Биосинтеза гвајанолида код цикорије (Cichorium intybus L.) - утицај трансформације на продукцију и испитивање активности гермакрен А-синтазе и -оксидазе коришћењем промоторских фузија и утишавања гена
Guaianolide biosynthesis in chicory (Cichorium intybus L.) - influence of transformation on the production and study of germacrene A synthase and oxidase activity using promoter fusions and gene silencing
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Chicory (Cichorium intybus L.) is traditionally used medicinal plant rich in guaianolides - bioactive sesquiterpene lactones. In this research, chicory was transformed with Agrobacterium rhizogenes in order to evaluate the production of guaianolides in transgenic root cultures and regenerated plants, and to explore the influence of floral transition and bacterial oncogene expression on guaianolide accumulation. A. rhizogenes strain A4M70GUS was successfully used for chicory transformation, producing hairy root cultures, which spontaneously regenerated shoots and plants that were flowering in vitro. In transgenic plants, guaianolides were accumulated during floral transition, which was not the case in untransformed plants. Accumulation of these compounds was especially significant in transformed plants' roots, which correlated with the expression of RolC gene. Guaianolides originate from a common precursor - germacrene A, which is synthesized by germacrene A synthase (GAS). In subsequent steps, germacrene A is converted to germacrene A acid in a series of steps catalyzed by germacrene A oxidase (GAO), which is then converted into costunolide - precursor of other sesquiterpene lactones. Two GAS mRNAs have been previously isolated from chicory - CiGASlo (GAS long) and CiGASsh (GAS short). One promoter for GAS long, two for GAS short and one promoter for GAO have been isolated from chicory BAC library, and fused with a fluorescent reporter GFP. Strength and functionality of the promoters were characterized by lettuce agroinfiltration and by cotransformation of chicory by A4M70GUS strain containing the constructs. DsRED, another fluorescent reporter, was used as a marker for cotransformation. Due to incompatibility of DsRED and GFP, promoter activity was detected in regenerated transgenic plants by RT-PCR and qRT-PCR. The promoters were characterized by different strength and partial tissue specificity. GAS long promoter was less tissue specific than GAS short, which was active mainly in chicory roots...
Keywords:Cichorium intybus; Sesquiterpene lactones; Guaianolides; Germacrene A synthase; Germacrene A oxidase; Agrobacterium rhizogenes; Promoter fusions; RNA interference; DsRED; GFP
Source:University of Belgrade, Faculty of Biology, 2015, 1-261
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