欢迎访问作物学报，今天是
作物学报

作物学报 ›› 2026, Vol. 52 ›› Issue (3): 677-687.doi: 10.3724/SP.J.1006.2026.51081

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

小麦GSK激酶TaSK41的功能分析及互作蛋白的筛选

李灿1(), 张喜伟2(), 朱博涛1, 张沛沛1,*()   

  1. 1甘肃农业大学干旱生境作物学国家重点实验室, 甘肃兰州 730070
    2甘肃农业大学生命科学技术学院, 甘肃兰州 730070
  • 收稿日期:2025-09-01 接受日期:2025-11-18 出版日期:2026-03-12 网络出版日期:2025-11-26
  • 通讯作者: *张沛沛, E-mail: 13858061472@163.com
  • 作者简介:李灿, E-mail: lican@gsau.edu.cn;
    张喜伟, E-mail: zhangxw20002024@163.com

    **同等贡献

  • 基金资助:
    国家自然科学基金项目(32160487);甘肃省联合科研基金一般项目(24JRRA839);甘肃农业大学公招博士科研启动基金项目(GAU-KYQD-2018-41)

Functional characterization of wheat GSK kinase TaSK41 and screening for interacting proteins

Li Can1(), Zhang Xi-Wei2(), Zhu Bo-Tao1, Zhang Pei-Pei1,*()   

  1. 1State Key Laboratory of Aridland Crop Science, Gansu Agricultural University, Lanzhou 730070, Gansu, China
    2College of Life Science and Technology, Gansu Agricultural University, Lanzhou 730070, Gansu, China
  • Received:2025-09-01 Accepted:2025-11-18 Published:2026-03-12 Published online:2025-11-26
  • Contact: *张沛沛, E-mail: 13858061472@163.com
  • About author:

    **Contributed equally to this work

  • Supported by:
    National Natural Science Foundation of China(32160487);Natural Science Foundation of Gansu Provincial Joint Fund, China(24JRRA839);Scientific Research Start-up Funds for Openly-recruited Doctors of Gansu Agricultural University(GAU-KYQD-2018-41)

RichHTML

25

PDF

194

摘要：

植物糖原合成激酶3 (GSK3)家族成员SK41在籽粒发育和粒重形成过程中发挥重要作用。为进一步探究TaSK41调控小麦籽粒发育的生物学功能及其潜在的分子机制, 本研究分析了TaSK41在不同组织中的表达模式、亚细胞定位、过表达TaSK41水稻的籽粒表型, 以及与TaSK41相互作用的蛋白。qRT-PCR分析表明, TaSK41在各个组织中均有表达, 其中在穗部、早期发育的籽粒、子房以及种皮中的表达量较高。亚细胞定位显示, TaSK41-GFP融合蛋白主要定位于细胞质和细胞核中。TaSK41过表达转基因水稻株系的千粒重显著降低, 粒长和粒宽均显著减小。通过酵母双杂交系统筛选小麦籽粒cDNA文库, 共获得17个可能与TaSK41相互作用的候选蛋白。进一步对TaSK41与调控籽粒发育相关基因TaARF4和TaBSK3的全长互作验证, 结果显示, TaSK41能够与小麦生长素响应因子TaARF4的全长蛋白发生相互作用。利用荧光素酶互补系统在体内对其互作关系进行了验证, 结果表明, TaSK41-nLUC与TaARF4-cLUC共转化烟草叶片, 可观察到荧光信号, 这表明它们在体内确实存在互作关系。本研究结果为深入解析TaSK41调控小麦粒重形成的分子机制提供了重要理论依据。

关键词: 小麦, 粒重, TaSK41, 酵母双杂交, 互作蛋白

Abstract:

SK41, a plant glycogen synthase kinase 3 (GSK3) /SHAGGY-like kinases gene, plays an important role in grain development and grain weight formation. To further elucidate the biological function of TaSK41 in regulating wheat grain development and its underlying molecular mechanisms, this study examined the expression patterns of TaSK41 across different tissues, its subcellular localization, the grain phenotypes of transgenic rice overexpressing TaSK41, and its interacting proteins. qRT-PCR analysis revealed that TaSK41 was ubiquitously expressed, with particularly high expression in spikes, early developing grains, ovaries, and seed coats. Subcellular localization assays showed that the TaSK41-GFP fusion protein was predominantly localized in both the cytoplasm and the nucleus. Overexpression of TaSK41 in transgenic rice led to a reduction in thousand-grain weight, accompanied by significant decreases in both grain length and width. Through yeast two-hybrid screening, 17 candidate proteins were identified as potential interactors with TaSK41. Among these, full-length interaction validation was conducted for TaARF4and TaBSK3, both of which are associated with grain development. Point-to-point assays confirmed that TaSK41 interacts with the full-length TaARF4 protein. This interaction was further validated in vivo using a luciferase complementation assay, confirming that TaSK41 physically interacts with TaARF4 in plant cells. These findings provide a theoretical basis for future investigations into the molecular mechanisms by which TaSK41 regulates grain weight formation in wheat.

Key words: wheat, grain weight, TaSK41, yeast two-hybrid, interacting proteins

表1

引物名称及序列"

引物名称
Primer name		 引物序列
Primer sequence (5'-3')		 用途
Usage
TaSK41-OE-1A-Forward TCCTCTAGAGTCGACCTGCAGATGGCGTATTCTGGGCAGAG 水稻过表达
Over-expression in rice
TaSK41-OE-1A-Reverse AGAGCCCTGGCATGCCTGCAGCTAGGTGCGGAGCGCCATGA
TaSK41-GFP-Forward CACGGGGGACGAGCTCGGTACCATGGCGTATTCTGGGCAGAG 亚细胞定位
Subcellular localization
TaSK41-GFP-Reverse TGCTCACCATGTCGACTCTAGAGGTGCGGAGCGCCATGAACA
qRT-TaSK41-Forward ACCCACATACACACCAGCTT 实时荧光定量PCR
qRT-PCR
qRT-TaSK41-Reverse CAACTCCACTTTCCCCAGGA
TaGAPDH-Forward GACCCAGACAACTCGCAAC
TaGAPDH-Reverse GGAATCCATGACCACCTAC
BD-TaSK41-Forward ATGGCCATGGAGGCCGAATTCATGGCGTATTCTGGGCAGAGGC 酵母双杂交
Yeast two-hybrid assay
BD-TaSK41-Reverse CCGCTGCAGGTCGACGGATCCCTAGGTGCGGAGCGCCATGA
AD-TaARF4-Forward GTACCAGATTACGCTCATATGATGCCGCCCGCCGCCATGG
AD-TaARF4-Reverse ATGCCCACCCGGGTGGAATTCCTAGTTATCTAAATGGCTTG
AD-TaBSK3-Forward GTACCAGATTACGCTCATATGATGGGCGCCAGGGTGTCCAA
AD-TaBSK3-Reverse ATGCCCACCCGGGTGGAATTCTCAGCGTCCGCTGCTGCTACT
TaSK41-nLUC-Forward ACGGGGGACGAGCTCGGTACCATGGCGTATTCTGGGCAGAG 双荧光素酶互补
Dual-luciferase reporter
TaSK41-nLUC-Reverse GCCGGGCCCTCTAGAGGATCCGGTGCGGAGCGCCATGAACA
TaARF4-cLUC-Forward TACGCGTCCCGGGGCGGTACCATGCCGCCCGCCGCCATGGC
TaARF4-cLUC-Reverse GCCGGGCCCTCTAGAGGATCCGTTATCTAAATGGCTTGACGT

图1

不同物种SK41蛋白系统进化和多重序列比对 A: 不同物种TaSK41蛋白的进化分析。At: 拟南芥; Os: 水稻; Hv: 大麦; Ta: 小麦。B: TaSK41蛋白序列比对。"

图2

TaSK41-1A亚细胞定位 A: TaSK41-GFP融合蛋白在烟草叶片中的瞬时表达。B: GFP在烟草叶片中的瞬时表达。从左到右分别为GFP绿色荧光蛋白、明场和叠加图。标尺为20 μm。"

图3

TaSK41在不同组织中的表达模式 不同小写字母表示在0.05概率水平差异显著。DAA: 授粉后天数。"

图4

野生型和TaSK41-1A过表达水稻株系籽粒表型分析 A: TaSK41在转基因与野生型水稻中的基因表达水平。B: WT、TaSK41-OE1和TaSK41-OE2的籽粒表型。标尺为1 cm。C: 千粒重; D: 粒长; E: 粒宽。数据为平均值±标准差。**: P < 0.01, t检验。"

图5

诱饵载体pGBKT7-TaSK41自激活检测"

图6

在四缺培养基SD/-Trp/-Leu/-His/-Ade上筛选阳性克隆 A: 共转化pGADT7-cDNA文库质粒和pGBKT7-TaSK41的AH109在SD/-Trp/-Leu/-His/-Ade培养基上生长的克隆。B: 共转化pGADT7-cDNA文库质粒和pGBKT7-TaSK41的AH109在SD/-Trp/-Leu/-His/-Ade/X-α-gal培养基上生长的克隆。"

表2

TaSK41的候选互作蛋白"

序号
Number		 NCBI蛋白登录号
NCBI accession number		 基因注释
Gene annotation
1 XP_044359233.1 SCREAM2转录因子 Transcription factor SCREAM2
2 XP_044369315.1 葡聚糖内切β-1,3-葡聚糖酶前体 Glucanendo-1,3-beta-glucosidase precursor
3 XP_044399719.1 谷氨酰tRNA还原酶Glutamyl-tRNA reductase1,chloroplastic
4 XP_044325399.1 ARF4转录因子transcription factor ARF4
SH3结构域结合蛋白2 SH3 domain-containing protein 2
5 XP_044436880.1
6 XP_044376166.1 B3结构域蛋白B3 DNA binding domain containing protein
7 XP_044369954.1 SNF1相关蛋白激酶β-1亚基SNF1-related protein kinase regulatory subunit beta-1
8 XP_044459833.1 丝氨酸/苏氨酸蛋白激酶BSK3 Serine/threonine-protein kinase BSK3
9 XP_044408408.1 泛素融合降解蛋白UFD1 Ubiquitin fusion degradation protein UFD1
10 XP_044439125.1 E3泛素连接酶WAV3 E3 ubiquitin-protein ligase WAV3
11 XP_044347787.1 NADH-细胞色素b5还原酶NADH-cytochrome b5 reductase-like protein
12 XP_044349897.1 富含脯氨酸的延伸蛋白样受体激酶PERK9 Proline-rich receptor-like protein kinase PERK9
13 XP_044387058.1 MYB转录因子MYB transcription factor
14 XP_044347705.1 泛素羧基末端水解酶14 Ubiquitin carboxyl-terminal hydrolase 14
15 XP_044396272.1 保守的假设蛋白Os07t0213400-01 Conserved hypothetical protein Os07t0213400-01
16 XP_044403661.1 扩展蛋白B4 Expansin-B4
17 XP_044363920.1 DNA拓扑异构酶DNA topoisomerase

图7

酵母双杂交验证TaSK41与TaARF4和TaBSK3的互作 携带指定质粒的酵母细胞在生长培养基(SD/-Trp/-Leu)和选择培养基(SD/-Trp/-Leu/-His/-Ade/X-α-gal)上的生长情况。"

图8

荧光素酶互补验证TaSK41和TaARF4在体内存在相互作用 TaSK41-nLUC和TaARF4-cLUC共转化的烟草叶片中显示LUC荧光。以共表达TaSK41-nLUC和cLUC或TaARF4-cLUC和nLUC的叶片为阴性对照。"

[1] Cao S H, Xu D A, Hanif M, et al. Genetic architecture underpinning yield component traits in wheat. Theor Appl Genet, 2020, 133: 1811-1823.
doi: 10.1007/s00122-020-03562-8
[2] Alam I, Batool K, Huang Y Y, et al. Developing genetic engineering techniques for control of seed size and yield. Int J Mol Sci, 2022, 23: 13256.
doi: 10.3390/ijms232113256
[3] Youn J H, Kim T W. Functional insights of plant GSK3-like kinases: multi-taskers in diverse cellular signal transduction pathways. Mol Plant, 2015, 8: 552-565.
doi: 10.1016/j.molp.2014.12.006
[4] Mao J, Li W X, Liu J, et al. Versatile physiological functions of plant GSK3-like kinases. Genes (Basel), 2021, 12: 697.
doi: 10.3390/genes12050697
[5] Li C X, Zhang B, Yu H. GSK3s: nodes of multilayer regulation of plant development and stress responses. Trends Plant Sci, 2021, 26: 1286-1300.
doi: 10.1016/j.tplants.2021.07.017 pmid: 34417080
[6] Dornelas M C, Lejeune B, Dron M, et al. The Arabidopsis SHAGGY-related protein kinase (ASK) gene family: structure, organization and evolution. Gene, 1998, 212: 249-257.
doi: 10.1016/s0378-1119(98)00147-4 pmid: 9611268
[7] Groszyk J, Yanushevska Y, Zielezinski A, et al. Annotation and profiling of barley GLYCOGEN SYNTHASE3/Shaggy-like genes indicated shift in organ-preferential expression. PLoS One, 2018, 13: e0199364.
[8] Zhang P P, Zhang L H, Chen T, et al. Genome-wide identification and expression analysis of the GSK gene family in wheat (Triticum aestivum L.). Mol Biol Rep, 2022, 49: 2899-2913.
doi: 10.1007/s11033-021-07105-2
[9] Xia D, Zhou H, Liu R J, et al. GL3.3, a novel QTL encoding a GSK3/SHAGGY-like kinase, epistatically interacts with GS3 to produce extra-long grains in rice. Mol Plant, 2018, 11: 754-756.
[10] Li T T, Lei W, He R Y, et al. Brassinosteroids regulate root meristem development by mediating BIN2-UPB1 module in Arabidopsis. PLoS Genet, 2020, 16: e1008883.
[11] Tong H N, Liu L C, Jin Y, et al. DWARF AND LOW-TILLERING acts as a direct downstream target of a GSK3/SHAGGY-like kinase to mediate brassinosteroid responses in rice. Plant Cell, 2012, 24: 2562-2577.
doi: 10.1105/tpc.112.097394
[12] Gao X Y, Zhang J Q, Zhang X, et al. Rice qGL3/OsPPKL1 functions with the GSK3/SHAGGY-like kinase OsGSK3 to modulate brassinosteroid signaling. Plant Cell, 2019, 31: 1077-1093.
doi: 10.1105/tpc.18.00836
[13] Kloc Y, Dmochowska-Boguta M, Zielezinski A, et al. Silencing of HvGSK1.1—A GSK3/SHAGGY-like kinase-enhances barley (Hordeum vulgare L.) growth in normal and in salt stress conditions. Int J Mol Sci, 2020, 21: 6616.
doi: 10.3390/ijms21186616
[14] Wang Y, Xu J H, Yu J J, et al. Maize GSK3-like kinase ZmSK2 is involved in embryonic development. Plant Sci, 2022, 318: 111221.
doi: 10.1016/j.plantsci.2022.111221
[15] Cheng X J, Xin M M, Xu R B, et al. A single amino acid substitution in STKc_GSK3 kinase conferring semispherical grains and its implications for the origin of Triticum sphaerococcum. Plant Cell, 2020, 32: 923-934.
doi: 10.1105/tpc.19.00580
[16] Hu Z J, Lu S J, Wang M J, et al. A novel QTL qTGW3 encodes the GSK3/SHAGGY-like kinase OsGSK5/OsSK41 that interacts with OsARF4 to negatively regulate grain size and weight in rice. Mol Plant, 2018, 11: 736-749.
doi: 10.1016/j.molp.2018.03.005
[17] Ma M, Shen S Y, Bai C, et al. Control of grain size in rice by TGW3 phosphorylation of OsIAA10 through potentiation of OsIAA10-OsARF4-mediated auxin signaling. Cell Rep, 2023, 42: 112187.
doi: 10.1016/j.celrep.2023.112187
[18] Shen S Y, Ma M, Bai C, et al. Optimizing rice grain size by attenuating phosphorylation-triggered functional impairment of a chromatin modifier ternary complex. Dev Cell, 2024, 59: 448-464.
doi: 10.1016/j.devcel.2023.12.013
[19] 张令慧, 张沛沛, 刘媛, 等. 小麦TaGSK5基因的克隆与表达分析. 甘肃农业大学学报, 2021, 56(6): 64-72.
Zhang L H, Zhang P P, Liu Y, et al. Cloning and expression analysis of TaGSK5 gene in wheat. J Gansu Agric Univ, 2021, 56(6): 64-72.
[20] Tian P, Liu J F, Yan B H, et al. OsBSK3 positively regulates grain length and weight by inhibiting the phosphatase activity of OsPPKL1. Plants, 2022, 11: 1586.
doi: 10.3390/plants11121586
[21] Hu J, Wang Y X, Fang Y X, et al. A rare allele of GS2 enhances grain size and grain yield in rice. Mol Plant, 2015, 8: 1455-1465.
doi: 10.1016/j.molp.2015.07.002
[22] Liu H, Li H F, Hao C Y, et al. TaDA1, a conserved negative regulator of kernel size, has an additive effect with TaGW2 in common wheat (Triticum aestivum L.). Plant Biotechnol J, 2020, 18: 1330-1342.
doi: 10.1111/pbi.v18.5
[23] Wang W, Simmonds J, Pan Q L, et al. Gene editing and mutagenesis reveal inter-cultivar differences and additivity in the contribution of TaGW2 homoeologues to grain size and weight in wheat. Theor Appl Genet, 2018, 131: 2463-2475.
doi: 10.1007/s00122-018-3166-7 pmid: 30136108
[24] Zhang Y, Li D, Zhang D B, et al. Analysis of the functions of TaGW2 homoeologs in wheat grain weight and protein content traits. Plant J, 2018, 94: 857-866.
doi: 10.1111/tpj.2018.94.issue-5
[25] Ishimaru K, Hirotsu N, Madoka Y, et al. Loss of function of the IAA-glucose hydrolase gene TGW6 enhances rice grain weight and increases yield. Nat Genet, 2013, 45: 707-711.
doi: 10.1038/ng.2612 pmid: 23583977
[26] Farooq H, Asghar H N, Khan M Y, et al. Auxin-mediated growth of rice in cadmium-contaminated soil. Turk J Agric For, 2015, 39: 272-276.
doi: 10.3906/tar-1405-54
[27] Tian P, Liu J F, Yan B H, et al. BRASSINOSTEROID- SIGNALING KINASE1-1, a positive regulator of brassinosteroid signalling, modulates plant architecture and grain size in rice. J Exp Bot, 2023, 74: 283-295.
doi: 10.1093/jxb/erac429
[28] Yuan H, Xu Z Y, Chen W L, et al. OsBSK2, a putative brassinosteroid-signalling kinase, positively controls grain size in rice. J Exp Bot, 2022, 73: 5529-5542.
doi: 10.1093/jxb/erac222
[1] 毛嘉琦, 黄朋雨, 赵佳佳, 郑兴卫, 武棒棒, 郝宇琼, 屈非, 刘成, 马朋涛, 郑军. 山西小麦品种白粉病抗性评价及抗病基因分子检测[J]. 作物学报, 2026, 52(6): 1669-1681.
[2] 胡川, 赵凯男, 黄修利, 吴金芝, 任开明, 王贺正, 付国占, 黄明, 李友军. 一次灌溉下耕作方式和氮肥用量对旱地小麦产量和品质的影响[J]. 作物学报, 2026, 52(6): 1830-1846.
[3] 陈雪燕, 何华川, 李政嘉, 董新盼, 李藕琪, 刘小云, 李丹萍, 陈志伟, 刘国霞, 吕胜源, 吴印莹, 赵振东, 曹新有, 万何平. 水培盐碱复合胁迫下‘济麦60’苗期根系有机酸分泌动态变化及其转录调控机制[J]. 作物学报, 2026, 52(6): 1859-1875.
[4] 左同鸿, 张贺翠, 曾静, 朱利泉. 甘蓝自交不亲和相关基因BoPUB3L的克隆与表达分析[J]. 作物学报, 2026, 52(6): 1698-1710.
[5] 高沛阳, 李瑾璇, 董宇奎, 石玉, 张振, 张永丽. 测墒补灌下小麦分蘖发生和成穗对施氮量的响应[J]. 作物学报, 2026, 52(6): 1847-1858.
[6] 张献丰, 郭利建, 李康春, 孔斌雪, 刘玉芳, 车卓, 杨德龙. 小麦ABHD6基因家族鉴定与粒重功能标记开发[J]. 作物学报, 2026, 52(6): 1711-1727.
[7] 翟胜男, 曹新有, 李豪圣, 李吉虎, 李法计, 刘金栋, 夏先春, 吕莹莹, 马瑞峰, 王颖, 耿洪伟, 刘建军. 小麦Pod-A1、Pod-D1和Pod-2D位点等位变异对籽粒过氧化物酶活性的遗传效应分析[J]. 作物学报, 2026, 52(6): 1593-1603.
[8] 习千辉, 徐梓瑗, 刘梦梦, 王宏艺, 郎凯琳, 井震海, 陈锋, 赵磊. 小麦籽粒铜含量的全基因组关联分析及候选基因预测[J]. 作物学报, 2026, 52(6): 1604-1617.
[9] 王壮壮, 武紫君, 张永新, 张芯源, 袁丽雪, 陈如雪, 刘世举, 段剑钊, 冯伟, 王同朝, 王永华. 豫东南黏壤潮土区水氮优化协同提高冬小麦产量和氮素利用效率[J]. 作物学报, 2026, 52(5): 1501-1521.
[10] 何万龙, 耿洪伟, 张飞飞, 米克热阿依·阿巴白克热, 罗紫洋, 李鹏程, 周钊宇, 程宇坤. 基于深度学习的小麦重要病害图像识别系统的研究[J]. 作物学报, 2026, 52(5): 1401-1417.
[11] 张振, 冯连杰, 石玉, 于振文, 张永丽. 节水补灌下不同穗型小麦产量形成差异研究[J]. 作物学报, 2026, 52(5): 1522-1535.
[12] 侯思宇, 王国璀, 韦金贵, 谢玮欣, 殷文, 樊志龙, 柴强, 胡发龙. 绿肥配施化学氮肥对西北干旱灌区小麦干物质积累及产量形成的影响[J]. 作物学报, 2026, 52(4): 1208-1219.
[13] 尚云秋, 赵竹, 陈欢, 丁永刚, 乔玉强, 李玮, 张向前, 曹承富, 杜世州. 长期定位耕作方式对雨养小麦籽粒灌浆和产量形成的影响[J]. 作物学报, 2026, 52(4): 1236-1250.
[14] 乔宇馨, 李程越, 康晓玉, 张鑫琪, 贾绍辉, 刘倩, 曹亚丽, 史鑫蕊, 郝兴宇, 李萍. 基于APSIM模型的长期免耕秸秆覆盖对旱地小麦增产效应研究[J]. 作物学报, 2026, 52(4): 1181-1192.
[15] 侯洁, 付朵朵, 武海峰, 郝宇琼, 郑兴卫, 武棒棒, 周凯, 李晓华, 郑军, 赵佳佳. 山西省小麦地方品种的染色体多样性及遗传效应分析[J]. 作物学报, 2026, 52(3): 746-763.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备15008789号-2