||Eucalyptus robusta におけるフェニルアラニンアンモニアリアーゼ遺伝子の解析
Analysis of the Phenylalanine Ammonia-Lyase Gene in Eucalyptus robusta
植木, 悠貴 ,
秋元, 真也 ,
寺田, 珠実 ,
鮫島, 正浩鴨田, 重裕
43 , 2016-08-23 , 東京大学大学院農学生命科学研究科附属演習林
Phenylalanine ammonia-lyase (PAL) is the first enzyme of phenolics (secondary metabolites) biosynthesis. So it may play an important role during growth and in the defense mechanism in eucalypts. Using PCR, TA cloning, and protein expression technologies, five of the PAL-like genes were cloned from genomic DNA of Eucalyptus robusta (Er) suspension cultured cells, and all new genes. These base sequences consisted of an intron (various sizes) and two exons (same sizes, first: 395 bp + second: 1750 bp = 2145 bp). This was a typical characteristic in plant PAL genes. Also four of the PAL-like genes were cloned from synthesized cDNA using mRNA of Er cells. The full-length sequences contained 2145bp ORF encoding 714 amino-acid polypeptides. Sequence alignment indicated nine PAL-like genes shared more than 80% identity with ErPAL1, while these nine sequences possessed 96-99% and very high homology with each other. Predicted PAL-like sequences contained active site motives and some well conserved amino-acid residues. Although new recombinant ErPAL proteins were not purified this time, it was possible to confirm coarse enzyme activities. Further consideration is necessary for enzyme purification and functional analysis.