| Artemisia annua L.is a traditional Chinese medicinal material,which belongs to the Asteraceae family.Artemisinin,a sesquiterpene lactone compound with an endoperoxide bridge structure,it is an effective monomer isolated from A.annua which extensively used in the treatment of chloroquine-resistant and brain malaria.World Health Organization(WHO)has recommended artemisinin combination therapies(ACTs)as the first-line therapy for malaria worldwide.As a result,the demand for artemisinin is very huge and still increasing.A.annua is the only commercial source for producing artemisinin.Unfortunately,the content of artemisinin in A.annua is low and the resources are extremely limited,which greatly limits the market supply capacity of artemisinin.It is a common goal for the artemisinin industry to develop new varieties of A.annua with high-yield artemisinin.So,the development of new varieties of A.annua with high-yield artemisinin is a major problem improve the content of artemisinin in A.annua plant has become a major problem for the artemisinin industry and a hot point for the plant scientists.Basic helix-loop-helix proteins(bHLHs)are widely found in plants.In recent years,bHLH transcription factors have been involved in the regulation of plant secondary metabolites such as alkaloids,flavonoids and terpenoids,which have been discovered and reported in different medicinal plants.Two transcription factors,AaMYC1 and AabHLH1 were screened from A.annua successively,the results showed that the two transcription factors have positive effects on the biosynthesis of artemisinin in A.annua.In the model plant Arabidopsis thaliana,the bHLH TFs,especially phytochrome interacting factors(PIFs),play important roles in light signaling.Among these PIFs of Arabidopsis(AtPIFs),AtPIF3 is necessary for normal photoinduced signal transduction.Besides its key role in light signaling,AtPIF3 positively regulates anthocyanin biosynthesis,implying its involvement in regulating secondary metabolism.Based on these previous studies in A.annua and A.thaliana,it might deduce that the PIF3 homologue might be involved in regulating artemisinin biosynthesis in A.annua.The functions of AaPIF3 was characterized to regulate the artemisinin biosynthesis in A.annua at the levels of molecular biology,biochemistry and biotechnology.The main discoveries are as follows:1.AaPIF3 bind to the AaERF1 promoter transactivated the artemisininbiosynthesis genes indrectilyIn this study,a bHLH TF(AaPIF3),phylogenetic association with AtPIFs,was obtained using genome-wide analysis.AaPIF3 showed similar expression patterns to artemisinin biosynthesis genes and AaPIF3 localized in nucleus.Dual-Luciferase assay showed that AaPIF3 was able to activate the ADS,CYP71AV1,DBR2,ALDH1 and AaERF1 genes to expression.Yeast one-hybrid assay showed that there were no direct interaction between AaPIF3 and ADS,CYP71AV1,DBR2 and ALDH1 promoters.However,yeast one-hybrid assay confirmed that AaPIF3 had direct interaction with the AaERF1 promoter.Based on the dual-luciferase and yeast one-hybrid assays,it was found that AaPIF3 was able to directly transactivate AaERF1 and indirectly transactivate artemisinin biosynthesis.2.Overexpression of AaPIF3 promoted the biosynthesis of artemisininThe expression levels of ADS,CYP71AV1,DBR2,ALDH1 and AaERF1 in the AaPIF3-overexpressing transgenic plants of A.annua were significantly up-regulated(p<0.01).The content of artemisinin in the control was 9.63 mg/g DW(Dry weight),the artemisinin production was 15.91 mg/g DW in OE-10 line,15.02 mg/g DW in OE-21 line and 15.41 mg/g DW in OE-26 line.In another word,the artemisinin production of AaPIF3-overexpressing plants was increased by 65.21% in OE-10,55.97% in OE-21 and 60.02% in OE-26,compared with the artemisinin production in wild-type plants.Wild-type plants of A.annua produced dihydroartemisinic acid at the level of 0.76 mg/g DW.Transgenic lines with AaPIF3 overexpression produced dihydroartemisinic acid at the levels of 4.59 mg/g DW(OE-10),4.99 mg/g DW(OE-21)and 4.51 mg/g DW(OE-26),respectively.3.Suppression of AaPIF3 reduced the biosynthesis of artemisininIn the AaPIF3-suppression transgenic lines of RI-46,RI-53,RI-57,the expression levels of ADS,CYP71AV1,DBR2,ALDH1 and AaERF1 were significantly up-regulated(p<0.01).Compared with the control,the artemisinin and dihydroartemisinic acid contents were significantly decreased(p<0.01),the artemisinin contents of AaPIF3-RNAi plants were 46.72~51.81% of those in control,and the contents of dihydroartemisinic acid of AaPIF3-RNAi plants were 18.42~35.52% of those in control.In summary,AaPIF3 gene was cloned and analyzed,transgenic plants of A.annua,with overexpression or suppression of AaPIF3,were developed to reveal its function in regulating artemisinin biosynthesis which AaPIF3 gene can promote artemisinin biosynthesis. |
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