植物重金属响应可视化系统的构建与验证

doi:10.13560/j.cnki.biotech.bull.1985.2025-0804

摘要/Abstract

摘要：

目的开发植物体内重金属离子可视化监测系统，实现水稻重金属离子快速检测。方法基于金属响应转录因子（MTF-1）特异性结合金属响应元件（MREs）从而激活转录这一原理，构建了以β-葡萄糖醛酸酶（GUS）或增强绿色荧光蛋白（EGFP）为报告基因的重金属响应植物表达载体，利用农杆菌介导法导入水稻及拟南芥中；通过对转基因植株进行不同浓度重金属离子处理，观察其GUS染色或荧光信号强度；利用RNA-seq分析转基因水稻及野生型对照重金属处理前后的差异基因表达；通过抗氧化酶活性测定和表型验证，分析转基因水稻及野生型对照的金属耐受性。结果经不同浓度的As³⁺、Cd²⁺、Cu²⁺溶液处理后，转基因拟南芥或水稻可观察到明显的GUS染色或绿色荧光信号。此外，转基因水稻中重金属解毒相关基因的表达量显著高于对照，超氧化物歧化酶（SOD）和过氧化物酶（POD）活性增强。结论在水稻及拟南芥中初步构建了响应重金属离子的可视化系统，为实现水稻体内重金属快速检测提供技术支撑。

关键词: 重金属, 水稻, MTF-1, MREs, 可视化

Abstract:

Objective To develop a visual monitoring system for heavy metal ions inside plants and achieve rapid detection of heavy metal ions in rice (Oryza sativa). Method Based on the principle that metal-responsive transcription factors (MTF-1) specifically bind to metal-responsive elements (MREs) and activate transcription, a metal-responsive plant expression vector with β-glucuronidase (GUS) or enhanced green fluorescent protein (EGFP) as reporter genes was constructed, and it was introduced into rice and Arabidopsis thaliana using the Agrobacterium-mediated method. By treating the transgenic plants with different concentrations of heavy metal ions, the intensity of GUS staining or fluorescence signal was observed. RNA-seq was used to analyze the differential gene expression of transgenic rice and wild-type control before and after heavy metal treatment. The antioxidant enzyme activities and phenotypic verification were used to analyze the metal tolerance of transgenic rice and wild-type control. Result After being treated with different concentrations of As³⁺, Cd²⁺, and Cu²⁺solutions, obvious GUS staining or green fluorescence signals were observed in transgenic A. thaliana or rice. In addition, the expressions of heavy metal detoxification-related genes in transgenic rice were significantly higher than those in the control, and the activities of superoxide dismutase (SOD) and peroxidase (POD) were enhanced. Conclusion A visualization system responding to heavy metal ions was initially constructed in rice and A. thaliana, providing technical support for the rapid detection of heavy metals in rice.

Key words: heavy metals, rice, MTF-1, MREs, visualization

彭彦, 安晨, 韶也, 毛毕刚, 张学文, 赵炳然. 植物重金属响应可视化系统的构建与验证[J]. 生物技术通报, 2026, 42(7): 105-115.

PENG Yan, AN Chen, SHAO Ye, MAO Bi-gang, ZHANG Xue-wen, ZHAO Bing-ran. Construction and Validation of a Visualization System for Plants Heavy Metal Response[J]. Biotechnology Bulletin, 2026, 42(7): 105-115.

图/表 7

图1 重金属响应植物表达载体的构建A-D分别代表35S： MTF1-MRE8-mini35S： GUS、35S： MTF1-MRE8-mini35S： EGFP、Ubi： MTF1-MRE8-mini35S： GUS和Ubi： MTF1-MRE8-mini35S： EGFP载体示意图；Hyg（R）代表潮霉素抗性基因，MTF-1 core代表金属响应转录因子MTF-1核心序列，MRE（8×）代表8个拷贝的金属响应元件MRE，MRE（8×）与mini 35S启动子连接成金属响应人工启动子

Fig. 1 Construction of heavy-metal-responsive plant expression vectorsA-D refers to schematic diagrams of the 35S: MTF1-MRE8-mini35S: GUS, 35S: MTF1-MRE8-mini35S: EGFP, Ubi: MTF1-MRE8-mini35S: GUS,and Ubi: MTF1-MRE8-mini35S: EGFP vectors, respectively. Hyg (R) indicates the hygromycin resistance gene, MTF-1 core indicates the core sequence of the metal-responsive transcription factor MTF-1, and MRE (8×) indicates eight copies of the metal response element MRE. MRE (8×) is linked to the mini 35S promoter to form a metal-responsive synthetic promoter

图2 转基因拟南芥的GUS表达分析A-C： As3+、Cd2+和Cu2+分别处理7 d（A）、10 d（B）及12 d（C）的转基因拟南芥GUS染色，叶片比例尺500 μm，植株比例尺1 mm；D：As3+、Cd2+和Cu2+分别处理12 d的转基因拟南芥GUS染色液颜色强度；E：As3+、Cd2+和Cu2+分别处理7 d（I-III）、10 d（IV-VI）及12 d（VII-IX）的转基因拟南芥GUS基因相对表达量，柱状图表示平均值±标准差，n=3，柱状图上不同字母表示在0.05水平上存在显著差异； F：As3+、Cd2+和Cu2+分别处理7 d（I-III）、10 d（IV-VI）及12 d（VII-IX）的转基因拟南芥GUS活性，*P<0.05， **P<0.01，***P<0.001，****P<0.000 1，下同

Fig. 2 GUS expression analysis in transgenic A. thalianaA-C: GUS staining in transgenic A. thaliana at 7 d (A), 10 d (B), and 12 d (C)) after treated with As3+, Cd2+and Cu2+. Scale bars: 500 μm (leaf), and 1 mm (plant). D: The color intensity of the GUS staining solution at 12 d after treated with As3+, Cd2+and Cu2+. E: The relative expressions of the GUS gene in transgenic A. thaliana at 7 d (I-III), 10 d (IV-VI) and 12 d (VII-IX) after treated with As3+, Cd2+and Cu2+, respectively. The bar graph indicates the mean ± standard deviation (n = 3), the different letters on the bars indicate a statistically significant difference. F: GUS activity in transgenic A. thaliana at 7 (I-III), 10 (IV-VI), and 12 d (VII-IX) after treated with As3+, Cd2+and Cu2+, respectively. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.000 1. The same below

图3 转基因拟南芥的EGFP表达分析A： As3+、Cd2+和Cu2+分别处理7 d后的转基因拟南芥根尖绿色荧光强度，比例尺50 μm；B：As3+、Cd2+和Cu2+分别处理7 d（I-III）、10 d（IV-VI）及12 d（VII-IX）的转基因拟南芥EGFP基因相对表达量

Fig. 3 EGFP expression analysis in transgenic A. thalianaA: Green fluorescence intensity in the root tips of transgenic A. thaliana at 7 d after treated with As³⁺, Cd²⁺, and Cu²⁺, respectively. Scale bar = 50 μm. B: Relative expression of the EGFP gene in transgenic A. thaliana at 7 d (I-III), 10 d (IV-VI), and 12 d (VII-IX) after treated with As³⁺, Cd²⁺, and Cu²⁺, respectively

图4 重金属响应植物表达载体在水稻中表达验证A：重金属处理7 d的转基因水稻GUS活性；B：重金属处理3 d（I-III）、5 d（IV-VI）和7 d（VII-IX）的转基因水稻EGFP基因相对表达量；C：重金属处理7 d的转基因水稻GUS染色，比例尺1 mm；D-F：重金属处理3 d（D）、5 d（E）和7 d（F）的转基因水稻根尖绿色荧光强度，比例尺100 μm

Fig. 4 Verification of the expressions of heavy metal-responsive plant expression vectors inriceA: GUS activity of transgenic rice exposed to heavy metals for 7 d. B: Relative expression of the EGFP gene in transgenic rice exposed to heavy metals for 3 d (I-III), 5 d (IV-VI), and 7 d (VII-IX). C: GUS staining of transgenic rice exposed to heavy metals for 7 d, scale bars: 1 mm. D-F: Green fluorescence intensity emitted by the root tips of transgenic rice exposed to heavy metals for 3 d (D), 5 d (E), and 7 d (F), scale bars: 100 μm

图5 差异表达及GO聚类分析A：不同组之间的差异表达基因数量。MTF（As）、MTF（Cd）和MTF（Cu）分别代表As3+、Cd2+和Cu2+处理3 d的转基因水稻，MTF（-）代表未处理转基因水稻，WT（As）、WT（Cd）和WT（Cu）分别代表As3+、Cd2+和Cu2+处理3 d的野生型水稻，All指所有差异表达基因的集合，Up和Down分别代表显著上调基因和下调基因的数量。B-D：代表相同条件下野生型与转基因水稻之间差异表达基因GO聚类，横坐标GeneRatio表示差异基因数与差异基因总数的比值，气泡大小代表富集到对应通路的基因数量，气泡的颜色代表多重假设检验校正后的P值，Padj <0.05为显著富集

Fig. 5 Differential expression and GO clustering analysisA: Number of differentially expressed genes (DEGs) among different groups. MTF(As), MTF(Cd), and MTF(Cu) refer to transgenic rice treated with As³⁺, Cd²⁺, and Cu²⁺ for 3 d, respectively. MTF(-) refers to untreated transgenic rice. WT(As), WT(Cd), and WT(Cu) refer to wild-type rice treated with As³⁺, Cd²⁺, and Cu²⁺ for 3 d, respectively. “All” refers to the total set of DEGs. “Up” and “Down” indicate the number of significantly up-regulated and down-regulated genes, respectively. B-D: GO clustering of DEGs between wild-type and transgenic rice under the same conditions. The X-axis “GeneRatio” is the ratio of the number of DEGs to the total number of DEGs. The bubble size indicates the number of genes enriched in the corresponding pathway, and the bubble color indicates the P-value adjusted for multiple hypothesis testing, with Padj < 0.05 considered significantly enriched

图6 重金属解毒相关基因表达验证HZ：野生型水稻，MTF：转基因水稻；数据以均值±标准差表示，n=3；*和**分别表示野生型水稻和转基因水稻在0.05和0.01水平上差异显著（t检验）

Fig. 6 Verification of gene expression related to heavy metal detoxificationHZ: Wild-type rice. MTF: Transgenic rice. Data are expressed as mean ± standard deviation, n = 3; * and ** indicate significant differences between wild-type rice and transgenic rice at 0.05 and 0.01 levels, respectively (t-test)

图7 不同重金属胁迫下野生型和转基因水稻生理和表型特征A：野生型（HZ）和转基因水稻（MTF）在As、Cd或Cu胁迫下的株高、根长、鲜重、抗氧化酶（SOD、POD和CAT）活性，浓度梯度为0、50、100、200、300和500 µmol/L，均值±标准差，n=3，*表示野生型（HZ）和转基因水稻（MTF）在0.05水平上差异显著（t 检验）。B：野生型（白色虚线左侧）和转基因水稻（白色虚线右侧）表型性状，标尺=4 cm

Fig. 7 Physiological characteristics and phenotypic traits of wild-type and transgenic rice exposed to different heavy metalsA: Plant height, root length, fresh weight, and antioxidant enzyme (SOD, POD, and CAT) activities of wild-type (HZ) and transgenic rice (MTF) under As, Cd, or Cu stress. Concentration gradients are 0, 50, 100, 200, 300, and 500 µmol/L. Data are presented as mean ± standard deviation, n = 3. * indicates a significant difference between wild-type (HZ) and transgenic rice (MTF) at 0.05 level (t-test). B: Phenotypic traits of wild-type (left of the white dashed line) and transgenic rice (right of the white dashed line). Scale bar = 4 cm

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