Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2001-06-26
2004-04-27
Mehta, Ashwin (Department: 1638)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S320100, C435S471000, C536S023600, C800S278000, C800S286000, C800S298000
Reexamination Certificate
active
06727095
ABSTRACT:
DESCRIPTION
The present invention relates to the nucleotide sequence of a glutamine synthetase from sugarbeet, to a vector comprising this nucleotide sequence, to cells which are transformed with this vector, to proteins encoded by this nucleotide sequence, to plants which have been transformed with this nucleotide sequence, and to methods for the genetic modification of plants, in particular sugarbeet.
The accumulation of glutamine as the main &agr;-amino-N component which occurs in the storage root, that is to say the storage organ of the sugarbeet, and which is also referred to as harmful nitrogen, gives rise to considerable problems in sugar production. The compensation, generally by rendering alkaline, for the acidification of the beet juice which is caused,by this component is costly, leads to faster wear on the production systems and, finally, also involves considerable environmental pollution, which can likewise be prevented only by use of costly measures. This glutamine is synthesized in the plant through amidation of glutamic acid by NH
4
+
being bound, with consumption of ATP, to C-4 of glutamate. This step is catalyzed by the enzyme glutamine synthetase (abbreviated to GS hereinafter). This enzyme is present both in the chloroplast and in the cytoplasm of the plant cell. In the chloroplast, the enzyme occurs as a tetramer which is encoded by one gene and consists of up to five subunits (GS-2). In the cytoplasm, according to current knowledge, the enzyme normally occurs as a heterooligomer (GS-1) encoded by more than one gene. The various GS-1 isoenzymes known are usually heterooctamers. Six different isoforms of GS-1 have been found to date. The glutamine synthetase localized in the chloroplast, that is to say GS-2, has the main function of binding the NH
4
+
produced during photorespiration and converting the NH
4
+
derived from nitrate reduction into organic compounds. The function of GS-1 is, by contrast, mainly in catabolic degradation pathways during the course of which the NH
4+
resulting from protein degradation is fixed. Such degradation pathways are particularly important during aging of the leaf, that is to say during senescence, and the resulting glutamine is exported from the leaf into storage organs.
In contrast to GS-2 in the chloroplast, relatively little is known about GS-1 in the cytoplasm. This relates in particular to its function in the cell, but also to its regulation. In contrast to GS-2, it has been assumed to date that GS-1 is encoded by more than one gene, the number thereof being variable. The genes show homologies with one another but can be unambiguously separated from one another (Brears et al., Plant J. 1 (1991), 235 to 244; Edwards et at al., Plant Cell 1 (1989), 241 to 248). In addition, the GS-1 genes, which each encode one subunit of the octameric holoenzyme, appear to be regulated differently (Petermann and Goodman MGG 230 (1991), 145 to 154). Controlled external influencing of glutamine metabolism by modification of GS-1 is made difficult thereby.
The localization of GS-1 in the plant is also still substantially unknown. It is known from Edwards et al. (loc. cit.) that one isoform is thought to be expressed exclusively in the phloem, where it possibly plays a part in intercellular transport. Sakurai et al. (Planta 200 (1996), 306 to 311) reports that in rice one isoform of GS-1 is present in the conducting bundles and evidently plays a part in the export of glutamine from leaves. It is also known that tobacco and alfalfa plants which have been transformed with a GS-1 gene from Lotus corniculatus show expression in the flowers (Carrayol et al., Plant Sci 125 (1997), 75 to 85). It is additionally known that the composition and localization of GS-1 holoenzymes in the root nodules of Lotus corniculatus may vary greatly.
No targeted reduction, carried out by methods of molecular biology, in the glutamine content in plants, especially in storage organs of plants, has been disclosed to date. The essential difficulties occurring in the targeted reduction of the glutamine synthetase activity in plants and the eventually desired reduction in the nitrogen content in storage organs of plants derive from the fact that the glutamine synthetase activity must be restricted tissue- and time-specifically in such a way that the nitrogen content in the target organ, for example the storage root of a sugarbeet, is reduced. This must not involve any impairment of the other functions and properties of the sugarbeet. On the contrary, it must be ensured that the overall physiology of the sugarbeet remains intact and there is merely a specific reduction in the nitrogen content in the storage organ of the sugarbeet. Because of the problems described concerning the localization of GS-1 and the lack of clarity in relation to the time specificity of its activity, no successful experiments have been disclosed indicating that it was possible to reduce the nitrogen content in storage organs of sugarbeet via modulation of the GS-1 activity.
The technical problem on which the present invention is based is thus to provide means and methods which make it possible in a targeted manner, that is to say tissue- and time-specifically, to reduce the nitrogen content in the storage organ of a plant, in particular of a sugarbeet, without this involving impairment of the vital, growth and reproductive functions of the sugarbeet and its commercial value.
The present invention solves this problem by providing an isolated and purified nucleotide sequence and vectors comprising this nucleotide sequence, which code for one subunit of the GS-1 of sugarbeet. The present invention solves the technical problem also by providing the isolated and purified protein encoded by this nucleotide sequence, in particular the amino acid sequence of the GS-1 subunit from sugarbeet, and by methods for the genetic modification of plants, in particular sugarbeet, where the content of glutamine synthetase in a plant, in particular in its senescent leaves, is altered, in particular reduced, by transforming cells of this plant with said vectors, and regenerating from the transformed cells intact, propagatable, stably transformed transgenic plants in whose senescent leaves the activity of GS-1 is reduced or completely suppressed.
The invention is surprising and advantageous in particular because the subunit encoded by the nucleotide sequences of the invention represents the only subunit of the oligomeric GS-1 isoform occurring in the senescent leaves of the sugarbeet. Accordingly, the invention also provides the surprising information that this isoform of GS-1 is a homooctamer. This makes it possible in a surprising manner to influence the activity of this enzyme by influencing the expression of a single gene, namely the gene encoding the GS-1 subunit, in particular to prevent or reduce the expression thereof. The invention is also surprising inasmuch as the homooligomeric isoform of GS-1 provided by the invention occurs only in the stage of senescence and accordingly displays, besides the tissue specificity in relation to the localization in the leaf which has been mentioned above, also a time specificity in relation to the occurrence during senescence. The present invention surprisingly therefore provides means and methods for influencing the qualitative and/or quantitative occurrence of a homooctameric GS-1 isoform which can be found only in senescent leaves of the sugarbeet. The nucleotide sequences of the invention can also be used for cloning homologous genes, in particular the coding regions thereof, in other tissues and even other plants and organisms. It is possible in particular on use of the present nucleotide sequence as hybridization probe in homologous or heterologous systems also to identify and isolate endogenous regulatory noncoding nucleotide sequences which are associated with this sequence and which, for example, mediate time- and tissue-specific expression.
The invention solves the present technical problem in particular by providing a nucleotide sequence f
Hoffmann Guido
Tischner Rudolf
Mehta Ashwin
Ostrolenk Faber Gerb & Soffen, LLP
Sudzucker Aktiengesellschaft
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