Biosynthesis Of Anthocyanins And Their Impact On Artemisinin In Engineering Of Artemisia Annua

Anthocyanins and anthocyanidins are widely synthesized natural water-soluble pigments in plants.Anthocyanidins can scavenge reactive oxygen species(ROS)in plant cells and have strong antioxidant properties.In plants,the biosynthesis of anthocyanidins is regulated by the Production of the Anthocyanin Pigment1(PAP1)gene in Arabidopsis thaliana.PAP1 is a positive regulator of anthocyanin biosynthesis,and it can form an MBW complex with b HLH and WD40 to activate the biosynthetic pathway of anthocyanins.The biosynthesis of anthocyanins can be induced by various abiotic factors.Increasing research is using different abiotic factors to regulate the biosynthesis of anthocyanins in engineered cells during growth.Artemisia annua L.is the only medicinal crop that can produce artemisinin,an effective drug for the treatment of malignant malaria.The biosynthesis of artemisinin can occur under conditions of reactive oxygen species(ROS)and is formed by the spontaneous oxidation of dihydroartemisinic acid.It is unclear whether the strong antioxidant effect affects the biosynthesis of artemisinin.This study conducted a preliminary investigation into the biosynthesis of anthocyanins and its effect on artemisinin in A.annua cells,and the main results are as follows:1.PAP1,a transcription factor for anthocyanin in A.thaliana,was introduced into A.annua cells via transgenic technology,resulting in four types of TAPA purple-red cells.The expression levels of genes involved in anthocyanin biosynthesis pathways and the content of anthocyanin in the purple-red cells were analyzed by fluorescence quantification and determination,respectively.The results showed that the purple-red cells were activated by PAP1 expression and the expression of genes involved in anthocyanin biosynthesis pathways.2.LC-MS analysis revealed that at least 17 types of anthocyanins can be synthesized in the purple-red cells of A.annua.Four types of anthocyanins were obtained through organic extraction,column chromatography,high-performance liquid chromatography,and recrystallization.Mass spectrometry and nuclear magnetic resonance analysis showed that they were cyanidin,pelargonidin,Cyanidin 3-O-glucoside,and pelargonidin 3-O-glucoside.3.Anthocyanins have strong antioxidant properties.Through fluorescence quantitative analysis of the expression levels of artemisinin biosynthesis pathway genes and the content determination of artemisinin,artemisinic acid,and arteannuin B in the purple-red cells,the results show that under oxidative stress,the biosynthesis pathway of artemisinin is still expressed in the purple-red A.annua cells,and artemisinin and its precursor can still be produced.Among them,the content of artemisinin is the highest in the purple-red cells of TAPA1.4.In order to investigate whether PAP1 can activate the promoter of anthocyanin biosynthesis-related genes in purple-red A.annua cells,experiments were conducted to analyze the AC and G-box cis-elements in the promoter of anthocyanin pathway genes,analyze and clone the promoter,perform transient expression in Nicotiana benthamiana,and conduct dual-luciferase assays.The results showed that PAP1 binds to the cis-elements of structural genes in the anthocyanin biosynthesis pathway and activates the activity of anthocyanin-related gene promoters.5.To investigate whether other factors can regulate the biosynthesis of anthocyanins in purple-red A.annua cells,the effects of different concentrations of growth hormones(NAA,IAA,2,4-D),and exogenous nitrogen sources on cell growth,anthocyanin content,and production of cyanidin-3-O-glucoside were measured.The results showed that 2,4-D had an inhibitory effect on anthocyanin biosynthesis.HPLC analysis showed that 2,4-D did not affect the types of anthocyanins produced.Fluorescence quantification analysis indicated that 2,4-D also had an inhibitory regulatory effect on the expression of genes related to the anthocyanin biosynthesis pathway.In summary,the successfully cultivated engineering cell of A.annua provides a basis for further studying the biosynthesis and metabolic regulation of artemisinin and anthocyanins in A.annua.