Study Of Molecular Mechanism Of Secondary Metabolism In Artemisia Annua Affected By Cadmium

The sweet wormwood (Artemisia annua L.), the dry overground part of which is one of our traditional Chinese medicines (TCM), is a genus of Artemisia, annual herb in the family Compositae or Asteraceae. The main active ingredient is sesquiterpene lactones artemisinin, which is alaways used for Malaria. The mechanism is that artemisinin can change the structure of the parasite membrane system and eventually kill the parasite. At present, the prefer approach to get artemisinin still is A. annua of artificial cultivation, and then extract and separate from the plants. The early study in our laboratory found that low-dose cadmium could promote the accumulation of artemisinin. Under multiple doses cadmium stress, this paper aims to explore the regulation of accumulation of secondary metabolites in the A. annua affected by cadmium and the key enzyme gene expression involved in the pathway of synthesis of secondary metabolites. In addition, screening out the MAPK genes associated with cadmium stresses by the analysis of the differential expression, we preliminary investigate the MAPK cascade of A. annua playing a role in signal transduction of cadmium and its effect on accumulation of secondary metabolites.The main results are as follows:1Study on individual differences in seed of A.annuaIn order to evaluate individual differences of A. annua used in the experiments, we observed plant height, growth, color of leaf and content of secondary metabolites, such as Artemisinin, Arteannuin B, Artemisinic acid and Dihydroartemisinic acid. The result suggest that we should select the similar seedings in plant height, growth and the color of leaf as experiment materials.2Effect of cadmium on the accumulation of secondary metabolites of A. annuaAdding Cd(NO3)2·4H2O in nutrient solutions to cultivate A. annua, We use LC-MS to detect the content of Artemisinin, Arteannuin B, Artemisinic acid and Dihydroartemisinic acid in A. annua at1,3d after cadmium treatment and perform statistical analyses. The results displays that the content of Artemisinin (the treatment groups of60,80and120μM), Artemisinic acid (the treatment groups of60and120μM) and Arteannuin B (the treatment groups of120μM) have significant differences (p<0.05) compared with the control group, showing that cadmium can significantly improve the Artemisinin, Artemisinic acid and Arteannuin B.3Stability analysis of reference gene in A. annua under cadmium treatmentIn this study, we chose Actin,18S rRNA, PAL, GAPDH and CPR of A. annua as candidate reference genes, and designed their gene-specific primers for real-time PCR, then used geNorm, NormFinder, BestKeeper, Delta CT and RefFinder to evaluate their expression stability in the leaves of A. annua under treatment of different concentrations of Cd, with the purpose of finding a reliable reference gene to ensure the reliability of gene-expression analysis. The results showed that there were some significant differences among the candidate reference genes under different treatments and the order of expression stability of candidate reference gene was Actin>18S rRNA>PAL>GAPDH>CPR.4Study on the differential expression of the key enzymes in the biosynthesis of artemisinin under treatment of cadmiumAnalysis the differential expression of HMGR、 FPS、 ADS、 CYP71AV1, CPR、 ALDH1、DBR2、DXS and DXR—the key enzymes in the biosynthesis of artemisinin in A.annua under the treatment of cadmium by Real-Time PCR. The results shows that the expression of ADS、 CYP71A VI、DBR2and ALDH1increases greatly4and8hours after under the treatment of cadmium, indicating that these genes could quickly response to cadmium, and they may be the key enzymes in the biosynthesis of artemisinin under treatment of cadmium.5. Study on the differential expression of the signal transduction genes MAPKs under treatment of cadmiumSplice the EST of A.annua from SRA data to get Unigene, by BLAST and analysising the structural domain, we get17MAPKs of A.annua. Analysis the differential expression of MAPKs under treatment of cadmium by RT-PCR. The results shows that the expression of2MAPKs is repressed under treatment of cadmium, meanwhile4are increased, suggesting cadmium could activate MAPKs in A.annua, and pathway of MAPKs plays a role in the biosynthesis of artemisinin under treatment of cadmium.