OsbHLH069通过对LAX1-LAX2复合体的竞争性干扰负调控水稻穗发育

doi:10.13560/j.cnki.biotech.bull.1985.2025-1282

摘要/Abstract

摘要：

目的 LAX1 (LAX PANICLE 1)-LAX2 (LAX PANICLE 2)复合体是调控水稻穗侧生分生组织发育的关键模块。bHLH（basic helix-loop-helix）转录因子OsbHLH069能与LAX1互作，其功能获得突变体nsp1-D（no spikelet 1-Dominant）呈现稀穗表型。旨在解析三者间的分子互作模式，阐明其调控穗分枝发育的机制。方法利用水稻原生质体和酵母系统鉴定OsbHLH069的核定位信号与转录激活域。通过酵母双杂交、双分子荧光互补和体外pull-down实验，探究OsbHLH069与LAX2的互作关系。利用田间杂交技术，构建lax2-4 nsp1-D双突变体，采用实时荧光定量PCR分析其遗传关系。通过截短突变体和酵母双杂交试验确定3个蛋白互作区段。利用荧光素酶互补试验探究互作模型。结果 OsbHLH069是一个典型的核定位转录激活因子，其核定位信号位于266‒285 aa，转录激活域位于N端1‒120 aa。OsbHLH069与LAX2在体内外互作。OsbHLH069通过bHLH结构域与LAX1互作，又通过C端区域与LAX2互作，其互作区段与LAX1-LAX2互作区段重合。OsbHLH069竞争性抑制LAX1-LAX2的复合体稳定性。lax2-4 nsp1-D双突变体枝梗数和每穗粒数缺失表型强于任一单突变体，与lax1 nsp1-D双突变体表型结果类似。结论提出一个阶段性调控模型：在穗侧生分生组织发育早期，LAX1-LAX2复合体促进侧生分生组织起始；随着发育的进行，OsbHLH069通过竞争性拆解LAX1-LAX2复合体，实现对穗侧生分生组织的精细负调控。

关键词: 水稻, 穗发育, OsbHLH069, LAX1, LAX2, 蛋白互作

Abstract:

Objective The LAX1-LAX2 complex is a key module regulating axillary meristem development in the rice panicle. The bHLH (basic helix-loop-helix) transcription factor OsbHLH069 interacts with LAX1, and its gain-of-function mutant nsp1-D (no spikelet 1-dominant) exhibits a sparse panicle phenotype. This study aimed to elucidate the molecular interaction model among these three proteins and clarify the mechanism by which they regulate panicle branching development. Methods The nuclear localization signal and transcriptional activation domain of OsbHLH069 were analyzed using rice protoplasts and yeast systems. The interaction between OsbHLH069 and LAX2 was investigated through yeast two-hybrid assays, bimolecular fluorescence complementation, and in vitro pull-down assays. The lax2-4 nsp1-D double mutant was generated by field crossing, and genetic interactions were analyzed by quantitative real-time PCR. The interaction regions among the three proteins were mapped using truncation mutants and yeast two-hybrid assays. The interaction model was further explored via luciferase complementation assays. Results OsbHLH069 is a typical nuclear-localized transcriptional activator, with its nuclear localization signal located at amino acids 266–285 and its transcriptional activation domain at the N-terminal region (amino acids 1–120). OsbHLH069 directly interacted with LAX2 both in vivo and in vitro. OsbHLH069 interacted with LAX1 through its bHLH domain and with LAX2 through its C-terminal region; these interaction regions overlapped with the LAX1-LAX2 interaction interface. OsbHLH069 competitively inhibited the stability of the LAX1-LAX2 complex. The lax2-4 nsp1-D double mutant showed more severe defects in branch number and spikelets per panicle than either single mutant, resembling the previously reported phenotype of the lax1 nsp1-D double mutant. Conclusion A phased regulatory model is proposed: during early panicle axillary meristem development, the LAX1-LAX2 complex promotes axillary meristem initiation; as development progresses, OsbHLH069 achieves fine-tuned negative regulation of the panicle axillary meristem by competitively disassembling the LAX1-LAX2 complex.

Key words: rice, panicle development, OsbHLH069, LAX1, LAX2, protein interaction

杨群, 李京, 符德保, 许婷婷. OsbHLH069通过对LAX1-LAX2复合体的竞争性干扰负调控水稻穗发育[J]. 生物技术通报, 2026, 42(8): 113-122.

YANG Qun, LI Jing, FU De-bao, XU Ting-ting. OsbHLH069 Negatively Regulates Rice Panicle Development by Competitively Interfering with the LAX1-LAX2 Complex[J]. Biotechnology Bulletin, 2026, 42(8): 113-122.

图/表 5

图1 OsbHLH069核定位信号肽分析35S：OsAPI5-RFP为核定位标记蛋白。标尺=10 μm

Fig. 1 Analysis of the nuclear localization signal peptide of OsbHLH06935S:OsAPI5-RFP served as a nuclear localization protein marker. Scale bar=10 μm

图2 OsbHLH069截短蛋白转录活性分析A：酵母细胞中分析OsbHLH069截短蛋白转录活性；B：载体示意图；C：水稻原生质体中分析OsbHLH069不同截短蛋白转录活性。***：差异极显著（P<0.001），ns：差异不显著（P>0.05）

Fig. 2 Transcriptional activity analysis of OsbHLH069 truncated proteinA: Analysis of the transcriptional activity of truncated OsbHLH069 proteins in yeast cells. B: Schematic diagram of the vectors. C: Analysis of the transcriptional activity of truncated OsbHLH069 proteins in rice protoplasts. ***: A significant difference (P<0.001). ns: No significant difference (P>0.05)

图3 OsbHLH069与LAX2在体内和体外的互作验证A：酵母双杂交试验；B：双分子荧光互补试验；35S：OsAPI5-YFP为细胞核标记物；标尺=10 µm；C：体外pull-down试验

Fig. 3 Validation of the interaction between OsbHLH069 and LAX2 in vivo and in vitroA: Yeast two-hybrid assay. B: Bimolecular fluorescence complementation assay. 35S:OsAPI5-YFP serves as a nuclear marker. Scale bar = 10 µm. C: In vitro pull-down assay

图4 lax2-4 nsp1-D双突变体的表型分析A‒D：株型图，标尺为11 cm；E：穗型图，标尺为6 cm；F：分蘖数；G：一次枝梗数；H：二次枝梗数；I：一次枝梗上的小穗数；J：二次枝梗上的小穗数；K：总穗数；L：实时荧光定量PCR检测LAX2的表达量；M：实时荧光定量PCR检测OsbHLH069的表达量。不同小写字母表示组间差异显著（P<0.05），ns代表差异不显著（P>0.05）

Fig. 4 Phenotypic analysis of lax2-4 nsp1-D double mutantsA‒D: Plant architecture, bars=11 cm. E: Panicle morphology, bar=6 cm. F: Tiller number. G: Primary branch number. H: Secondary branch number. I: Spikelet number per primary branch. J: Spikelets number per secondary branch. K: Total spikelet number per panicle. L: Quantitative real-time PCR detection of LAX2 expression. M: Quantitative real-time PCR detection of OsbHLH069 expression. Different lowercase letters indicate significant differences between groups (P<0.05); ns refers to no significant difference (P>0.05)

图5 OsbHLH069、LAX1和LAX2互作模式研究A：LAX1、LAX2和OsbHLH069氨基酸截短示意图；B‒F：酵母双杂交分析，DDO：二缺培养基SD/-Leu/-Trp，QDO：四缺培养基SD/-Leu/-Trp/-His/-Ade；G：荧光素酶互补试验

Fig. 5 Study on the interaction modes of OsbHLH069, LAX1, and LAX2A: Schematic diagram of amino acid truncation for LAX1, LAX2, and OsbHLH069. B‒F: Yeast two-hybrid analysis. Double Dropout Medium (DDO) refers to SD/-Leu/-Trp. Quadruple Dropout Medium (QDO) refers to SD/-Leu/-Trp/-His/-Ade. G: Luciferase complementation assay

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