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Activity of hygromycin phosphotransferase marker gene optimised for genetic modifications of miscanthus
Journal of Fundamentals of Renewable Energy and Applications

Journal of Fundamentals of Renewable Energy and Applications
Open Access

ISSN: 2090-4541

Activity of hygromycin phosphotransferase marker gene optimised for genetic modifications of miscanthus


International Congress and Expo on Biofuels & Bioenergy

August 25- 27, 2015 Valencia, Spain

Karolina Malec1, Monika Kowalska1, Jan Podkowi??ski2, Magdalena Zieli??ska2, Joanna Cerazy1, Aurelia ??lusarkiewicz-Jarzina1, Aleksandra Ponitka1, Stanis??aw Je?¼owski1 and Tomasz Pniewski1

Posters-Accepted Abstracts: J Fundam Renewable Energy Appl

Abstract :

Biotechnological methods based on genetic engineering and plant transformation are essential complement of research on
biomass production of miscanthus and other energy plants. Hygromycin phosphotransferase (hpt) is one of commonly used
marker genes used for such manipulations. Preliminary tests showed that hygromycin B is an appropriate selection factor for
miscanthus species (Miscanthus x giganteus, M. sinensis, M. sacchariflorus). A vector with optimised hpt coding sequence can
be a promising tool for transformation. In order to increase its expression and consequently improve efficiency of miscanthus
transformation, the coding sequence of bacterial hpt gene (Streptomyces hygroscopicus) was optimised basing on codon usage
specific for maize (Zea mays, Codon Usage Database, www. cazusa.or.jp). Optimised hpt coding sequence (Ohpt) was introduced
into pCAMBIA 1201 vector to replace original hpt gene and achieve pCAOhpt. Both vectors were used for transformation of
tobacco (Nicotina tabacum) as a model plant. Regenerating explants were cultured on selection media containing 10, 20 and 50
mg/L of hygromycin (hyg). After transformation with pCAOhpt, plants regenerated on media with hyg 10 and 20 mg/L amounted
142% and 130%, respectively, in comparison to those obtained for pCAMBIA. In the case of hyg 50 mg/L, only pCAOhpt gave
positive results (4 plants regenerated). The Ohpt sequence was detected using PCR in 50%, 83% and 100% of plants for hyg 10,
20 and 50 mg/L, respectively, while hpt sequence was present in 62.5% and 66% of obtained plants, for hyg 10 and 20 mg/L,
respectively. Thus, it is estimated that the Ohpt sequence increases transformation efficiency by approx. 35% and decreases risk
of false positive transgenic plants (escapes). Further analyses confirming functionality of t, as RT-PCR and assay of enzymatic
activity of hygromycin phosphotransferase are in progress. However Ohpt sequence has been already used for contruction of
vector pCAHGA, containing Ohpt sequence under control of ubiquitin1 promoter (form maize) and GUS reporter gene under
control of actin1 promoter (rice) for development of miscanthus transformation method.

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