 [J]. 中国农业科学, 2004, 37(8): 1123-1129.
	Zhao BH, Zhang WJ, Chang EH, et al. Changes in activities of the key enzymes related to starch synthesis in rice grains during grain filling and their relationships with the filling rate and cooking quality [J]. Sci Agric Sin, 2004, 37(8): 1123-1129.
[22]	梁任繁, 李创珍, 张娟, 等. 山药块茎发育中物质积累及相关代谢酶变化 [J]. 作物学报, 2011, 37(5): 903-910.
	Liang RF, Li CZ, Zhang J, et al. Changes of matter accumulation and relative enzymatic activity during yam Tuber development [J]. Acta Agron Sin, 2011, 37(5): 903-910.
[23]	Batra R, Saripalli G, Mohan A, et al. Comparative analysis of AGPase genes and encoded proteins in eight monocots and three dicots with emphasis on wheat [J]. Front Plant Sci, 2017, 8: 19.
[24]	晁毛妮, 董洁, 胡根海, 等. 陆地棉AGPase基因家族的鉴定及表达分析 [J]. 棉花学报, 2022, 34(4): 299-312.
	Chao MN, Dong J, Hu GH, et al. Identification and expression analysis of AGPase gene family in upland cotton (Gossypium hirsutum L.) [J]. Cotton Sci, 2022, 34(4): 299-312.
[25]	何芳练, 刘莉莉, 蒋慧萍, 等. 芋ADP-葡萄糖焦磷酸化酶(AGPase)基因家族的克隆、生物信息学及表达分析 [J]. 热带作物学报, 2022, 43(8): 1554-1564.
	He FL, Liu LL, Jiang HP, et al. Cloning, bioinformatics and expression analysis of ADP-glucose py-rophosphorylase gene family in Colocasia esculenta [J]. Chin J Trop Crops, 2022, 43(8): 1554-1564.
[26]	Dickinson DB, Preiss J. ADP glucose pyrophosphorylase from maize endosperm [J]. Arch Biochem Biophys, 1969, 130: 119-128.
[27]	石桃雄, 唐彬, 任蓉蓉, 等. 苦荞AGPase编码基因FtAGPL和FtAGPS全基因组鉴定和表达分析 [J]. 贵州师范大学学报: 自然科学版, 2021, 39(4): 52-57.
	Shi TX, Tang B, Ren RR, et al. Genome-wide identification and gene expression analyses of AGPase encoding genes FtAGPL and FtAGPS in Tartary buckwheat (Fagopyrum tataricum) [J]. J Guizhou Norm Univ Nat Sci, 2021, 39(4): 52-57.
[28]	Dong MY, Fan XW, Li YZ. Cassava AGPase genes and their encoded proteins are different from those of other plants [J]. Planta, 2019, 250(5): 1621-1635.
[29]	Miao HX, Sun PG, Liu Q, et al. The AGPase family proteins in banana: genome-wide identification, phylogeny, and expression analyses reveal their involvement in the development, ripening, and abiotic/biotic stress responses [J]. Int J Mol Sci, 2017, 18(8): 1581.
[30]	La Cognata U, Willmitzer L, Müller-Röber B. Molecular cloning and characterization of novel isoforms of potato ADP-glucose pyrophosphorylase [J]. Molec Gen Genet, 1995, 246(5): 538-548.
[31]	Chao MN, Zhang QF, Huang L, et al. ADP-glucose pyrophosphorylase gene family in soybean and implications in drought stress tolerance [J]. Genes Genom, 2024, 46(10): 1183-1199.
[32]	Akihiro T, Mizuno K, Fujimura T. Gene expression of ADP-glucose pyrophosphorylase and starch contents in rice cultured cells are cooperatively regulated by sucrose and ABA [J]. Plant Cell Physiol, 2005, 46(6): 937-946.
[33]	董洁, 李鸽子, 韩巧霞, 等. 小麦TaAGPL1基因上游转录因子TabHLH39的分离及其功能研究 [J]. 中国农业科技导报, 2020, 22(10): 18-26.
	Dong J, Li GZ, Han QX, et al. Isolation and function of TabHLH39 transcription factor regulating expression of the TaAGPL1 gene in bread wheat [J]. J Agric Sci Technol, 2020, 22(10): 18-26.
[34]	Meng Q, Zhang WQ, Hu X, et al. Two ADP-glucose pyrophosphorylase subunits, OsAGPL1 and OsAGPS1, modulate phosphorus homeostasis in rice [J]. Plant J, 2020, 104(5): 1269-1284.
[35]	Kaur V, Madaan S, Behl RK. ADP-glucose pyrophosphorylase activity in relation to yield potential of wheat: Response to independent and combined high temperature and drought stress [J]. Cereal Res Commun, 2017, 45(2): 181-191.
[36]	Ku AT, Huang YS, Wang YS, et al. IbMADS1 (Ipomoea batatas MADS-box 1 gene) is Involved in Tuberous Root Initiation in Sweet Potato (Ipomoea batatas) [J]. Ann Bot, 2008, 102(1): 57-67.
[37]	Xia YF, Jiang SX, Ren YY, et al. Genome-wide identification of MADS-box gene family members in poplar and functional characterization of PagMADS23a in the regulation of stomatal density [J]. Ind Crops Prod, 2026, 241: 122735.