U.S. patent application number 08/690237 was filed with the patent office on 2002-07-11 for expression cassette and plasmids for a guard cell specific expression and their use for the introduction of transgenic plant cells and plants.
Invention is credited to MULLER-ROBER, BERND, SONNEWALD, UWE, WILLMITZER, LOTHAR.
Application Number | 20020090726 08/690237 |
Document ID | / |
Family ID | 6453545 |
Filed Date | 2002-07-11 |
United States Patent
Application |
20020090726 |
Kind Code |
A1 |
MULLER-ROBER, BERND ; et
al. |
July 11, 2002 |
EXPRESSION CASSETTE AND PLASMIDS FOR A GUARD CELL SPECIFIC
EXPRESSION AND THEIR USE FOR THE INTRODUCTION OF TRANSGENIC PLANT
CELLS AND PLANTS
Abstract
A method of and plasmids for producing specific gene expression
in the guard cells of plants. In particular, 5' transcriptional
regulatory promoter regions are provided for guard cell specific
expression. In addition, methods for preparing an expression
cassette, plasmids, and the use of the cassettes and plasmids to
produce transgenic plant cells and plants are described.
Inventors: |
MULLER-ROBER, BERND;
(BERLIN, DE) ; SONNEWALD, UWE; (BERLIN, DE)
; WILLMITZER, LOTHAR; (BERLIN, DE) |
Correspondence
Address: |
FROMMER LAWRENCE & HAUG
745 FIFTH AVENUE- 10TH FL.
NEW YORK
NY
10151
US
|
Family ID: |
6453545 |
Appl. No.: |
08/690237 |
Filed: |
July 19, 1996 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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08690237 |
Jul 19, 1996 |
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08140180 |
Jan 24, 1994 |
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5538879 |
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Current U.S.
Class: |
435/468 ;
435/320.1; 536/24.1; 800/278; 800/287 |
Current CPC
Class: |
C12N 15/8245 20130101;
C12N 9/1241 20130101 |
Class at
Publication: |
435/468 ;
435/320.1; 536/24.1; 800/278; 800/287 |
International
Class: |
C12N 015/63; C07H
021/04; C12N 015/82; A01H 001/00 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 4, 1992 |
DE |
P 4207358.8 |
Claims
1. An expression cassette characterised in that it mediates a DNA
sequence with a transcriptional regulatory starter region that
ensures a guard cell specific gene expression in the closed cells
of leaves of plants and no expression in mesophyllic cells or
epidermal cells of the leaves.
2. An expression cassette according to claim 1 characterised in
that there is contained on the DNA sequence an approximately 3.2 kb
size HincII/BglII promoter fragment of the ADP glucose
pyrophosphorylase gene GS6-11 or a part thereof.
3. An expression cassette according to claim 2 characterised in
that there is contained on the DNA sequence of the guard cell
specific promoter the sequence: Seq ID No:1
2 HindIII AAGCTTCGTA AAGAATATTT TATCATAGTA AAACATGATT ATCAAGTAAA
AGTGAACAAA GGGAGTAATA TGAAGATTTA TCATGTATTT AAAAGCTCAA TAGTGATTAT
AATTTGAGGG ACTAAATAAA TTTAAGGAGT TGTTAATATA TTCCGAGAAA ATAAAATATT
GTTTAAGTAG AAAAGTTATG GGGTGTATAA GTTAAATAAT AATATTTTGT AAATAGGGAT
ATGGAAATGA GTATAAATAG AAAGATAGCA AGGTTTCTCG TGAGACTTCA CAAGCCAATA
AAGCTGATCA CACTCCCCTT TGTATGTCCA CTCAACAACA CAACTTCTTG TGATTCACTT
TCAATTCTAG ATCGGGGATC C BamHI.
4. An expression cassette according to any one of the preceding
claims characterised in that the DNA sequence contained therein,
which is fused to suitable coding DNA sequences, changes the plant
guard cells therein so that a permanent opening or closing or
lengthening or shortening of the opening period of these cells is
made possible.
5. An expression cassette according to any one so the preceding
claims characterised in that the transcriptional regulatory starter
region for a guard cell specific gene expression can be inserted
after a DNA sequence, that contains the information for the
formation of endogenous products or the formation of heterologous
expression products in crops.
6. Plasmids containing an expression cassette according to any one
or claims 1 to 5.
7. Plasmid pAS (DSM 6906) consisting of an approximately 13.2 kb
size DNA sequence in which there is contained an approximately 2.0
kb size kanamycin resistance gene, and an approximately 3.2 kb size
HincII/BglII promoter fragment of the regulatory starter area of
the ADP glucose pyrophosphorylase gene GS6-11 of Solanum tuberosum,
a polylinker and the transcription terminator of the nopaline
synthase gene.
8. Plasmid pAS-GUS (DSM 6907), consisting of an approximately 15.2
kb size DNA sequence in which there is contained an approximately
2.0 kb size kanamycin resistance gene, an approximately 3.2 kb size
HincII/BglII promoter fragment of the regulatory starter area of
the ADP glucose pyrophosphorylase gene GS6-11 from Solanum
tuberosum, the approximately 2 kb size coding region
.beta.-glucuronidase and the transcription terminator of the
nopaline synthase gene.
9. Plasmid pS1-D4GUS or derivatives thereof containing the DNA
sequence Seq ID No:1, according to claim 3 or parts thereof.
10. Process of genetic manipulation of plant cells containing an
expression cassette with a DNA sequence for the closed cell
specific gene expression that consists of the following steps, a.
Isolating clone GS6-11, b. preparing plasmid pSF-6, using the
approximately 12 kb size SalI-fragment of the clone GS6-11, c.
preparing the plasmid pH6-1 using an approximately 5.3 kb size
HincII-fragment from the plasmid pSF-6, d. preparing the plasmids
pAS (DSM 6906), pAS-GUS (DSM 6907) and/or pS1-D4GUS using the
approximately 3.2 kb size HincII/BglII-fragment of the regulatory
starter area of the ADP glucose pyrophosphorylase gene of Solanum
tuberosum from the plasmid pH 6-1, and e. transferring the plasmid
pAS (DSM6906), pAS-GUS (DSM6907) and/or pS1-D4GUS into a plant
cell.
11. A plant cell prepared according to the process of claim 10.
12. A plant or plant tissue regenerated from a plant cell according
to claim 11.
13. A plant cell containing an expression cassette according to any
one of claims 1 to 5.
14. Use of an expression cassette according to any one of claims 1
to 5 for the expression of genes in guard cells that change the
regulation of gas exchange and transpiration.
15. Use of expression cassette to any one of claims 1 to 5 for the
expression of homologous or heterologous genes in guard cells of
transformed plants.
16. Use of an expression cassette to any one of claims 1 to 5 for
the expression of genes in guard cells that cause a local change of
the phytohormone level.
17. Use of the plasmid pAS, pAS-GUS or pS1-D4GUS or derivatives
thereof for the transformation of crops.
18. Use of a plasmid pAS, pAS-GUS or pS1-D4GUS pAS or derivatives
thereof for the regulation of endogenous processes or for the
preparation of heterogenous products in crops.
19. A plant according to claim 12 characterised in that it is a
tobacco plant.
20. A plant according to claim 12 characterised in that it is a
potato plant.
21. A plant according to claim 12 characterised in that it is a
tomato plant.
22. A plant according to claim 12 characterised in that it is a
sugar beet plant.
Description
[0001] The present invention relates to an expression cassette and
plasmids containing this expression cassette. The DNA sequence of
the expression cassette contains a transcriptional regulatory
starter region, that ensures a specific gene expression in the
guard cell of the leaves of plants, and no expression in
mesophyllic cells or epidermal cells of the leaves. The invention
further relates to a process for the preparation of transgenic
plant cells, which contain sequences of the expression cassette as
well as the use of the plasmids containing expression cassette for
the preparation of transgenic plants.
[0002] Because of the continual growth in world population, there
is a continual growing demand for nutrient and raw materials and,
because of the foreseeable long-term limitation of agricultural
land, it is the continuing task of biotechnological research to
strive for the production of high yielding ecologically acceptable
crops. To achieve this, the metabolism of plants has to be
modified. This can be achieved, amongst other ways, by altering the
DNA in the cell nucleus. The process for genetic modification of
dicotyledenous and monocotyledenous is already known, (see for
example Fraley, R. T. (1989) Science 244: 1293-1299; and Potrykus
(1991) Ann Rev Plant Mol Biol Plant Physiol 42: 205-225).
[0003] With the present invention it is possible to influence the
transpiration process and the gas exchange of a plant in regard to
the increase of yield through manipulation of the genes which are
expressed specifically in guard cells. This is not practicable
using known expression systems because of the wide-ranging
consequences of a non-guard cell specific gene expression for the
total material exchange of the plant. An increase in yield of crop
plants can be achieved by changing the photosynthesis loading of
plants or by reducing the water needs. For this the guard cells of
the epidermis of the leaf tissue are a valuable starting point.
[0004] Guard cells are specific kidney-shaped cells of that part of
the epidermis which is above ground and in the air surrounding the
green parts of higher plants and which are arranged in pairs and
have a gap (hole) between each other. The hole interrupts the
other-wise continuous film of epidermis cells and causes the
connection between the outer air and the intercellular system. In
most plants the pore area resulting from the open holes occupies
around 0.5 to 1.5% of the leaf area. Owing to their special
formation, guard cells can so regulate their shape through active
turgidity changes that the hole between them closes or opens. The
openings are thus regulators of the exchange, especially the
transpiration. The guard cells therefore have the task to so
regulate the diffusion resistance, that the water demand through
transpiration and CO.sub.2 uptake for photosynthetic or
darkness-CO.sub.2 fixation, is in an appropriate relationship for
the particular requirements. The opening or closing of the guard
cells is led back to a change in the turgidity in the guard cells
themselves and with it to the building up of a difference of the
turgidity in the guard cells to that in the bordering epidermal
cells (neighbouring cells).
[0005] Guard cells control the gas exchange between carbon dioxide
uptake and transpiration (expiration of water vapour). Besides the
concentration of carbon dioxide and the phytohormones, a
constituent of the control system is the concentration of dissolved
substances since these lead via changes in turgidity to the opening
and closing of the holes. Since the changes of concentration in
dissolved substances have wide-reaching consequences for the
metabolism of plants, whether this occurs in tissues or cells, it
is desirable to allow these changes only in certain areas of plants
or during a certain time period in the plant growth cycle. It
should be especially possible to have a change influencing the gas
exchange of the carbon dioxide or also the content in osmotically
active substance of guard cells which is independent of the
concentration of these substances in mesophyllic cells. For this
reason, there is a great interest in the identification and the
economic use of regulatory DNA sequences of plant genomes which are
responsible for the specific transcription in guard cells.
[0006] With the help of guard cell specific regulatory sequences,
by expression of suitable, newly introduced genes or by modulation
of endogenous gene products, a permanent opening or closing or
lengthening or shortening of the opening period of guard cells can
be introduced.
[0007] The present invention provides a guard cell specific
expression system with which such effect are possible.
[0008] Permanent opening of the guard cells should, on account of
an increased transpiration, lead to a higher water loss in the
plants which can however, for example, in late phases of the growth
cycle be desirable for the acceleration of the ripening of crops.
Besides this, one can conceive in the production of strongly
transpirationally active plants for thus purpose a controlled
drainage of soil. Plants rooting above ground can cause, by a
lowering of the heat capacity of the soil, a year long earlier
warming of growing ares and so make possible an earlier harvesting
of temperature-sensitive crops such as, for example, sugar beet.
Since the first introduced genetically modified help plants are
handicapped in the later phases of the growth cycle, they do not
lead to a hindering of the development of crops, but on the
contrary through an extension of the vegetation phase to an
increase in yield.
[0009] A wide-ranging closed holding of the hole openings should
cause through a reduced gas exchange, a delayed growth and, as a
result, timely extension of the vegetation phase. This can be of
interest in ornamental crops. Further, agricultural help crops can
be produced which as transpiration weak soil cover, prevent an
erosion of useful land in the quiet phase or at the beginning of
the vegetation phase of crops.
[0010] A change in the opening period of the holes has been shown
to be valuable in crops. For example, in green harvested plants,
especially fodder crops, by retention of water drying out of older
leaf material can be prevented and so the yield increased. Further,
an adaptation to an area with increased atmospheric carbon dioxide
concentraton (for example adding CO.sub.2 in greenhouses), can be
envisaged. Through artificial lengthening of the opening period,
effects such as mineral deprivation in reduced transpiration,
following a higher carbon dioxide partial pressure in the plants,
can be compensated for.
[0011] By combination of guard cell specific regulatory elements
with other control systems, further use possibilities can be seen.
For example, through a coupling of chemical sensors, a closing of
the hole openings induced from outside of the hole openings can be
produced, and so that for example a growth stop of certain plants,
the so-called agricultural help plants, could be produced at the
desired time.
[0012] In order to specify regulatory DNA sequences, one must first
seek products that appear only at certain times in the cell growth
cycle or in a certain part of the plant such as here in guard
cells. Once the responsible gene is identified and isolated, it is
necessary to have a basic investigation of the sequence and above
all the identification and isolation of the desired transcriptional
regulatory region. Then a suitable model must be prepared whose
validity can be proven by experiment.
[0013] There is now provided an expression cassette whose DNA
sequence is supplied with a transcriptional regulatory starter
region for guard cell specific gene expression which causes no
expression in mesophyllic cells or epidermal cells of the leaves.
With the expression cassette, a gene manipulating of plants is
possible which causes a permanent opening or closing or a
lengthening or shortening of the opening period of the guard
cells.
[0014] The DNA sequence contains the transcriptional regulatory
starting region for a guard cell specific gene expression.
[0015] On the expressions cassette there is located the DNA
sequence of the guard cell specific promoter having the sequence
(Seq. ID No:1)
1 HindIII AAGCTTCGTA AAGAATATTT TATCATAGTA AAACATGATT ATCAAGTAAA
AGTGAACAAA GGGAGTAATA TGAAGATTTA TCATGTATTT AAAAGCTCAA TAGTGATTAT
AATTTGAGGG ACTAAATAAA TTTAAGGAGT TGTTAATATA TTCCGAGAAA ATAAAATATT
GTTTAAGTAG AAAAGTTATG GGGTGTATAA GTTAAATAAT AATATTTTGT AAATAGGGAT
ATGGAAATGA GTATAAATAG AAAGATAGCA AGGTTTCTCG TGAGAGTTCA CAAGCCAATA
AAGCTGATCA CACTCCCCTT TGTATGTCCA CTCAACAACA CAACTTCTTG TGATTCACTT
TCAATTCTAG ATCGGGGATC C BamHI
[0016] (see also Example 4)
[0017] Plant cells, that contain a regulatory region for a guard
cell specific gene expression, can be prepared by the following
process:
[0018] a) Isolation of the clone GS 6-11 as described in Examples 1
and 2. The clone contains an approximately 12 kb size DNA-fragment,
that contains a guard cells specific regulator element (see FIG.
1).
[0019] b) preparation of the plasmid pSF-6 corresponding to Example
3 using the approximately 12 kb size SalI-fragment of the clone GS
6-11.
[0020] c) preparation of the plasmid pH 6-1 using the approximately
5.3 kb size HincII-fragment of the plasmid pSF-6 corresponding to
Example 3.
[0021] d) preparation of the plasmid pAS (DSM 6906), pAS-GUS (DSM.
6907) and/or pS1-D4GUS, using the approximately 3.2 kb size
HincII/BglII-fragment of the regulatory starter-region of the ADP
glucose pyrophosphorylase gene of Solanum tuberosum from the
plasmid pH 6-1 (Example 3). The plasmid pAS-GUS contains in
addition to the sequence of the plasmid pAS the coding region of
the .beta.-glucuronidase gene.
[0022] e) transfer of the T-DNA of the plasmid pAS (DSM 6906),
pAS-GUS (DSM 6907) and/or pS1-D4GUS, in a plant cell.
[0023] For the introduction of DNA into a plant host cell there
many techniques available. These techniques include the
transformation with T-DNA using Agrobacterium tumefaciens or
Agrobacterium rhizogenes as transformation agent, fusion of
protoplasts, injection or electroporation of DNA etc. Where
Agrobakteria are used for the transformation, the DNA being
introduced must be cloned in special plasmids, and that means
either in an intermediary or binary vector. The intermediary
vectors, on the basis of sequences, which are homologous to
sequences in the T-DNA, can be integrated by homologous
recombination in the Ti- or Ri-plasmid. This contains also the
necessary vir-region for the transfer of the T-DNA. Intermediary
vectors cannot be replicated in Agrobakteria. Using a helper
plasmid, the intermediary vector can be transferred to
Agrobacterium tumefaciens (conjugation). Binary vectors can be
replicated in E. coli as well as in Agrobakteria. They contain a
selection marker gene and a linker or polylinker, which are framed
by the right and left T-DNA border regions. They can be directly
transformed in the Agrobakteria (Holsters et al.(1978) Mol gene
geneet 163: 181-187). The Agrobakteria serving as host cells should
contain a plasmid, that carries a vir-region. The vir-region is
necessary for the transfer of the T-DNA in the plants cells.
Additional T-DNA can be contained. The so transformed bacterium is
used for the transformation of plant cells. For the transfer of the
DNA in the plant cells plant-explantate can suitably be
cocultivated with Agrobacterium tumefaciens or Agrobacterium
rhizogenes. From the infected plant material (e.g. leaf pieces,
stem segments, roots and also protoplasts or suspensions of
cultivated cells) whole plants can be regenerated in a suitable
medium, which contain antibiotics or biocides for the selection.
The resulting plants can then be tested for the presence of
introduced DNA. No special demands are placed an the plasmids in
the injection and electroporation. Simple plasmids such as e.g.
pUC-derivatives can be used. Should however whole plants be
regenerated from such transformed cells the presence of a
selectable marker gene is necessary.
[0024] The plasmids pAS and pAS-GUS have a size of around 13.3 and
15.2 kb respectively and contain the approximately 2.0 kb size
kanamycin resistance gene, the approximately 3.2 kb size
HindII/BglII-promoter-frag- ment of the regulatory starter-region
of the ADP-glucose-pyrophosphorylase- -gene GS6-11 of Solanum
tuberosum, a linker and a transcription terminator of the nopaline
synthase gene. The plasmid pAS-GUS contains also the approximately
2 kb size .beta.-glucuronidase gene.
[0025] For the preparation for the introduction of foreign genes in
higher plants, a large number of cloning vectors are available,
which contain a replications signal for. E. coli and a marker,
which allows a selection of the transformed cells. Examples of
vectors are pBR 332, pUC-series, M13 mp-series, PACYC 184 etc. The
desired sequence can be introduced in the vector on a suitable
restriction position. The resulting plasmid is used for the
transformation in E. coli. The E. coli cells are cultivated in a
suitable nutrient medium, then harvested and lysed. The plasmid is
reextracted. For analysis general, a sequence analysis, a
restriction analysis, electrophoresis and other
biochemical-molecular biological methods can be used. After each
manipulation the used DNA-sequence can be split and attached to
another DNA-sequence. Each plasmid sequence can be cloned in the
same or different plasmids. Depending on the method of
introductions of the desired gene in the plant, further
DNA-sequences may be necessary. Should for example the Ti- or
Ri-plasmid be used for the transformation of the plant cells, at
least the right boundary, and often however, the right and the left
boundary of the Ti- and Ri-plasmid T-DNA, should be linked as a
flanking area of the introduced gene.
[0026] The use of T-DNA for the transformation of plants cells has
been intensively researched and is well described in EP 120 516;
Hoekema, In: The Binary Plant Vector System, Offset-drukkerij
Kanters B. V., Alblasserdam, (1985), Chapter V; Fraley, et al.,
Crit. Rev. Plant Sci., 4:1-46 and An et al.(1985)-MO J. 4:
277-287.
[0027] Once the introduced DNA is integrated in the genome, it is
as a rule stable there and remains also in the offspring of the
original transformed cells. It normally contains a selection
marker, which induces resistance in the transformed plant cells
against a biocide or antibiotic such as kanamycin, G 418,
bleomycin, hygromycin or phosphinotricin etc. The individual marker
employed should therefore allow the selection of transformed cells
from cells, which lack the introduced DNA.
[0028] The transformed cells grow within the plants in the usual
manner (see also McCormick et al. (1986) Plant Cell Reports 5:
81-84). These plants can be grown normally and crossed with plants,
that possess the same transformed genes or different. The resulting
hybrid individuals have the corresponding phenotypical
properties.
[0029] Two or more generations should be grown to ensure that the
phenotypic characteristic remains stabile and inherited. Also seeds
should be harvested to ensure that the corresponding phenotype or
other individualities remain. All plant species are suitable as
host plants for the guard cell specific expression, but especially
crop species.
[0030] Suitable crops are e.g. potatoes, tobacco, tomatoes and
sugar beet as well as species agricultural supplementary crops e.g
nitrogen providing crops.
[0031] The DNA sequence contained in the expression cassette of the
invention can be inserted after further DNA sequences, that contain
information for the formation and quantitative distribution of
endogenous products or the formation of heterologous expression
products in crops.
[0032] Endogenous expression products are e.g. phytohormones,
carbohydrates and other metabolites. Heterologous products are e.g.
carbohydrates not naturally formed in plants or enzymes for
liberation of substances with phytohormone activity as a primary
step, which are not naturally converted to phytohormones in
plants.
[0033] The DNA sequences that can be inserted after the regulatory
sequence of the expression cassette should contain all possible
open reading screens for a preferred peptide as well as one or more
introns. As examples can be named: sequences for enzymes, sequences
which are complementary:
[0034] a) to a genome sequence, whereby the genome sequence should
be an open reading screen;
[0035] b) to an intron;
[0036] c) to a non-coded reading sequence;
[0037] d) to each sequence,
[0038] which inhibits, complementarily to integration in the
genome, the transcription, mRNA working up (for example splicing)
or the translation.
[0039] The desired DNA sequence can be prepared synthetically or
extracted naturally or can consist of a mixture of synthetic and
natural DNA constituents. In general, synthetic DNA sequences are
produced with codons, which are preferred for plants. These
preferred codons for plants can be fixed from codons with the
highest protein abundance, which are expressed in the most
interesting plant species. In the preparation of an expression
cassette, various DNA fragments can be manipulated in order to
obtain DNA sequence that reads suitably in the correct direction
and which is supplied with the correct reading screen. For the
combination of DNA fragments with each other, adaptors or linkers
can be put onto the fragments.
[0040] Suitably, a transcription start and termination region in
the transcription direction should be seen through a linker or
polylinker which contains one or more restriction positions for the
insertion of this sequence. As a rule, the linker has 1 to 10,
mostly 1 to 8 and preferably 2 to 6 restriction positions. In
general the linker has, within the regulatory area, a size of less
than 100 bp, generally less than 60 bp and at least however 5 bp.
The transcriptional start area as well as being native and/or
homologous can also be foreign and/or heterologous to the host
plants. The expression cassette comprises in the 5'-3'
transcription direction, a representative region for the plant, for
the transcription initiation, a preferred sequence and a region for
the transcriptional termination. Various termination areas are
exchangeable, preferably with each other.
[0041] Further, manipulations which prepare suitable restriction
cutting sites or separate the excess DNA or cutting positions, can
be carried out. Where insertions, deletions or substitutions, for
example transitions and transversions are to be considered, in
vitro-mutagenase, primer repair, restriction or ligation can be
used. In suitable manipulations, such as for example restriction,
chewing back or filling in of overhangs for blunt ends,
complementary ends of the fragments for the ligation can be used.
For carrying out the various stages, a cloning is necessary for an
enlargement of the DNA amounts and for the DNA analysis which
ensures the expected success of the intervention.
[0042] For the introduction of foreign genes into plants by using
the guard cell specific regulatory sequence a number of
possibilities are available, but especially interesting is however,
the expression of genes which change the regulation of the guard
cell in relation to the gas exchange as well as the expiration of
water vapour (transpiration).
[0043] For the expression of such genes the use of the expression
cassette of the invention is especially valuable.
[0044] A modification of the guard cells with effects on the
opening condition of the stomata can be achieved in two ways:
through a modulation of the content of endogenous proteins,
enzymes, carriers or pumps which transport the metabolite through
membranes, present in the guard cells; or through transfer and
expression of genes of homologous or heterologous or of synthetic
origin, which code the proteins, enzymes, carrier or pumps, which
do not belong to the normal constituents of a guard cell, but whose
activity or presence influences the state of the cells in relation
to the opening of the stomata.
[0045] For regulation of the transpiration three sensor systems are
concerned: sensors for measuring the carbon dioxide concentration,
phytohormone sensors and a measuring system for turgidity gradients
between guard cells and bordering epidermal cells. Turgidity
changes are for example possible through influencing the activity
of enzymes or transport systems of the carbohydrate metabolism.
Besides a direct change of the concentration of the osmotic
activity of the carbohydrate, for example by means of cytosolic or
vacuolar invertases or of the chloroplast triose phosphate
translocators, consequences for the osmo regulation to be expected,
through an enrichment or reduction of precursors of osmotically
active substances. For example, by modifying the ADP glucose
pyrophosphorylase activity, starch synthesis can be influenced, and
so the amounts on substrates for glycolyse and citrate cycle can be
modified, whereby lastly the concentration of the malate, a
substance of importance in relation to the cell turgidity, is
concerned. An increase of the apoplastic invertase activity in
guard cells can raise the starch concentration by an increased
uptake of hexoses. Such an introduction must follow a guard cell
specificity since an increase of apoplastic invertase activity in
the strongly photosynthetic active mesophyllic cells leads to a
reduction of the assimilate transport and with it a higher osmotic
loading of the tissues (see Schaewen et al 1990 EMBO J 9:
3033-3044). Further, an increase of the expression or the
expression of a modified phospho-enol-pyruvate carboxylase can be
envisaged. The enzyme catalyses the reaction of
phospho-enol-pyruvate to oxaloacetate, that again is a predecessor
for malate. Besides, proton pumps (proton-ATPases) can contribute
to a change of the osmotic potential, which makes possible the ion
transport process for the electrochemical gradients.
[0046] Further possibilities of influence are seen in the activity
of the carbonate dehydratase, which controls the equilibrium
between dissolved carbon dioxide and carbonate in so far as it thus
influences the cellular carbon dioxide concentration; but also for
example in fructose-biphosphotase which contributes as an enzyme in
the regenerative section of the Calvin cycle in the fixing of
carbon dioxide.
[0047] The expression cassette of the invention can also be used
for guard cell specific expression of genes which regulate the
phytohormone level of plants.
[0048] Local changes to the phytohormone level are especially
interesting for the influence of the properties of guard cells:
whilst for example auxins cause an opening of the holes,
application of abscisic acid leads to a fast hole closure. Numerous
genes whose products have influence in the hormone make-up of
plants, have already been cloned, (see e.g. Klee, H. & Estelle,
M. (1991) Ann Rev Plant Physiol Plant Mol Biol 42: 529-551). The
genes 1 and 2 of the T-DNA of Agrobacterium tumefaciens code for
auxin synthesis enzyme, whilst the indole acetate lysine synthetase
(Romano et al. (1991) Genes & Development 5: 438-446) catalyses
the inactivation of auxins. Cytokinins are liberated through a vom
rol c gene of the T-DNA from Agrobacterium rhizogenes coded
glucosidase (Estruch et al. (1991) EMBO J 10: 3125-3128); the gene
product of the T-DNA gen 6b von A. tumefaciens reduces cytokinin
activity (Spanier et al. (1989) Mol Gen Genet 219: 209-216).
Ethylene production can be stimulated in plants by expression of
the aminocyclopropane carboxylate synthase (Huang et al. (1991)
Proc Nat Acad Sience 88: 7021-7025); inhibition of the
aminocyclopropane carboxylate oxidase by expression of
antisense-RNA depresses the ethylene liberation.
[0049] Because of the wide-reaching consequences of hormonal
activity for the plants an influence of the guard cell activity is
feasible only by using the expression cassette of the invention for
a guard cell specific gene expression.
[0050] Processes for the reducing the activity and/or the amount of
a specified enzyme are already known in the art. As a rule a
chimeral genes is introduced into the plant. It consists of a
promoter active in the plant, a coding sequence of the enzyme
attached to the promoter in the anti-sense direction, whose
activity is reduced (3'-end of the coding sequence to the 3'-end of
the promoters) and a termination signal, functional in plants, for
the transcription. For introduction of genes of this kind in plant
cells there are various processes available. From the transformed
cells, intact transgenic plants can be regenerated for which the
desired phenotype (lowering of the activity of the target enzyme)
must be determined.
[0051] The identification of necessary transcriptional starting
areas can be achieved in a number of ways. As a rule mRNA is dealt
with, which is isolated from certain parts of the plants (here,
guard cells) (see also Example 1). For additional increase in
concentration of the mRNA characterising for the tissue or the
plant condition, cDNA can be prepared whereby unspecific cDNA is
attached to the mRNA or the cDNA from other tissues (mesophyllic
cells) or plant states. The remaining cDNA can be used then for the
probing of the genome of the complementary sequences using a
suitable plant DNA library.
[0052] Where a protein, appearing in a specific cell or tissue
type, is isolated it can be partially sequenced so that a probe can
be prepared for direct identification of the corresponding
sequences in a plant DNA library (see Example 2). Then the
sequences that hybridise with the probe can be isolated and
manipulated. Further, the non-translated 5' area that is associated
with the coded area can be isolated and tested in expression
cassettes for transcriptional activity.
[0053] The expression cassettes obtained, which Use the
non-translated 5' regions can be transformed in plants (see above)
where their functionality as transcriptional regulators in
combination with a heterologous structure gene (other than the open
reading screen of the wild type genes that is associate d wit h the
non-translated 5' region) a s well as the guard cells specificity
can be tested. In this way sequences, which are necessary for the
guard cell specific transcription, can be identified.
[0054] The following plasmids were deposited at the Deutschen
Sammlung von Mikroorganismen (DSM) in Braunschweig, Germany on the
13.02.1992 (deposit number):
[0055] Plasmid PAS (DSM 6906);
[0056] Plasmid pAS-GUS (DSM 6907).
DESCRIPTION OF THE FIGURES
[0057] FIG. 1 shows the restriction map of the genomic clone
GS6-11, which codes the S-subunit of the ADP glucose
pyrophosphorylase of Solanum tuberosum. Above the restriction man
are the designations of genes of the phages Lambda as well as a
scale for estimating the length of the DNA sequences in kilobases
(kb).
[0058] FIG. 2 shows the promoter GUS fusion of the plasmid pAS-GUS,
that contains the nucleic acid sequence of a part of the important
areas for transcriptional regulation of the ADP glucose
pyrophosphorylase gene GS6-11, which is also contained in Plasmid
pAS. ATG signifies the translation start for the
.beta.-glucuronidase. The Bgl II cutting site, where the clone
pH6-1 is cut and attached with the linearised plasmid pUC 19 to the
SmaI cutting site, is shown. The sequence up to the BamHI cutting
site stems from the polylinker of von pUC 19 and the following
nucleotide from the plasmid pBI101.1 (Jefferson et al.(1987) Plant
Mol Biol Rep 5: 387-405). The corresponding area with der cDNA is
shown in bold lettering. The two first codons of
.beta.-glucuronidase are bracketed. A sequence, which possibly
represents a Hogness box, is underlined.
[0059] FIG. 3 shows the approximately 13.2 kb size Plasmid pAS. It
contains the approximately 2.0 kb size kanamycin resistance gene as
a constituent of the starting vector pBIN 19 (Bevan, M.(1984) Nucl
Acids Res 12:-8711-8721), the approximately 3.2 kb size regulatory
area for a guard cell specific gene expression from the ADP glucose
pyrophosphorylase gene from Solanum tuberosum from the clone GS6-11
(A), inserted between nucleotide position (nt) 2525 and 2544 of
PBIN 19 as well as the transcription terminator from the nopaline
synthase gene (C), inserted at nt 2494 of pBIN 19.
[0060] A=Fragment A (approximately 3.2 kb): contains:
[0061] nt 400-414 from pUC 19
[0062] approximately 3.2 kb from GS6-11
[0063] nt 415-421 from pUC 19
[0064] C=Fragment C (192 bp): contains nt 11749-11939 from pTiACH5
(Gielen et al. (1984) EMBO J 3: 835-846)
[0065] FIG. 4 shows the approximately 15.2 kb size plasmid pAS-GUS,
that is a derivative of pBI101.1 (Jefferson et al.(1987) Plant Mol
Biol Rep 5: 387-405). The plasmid pBI101.1 again is a derivative of
PBIN 19, that contains, at nt 2534, an insertion of 1.87 kb of the
coding region of .beta.-glucuronidase (B). Plasmid PAS-GUS is even
differentiated from pAS by this insertion. In detail pAS-GUS
contains the following fragments:
[0066] A=Fragment A (approximately 3.2 kb): contains:
[0067] nt 400-414 from pUC 19
[0068] approximately 3.2 kb from GS6-11
[0069] nt 415-421 from pUC 19
[0070] B=Fragment B (1.87 kb): coding region of the
.beta.-Glucuronidase.
[0071] C=Fragment C (192 bp): contains nt 11749-11939 from pTiACH5
(Gielen et al. (1984) EMBO J 3: 835-846)
[0072] FIG. 5 shows the expression of .beta.-glucuronidase in the
leaf of a transgenic potato plant transformed with the plasmid
pAS-GUS. It can clearly be seen, that no expression in the
mesophyllic cells or epidermis cells is detectable but only a
specific expression in the guard cells.
[0073] In order to understand the examples forming the basis of
this invention all the processes necessary for these tests and
which are known per se will first of all be listed:
[0074] 1. Cloning Process
[0075] The vectors pUC 18/19 and M13mp10 series (Yanisch-Perron et
al. (1985) genee 33: 103-119), as well as the vector EMBL 3
(Frischauf et al. (1983) J Mol Biol 170: 827-842) were used for
cloning.
[0076] The gene construct ons in the binary vector BIN 19 (Bevan
(1984) Nucl. Acids Res 12: 8711-8720) were cloned for the plant
transformations
[0077] 2. Bacterial Strains
[0078] The E. coli strain BMH71-18 (Messing et al., Proc. Natl.
Acad. Sci. USA (1977), 24, 6342-6346) or TB1 was used for the pUC
and M13 mP vectors.
[0079] For the vector BIN19 exclusively the E. coli strain TB1 was
used. TB1 is a recombinant-negative, tetracycline-resistant
derivative of strain JM101 (Yanisch-Perron et al., genee (1985),
33, 103-119).
[0080] The genotype of the TB1 strain is (Bart Barrel, personal
communication): F' (traD36, proAB, lacI, lacZ.DELTA.M15),
.DELTA.(lac, pro), SupE, this, recA, Sr1::Tn10(TcR).
[0081] The transformation of the plasmids into the potato plants
was carried out by means of the Agrobacterium tumefaciens strain
LBA4404 (Bevan, M., Nucl. Acids Res. 12, 8711-8720, (1984); BIN19
derivative).
[0082] 3. Transformation von Agrobacterium tumefaciens
[0083] In the case of BIN19 derivatives, the insertion of the DNA
into the agrobacteria was effected by direct transformation in
accordance with the method developed by Holsters et al., (Mol.
gene. geneet. (1978), 163, 181-187). The plasmid DNA of transformed
agrobacteria was isolated in accordance with the method developed
by Birnboim and Doly (Nucl. Acids Res. (1979), 7, 1513-1523) and
was analysed by gel electrophoresis after suitable restriction
cleavage.
[0084] 4. Plant Transformation
[0085] Ten small leaves, wounded with a scalpel, of a sterile
potato culture were placed in 10 ml of MS medium with 2% sucrose
containing from 30 to 50 .mu.l of an Agrobacterium tumefaciens
overnight culture grown under selection. After from 3 to 5 minutes
gentle shaking, the Petri dishes were incubated in the dark at
25.degree. C. After 2 days, the leaves were laid out on MS medium
with 1.6% glucose, 2 mg/l of zeatin ribose, 0.02 mg/l of
naphthylacetic acid, 0.02 mg/l of gibberellic acid, 500 mg/l of
claforan, 50 mg/l of kanamycin and 0.8% Bacto agar. After
incubation for one week at 25.degree. C. and 3000 lux, the claforan
concentration in the medium was reduced by half.
[0086] 5. Analysis of Genomic DNA from Transgenic Potato Plants
[0087] The isolation of genomic plant DNA was effected in
accordance with Rogers and Bendich (Plant Mol. Biol. (1985), 5,
69-76. After suitable restriction cleavage, 10 to 20 .mu.g of DNA
were tested by means of Southern blots for the integration of the
DNA sequences to be introduced.
[0088] 6. .beta.-Glucuronidase-Activity Test (GUS-Assay)
[0089] The .beta.-glucuronidase is a bacterial enzyme, that
hydrolyses .beta.-glucuronide and accessible to quantitative as
well as histochemical activity determinations. Activity measurement
were carried out by the method of Jefferson et al. (1987) EMBO J 6:
3901-3907. Tissue probes incubated in 1 mM X-Gluc, 50 mM
Na-phosphate pH 7.0 and 0.1% Tween 20 bis to give the desired
intensity of blue colouring.
[0090] The following examples illustrate the preparation of the
expression cassette, whose DNA sequence contains a regulatory
sequence for a guard cell specific gene expression. The
introduction of the sequence in a plasmid is also illustrated with
which a transformation of plant cells is possible as well as the
transformation of plant cells, the regeneration of transgenic
plants and the investigation of the function of the expression
cassette in transgenic plants.
EXAMPLE 1
[0091] Cloning and Structure Analysis of an ADP Glucose
Pyrophosphorylase Gene from Solanum tuberosum
[0092] cDNA clones, that code for the S-subunit of ADP glucose
pyrophosphorylase of potato, were isolated from the potato variety
"Deasire" and sequenced (Muller-Rober et al. (1990) Mol gene geneet
224:136-146; EP 455 316). These cDNA clones are used to isolate a
homologous, genomic, ADP glucose pyrophosphorylase clone from the
potato variety AM 80/5793 (Max-Planck-Institut for
Zuchtungsforschung, Koln).
EXAMPLE 2
[0093] Cloning Identification and Primary Structure of an ADP
Glucose Pyrophosphorylase Gene as a Fragment of Genomic DNA from
Solanum tuberosum
[0094] A genomic library of the nuclear DNA from the potato variety
AM 80/5793, which has been established in vector EMBL 3 derived
from Lambda phages, was probed with the help of the ADP glucose
pyrophosphorylase cDNA S25-1 (see. Example 1). Several independent
clones were obtained, from which the clone GS6-11 was used for the
further working. The restriction map of the clone GS6-11 is shown
FIG. 1. Part of the gene was sequenced and the sequence is shown in
FIG. 2.
EXAMPLE 3
[0095] Identification of the Regulatory Areas Responsible for the
Guard Cell Specific Expression of the ADP Glucose Pyrophosphorylase
Gene
[0096] The greater than 12 kbp size Sal I insertion of the genomic
clone GS6-11 was cloned in the Sal I cutting position of the
vectors pUC 19 and the Plasmid SF-6 resulted. An approximately 5.3
kbp long HincII fragment of the plasmid SF-6 was sub-cloned in the
HincII cutting position of the vectors pUC 19 and the Plasmid pH6-1
resulted. The approximately 3.2 kbp long HincII/Bgl II of the
plasmid pH6-1 was cloned after filling in of the Bgl II cutting
position in the SmaI cutting position of the vector pUC 19. In this
way the 5'end for the EcoRI cutting position of the polylinker was
orientated, so that the plasmid pSA was obtained. Then the
EcoRI/BamHI fragment was cut from the pUC 19 derivative pSA and
after filling in the EcoRI cutting position, was put in the vector
pBI101.1 (Jefferson et al.(1987) Plant Mol Biol Rep 5: 387-405),
whereby PAS-GUS was obtained (see FIG. 4). The vector pBI101.1 was
previously cut HindIII/BamH I and the HindIII cutting position
filled in. The vector pBI101.1 contains the coding region of the
.beta.-glucuronidase gene as reporter gene. The
.beta.-glucuronidase is indicative of a histological determination
of its activity and can thus be introduced for the analysis of the
cell of a regulatory region of an expression system to be tested.
in parallel with the cloning of pAS-GUS the expression vector pAS
was prepared, which contains a polylinker between the promoter
fragment of the ADP glucose pyrophosphorylase and the terminator of
the octopine synthase gene (see. FIG. 3). With the help of this
plasmid an expression of preferred genes under the control of the
ADP glucose pyrophosphorylase promoters is possible.
[0097] The construct pAS-GUS, with the coding region of
.beta.-glucuronidase as reporter gene, was transferred into the
Agrobacteria strain LBA 4404 (Bevan, M.(1984) Nul Acids Res 12:
8711-8721) and the Agrobacteria containing the chimeral ADP glucose
pyrophosphorylase/5-glucuronidase gene is introduced for
transformation of potato and tobacco leaves.
[0098] Of ten resulting independent transformands, in which the
presence of the intact, unrearranged chimeral ADP glucose
pyrophosphorylase/.beta.- -glucuronidase gene had been proven with
the help of southern blot analyses, leaves, stems, tubers and roots
were tested for .beta.-glucuronidase activity. The results are
shown in FIG. 5. From these data is clearly shown, that the HincII
fragment of the ADP glucose pyrophosphorylase gene GS6-11, which
was fused with the .beta.-glucuronidase gene, had induced in the
leaf a guard cell specific activity of the .beta.-glucuronidase.
This activity could not been seen in the mesophyllic and epidermis
cells.
EXAMPLE 4
[0099] Further Concentration of the Regulatory Areas Responsible
for the Guard Cell Specific Expression of the ADP Glucose
Pyrophosphorylase Gene
[0100] The 0.35 kbp size HindIII/BamHI fragment was isolated from
Plasmid pSA described in Example 3 and inserted in vector pBI101.1
which had been linearised with restriction enzymes BamHI and
HindIII. The resulting plasmid bears the designation pS1-D4GUS. The
0.35 kbp size HindIII/BamHI insertion of the plasmid is shown in
FIG. 2 as well as in Seq ID No 1. The vector pBI101.1 contains the
coding region of the .beta.-glucuronidase gene as reporter gene.
The .beta.-glucuronidase is indicative of a histological
determination of its activity and can thus be introduced for the
analysis of the cell specificity of a regulatory region of an
expression system to be tested. The construct pS1-D4GUS with the
coding region of .beta.-glucuronidase as reporter gene, was
transferred into the Agrobacteria strain LBA 4404 (Bevan, M.(1984)
Nucl Acids Res 12: 8711-8721) and the Agrobacteria containing the
chimeral ADP glucose pyrophosphorylase/.beta.-glucuronidase gene is
introduced for transformation of potato and tobacco leaves.
[0101] Of ten resulting independent transformands, in which the
presence of the intact, unrearranged chimeral ADP glucose
pyrophosphorylase/5-gluc- uronidase gene had been proven with the
help of southern blot analyses, leaves, stems, tubers and roots
were tested for .beta.-glucuronidase activity. It was shown that
the 0.35 kbp size HindIII/BamHI promoter fragment of the ADP
glucose pyrophosphorylase gene has a comparable activity to that of
the EcoRI/BamHI promoter fragment of the plasmid pSA (see. FIG. 5)
in the stomata cells of leaves, whilst for vascular tissues, tubers
and stolons of potato plants and roots no activity was seen.
Sequence CWU 1
1
* * * * *