Scientia Agricultura Sinica ›› 2025, Vol. 58 ›› Issue (8): 1463-1478.doi: 10.3864/j.issn.0578-1752.2025.08.001

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

The Function of OsDREB1J in Regulating Rice Grain Size

WEI Ping1(), PAN JuZhong1, ZHU DePing1, SHAO ShengXue1, CHEN ShanShan1, WEI YaQian1, GAO WeiWei1,2()   

  1. 1 College of Agriculture, Guangxi University, Nanning 530004
    2 State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University/Guangxi Key Laboratory of Agro-Environment and Agric-Products Safety, Nanning 530004
  • Received:2024-08-28 Accepted:2024-10-17 Online:2025-04-21 Published:2025-04-21
  • Contact: GAO WeiWei

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Abstract:

【Objective】 The AP2/ERF (APETALA2/ethylene responsive factor) superfamily is a group of transcription factors that play important regulatory roles in plant growth and development, as well as in response to adverse environmental stressors. The AP2/ERF transcription factors are widely present and have many members in plants. Exploring the function of AP2/ERF family gene on grain size provides important genetic resources for regulating grain shape in rice. 【Method】OsDREB1J gene (LOC_Os08g43200) was cloned by homologous recombination, and its basic characteristics, tissue expression characteristics, and the relative expression patterns under plant hormones were analyzed by bioinformatics and qRT-PCR. The transactivation activity and subcellular localization of OsDREB1J were analyzed by yeast heterologous expression, transient expression of rice protoplasts and tobacco. The overexpression and knockout mutant transgenic rice plants of OsDREB1J were obtained by genetic transformation system, and the grain size phenotypes were analyzed by phenotypic analysis technology. 【Result】Subcellular localization analysis showed that OsDREB1J was localized in the nucleus. Bioinformatics showed that the full-length coding sequence of OsDREB1J was 711 bp, encoding 236 amino acids. OsDREB1J protein had no transmembrane structure, and the molecular weight of 27.47 kDa, the theoretical isoelectric point of 5.54, and had a conserved AP2 domain unique to the AP2/ERF family. The cis-acting elements analysis of OsDREB1J promoter showed that the promoter contained cis-acting elements related to hormone response, light and stresses response. The qRT-PCR analysis showed that OsDREB1J was expressed in different tissues of rice with no tissue specificity, and the relative expression level in panicle was the highest. At the same time, OsDREB1J was induced or reduced by different hormone. Transcriptional activation analysis showed that the full-length of OsDREB1J has no transcriptional activity, but the C-terminal fragment was sufficient for the transactivation ability. Phenotypic analysis showed that the grain length, length-width ratio and thousand grain weight of osdreb1j mutant were significantly higher than those of ZH11, OsDREB1J overexpression transgenic rice plants displayed opposite phenotypes, while changing the expression of OsDREB1J did not affect rice grain width. These results show that OsDREB1J may affect grain size by regulating cell length rather than cell proliferation and cell expansion. 【Conclusion】In conclusion, OsDREB1J may be involved in regulating rice grain size through hormone signaling pathway.

Key words: rice, OsDREB1J, transcription factor, grain size, subcellular localization, expression pattern

Table 1

Primers and sequences in this study"

引物名称Primer name 正向引物Forward primer (5′-3′) 反向引物Reverse primer (5′-3′)
OsDREB1J-GFP ACTAGTGGATCCGGTACCATGGAGAAGAACACCACCGC CTTGCTCACCATGGTACCGCTCCACAGCTCCATTTCAG
OsDREB1J-ox TCTCTCTCAAGCTTGGATCCATGGAGAAGAACACCACCGC ATACCGTCACTAGTGGATCCGCTCCACAGCTCCATTTCAG
BD-DREB1J GCCATGGAGGCCGAATTCATGGAGAAGAACACCACCGC CCGCTGCAGGTCGACGGATCCCGCTCCACAGCTCCATTTCAG
BD-DREB1J-N GCCATGGAGGCCGAATTCATGGAGAAGAACACCACCGC CCGCTGCAGGTCGACGGATCCCGAGCAGCCAGGCGGAGTC
BD-DREB1J-C GCCATGGAGGCCGAATTCATGCACGTCCCCCGCGC CCGCTGCAGGTCGACGGATCCCGCTCCACAGCTCCATTTCAG
OsDREB1J-q CATGACCAGCTGCCCGACGT GTGACAGAACGGGCGACGAC
DREB1J-62-sk CGCCGTCTAGAACTAGTGGATCCATGGAGAAGAACACCACCGC TCGATAAGCTTGATATCGAATTCCTAGCTCCACAGCTCCATTTC
DREB1J-N-62-sk CGCCGTCTAGAACTAGTGGATCCATGGAGAAGAACACCACCGC TCGATAAGCTTGATATCGAATTCCTAGAGCAGCCAGGCGGAGTC
DREB1J-C-62-sk CGCCGTCTAGAACTAGTGGATCCATGCACGTCCCCCGCGC TCGATAAGCTTGATATCGAATTCCTAGCTCCACAGCTCCATTTC
e-EF-1α GCACGCTCTTCTTGCTTTC AGGGAATCTTGTCAGGGTTG

Fig. 1

Bioinformatics analysis of OsDREB1J A: Prediction of protein hydrophilic/hydrophobic; B: Prediction of transmembrane domains; C: Schematic diagram of protein structure; D: Prediction of protein phosphorylation site"

Table 2

The promoter elements of OsDREB1J"

名称
Name		 序列
Sequence		 功能
Function		 数量
Number
ACE GACACGTATG 参与光响应的顺式调节元件 Cis-acting element involved in light responsive ness 9
Sp1 GGGCGG 光响应元件 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
TGACG-motif TACAAAA 茉莉酸甲酯响应元件 Cis-acting regulatory element involved in the MeJA-responsiveness 5
CGTCA-motif CGTCA 茉莉酸甲酯响应元件 Cis-acting regulatory element involved in the MeJA-responsiveness 5
TCA-element CCATCTTTTT 水杨酸响应元件 Cis-acting element involved in salicylic acid responsive ness 9
G-Box TGACG 参与光反应响应的顺式作用元件 Cis-acting regulatory element involved in light responsive ness 5
ABRE ACGTG 脱落酸响应元件 Cis-acting element involved in the abscisic acid responsive ness 6
ABRE CGCACGTGTC 脱落酸响应元件 Cis-acting element involved in the abscisic acid responsive ness 9
ABRE CACGTG 脱落酸响应元件 Cis-acting element involved in the abscisic acid responsive ness 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
MBS CAACTG 拟南芥中参与干旱诱导的MYB结合位点
Arabidopsis thaliana MYB binding site involved in drought-inducibility		 6
GARE-motif TCTGTTG 赤霉素响应元件 Gibberellin-responsive element 7
TCT-motif TCTTAC 光响应元件 Partofa light responsive element 6

Fig. 2

Analysis of conserved motifs, domains, and gene structure of OsDREB family genes A: The conserved motif analysis of OsDREB proteins. Different colored boxes indicate differently conserved motif, black lines indicate protein length; B: The gene structure analysis of OsDREB genes. Different colored boxes represent different domains, the green boxes represent 5′ and3′ untranslated regions (UTRs), the yellow boxes indicate the coding sequence (CDS), the red boxes indicate the AP2 domain, the black line indicates introns"

Fig. 3

Nucleic acid structure and cloning of OsDREB1J A: Nucleic acid structure of OsDREB1J, Filled blue bars indicate exons of the OsDREB1J gene; B: Amplification of OsDREB1J. M: DL2000 Maker, 1: OsDERB1J"

Fig. 4

Subcellular localization of OsDREB1J protein A: Subcellular localization of OsDREB1J protein in rice protoplasts, bar=3 μm; B: Subcellular localization of OsDREB1J protein in Nicotona benthamiana, bar=20 μm"

Fig. 5

Transcriptional activation analyses of OsDREB1J A, B: Transcriptional activation of OsDREB1J in yeast; C, D: Transcriptional activation of OsDREB1J in Nicotona benthamiana. A and C indicate the schematic diagrams of various constructs. *P<0.05, **P<0.01. The same as below"

Fig. 6

Expression patterns of OsDREB1J gene in different tissues A, B: Expression patterns of OsDREB1J 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 OsDREB1J in various organs of ZH11 by qRT-PCR"

Fig. 7

Effects of exogenous hormones on the expression of OsDREB1J A：10 μmol·L-1 JA；B：50 mmol·L-1 ABA；C：10 μmol·L-1 GA"

Fig. 8

The rice grain size phenotype of OsDREB1J transgenic rice plants A: Expression levels of OsDREB1J in OsDREB1J-ox lines by qRT-PCR; B: Generation of target site mutations in representative knockout lines of OsDREB1J rice plants using the CRISPR/Cas9 system. Filled blue bars indicate exons of the OsDREB1J gene. Cyan letters indicate the target sequence, the deletion line represents the missing base and the red letter represents the mutant base; C, D: Comparison of grain shape between wild-type (ZH11) and transgenic rice plants. E-H: The grain length, grain width, grain length to width ratio and thousand grain weight of ZH11 and transgenic rice plants. Bar=1 cm"

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