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Molecular cloning, characterization and expression of the phenylalanine ammonia-lyase gene from Juglans regia - PubMed

  • ️Sun Jan 01 2012

Molecular cloning, characterization and expression of the phenylalanine ammonia-lyase gene from Juglans regia

Feng Xu et al. Molecules. 2012.

Abstract

Phenylalanine ammonia-lyase (PAL) is the first key enzyme of the phenypropanoid pathway. A full-length cDNA of PAL gene was isolated from Juglans regia for the first time, and designated as JrPAL. The full-length cDNA of the JrPAL gene contained a 1935bp open reading frame encoding a 645-amino-acid protein with a calculated molecular weight of about 70.4 kD and isoelectric point (pI) of 6.7. The deduced JrPAL protein showed high identities with other plant PALs. Molecular modeling of JrPAL showed that the 3D model of JrPAL was similar to that of PAL protein from Petroselinum crispum (PcPAL), implying that JrPAL may have similar functions with PcPAL. Phylogenetic tree analysis revealed that JrPAL shared the same evolutionary ancestor of other PALs and had a closer relationship with other angiosperm species. Transcription analysis revealed that JrPAL was expressed in all tested tissues including roots, stems, and leaves, with the highest transcription level being found in roots. Expression profiling analyses by real-time PCR revealed that JrPAL expression was induced by a variety of abiotic and biotic stresses, including UV-B, wounding, cold, abscisic acid and salicylic acid.

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Figures

Figure 1
Figure 1

Sequence multialignment of the deduced JrPAL protein with other PALs. The completely identical amino acids are indicated with white foreground and black background. The conserved amino acids are indicated with black foreground and background. Non-similar amino acids are indicated with black foreground and white background. The active sites residues are indicated in asterisk (*), and residues Ala-Ser-Gly forming MIO group are boxed in the alignment. AtPAL, PAL from A. thaliana (NP181241); BnPAL, PAL from Brassica napus (ABC69916); GbPAL, PAL from Ginkgo biloba (ABU49842); OsPAL, PAL from Oryza sativa (CAA34226); MsPAL, PAL from Medicago sativa (CAA41169); LePAL, PAL from Lycopersicon esulentum (AAA34179); NtPAL, PAL from N. tabacum (BAA22948).

Figure 2
Figure 2

The 3D structure of JrPAL established by homology-based modeling. The helix, sheet and random coil are indicated by the column, arrow plate and rope shape, respectively. The MIO and active site residues of JrPAL are shown.

Figure 3
Figure 3

The phylogenetic analysis of PALs from J. regia and other plant species by MEGA 4 from CLUSTAL W alignments. The neighbor-joining method was used to construct the tree with p-distance. The number for each interior branch is the percent bootstraps value (100 replicates).

Figure 4
Figure 4

Expression pattern of JrPAL in different tissues of J. regia including leaves, stems and roots. Each tissue sample was individually assayed in triplicate. Values shown represent the mean reading from three independent analyses and the error bars indicate the standard error of the mean.

Figure 5
Figure 5

Relative quantities of JrPAL mRNA at various time points post-treatment with UV-B (A), wounding (B),SA (C), Cold (D) and ABA (E). Total RNA was isolated from the one-year old J. regia seedling leaves under the treatments of UV-B, wounding, cold, SA and ABA for various durations. The J. regia 18S gene was used as control. Each plant was individually assayed in triplicate. Values shown represent the mean reading from three plants and the error bars indicate the standard error of the means.

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