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Identification and Functional Characterization of Three Phenylalanine Ammonia-Lyase Genes from Fallopia multiflora (Thunb.) Harald.

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Abstract

Fallopia multiflora (Thunb.) Harald. is a commonly used traditional Chinese medicine that is rich in chemical constituents. Phenylalanine ammonia-lyase (PAL) is the first key enzyme in the phenylalanine pathway, catalyzing the deamination of L-phenylalanine to generate trans-cinnamic acid. Three PALs (FmPAL1, FmPAL2, and FmPAL3) were cloned and validated from F. multiflora for the first time, demonstrated the presence of a multigene family of PALs in F. multiflora. The predicted complete open reading frames (ORFs) of FmPAL1, FmPAL2, and FmPAL3 are 2118bp, 2109bp and 2160bp, encoding 705, 702 and 719 amino acids, respectively. The phylogenetic results indicated that FmPALs have significant evolutionary relatedness with the known PALs from dicotyledons. To further confirm their function, the three FmPALs were cloned into the pET-28a vector and expressed in Escherichia coli TransBL21 (DE3). The enzymatic activities of the FmPALs recombinant proteins were assayed and showed that FmPALs have a role in the d-catalyzed conversion of L-phenylalanine to trans-cinnamic acid. All three FmPALs showed tissue-specific expression. The expression of FmPAL1 and FmPAL2 genes were highest in stems and lowest in roots. Differently, the expression of FmPAL3 gene was highest in leaves and lowest in roots.

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Funding

This work was supported by the National Natural Science Foundation of China (no. 81973432), the Major Increase and Reduction Project at the Central Level “The Ability Establishment for Sustainable Utilization of Valuable Chinese Medicine Resources” (no. 2060302), the Research Unit of DAO-DI Herbs, Chinese Academy of Medical Sciences (no. 2019RU57), the Anhui University of Chinese Medicine Talent Support Program (2022rcyb018) and Innovation Team and Talents Cultivation Program of National Administration of Traditional Chinese Medicine (no. ZYYCXTD-D-202005).

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Authors and Affiliations

  1. School of Pharmacy, Anhui University of Chinese Medicine, 230012, Hefei, China

    Zhengyang Yang, Minzhen Yin, Mingen Cheng, Shanshan Chu, Liangping Zha, Yujiao Zhao & Huasheng Peng

  2. National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, 100700, Beijing, China

    Huasheng Peng

  3. Anhui Province Key Laboratory of Research and Development of Chinese Medicine, 230012, Hefei, China

    Mingen Cheng, Shanshan Chu, Liangping Zha & Yujiao Zhao

  4. Research Unit of DAO-DI Herbs, Chinese Academy of Medical Sciences, 2019RU57, Beijing, 100700, China

    Huasheng Peng

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  1. Zhengyang Yang
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Correspondence to Yujiao Zhao or Huasheng Peng.

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The authors declare that they have no conflicts of interest. This article does not contain any studies with human participants performed by any of the authors.

Additional information

Abbreviations: PAL, Phenylalanine ammonia-lyase; PCR, Polymerase Chain Reaction; RT-PCR, Reverse Transcription-Polymerase Chain Reaction; LB, Luria-Bertani; SDS-PAGE, Sodium Dodecyl Sulphate-polyacrylamide gel electrophoresis; ORF, Open Reading Frame.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1134/S1068162023030263.

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Zhengyang Yang, Yin, M., Cheng, M. et al. Identification and Functional Characterization of Three Phenylalanine Ammonia-Lyase Genes from Fallopia multiflora (Thunb.) Harald.. Russ J Bioorg Chem 49, 655–663 (2023). https://doi.org/10.1134/S1068162023030263

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