Cloning And Functional Characterization Of Prenyltransferase Genes In Psoralea Corylifolia L.

Psoralea corylifolia L.is an annual plant of the Leguminosae family,with a distribution in India,Myanmar,and Yunnan province of China.It has been originally used in traditional Indian medicine for the treatment of vitiligo and psoriasis,and was later introduced to China,where it has been used for hundreds of years.It reinforces kidney and strengthens yang,helps inspiration,and relieves asthma,warms spleen and stops diarrhea,and can be applied externally to get rid of freckles.P.corylifolia is rich in prenylated polyphenols,with known compounds discovered from which including 22 coumarins,52 flavonoids,and 43 monoterpene phenols.Recent pharmacological studies have shown that these compounds exhibit a wide range of pharmacological activities such as anti-cancer,anti-biotic,and neuroprotection.However,the biosynthetic pathway by which these polyphenols bind isopentenyl groups in P.corylifolia is still unclear.So far,only one prenyltransferase gene PcM4DT has been identified from P.corylifolia,which could prenylate two compounds in trace amount(4-Dimethylallylmaackiain and Licoagrocarpin).Therefore,more in-depth studies are still needed to elucidate the biosynthetic pathway of prenylated polyphenols in P.corylifolia.In this study,candidate genes of the UbiA prenyltransferase superfamily were identified from the transcriptome data of P.corylifolia using bioinformatics approaches.After filtering out sequences less than 300 amino acids,11 prenyltransferase candidate genes(PcPTs)were screened,named as PcPT1 to PcPT11.Five candidate genes,PcPT4,PcPT5,PcPT8,PcPT9 and PcPT11,were screened by physicochemical properties,phylogenetic analysis,and expression profile analysis,and finally,PcPT4,PcPT8,PcPT9 and PcPT11 were obtained.These four genes were transferred into yeast expression strains for in vitro enzyme activity assays.The results revealed that PcPT11 possessed catalytic activity and could prenylate 23 substrates,including 22 flavonoid compounds and 1 coumarin,which is the only one PT with such high substrate promiscuity in plants identified so far.The compounds that can be prenylated by PcPT11 included biochanin A,genistein,2’-hydroxygenistein,apigenin,luteolin,tricin,isorhamnetin,kaempferol,quercetin,kaempferide,tamarixetin,galangin,butein,2’,4’,6’,3,4-pentahydroxychalcone,echinatin,isoliquiritigenin,naringenin chalcone,naringenin,eriodictyol,liquiritigenin,hesperetin,dihydrokaempferol,and coumestrol.PcPT11 acted in a highly regio-specific way to specifically catalyze the prenylation of flavonoids at C-6 position,and the catalytic characteristics of PcPT11 on flavonoid also include(1)PcPT11 preferred to prenylate isoflavones and flavonols,such as biochanin A(with a conversion rate of 86.18%)and quercetin(with a conversion rate of 78.48%);(2)The hydroxylation and O-methylation pattern of flavonoid B-ring had an effect on the activity of PcPT11;(3)5-OH may be essential for the enzymatic activities of PcPT11.Functional study of PcPT11 dissected the biosynthetic pathways of the active components in P.corylifolia,including corylifolinin,bavachin,psoralidin and wighteone.Both transcriptome expression profiling and qRTPCR showed that PcPT11 was highly expressed in fruits and leaves.Subcellular localization experiments showed that the GFP-PcPT11 fusion protein was localized in chloroplasts,which was consistent to the previously identified prenyltransferases.The innovation of this study is that PcPT11 may be a key gene in the formation of the diverse of secondary metabolites in P.corylifolia.The biosynthesis of several active components such as corylifolinin(product of 16)and bavachin(product of 20)may depend on the function of PcPT11.Prenylation could significantly improve lipophilicity of polyphenols and their affinity to biomembranes,thus enhancing the activity of modified compounds or generating new activities.Therefore,the discovery of this gene has great potential in the biosynthesis of a variety of prenylated compounds.