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Poplar with modified lignin
Prof.dr. Wout Boerjan
The genetically modified trees have an altered lignin composition
resulting from the downregulation of the cinnamyl alcohol
dehydrogenase (CAD) enzyme through RNA interference. CAD catalyzes
the last step in the monolignol synthesis. The remaining CAD
activity in the modified trees is about 15% of that in wildtype
trees. The altered lignin composition has a positive effect on the
ease with which the lignin can be broken down to gain access to the
valuable sugar content in the cellulose and hemicellulose in the
wood of the trees. A lowered CAD activity has been found to
naturally occur in the U.S. in loblolly pine (Pinus taeda)
and in wild black poplar in Europe with comparable effects on the
The modified trees also carry the NPT-II selection marker gene
allowing an easy selection of transformed plants using kanamycin.
The term Recipient organism refers to an organism (either already modified or non-modified) that was subjected to genetic modification, whereas Parental organisms refers to those that were involved in cross breeding or cell fusion.
- Agrobacterium-mediated DNA transfer
Cinnamyl alcohol dehydrogenase
Pyruvate orthophosphate dikinase, Intron 3
Cinnamyl alcohol dehydrogenase
Octopine Synthase Gene Terminator
Nopaline Synthase Gene Promoter
Neomycin Phosphotransferase II
Nopaline Synthase Gene Terminator
RNA interference cassette:
Transcription of the hairpin RNA (HpRNA) is under control of the
Cauliflower Mosaic Virus (CaMV) 35S promoter and the
Agrobacterium tumefaciens octopine synthase terminator.
The transcript is expected to contain an inverted repeat of the
Populus tremula x Populus alba cinnamyl alcohol
dehydrogenase (CAD) gene separated by a segment sourced from
Flaveria trinervia pyruvate orthophosphate dikinase intron
3, which is essential to form a loop that allows the inverted
repeats to base pair and form the RNA secondary structure after
transcription. Then the hpRNA is sufficient to trigger an RNAi
response. Due to the RNAi response, the hpRNA is processed into
small interfering RNA (siRNA) and thus no translation to protein is
expected to occur.
The pHellsgate8 vector also contains a selectable marker for
kanamycin resistance. The Escherichia coli neomycin
phosphotransferase II gene is under transcriptional control of the
A. tumefaciens nopaline synthase promoter and terminator.
- Changes in quality and/or metabolite content
- Resistance to antibiotics
- Selectable marker genes and reporter genes
Cinnamyl alcohol dehydrogenase - Populus tremula x Populus alba - Gray Poplar
Post-transcription, the inverted repeat segments of the poplar
cinnamyl alcohol dehydrogenase (CAD) basepair to form a double
stranded region separated a loop derived from the Flaveria
trinervia pyruvate orthophosphate dikinase intron 3. The
double strandedness of the hpRNA is recognized by host cell RNA
interference machinery; DICER processes the hpRNA into siRNA
molecules (21-24 base pairs in length), which then associate with
ARGONAUTE to form the RISC complex. The siRNA are then used to
guide host machinery to degrade transcripts with sequence homology.
Thus, the host machinery targets the endogenous CAD transcripts,
resulting in silencing of CAD gene expression.
Three lines have been created with the same genetics: pHG8-CAD4,
pHG8-CAD19 and pHG8-CAD24. Thus, the transformation event listed
above represents a placeholder and will be updated should more
information become available.