Cloning and expression of Amorpha-4, 11-Diene synthase gene in Artemisia annua

Abstract

Artemisinin is a new effective antimalarial drug present in the medical plant Artemisia annua L. Its content is generally low in cultivated plants. We, therefore, aimed to increase the yield of artemisinin in this plant by using techniques of gamma irradiation, plant tissue culture and molecular biology. Amorpha-4,11-diene synthase (ADS), the enzyme catalyzing the first step of the artemisinin biosynthetic pathway, was the target of this study and its activity was assayed in various γ–ray treated A. annua plantlets. Many of these irradiated plantlets showed a strong correlation between the enzyme activity of ADS and the artemisinin content, suggesting that gamma irradiation had significant effects on the ADS gene (ads) which is likely to be related to the enhancement of artemisinin content in A. annua. Moreover, when some of these selected plantlets were transferred from the in vitro culture to greenhouse or open-field, the mature plants obtained also contained artemisinin essentially in a similar content pattern and, interestingly, with observed individual correlation among the selected plantlets. These results suggested that stable high artemisinin-yielding plants of A. annua can be obtained by the technique of gamma irradiation. In addition, an attempt to enhance artemisinin content was also performed by creating transgenic plants of A. annua. For this approach, the full-length open reading frame of ads gene was first amplified by RTPCR using specific primer and was then inserted into the plant expression vector in A. annua via Agrobacterium tumifaciens-mediate transformation. By using the process of plant regeneration through transgenic callus to shoot, the transgenic plants of A. annua were obtained. These transgenic plants were subsequently detected for the presence of the recombinant ads gene, its functional enzyme activity and finally the possible effect on the increase of artemisinin content. The results showed that the shoots of A. annua transferred with the 35s promoter were PCR positive. The ADS specific activity assayed by radioisotope method showed its activity in the transgenic plants 2-3 times higher than the untransgenic plant. Interestingly, the production level of artemisinin in the transgenic plant also showed 2- 3 times higher than the untransgenic plant. These results indicated that transgenic plant of A. annua did contain ads gene and could be expressed by the expression vector leading to the apparent high enzyme activity of ADS which, in turn, caused the increase in the overall yield of artemisinin in the transgenic A. annua plants.