U.S. patent application number 10/773412 was filed with the patent office on 2004-07-22 for genetically modified sugarbeet.
This patent application is currently assigned to Sudzucker Aktiengesellschaft. Invention is credited to Hoffmann, Guido, Tischner, Rudolf.
Application Number | 20040143873 10/773412 |
Document ID | / |
Family ID | 7880209 |
Filed Date | 2004-07-22 |
United States Patent
Application |
20040143873 |
Kind Code |
A1 |
Tischner, Rudolf ; et
al. |
July 22, 2004 |
Genetically modified sugarbeet
Abstract
The present invention relates to methods for reducing glutamine
metabolism in sugarbeet.
Inventors: |
Tischner, Rudolf;
(Gottingen, DE) ; Hoffmann, Guido; (Gottingen,
DE) |
Correspondence
Address: |
OSTROLENK FABER GERB & SOFFEN
1180 AVENUE OF THE AMERICAS
NEW YORK
NY
100368403
|
Assignee: |
Sudzucker
Aktiengesellschaft
|
Family ID: |
7880209 |
Appl. No.: |
10/773412 |
Filed: |
February 5, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10773412 |
Feb 5, 2004 |
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09786534 |
Jun 26, 2001 |
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6727095 |
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09786534 |
Jun 26, 2001 |
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PCT/EP99/06522 |
Oct 17, 2000 |
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Current U.S.
Class: |
800/287 ;
435/419; 435/468; 536/23.2 |
Current CPC
Class: |
Y02A 40/146 20180101;
C12N 15/8243 20130101; C12N 9/93 20130101; C12N 15/8251 20130101;
C12N 15/8261 20130101 |
Class at
Publication: |
800/287 ;
536/023.2; 435/468; 435/419 |
International
Class: |
A01H 001/00; C12N
015/82; C07H 021/04; C12N 005/04 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 8, 1998 |
DE |
198 40 964.8 |
Claims
1. An isolated nucleotide sequence for reducing or preventing the
expression of a protein having the activity of a glutamine
synthetase in the senescing leaves of a transgenic plant selected
from the group consisting of: a) the DNA sequence of SEQ ID Nos: 1
and 3; b) a nucleotide sequence which encodes the amino acid
sequence SEQ ID No: 2; c) a nucleotide sequence which is
complementary to the nucleotide sequence of a) or b), and d) a
nucleotide sequence which hybridizes with a nucleotide sequence of
a) to c).
2. A vector comprising the nucleotide sequence as claimed in claim
1.
3. The vector as claimed in claim 2, where the vector is a plasmid
or a viral vector.
4. The vector as claimed in claim 2, where the nucleotide sequence
is operatively linked to at least one regulatory nucleotide
sequence.
5. The vector as claimed in claim 4, where a promoter controlling
the expression of the nucleotide sequence is arranged 5'-wards of
the nucleotide sequence.
6. The vector as claimed in claim 4, where a 3'-polyadenylation
signal is arranged 3'-wards of the nucleotide sequence.
7. The vector as claimed in claim 4, where the regulatory sequence
is inducible.
8. The vector as claimed in claim 4, where the regulatory sequence
confers tissue specificity and/or time specificity to the
expression of the nucleotide sequence.
9. The vector as claimed in claim 2, where the nucleotide sequence
has antisense orientation to the promoter.
10. A transgenic bacterial or plant cell comprising the vector as
claimed in claim 2.
11. The cell as claimed in claim 10, which is a sugarbeet cell.
12. A plant comprising at least one cell as claimed in claim
10.
13. A seed of a plant, wherein said seed comprises at least one
plant cell as claimed in claim 10.
14. A method for altering gluatmine metabolism in a sugarbeet,
where synthesis of glutamine synthetase in senescing leaves in the
sugarbeet is prevented or reduced by transforming at least one
plant cell with the vector as claimed in claim 3, and regenerating
the sugarbeet.
15. A method for producing a transgenic sugarbeet which shows
altered glutamine metabolism, where the latter is based on a
reduction of the content of glutamine synthetase in senescing
leaves and where at least one sugarbeet plant cell is transformed
with the vector as claimed in claim 9, and the plant cell is
regenerated to an intact plant.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a continuation of application Ser. No. 09/786,534,
filed Jun. 26, 2001, which is based upon PCT International
Application No. PCT/EP99/06522, filed Oct. 17, 2000, claiming
priority of German Application No. 198 40 964.8, filed Sep. 8,
1998.
DESCRIPTION
[0002] 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.
[0003] The accumulation of glutamine as the main .alpha.-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.sub.4.sup.+ 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.sub.4.sup.+
produced during photorespiration and converting the NH.sub.4.sup.+
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.sub.4.sup.+ 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.
[0004] 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.
[0005] 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.
[0006] 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.
[0007] 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.
[0008] 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.
[0009] 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.
[0010] The invention solves the present technical problem in
particular by providing a nucleotide sequence for modulating the
expression, in particular for suppressing the expression, of a
protein having the activity of a glutamine synthetase, in
particular the activity of a GS-1, which is selected from the group
consisting of
[0011] a) the DNA sequence of SEQ ID No. 1, 3 or a part
thereof,
[0012] b) a nucleotide sequence which encodes the amino acid
sequence of SEQ ID No. 2 or a part thereof,
[0013] c) a nucleotide sequence which is complementary to the
nucleotide sequences of a) or b), or a part thereof, and
[0014] d) a nucleotide sequence which hybridizes with the
nucleotide sequences of a) to c).
[0015] The nucleotide sequence of the invention is functionally
characterized in that, in a cell transformed therewith and having
an endogenous GS-1-encoding sequence, it modulates the activity of
the GS-1 activity of this transformed cell, for example increases
the GS-1 activity, for example by overexpression, or reduces or
completely suppresses the GS-1 activity, for example through the
nucleotide sequence of the invention being transformed in the form
of an antisense construct which inhibits endogenous GS-1
translation.
[0016] The invention provides in a particularly preferred
embodiment for the nucleotide sequence to be derived from the
sugarbeet Beta vulgaris.
[0017] The nucleotide sequence of the invention may be a DNA
sequence, for example a genomic, where appropriate
intron-interrupted DNA sequence or cDNA sequence, but it can also
be an RNA sequence, for example an mRNA sequence or synthetically
prepared. The present invention relates both to the sense and to
the antisense nucleotide sequences. The nucleotide sequences of the
invention can be so-called full-length sequences, that is to say
sequences which encode a complete protein having the activity of a
glutamine synthetase, in particular of the GS-1 of sugarbeet, where
appropriate including the translation initiation site. However, the
invention also relates to partial sequences of such nucleotide
sequences, in particular those which serve to modulate the
expression of the protein having the activity of a glutamine
synthetase, in particular of a GS-1 from sugarbeet. Accordingly,
the nucleotide sequence of the invention may also form a fusion
gene in a transcription or translation unit with other nucleotide
sequences. The invention relates in an advantageous refinement also
to nucleotide sequences which hybridize with the DNA sequence
specified in SEQ ID No. 1 or 3 or hybridize with a nucleotide
sequence which encodes the amino acid sequence of SEQ ID No. 2, and
nucleotide sequences which hybridize with nucleotide sequences
complementary to the two sequences mentioned.
[0018] In connection with the present invention, hybridization
means a prehybridization, a hybridization and subsequent washing.
The prehybridization preferably takes place in an aqueous solution
composed of 6.times.SSPE, 0.1% SDS and 5.times. Denhardt's reagent,
and 500 .mu.g/ml denatured herring sperm at 60.degree. C.,
particularly preferably at 65.degree. C., for 3 hours. The
hybridization preferably takes place in an aqueous solution
composed of 3.times.SSPE, 0.1% SDS, 5.times. Denhardt's reagent and
500 .mu.g/ml denatured herring sperm at 60.degree. C., particularly
preferably at 65.degree. C., for 16 hours. The washing preferably
takes place in an aqueous solution composed of 2.times.SSPE and
0.1% SDS at room temperature for 10 minutes, this being followed by
another washing step in an aqueous solution composed of
2.times.SSPE and 0.1% SDS at 60.degree. C., particularly preferably
at 65.degree. C., for 15 minutes and by a final washing step with
an aqueous solution composed of 0.4.times.SSPE and 0.02% SDS at
60.degree. C., particularly preferably at 65.degree. C., for 15
minutes.
[0019] In a particularly preferred embodiment, a prehybridization
is carried out in an aqueous solution composed of 6.times.SSPE,
0.1% SDS and 5.times. Denhardt's reagent, and 500 .mu.g/ml
denatured herring sperm at 65.degree. C. for 3 hours. The
hybridization takes place in an aqueous solution composed of
3.times.SSPE, 0.1% SDS, 5.times. Denhardt's reagent and 500
.mu.g/ml denatured herring sperm at 68.degree. C. for 16 hours. The
washing takes place in an aqueous solution composed of 2.times.SSPE
and 0.1% SDS at 68.degree. C. for 15 minutes, this being followed
by another washing step in an aqueous solution composed of
1.times.SSPE and 0.1% SDS at 68.degree. C. for 15 minutes and by a
final washing step with an aqueous solution composed of
0.1.times.SSPE and 0.1% SDS at 68.degree. C. for 15 minutes.
[0020] The present invention also relates, of course, to
modifications of the aforementioned sequences, in particular those
which display, by comparision with the sequences shown in SEQ ID
No. 1 or 3, nucleotide additions, deletions, inversions,
substitutions or the like, including chemical derivatizations or
replacement, exchange or addition of unusual nucleotides.
[0021] The invention also relates to nucleotide sequences which
have a degree of homology of at least 80%, preferably 90%, to the
sequences shown in SEQ ID No. 1 or 3.
[0022] The nucleotide sequences of the present invention are
advantageous in particular inasmuch as they serve to modulate the
expression of a protein having the activity of a glutamine
synthetase, in particular the GS-1 from sugarbeet. The nucleotide
sequences of the invention can be employed for altering, in
particular reducing, and in a particularly preferred manner
completely suppressing, the amount of glutamine synthetase formed,
in particular the GS-1 from sugarbeet. In a particularly preferred
manner, the invention makes it possible through modulation of the
expression in a tissue- and time-specific manner for the deposition
of glutamine in the storage organ of the sugarbeet to be suppressed
without this inevitably entailing the need to employ tissue- and
time-specific regulatory elements for the transgene, that is to say
the nucleotide sequence of the invention. This is because the
nucleotide sequences of the invention encode the GS-1 of sugarbeet,
which occurs specifically only in senescent leaves, and accordingly
shows site specificity in relation to the expression in leaves and
time specificity in relation to the expression during senescence.
The nucleotide sequences of the invention make it possible to
inhibit this glutamine synthetase, which occurs specifically at
leaf senescence, by means of a single gene construct because the
GS-1 of sugarbeet is a homooctamer and accordingly the formation of
all the GS-1 subunits can be switched off by means of a single gene
construct. Accordingly, the invention provides in a particularly
preferred manner for the use of antisense constructs which
specifically suppress the formation of protein, that is to say
GS-1, in the senescent leaves of sugarbeet. It is possible by use
of antisense constructs to inhibit the expression of the GS-1 of
sugarbeet which occurs specifically at leaf senescence, so that
glutamine formation and deposition of glutamine in the storage root
is prevented. The growth of the sugarbeet is advantageously not
impaired in this process because GS-2 is not affected by the
genetic manipulation of the plant cell.
[0023] In connection with the present invention, the activity of a
protein having the activity of a glutamine synthetase means an
activity by which NH.sub.4.sup.+ is bound enzymatically to C-4 of a
glutamate molecule with use of ATP.
[0024] In connection with the present invention, modulation of the
expression of a protein means a deliberate change, that is to say
increase or reduction, achieved by genetic engineering methods, in
the amount of protein in a cell compared with the amount of protein
naturally present in the relevant cell at the relevant time.
[0025] The modulation of expression can take place by influencing
the translation or transcription of the endogenous nucleotide
sequence encoding the protein, for example by introducing an
antisense construct which partially or completely reduces the
amount of translatable mRNA. An increase in the amount of protein
may take place, for example, by introducing a nucleotide sequence
which encodes the protein and is under the control of
overexpressing regulatory elements, or by introducing multiple gene
copies.
[0026] Further alternative or additional modulations can be
achieved by employing tissue- or time-specific, inducible or
constitutively expressed regulatory elements which lead to an
expression pattern which is altered by comparison with the natural
expression pattern in the relevant cell and at the relevant time or
at the relevant stage of development of the cell or the plant.
[0027] The present invention relates in a further embodiment to
vectors comprising at least one of the nucleotide sequences of the
invention. In a particularly preferred embodiment of the invention,
such a vector is embodied as plasmid or viral vector.
[0028] The present invention also relates to vectors of the
aforementioned type, where the at least one nucleotide sequence of
the present invention is under the control of regulatory nucleotide
sequences which are likewise present in the vector and which are
arranged, for example, 5', 3', 5' and 3' or else within the
nucleotide sequence. These regulatory nucleotide sequences may be
heterologous to the nucleotide sequence of the invention, that is
to say be derived from a different organism or from a different
gene, or homologous, that is to say also naturally occurring
together with the nucleotide sequences of the invention in a
regulatory unit.
[0029] The invention accordingly also relates to vectors of the
aforementioned type, where a nucleotide sequence controlling the
expression of the nucleotide sequence of the invention, in
particular a promoter, is located 5'-wards of the nucleotide
sequence of the invention. In a particularly preferred embodiment
of the invention, the promoter is the 35 S promoter of CaMV or a
promoter of the T-DNA of Agrobacterium tumefaciens, for example the
promoter of the nopaline synthetase or octopine synthetase
gene.
[0030] The invention provides in a further embodiment for a
transcription termination unit, in particular a 3'-polyadenylation
signal, to be located 3'-wards of the nucleotide sequence of the
invention, particularly preferably the polyadenylation signal of
the NOS gene of Agrobacterium tumefaciens.
[0031] The present invention provides in another preferred
embodiment for the regulatory sequences to be inducible, for
example by external factors.
[0032] The invention provides in another preferred embodiment for
the regulatory sequences of the expression of the nucleotide
sequences of the invention controlled thereby to confer tissue
specificity and/or timespecificity, for example to bring about
expression of an antisense construct specifically in leaves during
senescence.
[0033] The invention also provides for the nucleotide sequences of
the invention, where appropriate in a unit with the regulatory
nucleotide sequences assigned to them, to be arranged in the vector
together with nucleotide sequences which assist transfer and
integration or recombination of the nucleotide sequences of the
invention where appropriate with the regulatory nucleotide
sequences assigned to them into the genome of a transformed cell.
The nucleotide sequences of the invention can therefore be
arranged, for example, between the left and right border region,
flanked by only one border region in each case and/or interrupted
by one or more border regions of Agrobacterium tumefaciens or
Agrobacterium rhizogenes.
[0034] The present invention also relates to cells comprising at
least one of the aforementioned vectors. In a particularly
preferred manner, such cells are bacterial cells, yeast cells or
plant cells, in particular plant cells from monocotyledonous or
dicotyledonous plants, in particular sugarbeet. The present
invention therefore particularly relates to a non-variety-specific
cell of a plant which has been transiently or stably transformed
with a nucleotide sequence of the invention, in particular which
has this nucleotide sequence in its genome, for example in the form
of an antisense construct. A plant means a photosynthetically
active organism including algae, mosses, ferns and higher
plants.
[0035] The invention also relates to cell assemblages, tissues,
organs, parts of organs, cell cultures, calli, differentiated or
undifferentiated cell aggregates, embryos, protoplasts etc. of an
organism which have, stably integrated into the genome or
transiently present, at least one cell transformed with the
nucleotide sequences of the invention. In a particularly preferred
manner, the invention relates to leaves, stalks, seeds, roots,
storage organs, petals, flower organs etc. of a plant, said organs
having at least one cell stably or transiently transformed with the
nucleotide sequences of the invention. In a particularly preferred
manner, the plants or the parts thereof are transformed in such a
way that the transformed nucleotide sequence is stably inherited
from generation to generation.
[0036] The present invention relates not only to cells, cell
assemblages, calli and plant organs but also, of course, to plants,
in particular intact fertile plants which have been transformed by
means of the nucleotide sequence of the invention and have in at
least one of their cells at least one of these sequences, in
particular stably integrated in their genome. The transformation
preferably takes place, as stated hereinafter, nonbiologically,
where agrobacterium-mediated gene transfer is understood to be
nonbiological. The resulting cells, and the plant tissues, plant
organs, plant parts or plants having these cells, are not
variety-specific. On the contrary, the invention is applicable to
virtually all plants, plant families or plant genera.
[0037] In a particularly preferred manner, the transformed
nucleotide sequence is heterologous to the transformed cell, that
is to say is not naturally present in the transformed cell, is not
present in the artificially generated high copy number, or is not
expressed at the place or time at which it is expressed according
to the invention. In cases in which the cell to be transformed
already has an endogenous identical or similar nucleotide sequence,
the cell obtained by the transformation of the invention differs
from the initial cell for example in that the introduced nucleotide
sequence is present in a different genetic context in the genome,
has different regulatory elements, is arranged in antisense
orientation to its regulatory elements and/or is present in
increased copy number.
[0038] The invention accordingly also relates to methods for
producing transgenic cells, where the nucleotide sequence of the
invention to be transformed is introduced into the cell to be
transformed by means of a conventional transformation method, for
example microprojectile bombardment, agrobacterium-mediated gene
transfer, electroporation, PEG-mediated transformation or the
like.
[0039] The invention also relates to methods for producing
transgenic plants having the nucleotide sequences of the invention,
where cells or cell assemblages transformed with the nucleotide
sequences of the invention are cultivated and regenerated to
intact, preferably fertile, plants. The cultivation and
regeneration take place by conventional methods.
[0040] The invention also relates to a protein having the activity
of a glutamine synthetase, in particular the GS-1 from sugarbeet,
the latter being encoded by the nucleotide sequences of the
invention, in particular the nucleotide sequence shown in SEQ ID
No. 1, particularly preferably an amino acid sequence shown in SEQ
ID No. 2. The invention also relates to proteins having the
activity of a glutamine synthetase, in particular the GS-1 from
sugarbeet, this sequence having modifications such as amino acid
exchanges, deletions, additions, inversions or the like, and the
protein having the activity of a GS-1 from sugarbeet. The invention
also relates to proteins which, at the amino acid level, have a
degree of homology (identical amino acids) of at least 90%,
preferably 95%, with the sequence shown in SEQ ID No. 2. Proteins
of this type can be prepared by employing the nucleotide sequences
of the invention as cloning probes or hybridization probes for
identifying and cloning homologous genes encoding these
proteins.
[0041] The invention relates in a further embodiment to monoclonal
or polyclonal antibodies against one of the aforementioned
proteins, these antibodies recognizing and binding one or more
epitopes of said proteins.
[0042] The invention relates in a further embodiment to methods for
altering glutamine metabolism in a sugarbeet, in particular for
modulating the expression, particularly preferably for repressing,
a protein having the activity of a glutamine synthetase, in
particular the GS-1 from sugarbeet, where the glutamine synthetase
content of the sugarbeet is altered by transforming at least one
sugarbeet cell with a vector of the present invention, in
particular transforming with a vector having the nucleotide
sequence of the invention in antisense orientation, and
regenerating a sugarbeet from the transformed cell or an
association of cells. A sugarbeet generated in this way is
advantageously characterized in that the glutamine synthetase GS-1
normally formed in its leaves during senescence is not formed
because the expression of glutamine synthetase GS-1 is prevented
because of the antisense construct present at least in the leaves
and expressed there, so that the formation of glutamine in the
leaves and, eventually, the deposition of the glutamine in the
storage root is prevented.
[0043] However, the invention also of course relates to methods for
altering glutamine metabolism in plants, in particular sugarbeet,
according to which the glutamine synthetase content in particular
cells or organs is increased, where appropriate at certain times,
in particular by transforming gene constructs which make
constitutive and/or enhanced expression of the nucleotide sequences
of the invention possible.
[0044] Further advantageous refinements of the invention are
evident from the dependent claims.
[0045] The invention is explained in detail by means of examples
and drawings belonging thereto.
[0046] SEQ ID No. 1 shows the translated region of the cDNA
sequence of the GS-1 from sugarbeet.
[0047] SEQ ID No. 2 shows the amino acid sequence of the GS-1 from
sugarbeet.
[0048] SEQ ID No. 3 shows the complete cDNA sequence of the GS-1
from sugarbeet.
[0049] The figures show:
[0050] FIG. 1 a Western blot of GS-1 obtained according to the
invention,
[0051] FIG. 2 an autoradiogram of the Western blot in FIG. 1
and
[0052] FIG. 3 a diagrammatic depiction of the results obtained in
FIGS. 1 and 2.
EXAMPLE 1
Cloning of the cDNA for GS-1
[0053] Complete RNA was extracted from senescent sugarbeet leaves.
This was done by grinding 20 g of leaf material from senescent
sugarbeet leaves in liquid nitrogen and transferring into 100 ml of
uptake buffer (50 mM Tris-HCl pH 9.0, 100 mM NaCl, 10 mM EDTA, 2%
w/v SDS and 0.2 mg/ml proteinase K). This mixture was phenolized
twice with phenol/chlorophorm/isoamyl alcohol (25/24/1),
precipitated ({fraction (1/10)} volume of 3M NaAc, pH 6.5, one
volume of isopropanol, 2 hours at -20.degree. C.) and washed with
70% ethanol. After taking up in 10 ml of H.sub.2O, 10 .mu.g/ml
proteinase K and 2.times. precipitating with 1/4 volume of 10 M
LiCl at 0.degree. C. for 16 hours, the complete RNA was taken up in
2 ml of H.sub.2O with 10 .mu.g/ml proteinase K. 5 mg of complete
RNA were obtained.
[0054] Poly (A).sup.+ mRNA was then isolated on an oligo-dT
cellulose column. This was done by incubating 2 ml of complete RNA
with 25 ml of binding buffer (400 mM NaCl, 10 mM Tris-HCl, pH 7.5
and 2% SDS) and oligo-dT cellulose (200 mg of oligo-dT cellulose)
at room temperature for 30 minutes while shaking gently. The
mixture was transferred into a glass column with cotton frit and
washed dropwise with a washing buffer (100 mM NaCl, 10 mM Tris-HCL,
pH 7.5, 0.2% SDS) until the OD.sub.260 was constant at 0.005. This
was followed by elution with 10 ml of elution buffer (10 mM
Tris-HCL, pH 7.5) at 55.degree. C. Precipitation was then carried
out with {fraction (1/10)} volume of 3 M NaAc, pH 6.5 and 2 volumes
of ethanol at -20.degree. C. for two hours, and the mixture was
taken up in 10 ml of binding buffer. The column purification was
then repeated, the eluate was phenolized before the precipitation,
and the mRNA pellet was taken up in TE buffer. 50 .mu.g of poly
(A)+ mRNA were obtained.
[0055] cDNA was prepared using a cDNA synthesis kit from Boehringer
Mannheim in accordance with a standard protocol (5 .mu.g of mRNA
employed). The resulting cDNA was ligated into lambda vectors (NM
1149). 4 .mu.g of NM 1149 (EcoR I digested) and 0.2 .mu.g of cDNA
with EcoR I linkers were employed for this. After the ligation, the
DNA was packaged in phages (NM 1149). The Gigapack.RTM. II gold
packaging extract from Stratagene was employed for this in
accordance with the standard protocol, using 4 .mu.g of DNA and
obtaining a titer of 140 000 pfu.
[0056] The resulting cDNA bank was screened, as was a cDNA bank
from sugarbeet root tissue (Lambda ZAP.RTM. II library, Stratagene,
cDNA inserted from EcoR I and Not I into the Lambda ZAP.RTM. II
vector system, titer: 250 000 pfu, vector pBluescript.RTM. SK (-)
with insertion isolated in accordance with standard protocol by in
vivo excision from Lambda ZAP.RTM. II) using a heterologous tobacco
probe. The heterologous tobacco probe is described in Becker at al
(1992) Plant. Molec. Biol. 19, 367-379. For the screening, E. coli
bacteria were infected with the lambda phages and plated out. The
phage DNA from lyzed bacteria was subsequently transferred to NC
membranes (Plaquelift), and the membrane-bound DNA was hybridized
with a radiolabeled tobacco GS-1 cDNA probe.
[0057] The screening of the cDNA bank with the heterologous tobacco
probe was carried out as follows. Firstly a prehybridization was
carried out with 6.times.SSPE, 0.1% SDS, 5.times. Denhardt's
reagent and 500 .mu.g/ml denatured herring sperm at 61.degree. C.
for two hours. The hybridization was then carried out at 61.degree.
C. for 16 hours with a solution of 3.times.SSPE, 0.1% SDS, 5.times.
Denhardt's reagent and 500 .mu.g/ml denatured herring sperm. The
washing was carried out with 2.times.SSC and 0.1% SDS at 61.degree.
C. for 2.times.15 minutes. A washing step was then carried out with
1.times.SSC and 0.1% SDS at 61.degree. C. for 15 minutes.
[0058] An autoradiogram of the membrane filters was developed,
positive phages were isolated, and the corresponding DNA was
extracted. The cDNA found was subcloned into the plasmid
pBluescript SK (Stratagene) and sequenced. The nucleotide sequence
of the translated region of the cDNA, including the translation
start codon ATG, is depicted in SEQ ID No. 1 and has a length of
1068 bp. The complete sequence of the cDNA is depicted in SEQ ID
No. 3 and has a length of 1543 bp. The start codon is located in
position 199 to 201. The translated region terminates at position
1266. A polyadenylation signal is located in the region of
nucleotides 1478 to 1508.
[0059] The amino acid sequence derived from SEQ ID No. 1 has a
length of 356 amino acids and is depicted in SEQ ID No. 2. The
amino acid sequence represents the amino acid sequence of subunit P
of the GS-1 from sugarbeet. The protein is about 42 kDa in size and
represents the only subunit of the GS-1 isoform which is present in
the form of a homooctamer in senescent sugarbeet leaves.
EXAMPLE 2
In Vitro Transcription and Translation of the P Subunit
[0060] The nucleotide sequence depicted in SEQ ID No. 1 was
transcribed and translated in vitro. The cloned GS-1 DNA sequence
employed for this was derived from the sugarbeet cDNA from root
tissue mentioned in example 1. In order to establish which GS-1
subunit this DNA sequence codes for, an in vitro transcription and
translation was carried out with the "Linked in vitro SP 6/T7
Transcription/Translation Kit-radioactive" kit from Boehringer
Mannheim. This was done by incubating 0.5 .mu.l (0.5 .mu.g) of
plasmid DNA (pBluescript.RTM. SK (-) with the GS-1 insert), 5 .mu.l
of T7 transcription buffer and 14.5 .mu.l of H.sub.2O at 30.degree.
C. for 15 minutes. Then 10 .mu.l of transcription reaction
solution, 1.6 .mu.l of .sup.35S-methionine and 38.4 .mu.l of
translation mix were incubated at 30.degree. C. for 1 hour.
[0061] In addition, a protein extract was prepared from 5 g of
sugarbeet leaves of various ages (in order to obtain all the GS-1
subunits for unambiguous identification). This extract was purified
by FPLC, and the fractions with the highest GS-1 activities were
concentrated. Protein was determined by the method of Bradford and
revealed a protein concentration of about 1 .mu.g/.mu.l. Both this
extract and the reaction mixture from the in vitro translation
(with the radiolabeled GS-1 protein) were mixed with the same
volume of urea loading buffer. 20 .mu.l of each of these were
together put as sample for an isoelectric focusing (IEF) on an
acrylamide capillary gel (1st dimension). Isoelectric focusing took
place at 190 V for 16 h.
[0062] Together with a protein size standard, the capillary gel was
transferred to an SDS gel in order to fractionate the proteins
according to their size (2nd dimension). An SDS-PAGE took place at
140 V for 2 h. A Western blot (500 mA; 1 h) was prepared from this
gel.
[0063] The nitrocellulose membrane was stained with Ponceau Red and
the bands of the size standard were marked with a pencil.
[0064] After a blocking treatment (1 h), the membrane was incubated
(16 h; RT) with the 1st antibody (anti-GS; antibody against barley
GS, Roger Wallsgrove, Rothamsted Experimental Station, Harpenden,
UK), 1:3 000 in blocking solution). The membrane was then washed
3.times. with TBS and incubated (3 h; RT) with the second antibody
(1:2 000 in blocking solution). After washing three more times, the
color reaction with NBT and BCIP was effected by the alkaline
phosphatase (10-20 min; 37.degree. C.; dark). The dried membrane
with the color-marked spots for the GS-1 subunits was exposed to an
X-ray film (exposure: 16 h; RT; dark).
[0065] FIG. 1 shows the membrane filter (Western blot) treated with
GS antibodies and colored. The protein size standard is loaded on
the left. The pH gradient in this case runs from pH 6 on the left
to pH 4 on the right. The 4 spots for the GS-1 subunits are evident
at the level of the 43 kDa band (compare FIG. 3, although the sides
are reversed in this case). The spot for the P subunit is marked
(arrow).
[0066] FIG. 2 shows the autoradiogram of the membrane on which the
bands of the size standard are indicated on the left and a spot
appears on the right at the level of the 43 kDa band. This spot was
produced by the radiolabeled protein which had been transcribed and
translated in vitro. It is possible by comparing the autoradiogram
with the membrane to assign a spot on the membrane to the single
spot on the film. This spot was identified as subunit "P" (compare
FIG. 3).
[0067] FIG. 3 represents a diagram of the proteins which can be
labeled by GS antibodies. The direction of the pH gradient is
indicated at the bottom (1st dimension) from pH 4 on the left to pH
6 on the right. The bands of the size standard (2nd dimension) are
depicted on the right from 43 kDa at the top to 30 kDa at the
bottom. Spots a, b, c and d, which are white in this diagram, show
the positions of the GS-2 subunits, which are likewise recognized
by the antibody but can be separated from GS-1 by FPLC. At the
level of the 43 kDa band there are four black spots s, i, p and w,
which were identifiable on the basis of their size as the subunits
forming the octamer of GS-1. The other black spots u, v, x.sub.1,
x.sub.2, y and z are possibly degradation products of the GS
proteins.
EXAMPLE 3
Production of Transgenic Sugarbeet
[0068] A series of constructs each comprising a promoter which can
be expressed in plants, namely the CaMV 35 S promoter, each
comprising a section of the sugarbeet GS-1 subunit gene of the
invention in antisense orientation, and each comprising the NOS
terminator was produced. The sections of the gene of the invention
employed differed from one another. The gene cassettes produced in
this way were ligated into the binary vector (BIN19 with kanamycin
resistance), and Agrobacterium tumefaciens (with rifampicin
resistance) was transformed with the resulting binary vectors by
electroporation. The transformants underwent rifampicin and
kanamycin selection. Subsequently, sugarbeet leaf disks and leaf
stalks were transformed in a suspension with transformed
agrobacteria, and callusing and shooting were induced with the
plant hormones NAA and BAP. After selection on kanamycin-containing
medium and regeneration to intact plants by conventional protocols,
it was possible by means of measurements of the GS1 enzyme
activities, SDS gel electrophoreses, 2D PAGE and Northern blot
analyses to demonstrate that all the constructs employed, with the
various gene sections, were present and active in the leaves of the
transgenic, regenerated plant, and led to suppression of glutamine
synthetase activity and formation in senescent sugarbeet
leaves.
* * * * *