Microalgae are a promising, but widely unexploited source for the sustainable produc¬tion of novel compounds, which find application in food, feed, pharma¬ceutical and cosmetic industries, as well as for biofuel production. The haptophyte Pavlova lutheri possesses a great nutritional value as it synthesizes and accumulates higher amounts of very long chain polyunsaturated fatty acids (vLC-PUFAs). Also, the rhodophyte Porphyridium purpureum is considered as one of the economically important strain since it produces fluorescent phycobiliproteins, exopolysaccharides, vLC-PUFAs, carotenoids and vitamins. However, successful nuclear transformation has not been for realized for these industrially important microalgae for exploiting their potential into a sizeable market. This study reports an efficient nuclear transformation protocol for the metabolic engineering of P. lutheri and P. purpureum. Initial studies were performed to standardize co-cultivation medium and determine the sensitivity of the microalgae to selective agents. Both cultures bleached in artificial seawater (ASW) medium containing micromolar concentrations of the herbicide norflurazon. A mutated genomic phytoene desaturase (pds) gene from Haematococcus pluvialis was cloned as a selectable marker into the binary vectors pCAMBIA 1301 and 1380 for the genetic transformation of microalgae. Agrobacterium tumefaciens-mediated transformation of P. lutheri and P. purpureum with the vector pCAMBIA 1301-pds-L504R and pCAMBIA 1380-pds-L504R yielded norflurazon-resistant cells. The effects of different factors that are known to influence the efficiency of Agrobacterium-mediated transformation were accessed: co-cultivation medium, acetosyringone concentration and co-cultivation period. Co-cultivation of P. lutheri with A. tumefaciens harboring pCAMBIA 1301-pds-L504R in ASW medium containing 100 µM acetosyringone at 25 ± 1ºC and under 10 ± 2 µmol m-2 s-1 of light intensity for 24 h resulted in the highest number of norflurazon-resistant P. lutheri cells. For P. ...
Agrobacterium-mediated Nuclear Transformation of Haptophyte and Rhodophyte Species ; Agrobacterium-vermittelte Transformation von Algen der Abteilungen Haptophyta und Rhodophyta
2017-01-01
Theses
Electronic Resource
English
DDC: | 629 |
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