拟南芥种子的胚与胚乳中pOp6/LhG4AtO表达系统的构建与验证

doi:10.13560/j.cnki.biotech.bull.1985.2026-0021

摘要/Abstract

摘要：

目的构建适用于拟南芥种子的胚与胚乳中基因功能研究的pOp6/LhG4^AtO表达系统，并验证该系统的可行性，为基因组织特异性表达提供可靠的操作工具。方法基于pOp6/LhG4^AtO的二元转录激活系统，选取已报道的胚（TWS1）和胚乳（FWA、ESH1）特异性启动子构建“驱动”载体。同时，选用增加核定位的荧光报告基因（H2B-Clover）构建效应载体。采用农杆菌介导的花序浸染法进行遗传转化，获得目标转基因阳性株系。通过将驱动系和效应系进行杂交，在其F₁代种子中，观察授粉后2‒5 d报告基因的组织特异性表达情况，并与公共芯片转录组数据（NCBI GEO；accession number GSE12404）进行比对分析。结果成功构建pOp6/LhG4^AtO表达系统，并获得所需的独立转基因株系，包括驱动系（pTWS1::LhG4^AtO 、pFWA::LhG4^AtO 、pESH1::LhG4^AtO ）和效应系（pOp6::H2B-Clover）。驱动系与效应系杂交后，pTWS1::LhG4^AtO 驱动的效应系仅在3‒5 d的胚细胞核中观察到特异且持续高表达的绿色荧光信号；pFWA::LhG4^AtO 和pESH1::LhG4^AtO 驱动的效应系在授粉后2‒5 d的胚乳细胞核中观察到特异且持续高表达的绿色荧光信号。表明pOp6/LhG4^AtO表达系统驱动的效应系荧光表达模式，与已发表的芯片转录组数据高度一致。结论建立并验证了pOp6/LhG4^AtO系统在拟南芥种子的胚和胚乳中组织特异性驱动基因表达的能力，为种子发育过程中关键基因的功能研究提供了可靠的操作工具。

关键词: pOp6/LhG4^AtO系统, 组织特异性表达, 胚, 胚乳, 种子发育

Abstract:

Objective To construct the pOp6/LhG4^AtO expression system for studying gene function in the embryo and endosperm of Arabidopsis seeds, and to validate its feasibility to provide a reliable tool for tissue-specific gene expression. Method Based on the pOp6/LhG4^AtO binary transcriptional activation system, previously reported embryo-specific (TWS1) and endosperm-specific (FWA, ESH1) promoters were selected to construct the driver vectors. Meanwhile, a fluorescent reporter gene with enhanced nuclear localization (H2B-Clover) was employed to generate the effector vector. Genetic transformation was performed via Agrobacterium-mediated floral dip method to obtain positive transgenic lines. By crossing the driver lines with the effector line, the tissue-specific expression of the reporter gene was observed in F1 seeds at 2–5 d after pollination, and the expression patterns were compared with publicly available microarray transcriptome data (NCBI GEO; accession number GSE12404). Result The pOp6/LhG4^AtO expression system was successfully constructed, and the required independent transgenic lines were obtained, including driver lines (pTWS1::LhG4^AtO, pFWA::LhG4^AtO, pESH1::LhG4^AtO ) and an effector line (pOp6::H2B-Clover). After crossing the driver line with the effector line, green fluorescence signals driven by pTWS1::LhG4^AtO, were specifically and continuously observed at high levels in the nuclei of embryos cells only at 3‒5 d after pollination (DAP). Similarly, green fluorescence signals driven by pFWA::LhG4^AtO and pESH1::LhG4^AtO were observed specifically and continuously at high levels in the nuclei of endosperm cells at 2‒5 DAP after pollination. These results demonstrate that the fluorescence expression patterns driven by the pOp6/LhG4^AtO system are highly consistent with published microarray transcriptome data. Conclusion This study established and validated the pOp6/LhG4^AtOsystem for tissue-specific driver gene expression in the embryo and endosperm of Arabidopsis seeds, providing a reliable tool for functional studies of key genes during seed development.

Key words: pOp6/LhG4^AtO system, tissue-specific expression, embryo, endosperm, seed development

付彩霞, 张瑞花, 祝云俊, 李靖, 杨科, 王轩鹏. 拟南芥种子的胚与胚乳中pOp6/LhG4^AtO表达系统的构建与验证[J]. 生物技术通报, 2026, 42(9): 120-127.

FU Cai-xia, ZHANG Rui-hua, ZHU Yun-jun, LI Jing, YANG Ke, WANG Xuan-peng. Construction and Validation of the pOp6/LhG4^AtO Expression System in the Embryo and Endosperm of Arabidopsis Seed[J]. Biotechnology Bulletin, 2026, 42(9): 120-127.

图/表 5

图1 pOp6/LhG4AtO 载体设计和杂交组合A：pop6/LhG4Ato载体设计结构；Promoter：启动子；Ω：翻译增强元件Omaga；Ter：终止子；TEV：烟草蚀纹病毒翻译增强子；pAt2S3：种子储藏清蛋白基因SESA3启动子；B：pop6/LhG4Ato体系中驱动系和效应系的杂交组合

Fig. 1 Vector design and hybrid combination of pOp6/LhG4AtOA: Design of pop6/LhG4Ato vector structure. Promoter: promoter. Ω: Omega, translation enhancement element. Ter: Terminator. TEV: Translation enhancer of tobacco etch virus. pAt2S3: Seed storage albumingene SESA3 promoter. B: Crossing combinations between driver lines and effector lines in the pop6/LhG4Ato system

图2 胚、胚乳启动子片段克隆和双元载体酶切鉴定A：组织特异性启动子克隆（M：2 000 DNA marker；1-4：克隆片段分别为pTWS1、pFWA、pESH1、H2B-Clover）；B：重组质粒酶切鉴定（M：2 000 DNA marker；1-4：酶切质粒分别为pTWS1::LhG4AtO 、pFWA::LhG4AtO 、pESH1::LhG4AtO 、pOp6::H2B-Clover）

Fig. 2 Cloning of embryo and endosperm promoter fragments and identification of binary vector digestionA: Cloning of tissue-specific promoter (M: 2 000 DNA marker. 1-4: Cloned fragments were, pTWS1, pFWA, pESH1, H2B-Clover). B: Restriction enzyme digestion identification of recombinant plasmid (M: 2 000 DNA marker. 1-4: Plasmids were pTWS1::LhG4AtO, pFWA::LhG4AtO, pESH1::LhG4AtO, pOp6::H2B-Clover, respectively)

图3 转基因材料的筛选及鉴定A：转基因阳性种子的筛选；标尺为1 mm；B：转基因株系阳性鉴定的琼脂糖凝胶电泳检测（M：2 000 DNA marker；1-4分别为pTWS1::LhG4AtO 、pFWA::LhG4AtO 、pESH1::LhG4AtO 、pOp6::H2B-Clover）

Fig. 3 Screening and identification of transgenic materialsA: Screening of transgenic positive seeds. Scale bar=1 mm. B: Detection of positive identification of transgenic lines by agarose gel electrophoresis (M: 2 000 DNA marker. 1-4: The fragments were pTWS1::LhG4AtO, pFWA::LhG4AtO, pESH1::LhG4AtO, pOp6::H2B-Clover, respectively)

图4 TWS1在种子不同发育阶段的表达模式及其驱动的H2B-Clover报告基因表达A：TWS1在种子发育阶段的表达模式（NCBI GEO； accession number GSE12404）；Stage pG：前球形期；Stage G：球形期；Stage H：心形期；Stage IC：幼嫩子叶期；Stage bC：弯曲子叶期；Em：胚；Mi En：珠孔端胚乳；Pe En：外周胚乳；Ch En：合点端胚乳；Ch SC：合点端种皮；Ge SC：常规种皮；Y轴数值是以10为基底的对数刻度值，下同；B：杂交F1 2‒5 d种子中TWS1驱动效应系的荧光表达情况；胚中核定位的绿色荧光信号为TWS1驱动的目标信号，而种皮及胚乳中质体的自发荧光呈现为红色荧光信号；DAP：授粉后的天数；标尺为50 μm。下同

Fig. 4 Relative expression of TWS1 at different developmental stages of seeds and the expression of H2B-Clover reporter gene driven by TWS1A: Expression pattern of TWS1 in seed development stage (NCBI GEO; accession number GSE12404). Stage pG: Pre-globular stage. Stage G: Globular stage. Stage H: Heart stage. Stage IC: Linage cotyledon stage. Stage bC: Bent cotyledon stage. Em: Embryo. Mi En: Micropylar endosperm. Pe En: Peripheral endosperm. Ch En: Chalazal endosperm. Ch SC: Chalazal seed coat. Ge SC: General seed coat. The Y-axis value is a logarithmic scale value based on 10. The same below. B: Fluorescence expression of the TWS1 driven effector line in F1 hybrid seeds at 2‒5 d after pollination. The nuclear-localized green fluorescent signal in the embryo indicates the TWS1 driven target signal, whereas the autofluorescence of plastids in the seed coat and endosperm appears as a red fluorescent signal. DAP is days after pollination. Scale bar, 50 μm. The same below

图5 FWA和ESH1在种子不同发育阶段的表达模式分析及其驱动的H2B-Clover报告基因表达A：FWA和ESH1在种子发育阶段的表达模式（NCBI GEO； accession number GSE12404）；B‒C：分别是FWA和ESH1在杂交F12‒5 d种子中驱动效应系的荧光表达情况。胚乳中核定位的绿色荧光信号为FWA和ESH1驱动的目标信号，而种皮、胚和胚乳中质体的自发荧光呈现为红色荧光信号；标尺为50 μm

Fig. 5 Expression pattern analysis of FWA and ESH1 genes at different developmental stages of seeds and the expression of H2B-Clover reporter gene driven by themA: Expression patterns of FWA and ESH1 during seed development (NCBI GEO; accession number GSE12404). B‒C: Fluorescence expression of the FWA and ESH1 effector lines in F1 hybrid seeds at 2‒5 d after pollination, respectively. The nuclear-localized green fluorescent signal in the endosperm represents the target signal driven by FWA and ESH1, whereas the autofluorescence of plastids in the seed coat, embryo, and endosperm appears as a red fluorescent signal. Scale bar, 50 μm

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