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Measuring Gene Expression in Bombarded Barley Aleurone Layers with Increased Throughput
Published on: March 30, 2018
Molecular characterization of a phenylalanine ammonia-lyase gene (BoPAL1) from Bambusa oldhamii
Lu-Sheng Hsieh1, Yi-Lin Hsieh, Chuan-Shan Yeh
1Institute of Microbiology and Biochemistry, Department of Biochemical Science and Technology, College of Life Science, National Taiwan University, 1 Roosevelt Road Section 4, Taipei, 106, Taiwan, ROC.
Abstract:
Phenylalanine ammonia-lyase is the first enzyme of general phenylpropanoid pathway. A PAL gene, designated as BoPAL1, was cloned from a Bambusa oldhamii cDNA library. The open reading frame of BoPAL1 was 2,139 bp in size and predicted to encode a 712-amino acid polypeptide. BoPAL1 was the first intronless PAL gene found in angiosperm plant. Several putative cis-acting elements such as P box, GT-1motif, and SOLIPs involved in light responsiveness were found in the 5'-flanking sequence of BoPAL1 which was obtained by TAIL-PCR method. Recombinant BoPAL1 protein expressed in Pichia pastoris was active. The optimum temperature and pH for BoPAL1 activity was 50°C and 9.0, respectively. The molecular mass of recombinant BoPAL1 was estimated as 323 kDa using gel filtration chromatography and the molecular mass of full-length BoPAL was about 80 kDa, indicating that BoPAL1 presents as a homotetramer. The Km and kcat values of BoPAL1 for L-Phe were 1.01 mM and 10.11 s(-1), respectively. The recombinant protein had similar biochemical properties with PALs reported in other plants.
