| 袁 晓 1,2，杨盼迪 1，王 斌 1.AOS1 基因克隆及其在鲜切芋头褐变中的表达模式分析[J].广东农业科学,2023,50(9):198-206 |
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AOS1 基因克隆及其在鲜切芋头褐变中的表达模式分析 |
| Analysis on Cloning of AOS1 Gene and Its Expression Pattern During the Browning Process in Fresh-cut Taros |
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| DOI：10.16768/j.issn.1004-874X.2023.09.021 |
| 中文关键词: 鲜切芋头 褐变 丙二烯氧化物合成酶 基因克隆 表达分析 |
| 英文关键词: fresh-cut taro browning allene oxide synthase gene cloning expression analysis |
| 基金项目:广东省粤北食药资源利用与保护重点实验室开放基金（FMR2022001M）；广东大学生科技创新培育专项资金（大学生科技创新培育）项目（pdjh2023a0470） |
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| 中文摘要: |
| 【目的】鲜切芋头即使在低温条件下贮藏，贮藏期间仍会出现变黄变褐的现象，严重影响鲜切芋头的商品性。从鲜切芋头中克隆丙二烯氧化物合成酶（Allene oxide synthase, AOS）基因 AOS1，并分析其表达与鲜切芋头褐变的关系，鉴定鲜切芋头褐变过程中的重要功能基因，为鲜切芋头褐变机制研究提供参考。【方法】以鲜切芋头 cDNA 为模板，采用 RT-PCR 法克隆 AOS1 基因编码序列（Coding sequence, CDS）；采用荧光定量 PCR 法分析鲜切芋头褐变过程中 AOS1 表达情况，利用转录组测序数据分析外源褐变抑制剂处理对 AOS1表达的影响，并分析 AOS1 启动子序列。【结果】20 ℃条件下，鲜切芋头贮藏 24 h 切面出现明显的变黄变褐症状，而 4 ℃下贮藏 12 d 才出现明显的褐变症状，表明贮藏温度是影响鲜切芋头褐变进程的重要因素，低温贮藏可有效抑制鲜切芋头褐变。芋头 AOS1 基因的 CDS 全长为 1 560 bp，编码 519 个氨基酸残基；AOS1 基因编码蛋白质的分子量为 57.63 kD，等电点为 8.68，定位在叶绿体。植物 AOS1 蛋白序列相似性较低，芋头 AOS1 与番茄AOS1、拟南芥 AOS1 蛋白的亲缘较远，但天南星科植物 AOS1 蛋白的序列同源性较高。芋头 AOS1 启动子中含有许多植物激素和逆境响应元件，AOS1 表达在鲜切处理后显著上调，表明切分处理诱导了 AOS1 的表达。随着鲜切芋头褐变进展，AOS1 表达量逐渐增加，外源乙醇酸和香草酸处理 AOS1 表达显著下调，表明 AOS1 表达与鲜切芋头褐变有关。【结论】鲜切芋头褐变与 AOS1 表达具有明显正相关性，切分去皮等处理可能通过诱导 AOS1表达促进鲜切芋头褐变。 |
| 英文摘要: |
| 【Objective】Fresh-cut taro is prone to turning yellow and brown during storage, even stored at low temperature, which greatly diminishes its commercial values. In order to identify the important functional genes involved in the browning process of fresh-cut taro, the allene oxide synthase (AOS) gene AOS1 was cloned from fresh-cut taros. The correlation between the expression of AOS1 and the browning development of fresh-cut taro was analyzed, with a view to offering references for the study of taro browning mechanism.【Method】With cDNA of fresh-cut taro as a template, the coding
sequence (CDS) of AOS1 gene was cloned by the RT-PCR method. The expression patterns of AOS1 during the browning of fresh-cut taro were analyzed by using qRT-PCR, the impact of exogenous browning inhibitors on AOS1 expression was examined by using transcriptome sequencing data, and the promoter subsequence was analyzed.【Result】Fresh-cut taros showed obvious yellowing and browning symptoms on the cut surface after 24 hours of storage at 20 ℃, while the obvious browning symptoms were occurred after 12 d of cold storage at 4 ℃. These results suggested that storage temperature was a crucial factor influencing the browning process of fresh-cut taros, with low temperature storage effectively inhibiting the browning. The CDS of taro AOS1 gene had a total length of 1 560 bp and encoded 519 amino acid residues. The molecular weight of the protein encoded by AOS1 gene was 57.63 kD, with an isoelectric point of 8.68, and located in chloroplast. The similarity of AOS1 protein sequences across different plant species was relatively low, with notable differences observed between taro, tomato, and Arabidopsis. However, high sequence homology was found among AOS1 proteins in Araceae family plants. The promoter region of taro AOS1 contained various plant hormone and stress response elements. The expression of AOS1 significantly increased after fresh-cut processing, indicating that the cutting treatment induced AOS1 expression. Moreover, as the browning of fresh-cut taro progressed, the expression of AOS1 gradually increased. Applications of exogenous glycolic acid and vanillic acid significantly decreased the expression of AOS1 in fresh-cut taros, showing its correlation with the browning process.【Conclusion】A significant positive correlation was observed between the browning of fresh-cut taros and the expression of AOS1 during cold storage. Treatments such as cutting and peeling may accelerate the browning process by inducing AOS1 expression. |
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