Cloning And Characterization Of Genes Involved In Tyrosine-branched Pathway Of Rosmarinic Acid Biosynthesis From Prunella Vulgaris L.

Prunella vulgaris L.,a perennial labiaceae plant,is one of the most popular traditional Chinese medicinal plants and gets its name because of withering after “summer solstice”.Its dry spikes are used as the medicinal materials.It is well known as “self-heal” or “heal-all”.Its property is spicy,bitter,and cold.It goes to the liver and gall bladder channels.Its main ingredients include rosmarinic acid(RA),caffeic acid,ursolic acid,oleanolic acid,flavonoids,cyanidin,and volatile oil.It has a wide application as a remedy for alleviating sore throats,reducing fever,and accelerating wound healing.RA is selected as the marker component for the quality control of P.vulgaris in 2015 Chinese Pharmacopeia,which stipulated that RA content should not be less than 0.2% in qualified medicinal materials.However,RA content in the intact P.vulgaris plant is relatively low compared with other members of Lamiaceae plants.Moreover,little information about enzymes involved in tyrosine-branched pathway is available in P.vulgaris.It is particularly important to increase the yield of RA of P.vulgaris using metabolic engineering methods.Here,we isolated and characterized a tyrosine aminotransferase gene(PvTAT,KM053278)and a hydroxyphenylpyruvate reductase gene(PvHPPR,KM053279)using homology-based cloning.On the basis of bioinformatics,we analyzed their tissue expreesion and elicitation expression,determined their subcellular localization,characterized the heterologously overexpressed protein in Escherichia coli and assayed the enzymatic activity in vitro,and showed PvTAT functional complementation for tyrosine auxotrophic mutant DL39 in vivo.Moreover,gene expression,enzyme activity,and RA accumulation were determined in Agrobacterium rhizogenes-mediated transgenic hairy root lines with PvTAT and PvHPPR overexpression or antisense expression.The main findings are shown below:1.The PvTAT cDNA contained a 1,236-bp ORF and encoded a predicted translation product of 411 amino acids with a molecular mass of 45.1 k Da and an isoelectric point of 5.97.The gene had a sequence of 2,634-bp with seven extrons and six introns.Based on the conserved domain,PvTAT belongs to PLP-dependent Asp aminotransferase superfamily(AAT-like proteins)and also belongs to the tyrosine aminotransferase(TAT)subfamily according to the Panther classification system.The PvHPPR cDNA contained a 942-bp ORF and encoded a predicted translation product of 313 amino acids with a molecular mass of 34.1 kDa and an isoelectric point of 5.66.Interestingly,there are two gene sequences containing 2,215-bp and 1,718-bp,each one has two exons and one intron,while the intron of the big fragment is 497-bp more than that of the small one.Using genome walking,we obtained a 2,273-bp sequence of the upstream of PvHPPR,which has motifs involved with light,salicylic acid,environmental stress responses,growth and development,and primary metabolism.The conserved domain has a typical NAD(P)H-binding domain and catalytic sites(arginine,glutamate,and histidine),belonging to the D-isomer-specific 2-hydroxyacid dehydrogenase family.2.qRT-PCR analysis of PvTAT and Pv HPPR at the complete flowering stage showed that the highest transcript abundances were observed in root tissues,followed by leaves.Both of them were regulated by exogenous ethephon and salicylic acid.3.By fusing GFP to the C-terminal of PvTAT and PvHPPR,we transiently expressed them in tobacco.Using confocal microscopy,cytoplasmic localization of PvTAT was observed in the tobacco protoplasts and endoplasmic reticulum localization of PvHPPR was observed in tobaco leaves.4.In vivo activity of Pv TAT was confirmed by functional complementation of the Escherichia coli tyrosine auxotrophic mutant DL39.Recombinantly expressed and purified PvTAT had a molecular mass of ~65 kDa,a substrate preference for L-tyrosine and phenylpyruvate,with apparent Km values of 0.40 and 0.48 mM,and favoured the conversion of tyrosine to 4-hydroxyphenylpyruvate.The PvHPPR was heterologously expressed in Escherichia coli and the protein was shown to catalyze the NAD(P)H-dependent reduction of 4-hydroxyphenylpyruvate to 4-hydroxyphenyllactate.The apparent Km values for the various substrates were at 0.31 mM for 4-hydroxyphenylpyruvate,at 0.73 mM for phenylpyruvate,and at 1.02 mM for pyruvate,showing a substrate preference for 4-hydroxyphenylpyruvate.5.Agrobacterium rhizogenes-mediated overexpression and antisense expression of PvTAT in P.vulgaris hairy roots were used to evaluate the contribution of PvTAT to RA synthesis.PvTAT transcript levels were reduced by 46–95% and RA levels were decreased by 36–91% with low catalytic activity in antisense transgenic hairy root lines;in contrast,PvTAT transcript levels were increased 0.77–2.6-fold with increased RA levels of 1.3–1.8-fold and strong catalytic activity in overexpressed transgenic hairy root lines compared with wild-type counterparts.The modest correlation between transcript level,TAT activity,and RA accumulation supports PvTAT important for RA biosynthesis in P.vulgaris.Overexpression of PvHPPR in P.vulgaris hairy roots leads to PvHPPR transcript levels increase by 0.7–9.7 folds with increased RA levels by 24–63% compared with controls.The modest correlation between transcript level and RA accumulation supports PvHPPR important for RA biosynthesis in P.vulgaris.6.Hairy root lines with RA content 10–30 times higher than the original plant were obtained.