Gene Expression and Growth Analyses in Populus RNAi lines Targeting Xylan Biosynthetic Gene Family GT 43
Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
Wood biomass is coming into focus as a good alternative renewable energy feedstock. Fast-growing woody plants, such as Populus species, offer potential as a bioenergy crop mediated by their abundance of energy containing cell wall polysaccharides.
Furthermore, the first tree genome sequencing of Populus trichocarpa (poplar) offers an opportunity for genetic manipulation of woody cell components in order to optimize it for instance as an energy crop. Plant cell walls are very complex structures consisting of biopolymers, which form a network in the secondary wall. Cellulose, for instance, is organized as microfibrils consisting of β‐1,4‐glucan chains, whereas the hemicelluloses present heterogeneous polysaccharide groups. Within the hemicelluloses, xylans are the most abundant. The biosynthesis of hemicelluloses is still poorly understood.
In order to investigate the biological role of GT43 genes during wood formation, transgenic hybrid aspen RNAi lines targeting GT43 have been created (Ratke et al., unpublished). Three GT43 genes (GT43B, GT43C and GT43F), and their double (GT43BC, GT43BF, GT43CF) and triple (GT43BCF) combinations, were selected to be targeted by RNAi. All the constructs were fused to the constitutive cauliflower 35 (CaM35S) and a wood-specific (WP) promoter and transformed into hybrid aspen (Populus tremula x tremuloides).
The first part of the study focuses on selecting the most down‐regulated GT43 RNAi lines by using real‐time quantitative PCR analysis. We used a stepwise screening strategy starting from 20 individual in vitro transformant lines per construct. During the two in vitro screening steps we used in vitro grown stem tissue as an RNA source. The first in vitro screening narrowed down the number of lines to eight per each construct. After second in vitro screening, four most down regulated lines per construct were selected in order to grow them in greenhouse conditions and obtain woody tissue for further analyses. Third screening was applied when the GT43 RNAi plants were growing under greenhouse conditions and it used petioles as an RNA source. After the third screening, three best lines were selected per construct based on RNA analysis and additional chemical analyses not presented here. For the final gene expression analysis of selected three best lines per construct, we used freeze-dried wood or bark powder as an RNA source. The GT43 gene expression analysis base on quantitative real‐time PCR.
These results are promising for developing Populus into a bioenergy crop. The use of molecular tools such a quantitative real-time PCR in stepwise screenings in order to select most down‐regulated lines of hybrid aspen resulted in selection of significantly altered lines as compared to the wild type. The future development of this technology should possibly use a higher number of transgenic lines as a starting point for selection.
Place, publisher, year, edition, pages
IdentifiersURN: urn:nbn:se:umu:diva-53558OAI: oai:DiVA.org:umu-53558DiVA: diva2:513467
Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences (SLU)
2012-03-30, KBF30, 13:00 (English)
UppsokLife Earth Science
Bako, LaszloMellerowicz, Ewa