title: [Molecular cloning and prokaryotic expression of phenylalanine ammonia-lyase gene FdPAL from Fagopyrum dibotrys].
abstract: OBJECTIVE
  To clone and characterize the DNA and cDNA sequences of phenylalanine ammonia-lyase gene (PAL) from Fagopyrum dibotrys, and investigate the biological activity of the obtained PAL.
  
  
  METHODS
  Using homology cloning and RT-PCR techniques, the DNA and full-length cDNA sequences of PAL gene were amplified from F. dibotrys. The obtained sequences were analyzed by bioinformatics software. The ORF of PAL gene was cloned into expression vector pET-30b(+) and transformed into Escherichia coli BL21 (DE3) for expression the recombined protein. The catalytic activity of the recombined protein was determined by Spectrophotometer and thin layer chromatography (TLC) methods.
  
  
  RESULTS
  The DNA sequence of PAL gene (designated as FdPAL, GenBank accession number: HM628904) was 2 583 bp in size, of which consisted two extrons and a single intron, and the full-length cDNA of FdPAL was 2 169 bp in size, which contained an ORF. The deduced protein of FdPAL contained 722 amino acids with calculated molecular weight (MW) of 78.31 kDa and an isoelectric point (pI) of 5.94. The SDS-PAGE results showed that the molecular weight of recombinant FdPAL protein was 75.37 kDa, which is consistent with the predictions. After 4 hours of induction, the enzymatic specific activity of FdPAL reached the summit, up to 4 386 nmol x g(-1) x min(-1). The reaction products were also identified by TLC, using L-Phe and trans-cinnamic acid as the internal standard.
  
  
  CONCLUSIONS
  The PAL gene (both DNA sequence and full-length cDNA sequence) was cloned from F. dibotrys, and it has the same classic characters as other PALs in plants. The recombinant FdPAL was efficiently expressed in E. coli and had the activity for catalyzing the conversion from L-phenylalanine to cinnamic acid.
authors.affiliation: College of Life Science, Sichuan Agricultural University, Ya'an 625014, China. lichenglei1998@yahoo.cn
authors.forename: Chenglei
authors.initials: C
authors.lastname: Li
authors.forename: Zhengyan
authors.initials: Z
authors.lastname: Feng
authors.forename: Yuechen
authors.initials: Y
authors.lastname: Bai
authors.forename: Hui
authors.initials: H
authors.lastname: Chen
authors.forename: Haixia
authors.initials: H
authors.lastname: Zhao
authors.forename: Qi
authors.initials: Q
authors.lastname: Wu
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D019612:Fagopyrum / Q000145:classification / Q000235:genetics*; D008969:Molecular Sequence Data; D010650:Phenylalanine Ammonia-Lyase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010802:Phylogeny; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D010650:Phenylalanine Ammonia-Lyase