The Study Of Regulating Artemisinin Biosynthesis In Artemisia Annua By Branch Pathway Blocking And Salicylic Acid

Artemisinin is the effective drug for curing malaria,but its content in the source plant Artemisia annua is low(0.1-1%).How to improve artemisinin content have long been the research focus in the study of artemisinin regulation.There exist several competitive branching pathways of artemisinin biosynthesis in A.annua.Previous studies indicate that blocking the branch pathway is a useful method for improving artemisinin content.There are high precentages of branch pathway metabolites including ?-caryophyllene,?-farnesene,squalene and germacrene A in volatile oil of A.annua,and the synthases in the branch pathways compete the substrate FPP with synthase in artemisinin biosynthesis,directly subtracting the metabolic flow to artemisinin.However,it is not clear blocking which branch pathway contributes most to artemisinin biosynthesis.Some earlier studies show that phytohormone SA can regulate artemisinin biosynthesis,but its regulatory mechanism is not clear.To study the influence of blocking competitive branch pathways and SA on artemisinin biosynthesis,research was carried out using the major Artemisia variety Youqing as the material,including transcriptome analysis,gene cloning,evolution analysis,expression and metabolic analyses,protein-DNA binding,protein-protein interation,and transgenic analysis.The major results are shown as follows:1 The influence of CPS(?-caryophyllene synthase gene),BFS(?-farnesene synthase gene),GAS(germacrene A synthase gene)or SQS(squalene synthase gene)on artemisinin content and metabolic flow in A.annuaTo study the relationship between genes encoding enzymes at branching points and artemisinin biosynthetic pathway,?-caryophyllene,?-farnesene and squalene were sprayed respectively on young seedlings of A.annua.Transient expression assays showed that spraying ?-caryophyllene could inhibit the expression of CPS,but upregulate the expression of BFS and artemisinin biosynthetic pathway gene ALDH1(aldehyde dehydrogenase 1 gene).Spraying ?-farnesene could inhibit BFS expression,but increase the expressions of CPS and CYP71AV1(amorphadiene 12 hydroxylase gene)by 2 and 4 folds respectively.Spraying squalene could reduce SQS expression,but upregulate the expressions of BFS,CPS,GAS,ADS(amorpha-4,11-diene synthase gene),CYP71AV1,DBR2(artemisinic aldehyde ?11(13)reductase gene)and ALDH1 by 1 to 3 folds.These results indicate that blocking the artemisinin competitive branching pathways can upregulate the expressions of artemisinin biosynthetic pathway genes,thus having the potential to improve artemisinin content in A.annua.To further study the influence of inhubiting CPS,BFS,germacrene A synthase gene(GAS)and SQS on artemisinin biosynthesis,antisense gene vectors containing anti-CPS,anti-BFS,anti-GAS and anti-SQS were constructed and introduced into Agrobacteria,the latter were used to transform A.annua.In anti-CPS transgenic plants,the expression levels of BFS and ADS were increased,while the contents of ?-farnesene,artemisinin and dihydroartemisinic acid(DHAA)were increased by 212%,38% and 77% respectively.In anti-BFS transgenic plants,the expression levels of CPS,GAS,SQS,ADS,CYP71AV1 and ALDH1 were increased while the contents of squalene,artemisinin and DHAA were increased by 235%,116% and 90%,respectively.In anti-GAS transgenic plants,the expression levels of CPS,BFS,SQS and ADS were increased while the contents of artemisinin and DHAA were enhanced by 25% and 83%,respectively.In anti-SQS transgenic plants,the expression levels of BFS,GAS,CPS,ADS,CYP71AV1 and ALDH1 were increased while the contents of ?-farnesene,artemisinin and DHAA were enhanced by 123%,71% and 223%,respectively.These results indicate that inhibition of competitive branching pathway genes can improve artemisinin content in A.annua,and the best results are achieved by inhibiting BFS and SQS.2 The study of SA on regulating artemisinin biosynthesisSA signaling pathway related genes NPR and TGA were identified through A.annua transcriptome sequencing.The phylogenetic analysis indicates that A.annua AaNPR1 is most closely related to AtNPR1,and A.annua AaTGA6 is most closely related to the clade II of the AtTGA2/5/6 in Arabidopsis.AaNPR1 is a gene encoding a protein containing ankyrin-like repeats.Yeast two-hybrid and BIFC assays indicate that AaNPR1 can interact with AaTGA6,and it interacts with the N-terminal of AaTGA6.An earlier study shows that an AP2/ERF transcription factor AaERF1 can promote artemisinin biosynthesis through direct binding of the promoters of artemisinin biosynthetic pathway genes ADS and CYP71AV1.In this study,dual-LUC assay indicates that AaTGA6 can activate the promoter of AaERF1.To further determine if AaTGA6 can bind the promoter of AaERF1,yeast one-hybrid and electrophoretic mobility shift assay(EMSA)were carried out.The results show that AaTGA6 can bind the core sequence “TGACG” of the AaERF1 promoter.EMSA and dual-LUC assay indicate AaNPR1 can promote AaTGA6 to bind AaERF1,thus promoting the transcription of AaERF1.Subsequently,AaTGA6 overexpressing and RNAi vectors were constructed and transferred into A.annua via Agrobacterium-mediated transformation.In AaTGA6 overexpressing transgenic A.annua plants,the expression levels of AaTGA6 and AaERF1 were increased by 11-15 folds and 1-2 folds respectively,while the expression levels of artemisinin biosynthetic pathway genes ADS and CYP71AV1,DBR2 and ALDH1 were increased by 1-6 folds,1-7 folds and 1-4 folds respectively.In AaTGA6 RNAi transgenic A.annua plants,the expression levels of AaTGA6 and AaERF1 were decreased by 70-90% and 25-50% respectively,while the expression levels of artemisinin biosynthetic pathway genes ADS and CYP71AV1,DBR2 and ALDH1 were decreased by 40-70%,20-60%,15-70% and 30-70% respectively.Artemisinin contents in AaTGA6 overexpressing or RNAi transgenic A.annua plants were increased by 90-120% or decreased by 20-60% respectively.This study indicates that AaTGA6 is a positive regulator of artemisinin biosynthesis,and SA,through passing signaling to AaNPR1,the latter can interact with AaTGA6,activating the promoter of AaERF1,thus enhance the expressions of artemisinin biosynthetic pathway genes,promotes artemisinin biosynthesis.