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Acta Agronomica Sinica ›› 2025, Vol. 51 ›› Issue (4): 900-913.doi: 10.3724/SP.J.1006.2025.42040

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

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 Online:2025-04-12 Published: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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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

Table1

Primers and sequences in this study"

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

Fig. 1

Bioinformatics analysis of OsERF104 protein A: prediction of hydrophilic/hydrophobic. B: prediction of transmembrane domains. C: schematic diagram of protein. D: prediction of phosphorylation site."

Fig. 2

Analysis of conserved motifs and domains of ERF family genes in rice and Arabidopsis A: analysis of conserved protein motifs, different colored boxes represent different conserved motifs, and the black line indicates protein length; B: protein domain analysis, different colored boxes represent different domains."

Table 2

The cis-acting elements of OsERF104 promotor"

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

Fig. 3

Subcellular localization of OsERF104 protein A: subcellular localization of OsERF104 protein in rice protoplasts. Scale bar: 3 μm. B: subcellular localization of OsERF104 protein in Nicotona benthamiana. Scale bar: 10 μm."

Fig. 4

Transcriptional activity assay of OsERF104"

Fig. 5

Expression patterns of OsERF104 A-B: expression patterns of OsERF104 in various organs of rice. Data are from the Rice eFP Browser. The shade of color represents the level of expression. C: expression patterns of OsERF104 in various organs of ZH11 by qRT-PCR. e-EF-1α was used as the reference gene. Values represent means ± SD of three biological replicates."

Fig. 6

Expression level of OsERF104 under hormone and stress treatment A: expression of OsERF104 under 50 mmol L-1 ABA treatment. B: expression of OsERF104 under 10 μmol L-1 JA treatment. C: expression of OsERF104 under 10 μmol L-1 GA treatment. D: expression of OsERF104 under 20% PEG-4000 treatment. E: expression of OsERF104 under 150 mmol L-1 NaCl treatment, Re: recovery. e-EF-1α was used as the reference gene. Values represent means ± SD of three biological replicates. CK represents the expression of OsERF104 under normal conditions."

Fig. 7

Nucleotide structure and DNA amplification bands of OsERF104 A: OsERF104 nucleotide structure. B: amplification and banding of OsERF104."

Fig. 8

OsERF104 mediate salt tolerance of rice A: expression levels of OsERF104 in OsERF104-ox lines. e-EF-1α was used as the reference gene. B: generation of target site mutations in representative knockout lines of OsERF104 rice plants using the CRISPR/Cas9 system. Filled black bars indicate exons of the OsERF104 gene. C-D: phenotypes and survival rates of OsERF104-ox transgenic rice seedlings under salt treatment. Scale bar is 6 cm. E-F: phenotypes and survival rates of oserf104 mutant transgenic rice seedlings under salt treatment. Scale bar is 6 cm. The experiments were performed in three replicates with similar results, each repeat was measured in 16-24 independent plants. G-H: germination phenotypes of OsERF104 transgenic rice after seven days under 5 μmol L-1 ABA and distilled water treatment. Scale bar is 0.5 cm. I-J: the length of shoot and root of OsERF104 transgenic rice after seven days under 5 μmol L-1 ABA and distilled water treatment. The experiments were performed in three replicates, each replicate containing 30-40 seeds. The values represent means ± SD of three biological replicates."

Fig. 9

Water loss rate of OsERF104 transgenic rice under salt treatment and the expression of key genes of ROS scavenging A: water loss rate of OsERF104 transgenic rice. B: expression of OsCatA, OsCatB and OsCatC in OsERF104 transgenic rice. e-EF-1α was used as the reference gene. Values represent means ± SD of three biological replicates."

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