Cloning And Prokaryotic Expression Of Phenylalanine Ammonia-lyase Gene From Fagopyrum Dibotrys

Fagopyrum dibotrys, one of Chinese perennial herbs of the genus Fagopyrum in the family Polygonaceaea, has prominent functions in relieving fever and cough, which also has a function in anti-infection and reducing inflammation. Studies have shown that roots, stems and leaves of F. dibotrys are rich in flavonoids. Flavonoids are the medicinal composition of F. dibotrys and are synthesized in phenylpropanes metabolism pathway. Phenylalanine ammonia-lyase(PAL) is the key enzyme in the pathway and the connection between primary and secondary metabolism pathway. In our study, the gene of phenylalanine ammonia-lyase (FdPal) was cloned from F. dibotrys, and the recombinant protein was successfully expressed in Escherichia coli. Our study provided some reference for further study on the structure, function and secondary metabolism of PAL in F. dibotrys. The results were as followed:1. The full DNA and cDNA sequences of Pal were obtained by the method of polymerase chain reaction (PCR). Bioinformatic analysis showed the full DNA sequence was 2622 bp in length. There were two exons and one "GU-AG" intron. The first exon was from 1 to 419bp, and the second was from 834 to 2622bp. The coding region of cDNA was 2169bp in length containing an open reading frame and encoding 722 amino acids. The molecular weight was predicted to be 78.31kDa with an isoelectric point at pH5.94, and the PAL protein was supposed to be stable. Amino sequence of FdPAL was analysed with 13 other PAL sequences using Clustal X1.81. The result indicated that the phenylalanine ammonia-lyase family had two important conservative regions. However, there was a big difference at the N-terminal, especially the region of the first 29 amino acid residues. Neighbor-joining method was proposed for constructing phylogenetic tree, which indicated that FdPAL shared 99% identities with the PAL sequence in Fagopyrum esculentum and 94% in Fagopyrum tataricum, respectively. Identities of FdPAL shared with other species were between 80%-82%.2. The expression vector pET30b-FdPal was constructed, and the recombinant protein was successfully expressed in E. coli. SDS-PAGE analysis indicated that the molecular weight of the new protein was about 75.37kDa. Enzyme activity analysis showed that the highest specific activity of FdPAL was up to 4386 nmol/g-min after IPTG induction for 4h. Thin-layer chromatography analysis demonstrated that the expression product had the ability to transform phenylalanine into cinnamic acid.