Molecular Cloning And Characterization Of The Promoter Of Aldehyde Dehydrogenase Gene From Artemisia Annua L.

Artemisinin, extracted from annual herb Artemisia annua Linn, is a new type of sesquiterpene lactone compound, containing peroxide bridges group structure, and it is a specific medicine treating cerebral malaria and chloroquine-resistant falciparum malaria. The World Health Organization(WHO) recommends artemisinin combination therapies(ACTs) as the preferred method for the treatment of malaria, however, Artemisinin is only found in the Artemisia annua L, the artemisinin content is very low in natural Artemisia annua L, accounting for only 0.01% ~ 0.6% of dry weight, and the artemisinin are far from meeting the demands of the market. Chemical synthesis of artemisinin has disadvantages of low yields, high cost, great toxicity and more complex synthetic routes, and is difficult to realize industrialization production. Therefore, it has been one of the hotspots in the field of plant secondary metabolism to increase the content of artemisinin and other sesquiterpenes in Artemisia annua L using genetic engineering techniques.Jasmonic acid and its derivatives, collectively called jasmonates(Jas), are important plant signal molecules, which can regulate plants growth, make the corresponding defense response to environment changes, and have a regulatory role on the accumulation of secondary metabolites. Meanwhile, it has reported that spraying Jas can increase the artemisinin content in natural Artemisia annua L.At the downstream of artemisinin anabolic pathways, Aldehyde dehydrogenase1 gene can catalytic artemisinic aidehyde and dihydroartemisinic aldehyde into artemisinic acid and dihydroartemisinic acid, and artemisinic acid and dihydroartemisinic acid can spontaneously under light into artemisinin B and artemisinin. It has been reported that there are the highest gene expression of ALDH1 in the glandular trichomes, scarcely in roots by semi-quantitative analysis. The gene sequences of ADS, CYP71AV1 and DBR2,which are key genes of artemisinin anabolism pathway, have been cloned, and their function has been studied. However, the role of ALDH1 gene in the artemisinin metabolic pathway has rarely been reported. Therefore, this experiment explore the function of ALDH1 gene in the artemisinin anabolism pathway more deeply, by studying ALDH1 gene promoter.In this study, according to ALDH1 gene sequence(registration number: FJ809784) reported by NCBI, we designed ALDH1 gene promoter specific primer from annua genome database, cloned and obtained ALDH1 gene promoter sequence by FPNI-PCR technology, carried on the bioinformatics analysis. The results show that ALDH1 gene promoter has 2100 bp, containing MeJA regulatory response element TGACG-motif, GA3 responsive element GARE-motifs and P-boxes, stress-responsive element TC-rich. Build pCAMBIA1391Z: pALDH1: GUS functional verification vector,GUS reporter gene is started by ALDH1 gene promoter. We transformed Arabidopsis thaliana stably using flower dip method, and collected seeds after being ripe, and then filtered the seeds on the filter medium plates containing Hygr resistance, finally obtained positive transgenic Arabidopsis thaliana detecting by PCR. After verified by GUS staining, it was proved that ALDH1 promoter was dyed blue in the top of foliage and young leaves of seedlings, and only the glandular trichome was dyed blue in the leaves, matching that ALDH1 was the highest gene expression in the glandular trichomes. After transgenic Arabidopsis thaliana was treated with MeJA and injury, the expression of the GUS gene was improved. Overexpression of Artemisia annua transcription factor can promote the accumulation of artemisinin. After verified by double-luciferase activity analysis, it was proved that overexpression of the transcription factor AaERF2 and Aa ORA, and the LUC / REN ratio significantly higher than the group of control, but no significant difference in Aa WRKY1 LUC / REN ratio with the control. Therefore, The results showed that transcription factors AaERF2 and AaORA can activate ALDH1 promoter expression, and Aa WRKY1 can’t, this matches that the promoter contain no W – box. In summary, through the experiment research, the role of ALDH1 gene in the process of artemisinin anabolic gets a better understanding,for further screening of artemisinin biosynthesis related transcription factors laid a solid foundation.