Cloning and Enzyme Catalytic Analysis in vitro of Camellia fascicularis Phenylalanine Ammonia-Lyase Gene CfPAL
Wang Heze
Mu Dejin
Chen Sihui
Tian Zineng
Tang Junrong
Abstract：This study aimed to provide gene source and basis for flavonoid molecular breeding of Camelli-a fascicularis.The phenylalanine ammonia-lyase gene obtained from early transcriptome sequencing data was cloned and analyzed by bioinformatics methods,and its function was identified by in vitro enzyme catalytic as-say.The CfPAL gene was cloned from C.fascicularis with GenBank accession number of PP502845.The full-length cDNA of CfPAL was 2 145 bp,encoding a protein with 714 amino acids and molecular weight of 77.76 kDa.The expression preferences of CfPAL gene were A and T,and the optimal codon was AGG.It was a hy-drophilic stable acidic protein located in cytoplasm and endoplasmic reticulum.The secondary structure was mainly composed of α-helix and irregular curl,and the tertiary structure showed typical'sea horse'shape.CfPAL belonged to the lyase I-Like superfamily having the conserved sequence of PAL(GTITASGDLVPLSY-IAG)and the similarity of 90.21%to 9 homologous PAL proteins.It had the most close evolutionary relation-ship with C.sinensis and C.lanceoleosa with the identity of 99.02%and 98.19%,respectively.The CfPAL protein was associated with the key enzyme CHS in flavonoid biosynthesis pathway.In vitro enzymatic reaction showed that CfPAL could catalyze phenylalanine to produce trans-cinnamic acid,but could not catalyze tyro-sine to produce P-coumaric acid.Overall,CfPAL was a typical phenylalanine ammonia-lyase gene,and played important roles in regulating flavonoid synthesis.
Keywords：Camellia fascicularisPhenylalanine ammonia-lyaseGene cloningSequence analysisFunctional verification
Publication Date：2025-05-30
Online Publishing Date：2026-05-22(First online date of this platform, not the publication date of the document)
Pages：10( 54-63 )