OsDREB1J调控水稻籽粒大小的功能研究

doi:10.3864/j.issn.0578-1752.2025.08.001

摘要/Abstract

摘要：

【目的】AP2/ERF（APETALA2/ethylene responsive factor）超家族是一类对植物生长发育以及响应逆境胁迫起重要调节作用的转录因子，在植物中广泛存在，且成员众多。探究AP2/ERF家族基因在水稻籽粒大小调控中的功能，为水稻粒形改良提供重要的基因资源。【方法】通过同源重组法克隆OsDREB1J（LOC_Os08g43200），利用生物信息学、实时荧光定量PCR（qRT-PCR）等技术对该基因的基本特性、组织表达特性及在激素下的表达模式等进行分析；利用酵母异源表达、水稻原生质体瞬时表达、烟草瞬时表达技术分析该基因的转录活性和亚细胞定位；利用遗传转化体系获得该基因的过表达和基因敲除水稻突变体，并运用表型分析技术对其粒型进行分析。【结果】亚细胞定位结果显示，OsDREB1J定位于细胞核。生物信息学分析结果显示，该基因序列全长为711 bp，编码236个氨基酸；OsDREB1J蛋白不具有跨膜结构域，相对分子质量为27.47 kDa，理论等电点为5.54，具有AP2/ERF家族特有的AP2保守结构域。启动子元件分析发现，该基因启动子的顺式作用元件包括与激素响应、光响应、逆境胁迫响应等相关的元件。实时荧光定量PCR（qRT-PCR）分析结果显示，OsDREB1J在水稻不同组织中均有表达，无组织特异性，但在穗中的表达量最高；同时，在不同激素处理下，OsDREB1J的表达受不同程度的诱导或抑制。转录活性分析结果显示，OsDREB1J全长无转录自激活活性，但C端是转录自激活所必需的结构域。表型分析结果表明，osdreb1j突变体的粒长、长宽比、千粒重显著高于ZH11，OsDREB1J过表达转基因水稻则相反，改变OsDREB1J的表达不影响水稻籽粒的粒宽。表明OsDREB1J可能是通过调控籽粒细胞的长度而非细胞增殖和细胞扩展来影响籽粒大小的。【结论】OsDREB1J可能通过激素信号通路调控水稻籽粒大小。

关键词: 水稻, OsDREB1J, 转录因子, 籽粒大小, 亚细胞定位, 表达模式

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

韦萍, 潘炬忠, 朱德平, 邵胜雪, 陈珊珊, 韦雅倩, 高维维. OsDREB1J调控水稻籽粒大小的功能研究[J]. 中国农业科学, 2025, 58(8): 1463-1478.

WEI Ping, PAN JuZhong, ZHU DePing, SHAO ShengXue, CHEN ShanShan, WEI YaQian, GAO WeiWei. The Function of OsDREB1J in Regulating Rice Grain Size[J]. Scientia Agricultura Sinica, 2025, 58(8): 1463-1478.

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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

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