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Cloning, Characterization and Expression of the Phenylalanine Ammonia-Lyase Gene (PaPAL) from Spruce Picea asperata
Phenylalanine ammonia-lyase (PAL) is the crucial enzyme of the phenylpropanoid pathway, which plays an important role in plant disease resistance. To understand the function of PAL in Picea asperata, in this study, the full-length cDNA sequence of the PAL gene from this species was isolated and named PaPAL. The gene contains a 2160-bp open reading frame (ORF) encoding 720 amino acids with a calculated molecular weight of 78.7 kDa and a theoretical isoelectric point of 5.88. The deduced PaPAL protein possesses the specific signature motif (GTITASGDLVPLSYIA) of phenylalanine ammonia-lyases. Multiple alignment analysis revealed that PaPAL has high identity with other plant PALs. The tertiary structure of PaPAL was predicted using PcPAL from Petroselinum crispum as a template, and the results suggested that PaPAL may have a similar function to that of PcPAL. Furthermore, phylogenetic analysis indicated that PaPAL has a close relationship with other PALs from the Pinaceae species. The optimal expression condition of recombinant PaPAL in Escherichia coli BL21 (DE3) was 0.2 mM IPTG (isopropyl β-D-thiogalactoside) at 16 °C for 4 h, and the molecular weight of recombinant PaPAL was found to be approximately 82 kDa. Recombinant PaPAL was purified and exhibited high PAL activity at optimal conditions of pH 8.6 and 60 °C. Quantitative real-time PCR (qRT-PCR) showed the PaPAL gene to be expressed in all tissues of P. asperata tested, with the highest expression level in the needles. The PaPAL gene was induced by the pathogen (Lophodermium piceae), which caused needle cast disease, indicating that it might be involved in defense against needle cast disease. These results provide a basis for understanding the molecular mechanisms of the PAL gene in the process of P. asperata disease resistance.
Cloning, Characterization and Expression of the Phenylalanine Ammonia-Lyase Gene (PaPAL) from Spruce Picea asperata
Phenylalanine ammonia-lyase (PAL) is the crucial enzyme of the phenylpropanoid pathway, which plays an important role in plant disease resistance. To understand the function of PAL in Picea asperata, in this study, the full-length cDNA sequence of the PAL gene from this species was isolated and named PaPAL. The gene contains a 2160-bp open reading frame (ORF) encoding 720 amino acids with a calculated molecular weight of 78.7 kDa and a theoretical isoelectric point of 5.88. The deduced PaPAL protein possesses the specific signature motif (GTITASGDLVPLSYIA) of phenylalanine ammonia-lyases. Multiple alignment analysis revealed that PaPAL has high identity with other plant PALs. The tertiary structure of PaPAL was predicted using PcPAL from Petroselinum crispum as a template, and the results suggested that PaPAL may have a similar function to that of PcPAL. Furthermore, phylogenetic analysis indicated that PaPAL has a close relationship with other PALs from the Pinaceae species. The optimal expression condition of recombinant PaPAL in Escherichia coli BL21 (DE3) was 0.2 mM IPTG (isopropyl β-D-thiogalactoside) at 16 °C for 4 h, and the molecular weight of recombinant PaPAL was found to be approximately 82 kDa. Recombinant PaPAL was purified and exhibited high PAL activity at optimal conditions of pH 8.6 and 60 °C. Quantitative real-time PCR (qRT-PCR) showed the PaPAL gene to be expressed in all tissues of P. asperata tested, with the highest expression level in the needles. The PaPAL gene was induced by the pathogen (Lophodermium piceae), which caused needle cast disease, indicating that it might be involved in defense against needle cast disease. These results provide a basis for understanding the molecular mechanisms of the PAL gene in the process of P. asperata disease resistance.
Cloning, Characterization and Expression of the Phenylalanine Ammonia-Lyase Gene (PaPAL) from Spruce Picea asperata
Yufeng Liu (author) / Lijuan Liu (author) / Shuai Yang (author) / Qian Zeng (author) / Zhiran He (author) / Yinggao Liu (author)
2019
Article (Journal)
Electronic Resource
Unknown
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