Molecular Cloning Of A Phenylalanine Ammonia-lyase Gene From Rhus Chinensis And The Study On Its Function And Quality

Rhus chinensis belonging to Anacardiaceae, Rhus, a small deciduous tree, is a major economic tree species in China. This tree can be used to make pharmaceuticals, extract oil and serve as the raw materials of industrial dye. At present the researches of the Rhus chinensis were about its composition, medicinal value, morphological characteristics, cultivation method and technique, but little attention has been paid to its defense enzyme.PAL is the key enzyme that connects the plant primary metabolism and benzene propane metabolism, catalyzes the first step reaction of benzene propane metabolic, also is the key enzyme of phenylpropanoid pathway. It is directly related to plant resistance, and is a defense enzyme.This paper used Rhus chinensis as research material to clone its PAL gene, build a recombinant expression vector, obtain its recombinant protein by the prokaryotic expression, and further research its function and quality. The main results were as follows:1. The study got the total RNA of Rhus chinensis,28S rRNA and18S rRNA had clear stripe, and showed no degradation phenomenon, suggesting it can be used in the subsequent molecular cloning.2. The study acquired the ORF sequence of RcPAL for the first time. The full-length cDNA of RcPAL was2491bp, including2124bp open reading box, which encoded707amino acids. Its5’end noncoding region was197bp (5’UTR) in length, while the3’end noncoding region was170bp (3’UTR). The cDNA sequence of Re PAL was the first report in Anacardiaceae.3. The study built the recombinant expression vector pET-28a-PAL, with a length was7500bp and encoding a77kDa protein. We got the pure and high activity RcPAL enzyme by50%imidazole elution fluid.4. RcPAL’s optimum pH was9.0, the optimum temperature was45℃, and Ea was8.03kcal/mol. Its dynamics curve was hyperbolic. Its activity increased with increasing concentration of the substrate L-phenylalanine. Its Km was7.9mM, and Kcat52.31s-1.5. The study got three mutants, Phe126â†'His126(TTCâ†'CAC) Ser194â†'Trp194(TCGâ†'TGG). Asp374â†'Ala374(GATâ†'GCT). The activity of mutants M1, M2, M3is25%,2%,20%of CK, which proved that the three sites in catalytic reaction played an important role. Phe126â†'His126, mutant M1’s PAL activity was only25%of CK, but its TAL activity was22times as much as that of CK. So Phe126was important substrate specificity site.