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作物学报 ›› 2025, Vol. 51 ›› Issue (4): 900-913.doi: 10.3724/SP.J.1006.2025.42040

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

水稻转录因子OsERF104的克隆和功能研究

潘炬忠1(), 韦萍1, 朱德平1, 邵胜雪1, 陈珊珊1, 韦雅倩1, 高维维1,2,3,*()   

  1. 1广西大学农学院, 广西南宁 530004
    2广西大学亚热带农业生物资源保护与利用国家重点实验室, 广西南宁 530004
    3广西农业环境与农产品安全重点实验室, 广西南宁 530004
  • 收稿日期:2024-08-26 接受日期:2025-01-23 出版日期:2025-04-12 网络出版日期:2025-02-07
  • 通讯作者: 高维维, E-mail: gaoweiwei@gxu.edu.cn
  • 作者简介:E-mail: 1728428229@qq.com
  • 基金资助:
    广西自然科学基金青年科学基金项目(2024GXNSFBA010324);广西自然科学基金青年科学基金项目(2025GXNSFAA069066);国家自然科学基金青年科学基金项目(32301749)

Cloning and functional analysis of OsERF104 transcription factor in rice

PAN Ju-Zhong1(), WEI Ping1, ZHU De-Ping1, SHAO Sheng-Xue1, CHEN Shan-Shan1, WEI Ya-Qian1, GAO Wei-Wei1,2,3,*()   

  1. 1College of Agriculture, Guangxi University, Nanning 530004, Guangxi, China
    2State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi University, Nanning 530004, Guangxi, China
    3Guangxi Key Laboratory of Agro-environment and Agric-products safety, Nanning 530004, Guangxi, China
  • Received:2024-08-26 Accepted:2025-01-23 Published:2025-04-12 Published online:2025-02-07
  • Contact: E-mail: gaoweiwei@gxu.edu.cn
  • Supported by:
    Young Scientists Fund of Guangxi Natural Science Foundation(2024GXNSFBA010324);Young Scientists Fund of Guangxi Natural Science Foundation(2025GXNSFAA069066);Young Scientists Fund of the National Natural Science Foundation of China(32301749)

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摘要：

乙烯响应因子(ERF)是植物中AP2/ERF转录因子超家族中的成员之一, 在植物的生长发育、响应逆境胁迫和调节激素信号转导等生物学过程中起重要调控作用。探究水稻ERF家族基因功能将为水稻育种提供重要的基因资源。本研究克隆了OsERF104 (LOC_Os08g36920)基因, 利用生物信息学分析发现该基因编码序列全长849 bp, 编码283个氨基酸。OsERF104蛋白含有AP2/ERF转录因子家族特有的AP2保守结构域, 与拟南芥中调控盐胁迫耐受性的AtERF96的蛋白序列相似性最高。亚细胞定位结果显示OsERF104定位于细胞核, 表明OsERF104是典型的核转录因子。OsERF104基因启动子顺式作用元件分析表明, 启动子含有与激素响应、逆境胁迫及光响应相关的顺式作用元件。为了探究OsERF104对非生物胁迫的响应, 利用实时荧光定量PCR (RT-qPCR)技术分析OsERF104的表达模式, 发现OsERF104在水稻不同组织中均有表达, 在叶鞘中的表达量最高, 且该基因的表达受ABA和GA抑制, 受JA、PEG和NaCl诱导。转录活性分析显示该基因全长和C端具有转录自激活活性, N端和AP2结构域不具有转录自激活活性。通过遗传转化获得OsERF104过表达和敲除的突变体转基因水稻。表型分析显示, 与野生型ZH11相比, OsERF104过表达植株对ABA的敏感性及苗期的耐盐性显著增加, oserf104突变体则相反。由此推测, OsERF104正调控水稻对盐胁迫的耐受性。本研究为进一步解析OsERF104的生物学功能及分子机制奠定了良好的基础。

关键词: 水稻, OsERF104, 转录因子, 盐胁迫, 表达分析, 亚细胞定位

Abstract:

Ethylene Responsive Factor (ERF), a subfamily of the APETALA2/Ethylene Responsive Factor (AP2/ERF) family, plays critical roles in regulating diverse biological processes, including plant growth and development, hormone signaling, and responses to abiotic stresses. Investigating the functions of the ERF family in rice (Oryza sativa L.) provides valuable genetic resources for rice breeding. In this study, the OsERF104 gene (LOC_Os08g36920) was cloned. Bioinformatic analysis revealed that the full-length coding sequence of OsERF104 is 849 bp, encoding a protein of 283 amino acids. OsERF104 contains a conserved domain characteristic of the AP2/ERF family and shares the highest sequence similarity with the AtERF96 protein in Arabidopsis thaliana, which is known to be involved in salt tolerance. Subcellular localization analysis confirmed that the OsERF104 protein is localized in the nucleus, indicating that it functions as a nuclear transcription factor. Cis-acting element analysis of the OsERF104 promoter identified elements associated with hormone responses, abiotic stress, and light responses. To examine the role of OsERF104 under abiotic stress, its expression pattern was analyzed using RT-qPCR. OsERF104 was expressed in various rice tissues, with the highest expression observed in the leaf sheath. Its expression was downregulated by ABA and GA but upregulated by JA, PEG, and NaCl treatments. Transcriptional activation assays showed that the full-length and C-terminal fragments of OsERF104 exhibit transcriptional activity, while the N-terminal fragment and the AP2 domain alone do not. Transgenic rice lines of overexpressing or knocking out OsERF104 were generated via genetic transformation. Phenotypic analysis demonstrated that OsERF104-overexpressing rice exhibited enhanced sensitivity to ABA and increased tolerance to salt stress during the seedling stage compared with the wild-type ZH11. In contrast, oserf104 mutant rice displayed the opposite phenotypes. In conclusion, OsERF104 positively regulates salt tolerance in rice. This study provides a strong foundation for further exploration of the biological functions and molecular mechanisms of OsERF104 in rice.

Key words: rice, OsERF104, transcription factor, salt stress, expression analysis, subcellular localization

表1

本研究所用引物及序列"

引物名称
Primer name		 正向引物
Forward sequence (5'-3')		 反向引物
Reverse sequence (5'-3')
OsERF104-GFP ACTAGTGGATCCGGTACCATGACCAACCGGATCTCC CTTGCTCACCATGGTACCATTCCAAGAATCTGACGACTG
OsERF104-ox TCTCTCTCAAGCTTGGATCCATGACCAACCGGATCTCC ATACCGTCACTAGTGGATCCATTCCAAGAATCTGACGACTG
BD-OsERF104 GCCATGGAGGCCGAATTCATGACCAACCGGATCTCC CCGCTGCAGGTCGACGGATCCCATTCCAAGAATCTGACGACTG
BD-OsERF104-N GCCATGGAGGCCGAATTCATGACCAACCGGATCTCC CCGCTGCAGGTCGACGGATCCCGTTCTTCTTCTTCCTCCTCCT
BD-OsERF104-C GCCATGGAGGCCGAATTCATGAAGTACCGCGGCGT CCGCTGCAGGTCGACGGATCCCATTCCAAGAATCTGACGACTG
BD-OsERF104-AP2 GCCATGGAGGCCGAATTCATGAAGTACCGCGGCGT CCGCTGCAGGTCGACGGATCCCCGGGCCGCGGAACTCG
OsERF104-q CGCACGACGACAGCAATG GGCTGGGATCTAATCACAACG
e-EF-1α GCACGCTCTTCTTGCTTTC AGGGAATCTTGTCAGGGTTG

图1

OsERF104蛋白的生物信息分析 A: 亲/疏水性预测。B: 跨膜结构域预测。C: 蛋白结构示意图。D: 磷酸化位点预测。"

图2

水稻和拟南芥ERF家族基因蛋白保守基序和结构域分析 A: 蛋白保守基序分析, 不同颜色方框表示不同保守基序, 黑线表示蛋白质长度; B: 蛋白结构域分析, 不同颜色方框表示不同结构。"

表2

OsERF04启动子顺式元件"

顺式作用元件
Cis-acting element		 序列
Sequence (5′-3′)		 功能
Function		 数量
Number
G-box ACACGTGGC 参与光响应的顺式调节元件
Cis-acting regulatory element involved in light responsiveness		 9
ATCT-motif AATCTAATCC 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		 10
GT1-motif GGTTAA 拟南芥光响应元件
Arabidopsis thaliana light responsive element		 6
TATA-box TACAAAA 转录起始位点-30附件的核心启动子元件
Core promoter element around -30 of transcription start		 7
CGTCA-motif CGTCA JA响应元件
Cis-acting regulatory element involved in the MeJA-responsiveness		 5
TCA-element CCATCTTTTT SA响应元件
Cis-acting element involved in salicylic acid responsiveness		 9
TGACG-motif TGACG JA响应元件
Cis-acting regulatory element involved in the MeJA-responsiveness		 5
MBS CAACTG 干旱响应元件
Arabidopsis thaliana MYB binding site involved in drought-inducibility		 6
ABRE GACACGTGGC ABA响应元件
Cis-acting element involved in the abscisic acid responsiveness		 9
P-box CCTTTTG GA响应元件
Oryza sativa gibberellin-responsive element		 7
DRE TACCGACAT 缺水、低温、盐响应元件
Cis-acting element involved in dehydration, low-temp, and salt stresses		 9
TGA-element AACGAC 生长素响应元件
Auxin-responsive element		 6
LTR CCGAAA 参与低温响应的顺式作用元件
Cis-acting element involved in low-temperature responsiveness		 6
CAAT-box CAAAT 启动子和增强子区域的通用的顺式作用元件
Common cis-acting element in promoter and enhancer regions		 5

图3

OsERF104亚细胞定位 A: 水稻原生质体中OsERF104的亚细胞定位。标尺为3 μm。B: 烟草中OsERF104的亚细胞定位。标尺为10 μm。"

图4

OsERF104转录活性分析"

图5

OsERF104的组织表达模式 A~B: eFP数据库中OsERF104在水稻不同组织中的表达模式。颜色深浅代表表达量的高低。C: qRT-PCR分析OsERF104在水稻不同组织中的相对表达量。水稻e-EF-1α为内参基因, 数值代表3次生物学重复的平均值±标准差。"

图6

外源激素和胁迫处理对OsERF104表达的影响 A: 50 mmol L-1 ABA处理下OsERF104的相对表达量。B: 10 μmol L-1 JA处理下OsERF104的相对表达量。C: 10 μmol L-1 GA处理下OsERF104的相对表达量。D: 20% PEG-4000处理下OsERF104的相对表达量。E: 150 mmol L-1盐处理下OsERF104的相对表达量, Re表示复水。水稻e-EF-1α为内参基因。数值代表平均值±标准差。CK表示正常条件下OsERF104的表达量。"

图7

OsERF104的基因结构和DNA扩增条带 A: OsERF104基因结构示意图。B: OsERF104的扩增和条带。"

图8

OsERF104参与调控水稻对盐胁迫的耐受性 A: qRT-RCR分析OsERF104-ox转基因水稻中OsERF104的表达量。水稻e-EF-1α为内参基因。B: 利用CRISPR/Cas9系统编辑OsERF104基因并产生靶标突变株系。黑色填充框代表OsERF104外显子。C~D: 氯化钠处理下OsERF104过表达转基因水稻的表型图及存活率统计。标尺为6 cm。E~F: 氯化钠处理下oserf104突变体水稻的表型图及存活率统计。标尺为6 cm。试验重复3次, 每次重复结果相似, 每次重复每个株系统计16~24个单株。G~H: 5 μmol L-1 ABA和水处理7 d后OsERF104转基因水稻萌发的表型图。标尺为0.5 cm。I~J: 5 μmol L-1 ABA和水处理7 d后OsERF104转基因水稻地上部和地下部长度统计。试验重复3次, 每次重复每个株系统计30~40粒种子。数值代表平均值±标准差。"

图9

盐胁迫下OsERF104转基因水稻失水率及活性氧清除酶关键基因的表达 A: OsERF104转基因水稻的失水率统计。B: OsERF104转基因水稻中OsCatA、OsCatB和OsCatC的表达。水稻e-EF-1α为内参基因, 数值代表3次生物学重复的平均值±标准差。"

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