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作物学报 ›› 2025, Vol. 51 ›› Issue (6): 1690-1700.doi: 10.3724/SP.J.1006.2025.42045

• 研究简报 • 上一篇    

水稻OsPUB4基因克隆、激素诱导表达分析与互作蛋白筛选

李福媛1(), 杨奕2(), 马继琼2, 许明辉2, 林良斌1,*(), 孙一丁2,*()   

  1. 1云南农业大学农学与生物技术学院, 云南昆明 650201
    2云南省农业科学院生物技术与种质资源研究所/云南省农业生物技术重点实验室/农业农村部西南作物基因资源与种质创制重点实验室 / 农业农村部云南稻种资源科学观测实验站, 云南昆明 650205
  • 收稿日期:2024-10-11 接受日期:2025-03-26 出版日期:2025-06-12 网络出版日期:2025-04-01
  • 通讯作者: *孙一丁, E-mail: yidingsun18@163.com;林良斌, E-mail: Linliangbin-63@163.com
  • 作者简介:李福媛, E-mail: 1642545302@qq.com;
    杨奕, E-mail: xiaoyang0326@126.com第一联系人：**同等贡献
  • 基金资助:
    本研究由国家自然科学基金项目(32260523);云南省科技计划项目(202101BD070001-002);云南省科技计划项目(202301BD070001-218);云南省科技计划项目(202405AC350096)

Cloning, hormone-induced expression analysis, and interaction protein screening of OsPUB4 in rice

LI Fu-Yuan1(), YANG Yi2(), MA Ji-Qiong2, XU Ming-Hui2, LIN Liang-Bin1,*(), SUN Yi-Ding2,*()   

  1. 1College of Agronomy and Biotechnology, Yunnan Agricultural University, Kunming 650201, Yunnan, China
    2Institute of Biotechnology and Germplasm Resources, Yunnan Academy of Agricultural Sciences / Yunnan Provincial Key Laboratory of Agricultural Biotechnology / Southwest Key Laboratory of Crop Gene Resources and Germplasm Creation, Ministry of Agriculture and Rural Affairs / Scientific Observation for Rice Germplasm Resources of Yunnan, Ministry of Agriculture and Rural Affairs, Kunming 650205, Yunnan, China
  • Received:2024-10-11 Accepted:2025-03-26 Published:2025-06-12 Published online:2025-04-01
  • Contact: *E-mail: yidingsun18@163.com;E-mail: Linliangbin-63@163.com
  • About author:First author contact:**Contributed equally to this work
  • Supported by:
    the National Natural Science Foundation of China(32260523);the Science and Technology Program of Yunnan Province(202101BD070001-002);the Science and Technology Program of Yunnan Province(202301BD070001-218);the Science and Technology Program of Yunnan Province(202405AC350096)

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摘要： 为探究E3泛素连接酶OsPUB4的功能, 阐明OsPUB4介导的调控机制, 以日本晴为材料, 克隆水稻U-Box型E3泛素连接酶基因OsPUB4, 且通过生物信息学在线网站对启动子顺式作用元件和编码区序列特征进行预测, 并构建系统发育树, 采用实时荧光定量PCR技术探究该基因在不同植物激素诱导下的表达特征, 同时利用酵母cDNA文库对OsPUB4的互作蛋白进行筛选。结果表明: (1) 水稻OsPUB4基因启动子区含多个与激素、光、温度等相关的响应元件, 编码区全长2187 bp, 无信号肽, 含1个跨膜结构域和62个磷酸化位点; (2) OsPUB4与乌拉尔图小麦PUB4蛋白的亲缘关系较近; (3) 通过外源激素诱导发现, 短时间内JA会抑制水稻叶片中OsPUB4基因的表达, 而IAA则相反; (4) OsPUB4蛋白与OsTPS5、Di19和THIC等存在相互作用关系。综上, OsPUB4基因受外源激素诱导表达, 且与多个胁迫响应相关蛋白存在互作关系, 为深入研究OsPUB4在水稻胁迫响应方面的功能提供理论依据。

关键词: 水稻, E3泛素连接酶, OsPUB4, 激素诱导表达, 互作蛋白

Abstract:

To investigate the function of the E3 ubiquitin ligase OsPUB4 and elucidate its regulatory mechanism, we cloned the coding sequence of OsPUB4, a U-box-type E3 ubiquitin ligase gene, from the rice cultivar “Nipponbare”. The cis-acting elements of the promoter and the sequence characteristics of the coding region were predicted using bioinformatics tools, and a phylogenetic tree was constructed. The expression patterns of OsPUB4 under different plant hormone treatments were analyzed by quantitative real-time PCR (qRT-PCR). Additionally, interaction proteins of OsPUB4 were identified using a yeast cDNA library screening approach. Our findings revealed that: (1) The promoter region of OsPUB4 contains multiple response elements associated with hormones, light, and temperature. The coding region is 2187 bp in length, lacks a signal peptide, and contains a transmembrane domain and 62 phosphorylation sites. (2) OsPUB4 is closely related to the PUB4 protein of Triticum Urartu. (3) Exogenous jasmonic acid (JA) treatment rapidly suppressed OsPUB4 expression in rice leaves, whereas indole-3-acetic acid (IAA) had the opposite effect. (4) OsPUB4 interacts with OsTPS5, Di19, and THIC. These results suggest that OsPUB4 is induced by exogenous hormones and interacts with multiple stress response-related proteins, providing a theoretical foundation for further investigations into the role of OsPUB4 in rice stress responses.

Key words: rice, E3 ubiquitin ligase, OsPUB4, inducible expression characteristics, interacting proteins

表1

本研究所用的引物序列"

引物
Primer		 序列
Sequence (5′-3′)
OsPUB4-F ATGGCGGCGGCGGCGTCGGC
OsPUB4-R CTAGGGCACCTGCACTGATA
QRT-Pub4-F CTACTGCCTATTCGCCGTGT
QRT-Pub4-R GAGAACTCCTTTGGCACCGA
OsActin1-F GCATCTCTCAGCACATTCCA
OsActin1-R ACCACAGGTAGCAATAGGTA
OsPUB4-BD-F TCTCAGAGGAGGACCTGCATATGTCGCT
OsPUB4-BD-R CGCCGCCTCGCTGGT
T7 GCGGCCGCTGCAGGTCGACGGATCCGGG
3BD CACCTGCACTGATATCG
TAATACGACTCACTATAGGGC
TTTTCGTTTTAAAACCTAAGAGTC

图1

水稻OsPUB4基因PCR扩增"

表2

OsPUB4基因启动子区主要顺式作用元件"

作用元件
Functional element		 核心系列
Core sequence		 位置
Location		 数量
Quantity		 功能
Function
ABRE ACGTG
CACGTG		 586(+)1149(-)696(+)1850(-)969(-)
695(+)		 5
1		 脱落酸响应元件
Cis-acting element involved in the abscisic acid responsiveness
ARE AAACCA 80(+)306(-) 2 厌氧诱导响应元件
Cis-acting regulatory element essential for the anaerobic induction
Box4 ATTAAT 411(+)1745(-) 2 参与光响应的保守DNA模块的一部分
Part of a conserved DNA module involved in light responsiveness
CAAT-box CAAAT
CCAAT
CAAAT		 131(+)
309(+)244(+)
264(+)320(-)369(-)		 4
2
3		 启动子和增强子区域中常见的顺式作用元件
Common cis-acting element in promoter and enhancer regions
CGTCA-motif CGTCA 843(-)861(-)846(-) 3 参与MeJA响应顺式作用调节元件
Cis-acting regulatory element involved in the MeJA-responsiveness
G-Box CACGTT
CACGAC
TACGTG
CAGACGTGGCA
CACGTG
CACGTC		 585(-)969(+)
684(+)
1850(-)
1146(-)
695(+)
1149(+)		 2
1
1
1
1
1		 光响应元件
Cis-acting regulatory element involved in light responsiveness
GA-motif ATAGATAA 547(+) 1 光响应元件一部分
Part of a light responsive element
GT1-motif GGTTAAT 420(+) 1 光响应元件
Light responsive element
LTR CCGAAA 826(-) 1 低温响应性的顺式作用元件
Cis-acting element involved in low-temperature responsiveness
MRE AACCTAA 1811(-) 1 光响应的MYB结合位点
MYB binding site involved in light
responsiveness
RY-element CATGCATG 278(+) 1 种子特异性调控的顺式作用调控元件Cis-acting regulatory element involved in seed-specific regulation
TATA-box TACAAAA 13(+)22(+)810(+)945(-) 4 -30 左右的核心启动子元件
Core promoter element around -30 of
transcription start
TCA-element CCATCTTTTT 1426(-) 1 水杨酸响应的顺式作用元件
Cis-acting element involved in salicylic acid responsiveness
TGA-element AACGAC 1140(-) 1 生长素响应元件
Auxin-responsive element
TGACG-motif TGACG 843(+)846(-)861(-) 3 参与MeJA响应顺式作用调节元件
Cis-acting regulatory element involved in the MeJA-responsiveness

图2

OsPUB4的生物信息学分析 A: 二级结构预测; B: 跨膜结构预测; C: 信号肽预测; D: 亲/疏水性预测; E: 磷酸化位点预测; F: 蛋白结构域预测。"

图3

OsPUB4与8个蛋白的进化树分析"

图4

不同激素处理后OsPUB4基因在水稻叶片(L)和根部(R)的表达水平分析 不同小写字母表示在0.05水平差异显著。IAA: 生长素; GA: 赤霉素; JA: 茉莉酸; SA: 水杨酸。"

图5

OsPUB4互作蛋白的筛选 A: PGBKT7-PUB4自激活检测(从左到右SD/-Leu/-Trp板中所添加的AbA浓度分别为0、100、200 ng mL-1, 当浓度为200 ng mL-1时酵母菌斑不再生长, 此浓度为自激活最佳抑制浓度); B: 诱饵载体毒性检测; C: 酵母菌株纯化培养。"

表3

OsPUB4互作蛋白在NCBI中的比对结果"

序号
No.		 名称
Name		 分子功能
Molecular function
6 Cell number regulator 未表征的富含Cys的结构域蛋白质
Uncharacterized Cys-rich domain containing protein
12 F-box protein SKIP22 含F-box及其他结构域蛋白
OsFBO13-F-box and other domain containing protein
13 Protein GID8 homolog 表达蛋白 Expressed protein
14 Uncharacterized protein LOC4346441 isoform X1 表达蛋白 Expressed protein
17 Trehalose-6-phosphate synthase 海藻糖-6-磷酸合酶活性 Trehalose-6-phosphate synthase
22 Isovaleryl-CoA dehydrogenase —
23 Aldehyde oxidase 3 醛氧化酶活性 Aldehyde oxidase
26 Protein early responsive to dehydration 15 表达蛋白 Expressed protein
34 6-phosphogluconate dehydrogenase 6-磷酸葡萄糖酸脱氢酶(脱羧)活性 6-phosphogluconate dehydrogenase
39 PHD-finger protein OsTITANIA PHD指蛋白; DNA转录因子活性
PHD-finger protein; transcription factor
40 NADH dehydrogenase 含有LYR基序的蛋白质; 催化活性
LYR motif containing protein; catalytic activity
42 Pyruvate dehydrogenase E1 component subunit alpha-1 含有脱氢酶E1组分结构域的蛋白质
Dehydrogenase E1 domain-containing protein
46 Uncharacterized protein LOC4337571 isoform X1 —
50 ADP-ribosylation factor GTPase-activating protein AGD12 生长素内向转运载体活性 Auxin inward transport carrier activity
59 Expressed 2-oxoisovalerate dehydrogenase subunit alpha 2 含有脱氢酶E1组分结构域的蛋白质
Dehydrogenase E1 domain-containing protein
68 Protein dehydration-induced 19 DNA转录因子活性; 蛋白同源二聚化活性; 蛋白异源二聚化活性;
转录激活因子结合
DNA transcription factor activity; protein homodimerization activity; protein heterodimerization activity; activator of transcription binding
74 Nucleoside diphosphate kinase 3 核苷二磷酸激酶 Nucleoside diphosphate kinase
76 UDP-arabinopyranose mutase 1 (OsUAM1; OsRGP1) UDP-阿拉伯糖变位酶活性 UDP-arabinose mutase activity
80 Homeobox protein knotted-1-like 8 表征蛋白 Characterize proteins
81 Xylanase inhibitor protein 1-like 水解酶活性 Hydrolase activity
83 Phosphomethylpyrimidine synthase 硫胺素生物合成蛋白 THIC Thiamine biosynthetic protein THIC

图6

OsPUB4-BD互作筛选回转验证结果"

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