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

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.