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Vnos in ugotavljanje markerskih genov pri hmelju (Humulus lupulus L.).
Brinovec, Maša (Author), Luthar, Zlata (Mentor) More about this mentor... This link opens in a new window

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Abstract
Internodijskim izsečkom hmelja in kontrolnim listnim izsečkom tobaka smo s posredno metodo transformacije z Agrobacterium tumefaciens vnesli markerske gene: gusA, ZsGreen in DsRed-Express. Poskus adventivne regeneracije netransformiranega hmelja šestnajstih genotipov je pokazal največji odstotek regeneriranih izsečkov pri sorti 'Tettnanger' in sicer 70 %. Ostali genotipi so imeli odstotek regeneriranih izsečkov 0,8-10 % ali pa so bili neodzivni. Po transformaciji regeneracija genotipov hmelja ni presegla 10 %. Najboljšo regeneracijo je ohranila sorta 'Tettnanger', ki je bila odzivna tudi po transformaciji vsakega od uporabljenih markerskih genov. Transformiranim regenerantom tobaka in hmelja smo fenotipsko določili izražanje vnešenih transgenov in vgraditev tujih genov v genom s pomočjo PCR in agarozne elektroforeze. Transgen gusA se je pri tobaku izražal pri 60 % regenerantov in 8,1 % regenerantov hmelja, ZsGreen pri 74 % regenerantov tobaka in 4,3 % hmelja ter DsRed-Express pri 38 % regeneranrov tobaka in 6,1 % hmelja. Od vsake skupine smo naključno izbrali 10 regenerantov in jih s PCR testirali na prisotnost značilno namnoženih fragmentov. Pri transgenu gusA so se značilni fragmenti namnožili pri devetih regenerantih tobaka ter štirih hmelja, pri ZsGreen pri osmih regenerantih tobaka in sedmih hmelja ter pri DsRed-Express devetih regenerantih tobaka in šestih hmelja.

Language:Slovenian
Keywords:biotehnologija, transformacija, markerski geni, selekcijski geni, hmelj, analiza transgenov
Work type:Master's thesis/paper (mb22)
Tipology:2.09 - Master's Thesis
Organization:BF - Biotechnical Faculty
Year:2017
Publisher:[M. Brinovec]
UDC:602.6:604.6:582.630.2(043.2)
COBISS.SI-ID:8811385 Link is opened in a new window
Views:285
Downloads:179
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Secondary language

Language:English
Title:Insertion and analysis of marker genes in hop (Humulus lupulus L.)
Abstract:
Using an indirect method of transformation with Agrobacterium tumefaciens, we introduced the marker genes gusA, ZsGreen and DsRed-Express into hop and tobacco. An attempt of adventive regeneration of non-transformed hop of sixteen genotypes showed the highest effectivness of regeneration in the cultivar 'Tettnanger', 70%. Regeneration efficiency in other genotypes was 0.8-10% while some genotypes were not regenerating. In the case of internode cuttings after transformation regeneration of all genotypes was low (<10%). The best regeneration was retained by the 'Tettnanger' variety, which regenerated after transformation with each of the marker genes used. Transformed tobacco and hop regenerates were phenotypically analysed by expression of transgenic gene and the insertion of marker gene into the genome determined by PCR and agarose electrophoresis. Transgene gusA was expressed in 60% of tested tobacco regenerants and 8.1% of hop regenerants, ZsGreen in 74% of tested tobacco regenerants and 4.3% of hop regenerants, and DsRed-Express in 38% of tested tobacco regenerants and 6.1% of hop regenerants. From each group we randomly selected 10 regenerants and tested them with PCR for the presence of significantly multiplied fragments. Using the gusA transgene, significant fragments were multiplied by nine tobacco regenerates and four hops, ZsGreen in eight tobacco and seven hops, and using DsRed-Express nine tobacco and six hop regenerates.

Keywords:biotechnology, transformation, marker genes, selection genes, hop, transgene analysis

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