Active Regulation Mechanism Study Of Phenylalanine Ammonia Lyase(FtPAL) In Tartary Buckwheat(Fagopyrum Tartarirum)

Phenylalanine ammonia lyase(PAL,EC 4.3.1.5)catalyzes the degradation of L-phenylalanine(L-Phe)amino group to form trans-cinnamic acid(t-CA).It is the first key enzyme and rate-limiting enzyme in the plant to connect primary metabolism and secondary metabolism of phenylpropanoids,and catalyze the metabolism of phenylpropanoids.Previous studies have focused on the PAL catalytic properties and transcriptional regulation in different plants,but little has been reported on the regulation of enzyme activity in cells.Studying the regulation mechanism of PAL activity of tartary buckwheat is important for elucidating the regulation mechanism of flavonoid metabolism pathway in tartary buckwheat and revealing the dynamic network of plant metabolism regulation.This study is based on the presence of high levels of secondary metabolites in tartary buckwheat leaves but the inability to detect PAL activity by classical methods.Based on the recombinant expression and purification of the tartary buckwheat FtPAL gene,the effects of the secondary metabolites of tartary buckwheat on PAL activity and the ubiquitination modification of tartary buckwheat were carried out under in vitro conditions.Mainly obtained the following results1.In this study,an unreported PAL gene was obtained by comparing the local buckwheat leaf transcriptome database and named FtPAL-N.In combination with the reported buckwheat PAL gene(FtPAL-B),prokaryotic expression vector and double gene co-expression vectors pET47b-PAL-N,pET47b-PAL-B and pRSF-duet1-PAL were constructed.Respectively,recombinant protein were successfully induced and purified.The crude extract of tartary buckwheat leaves and the effect of secondary metabolites of tartary buckwheat on PAL enzyme activity were determined under in vitro conditions.The effect of tetrameric enzyme homotypic/heterotetramer subunit association on enzyme activity was investigated.The results showed that the crude extract of tartary buckwheat leaves and the main secondary metabolites rutin,quercetin and caffeic acid had no inhibitory effect on the prokaryotic expression of tartary buckwheat FtPAL activity;the isoform/isotype subunit association of the enzyme had no significant effect on FtPAL activity.influences.2.PAL activity was detected in the crude extract of Tartary buckwheat leaves supplemented with the 26 S proteasome inhibitor MG132,and MG132 increased the activity of PAL in the crude extract of cotyledonary seedlings.By using Anti-FtPAL and Anti-Ub,It is proved that there is a high degree of ubiquitination modification in PAL in tartary buckwheat leaves.PAL in leaves is regulated by specific degradation of the ubiquitin-proteasome pathway.The enzyme activity analysis and Western Blotting results suggest that the existing classical PAL activity determination method needs to be corrected.3.A gene encoding the ubiquitin ligase(E3)gene(FtPUB26)was obtained by constructing the bait vector pGBKT7-FtPAL using yeast double hybrid.The interaction between PAL and FtPUB26 was verified by GST Pull-down and co-immunoprecipitation by constructing FtPUB26 prey vector pGADT7-FtPUB26 point-to-point yeast two-hybrid.The results showed a physical interaction between FtPAL and FtPUB26 protein.4.The tartary buckwheat ubiquitin activating enzyme gene(FtUBA1),ubiquitin-binding enzyme gene(FtUBC8)and ubiquitin gene(FtUb)were cloned.Expression vector pMAL-C4X-FtUBA1,pTWIN-1-FtUBC8 and pRSF-DUET1-FtUb were successfully constructed.The recombinant protein was purified by Amylose-Sepharose affinity column,chitin affinity column and cobalt ion chelate column,respectively,and its activity was verified by in vitro ubiquitination reaction.The results showed that the expression and purification of FtUBA1,FtUBC8 and FtUb proteins were achieved by prokaryotic expression system and affinity chromatography.The purification of the above proteins laid the foundation for the construction of PAL in vitro ubiquitination system.