Functional analysis of Solanum lycopersicum L. transcription factor SlERF.F4 against gray mold in tomato fruit
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摘要:
番茄（Solanum lycopersicum L.）是世界上种植最广泛的经济作物之一，具有极大的经济价值，但其极易受到灰葡萄孢（Botrytis cinerea）的影响，造成严重的经济损失。ERF转录因子在植物胁迫响应中发挥关键作用。本研究通过获得番茄SlERF.F4过表达材料，比较其与野生型果实的差异，研究其介导的番茄果实抗灰霉病的功能。结果显示，与野生型番茄品种‘AC’（‘Ailsa Craig’）果实相比，SlERF.F4过表达（SlERF.F4-OE）番茄果实对灰葡萄孢的抗性较强，表现在灰葡萄孢接种后果实病斑扩展速率较慢。生理生化分析结果表明，SlERF.F4-OE果实体内的活性氧（O2−.产生速率、H2O2含量）和MDA含量低于‘AC’果实，同时，抗氧化酶活性（POD、CAT和SOD酶）高于‘AC’果实。此外，SlERF.F4-OE番茄果实体内苯丙氨酸解氨酶（PAL）活性高于‘AC’果实。研究结果说明，SlERF.F4可通过调节番茄果实活性氧稳态及防御酶活性增强果实对灰葡萄孢的抵抗能力。
Abstract:Solanum lycopersicum L. is one of the most widely cultivated cash crops globally, but its susceptibility to gray mold (Botrytis cinerea) causes significant economic losses. ERF transcription factors play a key role in plant stress responses. In this study, SlERF.F4 overexpression lines were generated and compared with wild-type tomato variety ‘AC’ (‘Ailsa Craig’) fruits to investigate the role of SlERF.F4 in mediating resistance to gray mold in tomatoes. Compared with ‘AC’ fruits, SlERF.F4-overexpressed (SlERF.F4-OE) fruits exhibited enhanced resistance to B. cinerea, as indicated by a slower lesion expansion following inoculation. Physiological and biochemical analyses revealed that the production rate of reactive oxygen species (ROS), including O2-. and H2O2 content, as well as malondialdehyde (MDA) levels, were significantly lower in SlERF.F4-OE fruits than in ‘AC’ fruits. Concurrently, antioxidant enzyme activities, including peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD), were significantly elevated in SlERF.F4-OE fruits. In addition, phenylalanine ammonia-lyase (PAL) activity was higher in SlERF.F4-OE fruits than in ‘AC’ fruits. These results suggest that SlERF.F4 enhances resistance to B. cinerea in S. lycopersicum fruits by regulating reactive oxygen homeostasis and defense enzyme activity. This study provides novel insights into the function of SlERF.F4 in fruit disease resistance.
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Keywords:
- Solanum lycopersicum /
- Botrytis cinerea /
- ERF /
- ROS
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图 1 番茄SlERF.F4的系统进化树分析
Figure 1. Phylogenetic tree analysis of Solanum lycopersicum SlERF.F4
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图 2 灰葡萄孢侵染后SlERF.F4的表达模式
Figure 2. Expression patterns of SlERF.F4 after Botrytis cinerea infection
*：P<0.05.
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图 3 pCAMBIA1300-221-35S-SlERF.F4基因过表达植株PCR鉴定
M：DL 2000 Marker；N：阴性对照（非转基因植株）。1、2：SlERF.F4-OE-1；3、4：SlERF.F4-OE-2。
Figure 3. PCR identification of pCAMBIA1300-SlERF.F4 overexpressed plants
M: DL 2000 Marker; N: negative control; 1, 2: SlERF.F4-OE-1; 3, 4: SIERF. F4-OE-2.
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图 4 转基因植株实时荧光定量PCR检测
不同小写字母表示差异显著（P<0.05），下同。
Figure 4. Real-time fluorescence quantitative PCR detection of transgenic plants
Different lowercase letters indicate significant differences (P<0.05). Same below.
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图 5 SlERF.F4过表达番茄果实侵染灰葡萄孢的表型（A）、病斑面积（B）和发病率（C）
Figure 5. Phenotype (A), lesion area (B) and incidence rate (C) of SlERF.F4-overexpressed Solanum lycopersicum fruits after inoculation with Botrytis cinerea
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图 6 接种灰葡萄孢后‘AC’和SlERF.F4-OE番茄果实中O2-.（A）、H2O2（B）和MDA（C）生成量的变化
Figure 6. Changes in O2-. (A), H2O2 (B) and MDA (C) production in AC and SlERF.F4 -overexpressed Solanum lycopersicum fruits after inoculation with Botrytis cinerea
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图 7 接种灰葡萄孢后‘AC’和SlERF.F4-OE果实POD（A）、CAT（B）、SOD（C）和PAL（D）的活性变化
Figure 7. Changes in POD (A), CAT (B), SOD (C), and PAL (D) activities in ‘AC’ and SlERF.F4-overexpressed fruits after inoculation with Botrytis cinerea
表 1 引物序列
Table 1 Primer sequences used in this study
引物名称
Primer name引物序列（5’→3’）
Primer sequence (5’→3’)SlERF.F4-F AGAGGATCCATGGCTGTGAAAGATA SlERF.F4-R AATTCGAGCTCTTAAACTTCCATAGGT Actin-F CCTCAGCACATTCCAGCAG Actin-R CCACCAAACTTCTCCATCCC qPCR-SlERF.F4-F GAGTCATCCAGCGGAGAAACGG qPCR-SlERF.F4-R GACACCTCCACGTCCACCTTCA Hyg-F CTTGACATTGGGGAGTTTAGCGAGA Hyg-R CCCTTATCTGGGAACTACTCACACA 
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