TRANSFORMATION OF AMORPHA-4,11-DIEN SYNTHASE (ADS) AND PROTEIN 19 (P19) GENES INTOARTEMISIA ANNUA L. PLANT AND ITS EFFECT ON ARTEMISININ PRODUCTION AS AN ANTIMALARIA FANIMUTIACAHYANI

• Main Author: Mutia Cahyani, Fani
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/49160
• Institution: Institut Teknologi Bandung

Background and objectives: The low content of artemisinin related to the biosynthetic pathway that is influenced by the role of certain enzymes in the formation of artemisinin. Regulation of genes which are involved in artemisinin biosynthesis through genetic engineering is a choice to enhance the artemisinin content. Amorfa-4,11-diene synthase (ADS) is a key enzyme in the biosynthesis of artemisinin. The aims of this study were to transform ADS gene and P19 gene as an antisilencing intoA.annuaand to see their effect on artemisinin production in the leaves and hairy roots of A.annua. Presence of P19 and ADS genes were confirmed through PCR products and sequencing analysis. The recombinant plasmids containing ADS and, or P19 genes, were transformed into Agrobacterium tumefaciens strains AGL1 theninto the leaves and hairy roots of A.annua. Transformation process were mediated by A. tumefaciens AGL1 performed by vacuum infiltration and syringe infiltration methods with the successful confirmation of the transformation through the GUS histochemical test and the PCR analysis. Artemisinin levels in each sample was measured using high performance liquid chromatography (HPLC). Results:GUS histochemical test results showed that transformations were successfully done which were indicated by the appearance of blue color of the transformant leaves and hairy roots, whereas the color does not appear on the negative controls. Percentages of the area's blue using the syringe infiltration method of leaves without transformation, untransformed AGL1, ADS, ADS-P19, P19, co-transformed were 0; 0; 39.04; 89.82; 92.55; 65.22% respectively. For the vacuum infiltration method were 0; 0; 58.47; 97.13; 98.25; 85.92% respectively. While the transformant hairy root without transformation, untranformed AGL1, ADS, ADS-P19, P19, cotransformed blue area were 0; 0; 80.21; 96; 99.42; 91.17% respectively. HPLC analysis resulted that the ADS-P19 sample contained higher level of artemisinin content, which was 0.18% while the sample without transformation, untransformed AGL1, ADS, P19, co transformed were 0.073; 0,074; 0.08; 0.16; 0.09% respectively. While the hairy root samples, without transformation, transformant hairy roots by AGL I and ADS did not containartemisinin, but the samples of ADS-P19, P19 and co transformed contained artemisinin 0.056; 0.08; 0.095% respectively.Conclusion: Transformationof ADS and P19 genes into leaves and hairy roots of A.annuaplant have been succesfully done and influenced on the enhancement of artemisinin content on transformed leaves with ADS-P19 up to 2,57 folds compared to untransformed leaves, while for P19, co transformed and ADS were up to 2,25 ; 1,29 and 1,14 folds respectively.