Characterization and primary functional analysis of phenylalanine ammonia-lyase gene from Phyllostachys edulis
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Phenylalanine ammonia-lyase (PAL) catalyzes the first reaction in phenylpropanoid pathway leading to the production of phenolic compounds with a wide range of biological functions. The cDNA encoding PAL was isolated from Phyllostachys edulis by reverse transcription-polymerase chain reaction (RT-PCR) and by 5′ and 3′ rapid amplification of cDNA ends, and was designated as PePAL. The full length of PePAL was 2,503 bp which contained an open reading frame (ORF) encoding a peptide of 701 amino acids, with a theoretic isoelectric point of 6.49 and a calculated molecular mass of 75.7 kDa. PePAL was heterologously expressed in Escherichia coli and the recombinant proteins exhibited both PAL and tyrosine ammonia-lyase (TAL) activities. The optimum temperature and pH of the recombinant PePAL were 50 °C and 8.5–9.0, respectively. The Km and Vmax values for l-phenylalanine was calculated as 422 μM and 45.9 nM s−1, while for l-tyrosine were 78 μM and 7.09 nM s−1, respectively. Tissue-specific expression assay showed that PePAL expressed highest in stem and sheath, followed by leaf, and lowest in root. Though the accumulation of PePAL proteins was observed in all these four organs by Western blotting, the highest was detected in leaf. Immunohistochemistry study showed that PePAL was localized primarily in vascular bundles and part of sclerenchyma cells of leaf, sheath and root. These results suggested that PePAL had similar expression pattern and biochemical properties with PALs in other plants, which laid the basis for molecular engineering to improve the quality of bamboo products.
Key message PePAL was a protein with bifunctional enzyme activities of PAL and TAL as shown in vitro assays, and localized primarily in bamboo vascular bundles.
KeywordsPhyllostachys edulis Phenylalanine ammonia-lyase Expression analysis Enzymatic assay in vitro Immunohistochemistry
Ethylene diamine tetraacetie acid
Open reading frame
Reverse transcription-polymerase chain reaction
Rapid amplification of cDNA ends
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
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