The Study Of R2R3MYB Transcription Factors Regulating Glandular Trichome Initiation And Artemisinin Biosynthesis In Artemisia Annua L.

Artemisia annua L.is a Chinese traditional medicinal plant species with long history.Artemisinin extracted from A.annua plants,is the most effective compound in the anti-malaria therapy.So far,Artemisinin-based combination therapies,known as ACTs,are the most effective treatment for malaria as recommended by WHO which saves hundreds of thousands of lives each year.Unfortunately,the supply of artemisinin in A.annua is significantly restricted to its low content(0.01%-0.1% dry leaf weight,DW).Therefore,improving the content of artemisinin in A.annua is a hotspot research in the field of A.annua study.Artemisinin,mainly produced and stored in glandular secretory trichomes(GSTs),is a sesquiterpene lactone with an endoperoxide bridge.However,very few researches were reported on the mechanism of plant trichome development and transcriptional regulation of artemisinin biosynthetic pathway.MYB transcription factors belong to a large family of proteins in plants.R2R3 MYB is a specialized member of this family,which has been reported to bear vital functions including,response to biotic and abiotic stresses,secondary metabolites biosynthesis,growth and development as well as meristem proliferation.Although R2R3 MYB transcription factors hold various functions,its involvement in trichome initiation and artemisinin biosynthesis is not yet clearly studied.Based on A.annua genome and GSTs transcriptome data,candidate R2R3 MYB genes involved in regulating GSTs initiation and artemisinin biosynthesis were sorted out.Further experiments using bioinformatic tools,molecular biology,and transcriptomic analysis were used to study the function of the candidate genes,and the results were as follows:1.Cloning and identification of R2R3 MYB transcription factor AaMIXTA1 in A.annua.It can positively regulate the GSTs density and leaf surface cuticle accumulation.Through bioinformatic and coexpression analyses,and by screening R2R3 MYB transcription factor genes from GSTs transcriptome database,AaMIXTA1,which is predominately expressed in the basal cells of GSTs in A.annua was identified.Transgenic results showed that overexpression and repression of AaMIXTA1 resulted in an increase and decrease,in the number of GSTs as well as the artemisinin content in transgenic A.annua plants respectively,demonstrating that AaMIXTA1 can positively regulate GSTs initiation and artemisinin content.In addition,the content of cuticle in OE-AaMIXTA1 lines was significantly increased.On the contrary,the content of cuticle in AaMIXTA1-RNAi lines was also significantly lower than that of control.The expressions of cutin and wax related synthase genes AaCYP77A1,AaCYP86A1,AaABCG12,AaKCS5 and AaCER1 were confirmed to be significantly reduced in AaMIXTA1-RNAi lines.The results of dual luciferase(Dual-LUC)assay demonstrated that AaMIXTA1 significantly activated the expression of these genes.The results demonstrated that AaMIXTA1 could positively regulate the GSTs density and leaf surface cuticle accumulation.This study is important for further elucidating GSTs initiation and regulatory mechanism of cuticle biosynthesis mechanism.2.Cloning and identification of R2R3 MYB transcription factors AaMYB3 and AaMYB6.AaMYB3 negatively regulated artemisinin biosynthesis,while AaMYB6 positively regulated artemisinin biosynthesis.Based on transcriptome database,two MYB transcription factors,named as AaMYB3 and AaMYB6,were cloned and identified by coexpression analyses.AaMYB3 and AaMYB6 responsed to MeJA nagatively and positively,respectively.Transgenic results showed that AaMYB3 negatively regulated artemisinin biosynthesis,while AaMYB6 positively regulated its biosynthesis in transgenic A.annua plants.In AaMYB3-OE transgenic A.annua plants,the expression of artemisinin biosynthetic pathway genes was significantly reduced,and the artemisinin content was also significantly decreased.In AaMYB3-Anti transgenic A.annua plants,the expression of artemisinin biosynthetic pathway genes and the artemisinin content were significantly increased.In AaMYB3-EAR transgenic A.annua plants,the artemisinin content was not detectable.In another hand,in AaMYB6-OE transgenic A.annua plants,the expression of artemisinin biosynthetic pathway genes was significantly improved,and the artemisinin content was also significantly increased.In AaMYB6-Anti and AaMYB6-EAR transgenic A.annua plants,the expression of artemisinin biosynthetic pathway genes was significantly reduced,and the artemisinin content was also significantly decreased.Subsequently,AaMYB3 and AaMYB6 were used to screen the yeast two hybrid library of leaf cDNA for interactive proteins.AaTPL and AaSPL9 were identified to interact with AaMYB3,and AaSPL9 and AaERF2 were identified to interact with AaMYB6.The results demonstrated that AaMYB3 negatively regulated artemisinin biosynthesis,while AaMYB6 positively regulated artemisinin biosynthesis.This study proposes a possible model of a new secondary metabolism regulation based on the formation of MYB-SPL-ERF complex.