Relative Expression Of The Genes Involved In Artemisinin Biosynthesis In Different Tissues And Enhancement Of Artemisinin Biosynthesis By Overexpressing ADS In The Plants Of Artemisia Annua L.

Artemisinin, a sesquiterpene endoperoxide lactone isolated from the Chinese medicinal plant of Artemisia annua L., is an effective antimalarial agent, especially for multi-drug resistant and cerebral malaria. It is also recommended by the World Health Organization (WHO) as a preferred drug to treatment the falciparum malaria. There are more than 100 million people using to treat malaria every year. However, artemisinin content in wild type Artemisia annua L. is so limited owing to genetic or environmental factors, only 0.01 to 0.6% of dry weight, which lead to its short supply in world market. Although artemisinin could be synthesized chemically, it could not be put into production due to high cost and strong toxicity. Recently, growing number of key genes in the artemisinin biosynthesis are cloned, genetic engineering is the best way to improve the artemisinin content.In our study, the 7 functional genes(HMGR, DXR, FPS, ADS, CYP71AV1, CPR and DBR2) involved in artemisinin biosynthesis were detected at the level of expression by using qPCR, and simultaneously the content of artemisinin in the 4 investigated tissues was detected in parallel. The 3 genes, including HMGR, DXR and FPS which were involved in the upstream pathway (MEP and MVA) of artemisinin biosynthesis, showed highest expression levels in flowers, the 4 functional genes, including ADS, CYP71AV1, CPR and DBR2 which were involved in the artemisinin-specific biosynthetic pathway, were found expressed in all the 4 detected tissues, the highest expression level of ADS was found in leaves, then followed by flowers, and the lowest expression level of ADS was found in roots and stems, CYP71AV1 had highest expression level in flowers and lowest in leaves, CPR showed highest expression level in flowers, and DBR2 had lower expression levels in the other 3 artemisinin-specific pathway genes in all the tissues.HPLC-ELSD analysis showed that the artemisinin content in different tissues have a great significant difference. The highest content of artemisinin was found in leaves (0.34±0.077 mg·g-1 DW), then followed by flowers (0.15±0.017 mg·g-1 DW), roots (0.06±0.0026 mg·g-1 DW) and stems (0.06±0.0024 mg·g-1 DW). The 3 genes including HMGR, DXR and FPS which were involved in the upstream pathway of artemisinin biosynthesis showed more activily than others, therefore, we supposed that the flower is the major site of artemisinin precursor synthesis. The positive correlation of ADS expression and artemisinin content in tissues demonstrated that ADS played a very important role in artemisinin biosynthesis, which should be the ideal target to engineering the artemisinin bio synthetic pathway.According the results of qPCR and analysis of artemisinin content in different tissues, we supposed that ADS is a key gene in the artemisinin biosynthesis. Consequently, Plant expression vector p1304-ADS was constructed and introduced into Agrobacterium tumefaciens strain LBA4404 to generate engineered strain LBA4404-p1304-ADS. Through leaf-disc transformation method, recombinant vector p1304-ADS was introduced into Artemisia annua L. Followed by screening with 10 mg·L-1 Hygromycin to obain transgenetic seedling of Artemisia annua L. After 2-3 cm Hygromycin-resistant shoots were obtained, they were separated and subcultureed in MS+NAA(0.05 mg·L-1)+Cef(250 mg·L-1), which is suitable for rooting of shoots. Genomic PCR was applied to detect the integration of ADS into the genome of Artemisia annua L. To get rid of the pollution of Agrobacterium tumefaciens on detection results, the virDl gene was also detected in all the samples.5 independent transgenic lines were confirmed by PCR analysis for the presence of the exogenoeous ADS gene. These results indicated that the transgenic Artemisia annua L. plants were authentic transgenic plants. Subsequently,5 transgenic Artemisia annua L. lines were micropropagated in MS+NAA(0.05 mg·L-1) to induce adventitious root.5 cm rooted plantlet were acclimated in PinderStrap matrices and then transferred into the field. After 4 month growth in the field, transgenic and non-transgenic Artemisia annua L. were collected and subjected to artemisinin content analysis.Artemisinin content in A164, A26, A48, A41 and A32 transgenic plants are 0.73± 0.003 mg·g-1,1.13±0.080 mg·g-1,2.16±0.285 mg·g-1,2.56±0.100 mg-g-1 and 2.67± 0.285 mg·g-1 respectively. The result showed that only three transgenic lines (A41, A48, A32)artemisinin contents are improved. The artemisinin content of transgenic A32 was highest, which were 2.7 times higher than that of non-transgenic. In all, the artemisinin content and gene expression result demonstrated that over-expression of the endogenous genes of ADS can improve the artemisinin content. Through this transgenic method, transgenic plants were obtained which can be provide as the first-hand plant material for the industrial production of artemisinin and meet the urgent demand on the world market.