| 刘 明，刘海峰，周 慧，何焕清，肖自添，罗学梅.草菇多酚氧化酶基因的克隆与表达分析[J].广东农业科学,2021,48(9):91-97 |
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草菇多酚氧化酶基因的克隆与表达分析 |
| Cloning and Expression Analysis of Polyphenol Oxidase Gene Family in Volvariella volvacea |
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| DOI：10.16768/j.issn.1004-874X.2021.09.010 |
| 中文关键词: 草菇 多酚氧化酶 褐变 表达分析 基因编码 |
| 英文关键词: Volvariella volvacea polyphenol oxidase browning expression analysis gene encoding |
| 基金项目:广 东 省 重 点 领 域 研 发 计 划 项 目（2018B020206001）； 广 州 市 科 技 计 划 项 目（201803020023，201904010408）；广东省农业科学院院长基金（201840） |
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| 中文摘要: |
| 【目的】解析草菇多酚氧化酶基因家族序列信息，明确其特征和表达模式。【方法】通过转录组测序找出含有酪氨酸酶结构域的基因，并根据搜索得到的序列设计引物，以不同发育时期的草菇 v26 菌株，以及草菇蛋形期外菌膜、菌盖、菌柄等不同组织为材料提取 RNA，经反转录为 cDNA，扩增获得草菇多酚氧化酶基因序列。采用在线生物信息学网站预测草菇多酚氧化酶的理化性质。采用 RT-PCR 的方法，检测草菇不同发育时期和不同组织部位多酚氧化酶的表达模式。【结果】获得 4 个草菇多酚氧化酶基因家族的 CDS 序列，依次命名为 VvPPO1、VvPPO2、VvPPO3、VvPPO4，其中 VvPPO1 CDS 全长 1 596 bp、编码 531 个氨基酸，VvPPO2 CDS 全长 2 685 bp、编码 894 个氨基酸，VvPPO3 CDS 全长 1 593 bp、编码 530 个氨基酸，VvPPO4 CDS 全长2 067 bp、编码 688 个氨基酸。生物信息学分析表明，4 个基因编码的蛋白均含有 2 个铜离子结合区，为亲水性蛋白；RT-PCR 结果显示，4 个基因在草菇发育的菌丝期、原基期、蛋形期、成熟期均有表达，VvPPO1、VvPPO3 和VvPPO4 的表达量在子实体时期高于菌丝期，在蛋形期 VvPPO1 和 VvPPO4 在外菌膜的表达量比在菌柄和菌盖中的表达量高。【结论】在草菇中搜索得到 4 个多酚氧化酶基因，均含有 2 个铜离子结合区，4 个基因在各个发育时期均有表达。 |
| 英文摘要: |
| 【Objective】The study is presented to obtain the sequence information of polyphenol oxidase gene family in Volvariella volvacea, and identify their characteristics and expression patterns.【Method】The genes containing tyro sinase domains were found out through transcriptome sequencing, and their primers were designed according to the obtained sequences. By using the tissues at different developmental stages of the v26 strain, as well as the outer membrane,
cap, and stipe of the egg-shaped stage as materials, RNA was extracted and reversely transcribed into cDNA. The cDNAs were used as the template to amplify the polyphenol oxidase gene sequences. The online bioinformatics website was used to predict the physicochemical properties of V. volvacea polyphenol oxidase. The RT-PCR method was used to detect the expression patterns of polyphenol oxidase in different developmental stages and different tissues.【Result】A total of four CDS sequences of PPO genes were cloned from V. volvacea, named as VvPPO1, VvPPO2, VvPPO3 and VvPPO4. The VvPPO1, VvPPO2, VvPPO3 and VvPPO4 CDSs were 1 596 bp, 2 685 bp, 1 593 bp and 2 067 bp in length and encoded 531, 894, 530 and 688 amino acids, respectively. Bioinformatics analysis showed that the proteins encoded by the four genes were hydrophilic proteins. VvPPOs had typical characteristics of PPO protein, holding a tyrosinase central domain which could bind two copper ions. RT-PCR results showed that the 4 genes were expressed in the hyphae, primordium, egg-shaped and mature stages. The expression of VvPPO1, VvPPO3 and VvPPO4 in the fruiting body were higher than that in the hyphae. In the egg-shaped stage, the expression of VvPPO1 and VvPPO4 in the outer membrane were higher than that in the stipe and cap.【Conclusion】A total of four VvPPO genes were obtained in V. volvacea, all of which contained a tyrosinase central domain and two copper-binding sites, and VvPPO genes were expressed in all developmental stages. |
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