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    宋慕波, 帅良, 段振华, 等. 荸荠苯丙氨酸解氨酶基因的克隆和表达[J]. 华南农业大学学报, 2019, 40(1): 77-83. DOI: 10.7671/j.issn.1001-411X.201804020
    引用本文: 宋慕波, 帅良, 段振华, 等. 荸荠苯丙氨酸解氨酶基因的克隆和表达[J]. 华南农业大学学报, 2019, 40(1): 77-83. DOI: 10.7671/j.issn.1001-411X.201804020
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    SONG Mubo, SHUAI Liang, DUAN Zhenhua, et al. Cloning and expression of phenylalanine ammonia-lyase (PAL) gene in Chinese water chestnut[J]. Journal of South China Agricultural University, 2019, 40(1): 77-83. DOI: 10.7671/j.issn.1001-411X.201804020
    Citation: SONG Mubo, SHUAI Liang, DUAN Zhenhua, et al. Cloning and expression of phenylalanine ammonia-lyase (PAL) gene in Chinese water chestnut[J]. Journal of South China Agricultural University, 2019, 40(1): 77-83. DOI: 10.7671/j.issn.1001-411X.201804020
    shu

    荸荠苯丙氨酸解氨酶基因的克隆和表达

    Cloning and expression of phenylalanine ammonia-lyase (PAL) gene in Chinese water chestnut

      摘要
    • 摘要:
      目的  克隆荸荠Eleocharis tuberosa苯丙氨酸解氨酶基因(PAL)，分析其序列特征及其在荸荠不同组织中和鲜切荸荠贮藏过程中的表达情况，为揭示鲜切荸荠黄化机理提供理论依据。
      方法  通过RT-PCR和RACE技术从荸荠中克隆PAL基因的cDNA全长，采用生物信息学方法对其序列和所编码的蛋白进行预测分析，利用荧光定量PCR技术分析PAL基因在荸荠不同组织和鲜切荸荠贮藏过程中的表达情况。
      结果  克隆得到荸荠PAL基因全长cDNA，将其命名为CwPAL，该序列长度为2 485 bp，含有1个2 142 bp的完整开放阅读框，共编码713个氨基酸。CwPAL蛋白分子式为C3437H5514N944O1058S34，相对分子质量为78 079，等电点为5.97，原子总数为10 987个。CwPAL包含PAL-HAL和PLN02457结构域及典型的PAL酶活性中心序列(GTITASGDLVPLSYIAG)。CwPAL的二级结构以α–螺旋为主，其三维结构模型呈典型的“海马状”结构。系统进化分析表明，CwPAL与菠萝Ananas comosus和海枣Phoenix dactylifera的PAL蛋白亲缘关系较近。荧光定量PCR分析表明，CwPAL基因在荸荠皮中的表达量最高，鲜切荸荠贮藏过程中CwPAL基因表达量快速上升，水杨酸处理显著抑制了CwPAL基因的表达。
      结论  CwPAL属于典型的苯丙氨酸解氨酶家族，该基因可能通过调控苯丙烷代谢从而影响鲜切荸荠的黄化。

       

      Abstract:
      Objective  To clone full-length cDNA of phenylalanin ammonia-lyase (PAL) in Chinese water chestnut (Eleocharis tuberosa), investigate its sequence characteristics, analyze its expressions in different organs and during storage of fresh-cut E. tuberosa, and provide theoretical references for revealing the mechanism of E. tuberosa etiolation.
      Method  The full sequence of PAL gene was cloned from E. tuberosa by RT-PCR and RACE techniques. Bioinformatics method was used to analyze the cDNA sequence and the encoded amino acid sequence. Real-time PCR was used to analyze the expression profile of PAL gene in different organs and in fresh-cut E. tuberosa during storage.
      Result  The full-length cDNA of PAL gene in E. tuberosa was cloned and named CwPAL. The sequence consists of 2 485 bp with an intact open reading frame of 2 142 bp, encoding a polypeptide of 713 amino acids. The formula of CwPAL protein is C3437H5514N944O1058S34, the relative molecular weight is 78 079, pI is 5.97, and the total number of atoms is 10 987. CwPAL protein contains two functional domains including PAL-HAL and PLN02457, and the typical PAL active site sequence (GTITASGDLVPLSYIAG). The main structural element in CwPAL secondary structure is α-Helix, the three-dimension structure of CwPAL protein shows a typical “sea horse” shape. The phylogenetic analysis showed that CwPAL was very closely related to PAL proteins of Ananas comosus and Phoenix dactylifera. The Q-PCR analysis showed that the expression level of CwPAL gene was the highest in peel, CwPAL gene expression quickly increased during storage of fresh-cut E. tuberosa, and salicylic acid treatment significantly inhibited CwPAL gene expression.
      Conclusion  The cloned CwPAL gene is a typical member of PAL family, and CwPAL gene may affect E. tuberosa etiolation by regulating the phenylpropanoid pathway.

       

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