New Strategies To Enhance The Content Of Artermisinin In Transgenic Artemisia Annua L.

Malaria is one of the most serious parasitic diseases worldwide. Currently, the most effective anti-malaria drug is Artemisinin Combinational Therapy (ACT), the central component of which is the sesquiterpene lactone endoperoxide, artemisinin. Since artemisinin is mainly isolated and purified from the traditional Chinese medicinal plant Artemisia annua L. with low content (0.01%-1%), currently the yield of artemisinin cannot meet the demand of the world market. With the growing development of genetic engineering, enhancing the artemisinin content by over-expressing the key enzyme involved in the biosynthesis of artemisinin has been proved to be feasible.This research selected the transgenic A. annua plants that were co-transformed with HMGR and FPS as the starting material, and took polyploidy breeding and gene pyramiding hybrid breeding to explore novel strategies to further enhance the artemiainin content in transgenic A. Annua. The results are as follows:1.Molecular detection and artemisinin content test of the T1 generations of transgenic A. annua that was co-transformed with HMGR and FPS. PCR detection was used to select transgenic offsprings. Semiquantitative RT-PCR showed that except for the normal expression of FPS in the roots, both of HMGR and FPS over-expressed in the four different tissues (roots, stems, leaves and flowers) of transgenic plants. Comported well with the RT-PCR results, Western Blot also showed that transgenic plants had higher expression of FPS in leaves. Besides, the average level of artemisinin in the transgenic plant was increased by 17.4% compared to the non-transgenic one, proving that these two genes modulating the synthesis of artemisinin.2.Induction and investigation of tetraploid A. Annua.Tetraploid A. annua plants were successfully inducted by using colchicine, and confirmed by FCM. No significant differences in major agronomic traits appeared between transgenic and non-transgenic tetraploid plants, both showing lower plant growth in earlier growth stage and speeding up growth one to two months later. No significant differences in some agronomic traits such as plant height between tetraploid and diploid plants, but tetraploid plants had larger seeds, thicker and wider leaves, higher stomatal length but lower frequency.The average level of artemisinin in non-transgenic tetraploids was increased compared with non-transgenic diploids, by 39% to 56% during vegetation growth period. Gene expression of key enzymes related to artemisinin biosynthetic pathway in different ploidy level was analyzed by semiquantitative PCR, and significant up-regulation of FPS, HMGR and artemisinin-metabolite specific Aldh1 genes was revealed in tetraploids. Slightly increased expression of ADS was also detected.The average artemisinin content in transgenic tetraploids with exogenous FPS and HMGR was increased by 48.7% comparing to its trangenic diploid ancestors and further with an increase of 69% to the wild A. Annua.3.Pyramiding breeding of trangenic A. Annua plants with different exogenous genes.Two cross combinations were carried out between HMGR and FPS co-transformed transgenic A. annua plants and the other two transgenic plants with ADS and hpSQS respectively. HPLC analysis revealed that F1 hybrids of these two combinations with three target genes pyramiding had higher average levels of artemisinin compared to their parents and with an increase of 34% and 50% respectively to the wild A. Annua.Present study indicates tetraploid breeding and pyramiding breeding are the effective strategies to further increase artemisinin content in transgenic A. Annua plants. This study provides new strategies for developing high artemisinin content transgenic A. Annua varieties.