Analysis of the function and evolution of the cellulose synthase-like C gene family using the model organism Physcomitrella patens
The plant cell wall is an integral component of everyday life. A complete understanding of the biosynthesis of the various cell wall polysaccharides will help in areas of agricultural production, biofuel development, and other economic industries. A daunting area of plant cell wall research is to identify genes that encode proteins involved in the biosynthesis of the various cell wall polysaccharides. The structure and resilience of the cell wall makes this a challenging endeavor. Recent advances in genetics and biochemical techniques may pave the way for promising insights into the dynamics of the cell wall. The focus of this study was to test the role of the CELLULOSE SYNTHASE LIKE C (CSLC) gene family in the biosynthesis of xyloglucan, as suggested in current literature. The bryophyte model organism, Physcomitrella patens, was used as it occupies a basal level of land plant evolution, has a simple body plan, and effective homologous recombination. The approach was to produce knockout mutations of the CSLC1-3 genes and a global knockdown of all CSLCs transcripts, via RNAi technology. The knockdown constructs were designed to target homologous regions within the genome and to replace the wildtype gene with the construct via homologous recombination. Eight knockout lines representing 3 of the 7 PpCSLC genes were produced, CSLC1KO #14, CSLC1KO #15, CSLC2KO #1, CSLC2KO #10, CSLC2KO #30, CSLC3KO #1-1, CSLC3KO #1-5, and CSLC3KO #1-9. Polymerase chain reaction analysis confirmed the removal of the wildtype gene and the stable integration of the vector at the target site. Multiple copies of the vector were detected through PCR screening in lines CSLC1KO #14, CSLC1KO #15, and CSLC3KO 1-5. Southern blot analysis confirmed integration of two tandem copies of the vector at the target site. The transient RNAi assay employed specially designed moss line, NLS-4, in which a GUS:GFP fusion protein was localized to the nucleus. The RNAi construct consisted of a portion of the CSLC3 gene with high similarity to all CSLC genes, fused to GUS, as inverted repeats separated by a hairpin. Visual screening for loss of nuclear fluorescence indicated successful knockdown of CSLC transcripts. Quantitative morphometric analysis was used to measure growth characteristics of the knockdown and knockout lines. Both knockout and knockdown lines displayed growth abnormalities compared to wildtype lines. This study also investigated the effects of mannitol, an osmoticum, on cell wall polysaccharide composition detected by immuno-fluorescent labeling of polysaccharides and proteins. It was shown that levels of cellulose, xyloglucan, pectin and arabinogalactan protein were altered by osmotic stress. The results from fluorescence labeling is corroborated by microarray analysis of osmotically stressed P. patens and suggests differences in the osmotic stress responses of mosses and vascular plants.
Molecular biology|Genetics|Cellular biology
Michael John Budziszek,
"Analysis of the function and evolution of the cellulose synthase-like C gene family using the model organism Physcomitrella patens"
Dissertations and Master's Theses (Campus Access).