Introduction of alkali-labile units into lignin in transgenic plants by genetic engineering
© Ishikawa et al; licensee BioMed Central Ltd. 2011
Published: 13 September 2011
Because of codon usage is significantly different between genes in plants and SYK-6, we chemically synthesized open reading frame (ORF) of the ligD gene for improving its expression in the transgenic plants. After addition of nucleotide sequence for apoplast-targeting signal peptide to the synthesized ligD ORF, it was introduced into Arabidopsis thaliana, tobacco BY-2 and hybrid aspen under the control of cauliflower mosaic virus 35S promoter. LigD expression in the transgenic plants was monitored by Western blot analysis and enzymatic activity with crude extract prepared from each transgenic line. Preliminary analysis of lignin structure by 2D-NMR and nitrobenzene oxidation was also performed.
Results and discussion
At first we confirmed expression of the ligD transgene in Arabidopsis by Western blot analysis with antiserum against LigD polypeptide. Positive expressions of the ligD were detected in some of the transgenic plants analyzed. Enzymatic activities of LigD in crude extracts prepared from both cytosolic and apoplastic fractions of the transgenic Arabidopsis plants were also detected, but it was relatively higher in the latter case. As expected, 2D NMR (1H-13C HMQC) analysis suggests that the abundance of the alpha-keto (alpha carbonyl) structure in 8-O-4’ units of lignin in the transgenic plants is relatively higher than that in the wild-type plant. Chemical compositions of lignin (syringyl/guaiacyl ratio) and neutral sugars could not be distinguishable between the transgenic and wild-type plants. Generation of transgenic hybrid aspen and its analysis are now in progress.
This research project was supported partly by the New Energy and Industrial Technology Development Organization (NEDO).
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