Using Genetic Engineering To Increase Artemisinin Content And Provide Glyphosate Tolerance In Aretemisia Annua

Malaria is an infectious disease caused by plasmodium and epidemic.The main manifestations for malaria are emesia,sweating,and fever.In severe cases,it can cause death.According to World Health Organization(WHO)statistics,more than 214 million people face the risk of malaria and over 438,000 dead in 2015.Millions of people cannot get proper medicine to treat malaria.Artemisia annua L.is a traditional medicinal plant in China.Artemisinin is an important sesquiterpene lactone isolated from the aboveground parts of A.annua.Artemisinin-based Combination Therapies have been identified as the most effective method to treat malaria by WHO.Tu Youyou obtained the 2015 Nobel Prize in Medicine for the discovery of artemisinin.Currently,there exist two issues on agricultural production of A.annua.Firstly,artemisinin content is low in wild-type A.annua(0.01%–0.8% DW).Also,weeds cause yield losses and affect the mechanized harvest of A.annua's leaves.Therefore,it is important to enhance artemisinin content,in the meantime,provide glyphosate resistance in A.annua.In this study,three genes HMGR,FPS,DBR2 involved in artemisinin biosynthesis pathway and a glyphosate tolerant gene EPSPS were co-overexpressed in A.annua.Besides,we cloned a 1620-bp promoter region upstream of the ALDH1 start codon,and the expression pattern of the ALDH1 promoter was analyzed using a promoter-GUS fusion in A.annua.Results and conclusions of this study are as follows:(1)A plant expression vector containing four genes(HMGR,FPS,DBR2 and EPSPS)was constructed;(2)A plant transformation system selected by glyphosate was established,and the selected concentration was 3 mg/L.33 positive transgenic plants were confirmed by PCR.7 high-artemisinin producing transgenic plants were selected for further analysis;(3)Southern blot analysis showed that foreign DNA fragments have been successfully inserted into A.annua genome;(4)RT-Q-PCR analysis showed that the expressions of HMGR,FPS and DBR2 enhanced dramatically in most transgenic plants,and the expression of EPSPS varied in different lines;(5)HPLC analysis showed that the artemisinin content in transgenic lines got different increase,with the highest in E82(32.58 mg/g),which was 3.3 fold compared with that of control(10.02 mg/g);(6)Glyphosate assay showed that transgenic plants were more tolerant to glyphosate compared with control plants,and the optimum concentration of glyphosate application was 0.05%(v/v);(7)Shikimic acid analysis showed that the transgenic plants suffered less than non-transgenic plants;(8)A 1620-bp promoter region upstream of the ALDH1 start codon was cloned.A number of regulatory cis-acting elements were predicted in the promoter region using software;(9)The activity of the ALDH1 promoter was analyzed using a promoter-GUS fusion.The results demonstrated that the ALDH1 promoter was trichome-specific.RT-Q-PCR analysis revealed that ALDH1 promoter could be induced by methyl-jasmonate,gibberellin and salicylic acid.