The Study On The Enhancement Of Artemisinin Content In Artemisia Annua L. By Overexpressing Enzymes In Artemisinin Biosynthesis Pathway

Malaria, one of the three severest infectious diseases in the world,causes the death of1million people every year. And artemisinin, a typeof sesquiterpene containing endoperoxide bridge extracted from the aerialpart of Artemisia annua L., is now the most effective drug for malaria.However, the artemisinin content in A. annua is very low, ranging from0.01%-0.1%(DW, dry weight), which limits the supply of artemisinin forthe market demand and, in turn, has caused a high price of artemisininthat malaria patients in underdeveloped areas cannot afford. Because ofthis, it is of great practical significance to lower down the cost ofartemisinin and promote its wide employment by increasing theartemisinin content in A. annua through overexpressing the enzyme genesin artemisinin bio-synthesis.This thesis focuses on increasing artemisinin content throughoverexpressing the enzyme genes DXR, DBR2, ALDH1and the enzyme gene combination of CYP71AV1, CPR and FPS in artemisinin metabolicpathways.The results of the study are as follows:(1) Double T-border vectors containing enzyme genes DXR,DBR2and ALDH1respectively and the plant expression vectorcontaining enzyme gene combination of CYP71AV1, CPR and FPS wereconstructed; And the subcellular localization study showed DBR2andALDH1were located in cytoplasm;(2) Transgenic plants were obtained through Agrobacteriumtumefaciens-mediated transformation. PCR detections demonstrated thetransgenic statuses of14independent plants transformed with DXR,15with DBR2,15with ALDH1, and8with CYP71AV1, CPR and FPS;(3) The insertion copies of foreign genes in the8transgenic plantstransformed with vector containing CYP71AV1, CPR and FPS, revealedby Southern blot, ranged from1-4copies;(4) Real-time PCR showed that the expression levels ofCYP71AV1, CPR and FPS were up-regulated in7out of8transgenicplants analyzed;(5) HPLC-ELSD analysis showed that the artemisinin contents ofthe transgenic plants transformed with DXR, with DBR2, with ALDH1and with CYP71AV1, CPR and FPS were up to2.8-fold,3.4-fold,3.8-foldand3.6-fold of that of the control respectively.