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EcoTub:
An ecologically safe selection system for transgenic crops based on modified
plant-tubulin genes
Background
and objectives
There is substantial public concern about a potential spread of antibiotic
resistance genes in the soil and intestinal bacteria as a consequence
of the use of genetically modified organisms, and in particular of the
agricultural use of transgenic crops. A selection system for plant transformation
that is exclusively based on genetic information already present in the
host plant and that does not require antibiotic resistance genes would
avoid any such risk. This project, which has just started, aims to develop
such ecologically safe systems for cereals. The project is based upon
truncated tubulins lacking the binding site for carbamates. Plants carrying
the genes encoding such truncated proteins will be resistant to carbamate
herbicides thereby allowing selection. The production of the truncated
tubulins will be driven by the tubulin's own promoters within DNA fragments
devoid of foreign sequences. The suitability of this selection system
will be analysed using rice as a model cereal crop and the rice gene for
phytochrome as a test gene.
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Rice
panicle.
Photo: PBIU, University of Leeds. |
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Figure 1 shows the response of microtubules (MT) and actin microfilaments
(MF) to a depletion of the plant growth regulator auxin. One can
see that the microtubules reorient into a longitudinal direction,
with the consequence that the cellulose fibres in the cell wall
are laid down in the same direction, such that the length growth
of the plant is blocked (the plants become shortened). This is the
physiological response, where the tubulin gene TUBA1 is important.
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In Figure 2, the rice seedlings shown are from the wild type (WT)
that have been treated with carbamate herbicides (+EPC) - note the
short-stunted and malformed seedlings in comparison to the untreated
(-EPC) control, where the seedlings are straight and long. The carbamates
destroy microtubules and thus block cell elongation. If the wild
type tubulin is replaced by a mutated form, where only one base
is exchanged (leading to a precocious stop codon and a C-terminally
truncated product), the carbamate can bind no longer and the respective
seedling is straight and long as if it had not experienced the drug
(ER31).
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Such seedlings change their growth patterns, for instance, as shown
in Figure 3: they produce longer organs in the embryo depending
on whether they have one (ER31) or two (ER31*) copies of the mutated
gene.
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Figure 4 shows the response of microtubules to the plant growth regulator
auxin in the wild type (WT) and in plants with the mutated tubulin
gene (ER31). Microtubules made up of the mutated tubulin are able
to produce proper microtubules, but these microtubules no longer respond
to the growth regulator, and they maintain a more or less longitudinal
orientation. |
Approach and methodology
A selection marker cassette consisting of the tubulin promoter, coding
sequence for alpha-tubulin conferring resistance to carbamate herbicides,
and full-length beta-tubulin will be constructed. Selection and transformation
protocols based on carbamates will then be optimised. The selection marker
cassette will then be used for cotransformation with rice phytochrome
as model and the expression and functionality of the transgene will be
assayed.
Throughout this work, the approach will be based on “clean DNA”
technology, which involves avoiding the use of extraneous backbone and
vector sequences.
Main findings and outcome
This project which is just starting is expected to generate the following
results. The sequences of various different rice alpha- and beta-tubulins
will be determined and their promoters isolated. Variants of rice alpha-tubulin
with a truncated or modified C-terminus will be constructed. Vectors will
then be made from these elements.
Protocols for homologous transfection into rice embryos will be developed
involving tests for conferred carbamate resistance. The clean-DNA system
will then be validated using the rice phytochrome gene. Phytochrome expression
and functionality in transformed cells will be investigated and carbamate
resistance and phytochrome overexpression during development and propagation
of transgenic plants analysed. This will also include tests for potential
negative effects of tubulin expression on plant development.
Conclusions
This project will contribute to a clean DNA technology to minimise the
risks associated with genetically modified organisms. In addition to the
advantages of reduced risk, this should also improve public acceptance
of genetic modification technology. In particular, rice plants will be
produced that are resistant to a herbicide (carbamate) but that do not
contain any DNA sequences of foreign origin.
Major
publications
Nick P., Yatou O., Furuya M., Lambert A.M., "Auxin-dependent
microtubule responses and seedling development are affected in a
rice mutant resistant to EPC".
Plant J., 6, 1994, pp. 651-663.
Qin
X., Gianì S., Breviario D., "Molecular cloning of
three rice alpha-tubulin isotypes: differential expression in
tissues and during flower development".
Biochem. Biophys. Acta 1354, 1997, pp. 19-23.
Kohli
A., Leech M., Vain P., Laurie D.A., Christou P., "Transgene
organization in rice engineered through direct DANN transfer supports
a two-phase integration mechanism mediated by the establishment
of integration hot-spots".
Proc. Natl. Acad. Sci. USA, 95, 1998, pp. 7203-7208.
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Research
project
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Contract
number
QLK3-2000-00060
Period
October 2000 - September 2003
Coordinator
P. Nick
Institut für Biologie II
Freiburg (DE)
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Partners
D. Breviario
Centro Nazionale di Ricercha
Milano (IT)
P.
Christou
John Innes Centre
Norwich (UK)
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Research areas
