Vector construction for promoter analyses in chicory and fluorescence evaluation by agroinfiltration
2013
Аутори:
Bogdanović, MilicaTodorović, Slađana
Dragićević, Milan
Cankar, Katarina
Beekwilder, Jules
Bouwmeester, Harro
Simonović, Ana
Остала ауторства
Vinterhalter, DraganТип документа:
Конференцијски прилог (Објављена верзија)
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© 2013 by the Serbian Plant Physiology Society
Метаподаци
Приказ свих података о документуАпстракт:
Chicory (Cichorium intybus L.) is rich in sesquiterpene lactones, compounds known for their bitter taste and medicinal properties. Most enzymes involved in the biosynthetic pathway of these secondary metabolites have recently been discovered and characterized. The first step in their biosynthesis is catalyzed by germacrene A synthase (GAS), in chicory present in two forms - long and short, and several P450 mono-oxygenases. So far, promoters of these genes have not been studied, and little is known about the spatial and temporal regulation of their expression. To address this issue, four vectors for plant transformation containing promoter-reporter gene fusions were designed and constructed by Gateway cloning, including one for GAS long, two for GAS short, and one for the cytochrome P450, germacrene A oxidase. As a marker for co-transformation, DsRED, a red fluorescent protein, was used, while the studied promoters were inserted to drive GFP/GUS fusion, to allow for visualization of promoter activity. Integrity and function of the constructs were checked by agroinfiltration in lettuce (Lactuca sativa Cv. Olof) - a transient transformation assay. Infiltration was performed with Agrobacterium tumefaciens, carrying the promoter constructs. Transformation success was checked five days after infiltration by fluorescent stereomicroscopy, and both DsRED and GFP were detected, indicating that the chicory promoters were active in lettuce. DsRED had strong and uniform fluorescence in all samples, but GFP fluorescence varied among plants infiltrated with different constructs. The GAS long promoter had strongest expression, followed by the P450 and the two rather weak GAS short promoters. The fluorescence was visible only in the infiltrated parts of the leaves, in tissues between leaf veins, but not in the veins themselves. Both abaxial and adaxial leaf sides were fluorescing. There were no differences observed between spatial distribution of DsRED and GFP: all infiltrated parts showed both markers. Since these vectors were confirmed to be functional, stable transformants of chicory will be generated by transformation using A. rhizogenes carrying the same promoter constructs.
Кључне речи:
chicory; sesquiterpene lactones; Gateway cloning; germacrene A synthase; agroinfiltrationФинансирање / пројекти:
- Физиолошка, хемијска и молекуларна анализа диверзитета одабраних ретких и угрожених биљних врста у циљу еx ситу заштите и продукције биолошки активних једињења (RS-MESTD-Basic Research (BR or ON)-173024)
- Plant Terpenoids for Human Health: a chemical and genomic approach to identify and produce bioactive compounds (EU-FP7-227448)
У:
- Vinterhalter D, editor. Programme and Abstracts: 1st International Conference on Plant Biology and 20th Symposium of the Serbian Plant Physiology Society; 2013 Jun 4-7; Subotica, Belgrade. Belgrade: Serbian Plant Physiology Society; 2013. p. 60-1.