| 张明先 1，张芮豪 1,2，李平平 1，吴 睿 1，周慧丹 1，邓明华 1，吕俊恒 1.辣椒 CMB1 基因克隆与表达分析[J].广东农业科学,2023,50(11):50-58 |
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辣椒 CMB1 基因克隆与表达分析 |
| Cloning and Expression Analysis of CMB1 Gene in Pepper |
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| DOI：10.16768/j.issn.1004-874X.2023.11.005 |
| 中文关键词: 辣椒 类胡萝卜素 转录因子 基因克隆 CMB1 |
| 英文关键词: pepper carotenoid transcription factor gene cloning CMB1 |
| 基金项目:国家自然科学基金（32360744）；云南省生物种业与精深加工重大专项（202202AE090031) |
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| 中文摘要: |
| 【目的】CMB1 基因在植物的花序结构和果实成熟调控中发挥重要作用，但有关辣椒 CMB1 基因的功能和调控机制鲜有报道，因此研究辣椒中调控类胡萝卜素合成相关的转录因子及 CMB1 基因在辣椒中的作用，为辣椒选育提供参考。【方法】以皱皮红、皱皮黄两种辣椒果实为试验材料，利用同源序列法克隆 CMB1的编码序列并对其进行生物学信息分析，通过实时荧光定量 PCR 技术分析 CMB1 在红色与黄色辣椒果实中不同发育时期（绿熟期、转色期、完熟期）的表达模式。【结果】皱皮红与皱皮黄辣椒的 CMB1 基因 ORF 长度均为732 bp，可编码 243 个氨基酸残基。序列比对发现，皱皮红 CMB1 碱基序列的第 11 个位点发生突变，G 突变为T，导致氨基酸序列由 G（甘氨酸）突变为 V（缬氨酸）。生物信息学分析显示，皱皮红 CMB1 编码蛋白的分子质量为 20 923.21，理论等电点为 5.59，不稳定系数为 57.28，亲水性总平均值（重力）指数为 -1.001；皱皮黄CMB1 编码蛋白的分子质量为 27 958.47，理论等电点为 7.10，不稳定系数为 53.54，亲水性总平均值（重力）指数为 -0.809。皱皮红和皱皮黄的 CMB1 均为不稳定的疏水蛋白，含有 38 个磷酸化位点，无跨膜结构和信号肽，具有典型的 MADS-box 结构域和 K-box 结构域。进化分析结果发现，辣椒 CMB1 与茄科的亲缘关系最近，与豆科亲缘关系最远。荧光定量结果显示，CMB1 在辣椒果实绿熟期、转色期、完熟期 3 个时期的表达量依次递增，且 3 个时期 CMB1 的表达量在皱皮红中均显著高于皱皮黄。【结论】辣椒 CMB1 是 MADS-box 家族的成员，为不稳定的疏水蛋白，在辣椒果实中的表达量与辣椒中类胡萝卜素的积累趋势相似，推测 CMB1 主要参与调控辣椒红素等类胡萝卜素的合成。 |
| 英文摘要: |
| 【Objective】CMB1 gene plays an important role in the regulation of inflorescence structure and fruit ripening in plants, but the function and regulatory mechanism of CMB1 gene in pepper have been rarely reported. Therefore, the role of transcription factors related to the regulation of carotenoid synthesis and CMB1 gene in pepper was studied so as to provide a reference for pepper breeding.【Method】The coding sequences of CMB1 were cloned by the homology sequence method, and the expression patterns of CMB1 in fruits of red and yellow peppers with wrinkled skin at different development stages (green ripening stage, color change stage, mature stage) were analyzed by real-time quantitative PCR.【Result】The ORF length of CMB1 gene was 732 bp in both red pepper and yellow pepper, encoding 243 amino acid residues. Sequence alignment showed that the mutation occurred at the 11th site of CMB1 base sequence, G mutated to T, resulting in amino acid sequence mutated from G (Glycine) to V (Valine). Bioinformatics analysis revealed that the CMB1-encoded protein of wrinkled skin red pepper had a molecular mass of 20 923.21, a theoretical isoelectric point of 5.59, an instability coefficient of 57.28, and a total mean hydrophilicity (gravimetric) index of -1.001. The CMB1-encoded protein of wrinkled skin yellow pepper had a molecular mass of 27 958.47, a theoretical isoelectric point of 7.10, an instability coefficient of 53.54, and a total mean hydrophilicity (gravimetric) index of -0.809. Both CMB1 of wrinkled skin red and yellow peppers were unstable hydrophobic proteins, both contained 38 phosphorylation sites, both were devoid of transmembrane structures and signal peptides, and both had typical MADS-box structural domains and K-box structural domains. Evolutionary tree
analysis revealed that pepper CMB1 was most closely related to Solanaceae and most distantly related to Leguminosae. The results of fluorescence quantitative PCR showed that the expression of CMB1 increased sequentially in the three stages of green ripening, color change, and maturity of pepper fruits, and the expression of CMB1 in the three stages was significantly higher in wrinkled skin red pepper than in wrinkled skin yellow pepper.【Conclusion】Pepper CMB1 is a member of MADS-box, and its expression in pepper fruit is similar to that of carotenoid in pepper, suggesting that CMB1 is mainly involved in regulating the synthesis of carotenoid like capsanthin. |
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