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Title: Transfer of rol genes and Evaluation of Artemisinin Synthesis in Transgenic Artemisia annua L. and Artemisia dubia Wall.
Keywords: Natural Sciences
Issue Date: 2012
Publisher: Quaid-i-Azam University Islamabad, Pakistan
Abstract: Artemisinin is an important secondary metabolite of Artemisia annua and Artemisia dubia. It is a major constituent of Artemisia species. Chemically it is an endoperoxide sesquiterpene lactone. It is a potent antimalarial drug that has also been proven very effective in treatment of cancer. The rol genes have been known to enhance production of secondary metabolites in plants, possibly through stimulation of defense pathway. This study examines the effect of transformation of A.annua and A.dubia with the rol genes through Agrobacterium tumefaciens and Agrobacterium rhizogenes. The artemisinin content, trichome density and expression of key genes in the biosynthetic pathway of artemisinin were measured. Anticancerous activity of extracts of transformed and untransformed A.annua and A.dubia was also observed against MCF-7 breast cancer cell lines. Transcriptomic study of transformed and control A.annua and A. dubia was carried out as well. A number of factors like type of explants, effect of sterilization and co- cultivation period have been reported to affect the efficiency of A.tumefacienes mediated transformation. LBA4404 strain of A.tumefacienes containing pRT99 plasmid harboring rol ABC genes were used for the A.tumefacienes mediated transformation. Mercuric chloride 0.1% for 2 minutes showed the best results for seed surface sterilization giving 95% germination. Leaf and stem were found best explants for transformation. Explants were infected with bacterial culture for 5 minutes and cultured on co-cultivation medium (MS medium with 200 μM acetosyringone) for 48 hours. Explants when cultured on selection medium (MS medium containing 0.1mg /L BAP supplemented with 50mg/ml kanamycin), resulted in the maximum number of transformants. Regeneration of transgenic shoots was obtained from both stem and leaf explants on regeneration medium (MS medium containing 0.1mg /L BAP supplemented with 20mg/ml kanamycin and 500mg/l cefotaxime). Eighty percent of the transgenic A.dubia shoots showed rooting response on half MS medium with 0.025mg/L NAA, while transgenic shoots of A.annua produced roots on half MS medium with 0.1mg/L NAA. Control and transgenic plants were transferred to small pots and acclimatized. Morphological differences like increased size and broad leaves were observed. Confirmation of transformation was made through PCR for rol A, B and C genes. Southern blot analysis was performed to check the copy number of inserted genes.A.rhizogenes strain LBA9402 and LBA 8196 carrying rol genes were used for the A.rhizogenes mediated transformation. Transformation with A.rhizogenes was carried out with the plants growing in green house through their in-vitro propagation. Hairy roots were produced from A. rhizogenes strain LBA9402 infected stem portions of A. annua and A.dubia after seven days of infection but no hairy roots were produced from strain LBA8196. Transformed and control roots were cultured on solid B5 medium for further roots proliferation. Transformed roots showed better proliferation than control roots. Artemisinin content was significantly increased in transformed material of both Artemisia species when compared to un-transformed plants. The artemisinin content increased mostly five to ten times within leaves of transformed lines, hairy roots and roots of transformed shoots. It indicated the plant capability of synthesizing much higher amounts than has been achieved so far through traditional breeding. Similarly, amount of different derivatives of artemisinin i.e. artemether, arteether, dihydroartemisinin and artesunate was also significantly increased in transformed material of both Artemisia species when compared to un-transformed plants. Expression of all the tested genes involved in artemisinin biosynthesis pathway was significantly increased, although variation amongst the genes was observed. Cytochrome P450 (CYP71AV1) and aldehyde dehydrogenase 1 (ALDH1) expression levels were higher than that of amorpha-4, 11 diene synthase (ADS). Levels of the trichome development and sesquiterpenoid biosynthetic gene (TFAR1) expression were also found increased in all transgenic lines. Trichome density significantly increased in the leaves of transformed plants, but no trichomes were seen in control or transformed roots. Crude hexane and aqueous extracts of rol genes transformed plants revealed higher anticancerous activities against MCF-7 breast cancer cell lines. Hexane extracts of transgenic plants revealed higher anticancerous activity against MCF-7 breast cancer cell lines compared to aqueous extracts. Transcriptomic study of A.annua and A.dubia allowed sequencing of the transcriptome of these species for the first time. 16400 Contigs were generated by aligning different transcriptome sequences. Up and down regulation and Putative gene functions were predicted. BLAST of 500 contigs was performed, out of which 264 contigs showed homology with genomes of different organisms. Blast results of somecontigs showed that some species have genes that are similar to those involved in artemisinin biosynthesis pathway. It would be interesting to know what are the pathways in which those genes are involved in these species. Furthermore, divergence of A. annua and A.dubia from the common ancestors can be found through phylogenetic tree construction.
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