Recombinant Expression And Polyclonal Antibody Preparation Of Phenylalanine Ammonia Lyase And Anthocyanin Reductase From Tartary Buckwheat

Flavonoids,as the important active ingredients of buckwheat(Fagopyrum tataricum)have more biological functions including anti-free radical,anti-oxidation,anti-cancer,lowering blood pressure and so on,which play a role on improving people’s health.Cloning key genes and studying their functions involved in flavonoid synthesis is of important theoretical significance for the cultivation of high flavonoid buckwheat.In most plants,the flavonoid biosynthetic pathway has been nearly completed and the content proven to be regulated by related genes.we studied the two genes involved Fagopyrum tataricum flavonoid synthetic way: phenylalanine ammonia lyase(PAL) and anthocyanin reductase(ANR).PAL is the first and rate-limiting enzyme in metabolic pathways,providing the precursors“t-Cat”for the formation of flavonoid synthesis;ANR catalyzes the anthocyanins into epicatechin—one monomer of proanthocyanidins.The following work was done:(1) The complete coding cDNA sequence of PAL and ANR was amplified from Tataricum “Yu-621”seed mRNA by reverse transcription polymerase chain reaction(RT-PCR).Bioinformatics analysis revealed that the ORF of FtPAL gene was 2166 bp and encoded 722 amino acids.The sequence alignment showed that the target sequence had the high similarity of 99%, 98%, 97% with buckwheat,Fagopyrum dibotrys and common buckwheat.The FtANR contained a 1014 bp ORF and encoded a protein of 377 amino acids.Further analysis indicated that the sequence had 75~80% homology with ANR from Grapes,cotton,tea and other plants.So the PAL and ANR gene were cloned successfully from Tataricum.(2) Semi-quantitative RT-PCR was performed to determine the expression profiling of these two genes in different tissues from buckwheat.Judging from their expression characters,FtPAL and FtANR had some expression in seed,stem,leaf and flower.Ft PAL had a highest expression in the stem,next to leaf and flower and the lowest in the seed.FtANR gene expressed lower in the stem than seed,but it was higher expressed in leaf and flower.(3) Two prokaryotic expression vector pET47b-PAL and pET28a-ANR were transformed into BL21 Star(DE3) and then induced to express the target protein.The results showed that the two fusion proteins expressed in two forms of soluble and inclusions under the induction of 1mM IPTG at 25 ℃.Futhermore the respective polyclonal antibody were obtained using FtPAL and FtANR which were isolated and purified by cobalt ion chelating chromatography.Western blotting analysis showed the raised antibody could specifically bind with the antigen FtPAL and FtANR.(4) Enzymatic properties analysis indicated that the optimum temperature and pH for the purified recombinant protein FtPAL activity was 60 ℃ and 8.2 seperately.Its kinetic parameters Km was 0.172 mM /L, Vmax was 17.9U / mg.