西瓜ClESR2在维持叶片形态稳定中的功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2026-0243

摘要/Abstract

摘要：

目的解析西瓜ERF转录因子ClESR2在调控叶片形态稳定中的功能，为叶片发育调控机制解析提供理论依据和候选基因资源。方法利用农杆菌侵染获得西瓜ClESR2转基因株系，观察转基因株系的叶片形态，利用石蜡切片和扫描电镜等技术进一步观察并统计叶片细胞形态和数量，使用RNA-seq进行叶片转录组测序，使用RT-qPCR检测叶片发育相关基因的表达量，并检测转基因株系叶片中生长素的含量，最后利用CRISPR/Cas技术获得Clesr2突变体，并对其互作蛋白进行筛选和鉴定。结果西瓜ClESR2过表达后，叶片表面呈现突起、褶皱及叶边缘上卷的表型；下表皮细胞形态观察和叶表皮扫描电镜分析结果表明，过表达后植株叶片在单位面积内的细胞数量显著减少，而细胞面积显著增加；叶片石蜡切片结果表明，过表达后的叶片上表皮和下表皮细胞表面有明显突起的细胞组织；RNA-seq结果发现，大量与生长素途径及叶片发育相关的基因显著富集并差异表达；ClESR2突变后影响叶片形态，并且还与ClSAUR6存在蛋白水平互作。结论西瓜ClESR2是维持叶片发育的关键转录因子，可能通过影响生长素途径调控叶片形态。

关键词: 西瓜, ClESR2, 叶片形态, 转基因, 转录组分析, 生长素

Abstract:

Objective This study aimed to elucidate the function of the watermelon ERF transcription factor ClESR2 in regulating leaf morphological stability, providing a theoretical basis and candidate gene resource for understanding the regulatory mechanisms of leaf development. Method Transgenic lines of ClESR2 were obtained via Agrobacterium-mediated transformation. Leaf morphology of the transgenic lines was observed. Paraffin sectioning and scanning electron microscopy (SEM) were used to examine and quantify leaf cell morphology and number. RNA-seq was performed on leaves, and the expression levels of leaf development-related genes were validated by RT-qPCR. Additionally, auxin content in the leaves of transgenic lines was measured. Clesr2 mutant was generated using CRISPR/Cas9, and interacting proteins were screened and identified. Result Overexpression of ClESR2 led to leaves with protrusions, wrinkling, and upward-curling margins. Observation of lower epidermal cell morphology and SEM analysis revealed that the number of cells per unit leaf area significantly decreased, while cell area significantly increased. Paraffin sectioning showed that cells in both the upper and lower epidermis of overexpressing leaves displayed obvious protrusions. RNA-seq analysis revealed significant enrichment and differential expression of numerous genes related to the auxin pathway and leaf development. Mutation of ClESR2 affects leaf morphology, and ClESR2 interacts with ClSAUR6 at the protein level. Conclusion ClESR2 is a key transcription factor in watermelon that maintains leaf development and likely regulates leaf morphology through modulation of the auxin pathway.

Key words: watermelon, ClESR2, leaf morphology, transgene, transcriptome analysis, auxin

金维煜, 孟晗, 陈雅心, 康旗帅, 常佳佳, 李许真, 杨路明, 牛欢欢. 西瓜ClESR2在维持叶片形态稳定中的功能分析[J]. 生物技术通报, doi: 10.13560/j.cnki.biotech.bull.1985.2026-0243.

JIN Wei-yu, MENG Han, CHEN Ya-xin, KANG Qi-shuai, CHANG Jia-jia, LI Xu-zhen, YANG Lu-ming, NIU Huan-huan. Functional Analysis of ClESR2 in Maintaining Leaf Morphological Stability in Watermelon[J]. Biotechnology Bulletin, doi: 10.13560/j.cnki.biotech.bull.1985.2026-0243.

图/表 8

图1 ClESR2过表达植株鉴定及叶片形态观察A：过表达株系的荧光鉴定，标尺=2 mm；B：ClESR2表达量检测；C：植株表型观察，标尺=8 cm；D：相同节位叶片形态观察，标尺=4.5 cm。数据以平均值±标准差表示（n=3）

Fig. 1 Identification of ClESR2-overexpressing plants and observation of leaf morphologyA: Fluorescence identification of overexpression lines, bar=2 mm. B: Detection of ClESR2 expression level. C: Plant phenotypic observation, bar=8 cm. D: Leaf morphology at the same node position, bar=4.5 cm. Data are presented as mean ± standard deviation (n=3). **P<0.01. The same below

图2 ClESR2过表达植株叶片上表皮和下表皮细胞形态观察与统计分析A：WT与ClESR2过表达株系叶片下表皮细胞形态观察，标尺=20 μm；B：下表皮细胞数量；C：下表皮细胞大小；D：叶片上表皮细胞的扫描电镜观察，标尺=100 μm；E：上表皮细胞数量；F：上表皮细胞大小。数据以平均值±标准差表示（n=10）

Fig. 2 Morphological observation and statistical analysis of adaxial and abaxial epidermal cells in leaves of ClESR2-overexpressing plantsA: Morphological observation of abaxial epidermal cells in leaves of WT and ClESR2-overexpressing lines, bar=20 μm. B: Abaxial epidermal cell number. C: Cell size of abaxial epidermal cells. D: Scanning electron microscopy observation of adaxial epidermal cells in leaves, bar=100 μm. E: Adaxial epidermal cell number. F: Cell size of adaxial epidermal cells. Data are presented as mean ± standard deviation (n=10)

图3 ClESR2过表达植株叶片细胞组织切片观察与统计分析A：WT与ClESR2过表达株系叶片在甲苯胺蓝染色后石蜡切片结果，标尺=50 μm；B：WT与ClESR2过表达株系叶片近轴表皮细胞（ad-EC）和远轴表皮细胞（ab-EC）大小统计；PM：栅栏组织细胞；SM：海绵组织细胞；黄色：叶片近轴端；绿色：叶片远轴端。数据以平均值±标准差表示（n=10）

Fig. 3 Observation and statistical analysis of leaf tissue sections from ClESR2-overexpressing plantsA: Paraffin sections of leaves from WT and ClESR2-overexpressing lines stained with toluidine blue, Bar=50 μm. B: Cell size statistics of adaxial epidermal cells (ad-EC) and abaxial epidermal cells (ab-EC) in leaves of WT and ClESR2-overexpressing lines. PM: Palisade mesophyll cells. SM: Spongy mesophyll cells. Yellow: Adaxial side of leaf blade. Green: Abaxial side of leaf blade. Data are presented as mean ± standard deviation (n=10)

图4 ClESR2过表达植株叶片转录组测序分析A：PCA分析；B：火山图分析；C：KEGG分析；D：GO富集分析

Fig. 4 Transcriptome sequencing analysis of leaves from ClESR2-overexpressing plantsA: Principal component analysis (PCA). B: Volcano plot analysis. C: KEGG (Kyoto encyclopedia of genes and genomes) pathway analysis. D: GO (gene ontology) enrichment analysis

图5 ClESR2过表达植株叶片转录组中部分差异表达基因验证A：部分上调表达基因热图分析；B：部分上调表达基因RT-qPCR分析；C：部分下调表达基因热图分析；D：部分下调表达基因RT-qPCR分析。数据以平均值±标准差表示（n=3）

Fig. 5 Validation of selected differentially expressed genes in the transcriptome of leaves from ClESR2-overexpressing plantsA: Heatmap analysis of partially upregulated expressed genes. B: RT-qPCR analysis of partially upregulated expressed genes. C: Heatmap analysis of partially downregulated expressed genes. D: RT-qPCR analysis of partially downregulated expressed genes. Data are presented as mean±standard deviation (n=3)

图6 WT与ClESR2过表达株系叶片中生长素含量的检测

Fig. 6 Detection of auxin content in leaves of WT and ClESR2-overexpressing line

图7 ClESR2纯合编辑植株叶片形态观察A：ClESR2靶点位置及核苷酸序列；B：ClESR2突变形式鉴定；C：WT与ClESR2纯合编辑植株（Clesr2#6和Clesr2#8）田间定植30 d时的植株形态，标尺=12 cm；D：WT与ClESR2纯合编辑植株（Clesr2#6和Clesr2#8）相同节位侧枝形态，标尺=10 cm；E：对照植株与ClESR2纯合编辑植株相同节位叶片形态对比，标尺=6 cm

Fig. 7 Observation of leaf morphology in homozygous ClESR2-edited plantsA: Target site and nucleotide sequence of ClESR2. B: Identification of mutation types in ClESR2. C: Plant morphology of WT and homozygous ClESR2-edited plants (Clesr2#6 and Clesr2#8) at 30 days after field transplanting, bar=12 cm. D: Lateral branch morphology at the same node position of WT and homozygous ClESR2-edited plants (Clesr2#6 and Clesr2#8), bar=10 cm. E: Comparison of leaf morphology at the same node position between WT and homozygous ClESR2-edited plants, bar=6 cm

图8 ClESR2与ClSAUR6存在蛋白水平的互作A：酵母双杂交分析；DDO：二缺培养基（SD/-Leu/-Trp）；QDO：四缺培养基（SD/-Ade/-His/-Leu/-Trp/X-α-gal/30 mmol/L 3AT）；pGBKT7-53/pGADT7-T组合为阳性对照；pGBKT7-Lam/pGADT7-T组合为阴性对照；B：萤火虫荧光素酶互补成像分析

Fig. 8 Protein interaction between ClESR2 and ClSAUR6A: Yeast two‑hybrid assay. DDO indicates double dropout medium (SD/-Leu/-Trp). QDO indicates quadruple dropout medium (SD/-Ade/-His/-Leu/-Trp supplemented with X‑α‑gal and 30 mmol/L 3‑AT). The pGBKT7‑53/pGADT7‑T combination served as the positive control, and pGBKT7‑Lam/pGADT7‑T as the negative control. B: Firefly luciferase complementation imaging assay

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