DNA sequence from Arabidopsis thaliana encoding ammonium...

Details DNA sequence from Arabidopsis thaliana encoding ammonium... DNA sequence from Arabidopsis thaliana encoding ammonium...

1996-04-29

2003-09-16

Nelson, Amy J. (Department: 1638)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Fungi

C435S252300, C435S320100, C536S023600

Reexamination Certificate

active

06620610

ABSTRACT:

BACKGROUND OF THE INVENTION
The present invention relates to DNA sequences that contain the coding region of ammonium transporters, whose introduction into a plant genome modifies the uptake and transfer of nitrogen compounds in transgenic plants. As well as to plasmids, bacteria, yeasts, plant cells and plants containing these DNA sequences, and also to a process for the identification and isolation of DNA sequences that code for an ammonium transporter.
The supplying of growing plants with nitrogen compounds is a limiting factor in biomass production and is thus a limit on the yield of agricultural production. For this reason, nitrogen compounds, often in the form of mineral fertilisers, are added in agricultural biomass production.
An only partial uptake by the plants of the added nitrogen compounds makes it, on the one hand, necessary that the nitrogen fertiliser produced with high energy input is used in an excess, and, on the other hand, leads only to a partial uptake so that the nitrogen compounds are washed into the ground water which can lead to considerable ecological problems.
There is thus a great interest in plants which are capable of taking up large amounts of nitrogen as well as in the provision of the possibility of modifying the nitrogen uptake in plants.
For many plants there is provided information that the uptake of nitrogen is essentially in the form of nitrate salts. In strongly acid soils or in soils which follow intensive cultivation or have a strong tannin content, the nitrate formation (nitrification) is however strongly reduced and the uptake of nitrogen in the form of ammonium becomes the most important mechanism for the uptake of nitrogen compounds (Raven & Smith 1976 New Phytol 76: 415-431). Plants which are well adapted to acid soils, appear in part to favour ammonium rather than nitrate uptake and can tolerate ammonium ion concentrations that would be toxic for other plants. Examples of these plants are sugar cane,
Betula verucosa
or
Lolium rigidum
(Foy et al 1978, Ann Rev Plant Physiol 29: 511-566). The toxicity of the ammonium follows from a displacement of the ion balance: the uptake of the positively charged ammonium ion leads to an acidification of the cytoplasm, provided that no cations are secreted in counter-exchange. In an uptake of ammonium via a transport system whose uptake mechanism is based on an ammonium ion proton antiport, the ionic imbalance is not a problem.
There is thus a great interest in transport systems which work by the mechanism of an ammonium ion proton antiport and/or in systems which could be converted through techniques of protein engineering into ammonium ion proton antiports.
In spite of extensive efforts, it has not been possible up until now to isolate transport systems with whose help plants are protected against an ammonium ion loss caused by membrane diffusion (retrieval system).
By the term ammonium is also to be understood methylamine which is analogous to ammonium.
An active uptake system has been investigated in the fungus
Aspergillus nidulans
(e.g Arst et al., 1973, Mol Gen Genet 121: 239-245), as well as in
Penicillum chrysogenum
(Hackefte et al., J Biol Chem 245: 4241-4250). In these studies methylamine was used as the ammonium analogue. For
Aspergillus nidulans,
five genetic loci were established, which take part in the transport of methylamine (Pateman et al., 1973, J Bacteriol 114: 943-950). In biochemical experiments concerning the methylammonium transport in
Penicillum chrysogenum
and
Saccharomyces cerevisisae,
it has been shown that the transport is temperature and pH dependent, and that the pH optimum is 6.0 to 6.5 and the transport efficiency steadily rises up to a temperature of 35° C. (Roon et al., 1975, J Bacteriol 122: 502-509). The methylamine and/or ammonium transport in
Saccharomyces cerevisiae
is dependent on the supply of easily usable chemical energy, e.g. in form of glucose. The transport system consists of at least three independent transporters, which differ in transport capacity and affinity for the substrate: besides a high affinity transporter with low capacity (Km value=250 &mgr;M, maximum speed Vmax=20 nmol/min per mg cells (dry weight)) there is a low affinity system with high capacity (Km=2 mM, Vmax=50 nmol/min per mg cells) and a low affinity system with medium capacity (Km=20 mM, Vmax=33 nmol/min per mg cells) (Dubois & Grenson, 1979, Mol Gen Genet 175: 67-76). In the presence of glutamine or asparagine in the surrounding medium, the ammonium uptake is reduced by 60 to 70%, while other amines hardly have any influence (Roon et al., 1975, J Bacteriol 122: 502-509).
Nothing is known about the molecular nature of the ammonium transporter of the above-mentioned or different fungi and other organisms, such as for example bacteria. Equally nothing is known about systems, with whose help fungi or bacteria protect against ammonium ion loss by membrane diffusion (retrieval systems) at the molecular level. Further genes, which code for ammonium transporters are not known.
Various evidence suggests that the ammonium transport in
Saccharomyces cerevisiae
is accomplished by at least two functionally different transport systems (Dubois & Grenson, 1979, Mol Gen Genet 175: 67-76):
1. Kinetic analyses of the methylamine-uptake by Saccharomyces show an abrupt transition between apparent linear sections, whereby both functions are inhibitable by ammonium.
2. Both functions can be separately excluded by mutation. The resulting mutations mep-1 and mep-2 are genetically independent.
3. The mutants mep-1 and mep-2 can each grow in media with ammonium as the only nitrogen source, while a double mutant mep-1/mep-2 shows hardly any growth under these conditions (data taken from Dubois & Grenson, 1979).
A clarification of the ammonium transport processes in plants, that leads to similar detailed information such as for yeast, is not available and because of the difficulty of the molecular biological analysis of mutations is scarcely possible in a corresponding manner.
SUMMARY OF THE INVENTION
The object of the present invention is to provide coding for DNA-sequences of ammonium transporters which cause a change in the uptake and transfer of nitrogen compounds in transgenic plants.
The object of the present invention is further to provide DNA-constructs, such as plasmids, with which the ammonium transport in transgenic plants can be modified by introduction of the corresponding construct (plasmids) into the plant genome which leads upon transcription to the formation of a new ammonium transporter molecule in the transgenic plant and/or the suppression of the formation the plant's own ammonium transporter molecules.
There have now surprisingly been found DNA sequences, that contain the coding region of a plant ammonium transporter, whereby the information contained in the nucleotide sequence when integrated in a plant genome
a) under the control of a promoter in a sense orientation makes the expression of a translatable mRNA which leads to the synthesis of an ammonium transporter in transgenic plants possible or
b) under the control of a promoter in an anti-sense orientation makes the expression of a non-translatable mRNA which prevents the synthesis of an endogenous ammonium transporter in transgenic plants possible.
A further aspect of the invention is to provide DNA sequences which contain the coding region of a plant ammonium transporter.
In an analogous way, the ammonium transporter can also be used to modify animal cells.
The DNA sequences, which code for a plant ammonium transporter, can be identified and isolated by a process of determining which DNA sequences are able to complement specific mutations in the yeast
Saccharomyces cerevisiae.
In general, the mutations, are those which have the result that the corresponding strains cannot grow any further in media which contain ammonium as the only nitrogen source. Such a strain can be transformed with a plant cDNA-library and transformands can be selected which can grow in medi

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