Functional Characterization Of AaMED25 Regulating Artemisinin Biosynthesis

Artemisia annua L.is a traditional Chinese medicinal material,which belongs to the Asteraceae family.The secondary metabolite artemisinin which produces in A.annua,is a sesquiterpene lactone compound containing a peroxidation bridge structure,and has a very remarkable effect against chloroquine type malaria and cerebral malaria.The World Health Organization(WHO)recommends the artemisinin-based combination therapies as the preferred method of treating malaria.Since artemisinin is mainly extracted from the A.annua,the molecular mechanism of artemisinin biosynthesis and regulation has become a hot spot in the field of the secondary metabolism research.The biosynthesis of plant secondary metabolites is often induced by hormones.Several research teams have found that the treatment with methyl jasmonate(MeJA)in A.annua can significantly increase the expression of artemisinin biosynthesis genes and the artemisinin content.Subsequently,the researchers cloned a series of MeJA-inducible transcription factors from A.annua,such as AaWRKY1,AaORA,AabHLH1,AaERF1,AaMYC2,AaHD1 and AaGSW1.However,recent molecular studies on the initiation of transcription in eukaryotic organisms indicate that the transcription factors cannot directly bind to the RNA polymerase II during the initiation of transcription,requiring mediators as intermediate bridges to link transcription factors and RNA polymerase II,and recruiting some universal transcription factors to forming a transcriptional initiation complex.The studies on Arabidopsis mediators have shown that the mediator subunit AtMED25 interacts with multiple transcription factors of the jasmonic acid(JA)signaling pathway,thereby regulating the transcriptional activation activities of these transcription factors on target genes.In order to further analyze the molecular regulation mechanism of artemisinin biosynthesis,this study screened a candidate A.annua mediator subunit AaMED25 based on the genome wide analysis.It was demonstrated that AaMED25 interacts with the important transcription factor AaMYC2 of the JA signaling pathway and activates the transcriptional activation activity of AaMYC2 on the artemisinin biosynthesis gene,thereby improving the biosynthesis ability of artemisinin.The specific research results are as follows: 1.Screening the candidate mediator subunit AaMED25 based on the genome wideanalysisScanning the genome of A.annua by using the HMMER and BLASTP programs revealed that A.annua has six MED25 homologous genes.Subsequently,the expression analysis of the six candidate AaMED25 genes were performed in the eighteen published transcriptome databases of various tissue sites of A.annua.The results showed that only one of the six MED25 candidate genes expressed in the glandular secretory trichome,which is the main artemisinin biosynthesis site.Therefore,this study used this gene as the final candidate gene and named it AaMED25.2.AaMED25 interacts with the transcription factor AaMYC2The tissue expression analysis indicated that the AaMED25 gene was constitutively expressed in A.annua.Moreover,the AaMED25 gene expression level in the leaves and flower buds was significantly higher than that in the stems,which is very similar to the expression pattern of the key enzyme genes of artemisinin biosynthesis.The induced expression analysis indicated that the AaMED25 gene was strongly induced by the plant hormone MeJA.After MeJA treatment,the gene expression level of AaMED25 was 9 times higher than that of the control group.In addition,the expression of the AaMED25 gene was also induced by the low temperature.After 4?treatment,the gene expression level of AaMED25 was 1.7 times higher than the control group.Subsequently,The Yeast two-hybrid experiment showed that there was an interaction between the AaMED25 and the JA-responsive transcription factor AaMYC2.Further studies on the functional domain of AaMED25 found that when the middle domain and the activator interaction domain coexist,AaMED25 interacts with AaMYC2.Finally,the dual luciferase assays showed that AaMED25 can enhance the transcriptional activation activity of AaMYC2 on the key enzymes of artemisinin biosynthesis,thereby regulating the biosynthesis of artemisinin.3.Overexpression the AaMED25 gene increases artemisinin contentIn order to study the effect of AaMED25 gene on artemisinin biosynthesis in vivo,this study obtained the over-expressed AaMED25 transgenic A.annua by the Agrobacterium-mediated genetic transformation.In the transgenic plants,the real-time quantitative PCR analysis showed that the expression level of AaMED25 was 3-4 times higher than that of the control group,and the expression of the key enzyme gene of artemisinin biosynthesis was significantly improved.Moreover,the content of artemisinin was detected by HPLC.The results showed that the content of artemisinin in transgenic palnts was 8.06-11.4 mg/g(DW),and the content of artemisinin in the wild type was 6.48 mg/g(DW).Compared with the control group,the artemisinin content of the transgenic A.annua increased by 1.3-1.7 times.