Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02348888 2001-05-O1
New expression cassette for expression of arbitrary genes in
plant seeds
Description
The invention in question relates to an expression cassette
for expression of arbitrary genes in plant seeds and the
plasmids containing the expression cassette. The invention
also includes the production of transgenic plant cells con-
taming this expression cassette as well as the use of the
plasmids in this expression cassette for production of trans-
genic plants. Fields of application of the invention are bio-
technology, pharmacy and plant production.
For a long time now, there have been methods making it possi-
ble to integrate relevant genes into the genome of higher
plants. The objective of this work is the production of
plants with new properties, for example to increase agricul-
tural production, to optimise manufacture of foodstuffs and
to produce specific pharmaceuticals and other interesting in-
gredients. One prerequisite for the expression of the trans-
ferred genes in this context is that they possess plant-spe-
cific promoter sequences. For this purpose, so-called consti-
tutive promoters such as the promoter of the nopaline syn-
thase gene /1/, the TR double promoter /2/ or the promoter of
the 35S transcript of the cauliflower mosaic virus /3/ are
used. One disadvantage of these promoters is that they are
active in almost all the tissues of the manipulated plants.
In this way, a controlled and purposeful expression of the
foreign genes in the plants is not possible. It is better to
use promoters which function tissue-specifically and inde-
pendently of development. Genes with the matching promoters,
which are only active in anthera, ovaries, blooms, leaves,
deciduous leaves, stems, roots or seeds, have been isolated
/4/ . But they differ greatly in the strength and specificity
of the expression and only have a limited use. For the use of
the seeds as a source of nutrition and for production of in-
CA 02348888 2001-05-O1
gredients, it is above all the seed-specific promoters which
are of great interest. With the years of research into the
genes of the seed-storage proteins, some more or less spe-
cific promoters with differing strengths, for example that of
phaseolin /5/ or legumin and USP /6/ are available. As these
storage proteins are synthesised by gene families, fusions of
such promoters with foreign genes are in competition with the
endogenous numerous genes of the corresponding gene family.
For this reason, it is more favourable to use promoters from
unique, strongly and specifically expressing genes. For co
and multiple transformations, the use of differing regulatory
sequences is suitable, in order to make better use of the
development of the seed in time, to synthesise identical or
differing gene products in parallel and to avoid co
suppression.
Although a number of expression cassettes for expression of
arbitrary genes in plant seeds are already known, the expres-
sion rates in plant seeds achieved have not been optimal up
to now far the substantiation of a plant biotechnological
production of the required materials.
The invention therefore has the objective of placing the
seed-specific expression in transgenic plants on a basis
suitable for a production of materials. It is based on the
task of constructing an expression cassette with which a sta-
ble expression with a high expression rate of genes of the
materials to be produced can be achieved in plant seeds.
The objective of the invention is achieved with the expres-
sion cassette described in claim 1, with sub-claims 2-7 being
preferred variants.
The expression cassette according to the invention contains
the following essential component parts:
~ the promoter of the gene of the sucrose binding
protein (SBP)like protein
2
CA 02348888 2001-05-O1
~ if applicable, the DNA sequence of a signal peptide,
preferably the SBP signal peptide
~ a gene to be expressed
~ 3' termination sequences
The invention relates above all to a regulatory DNA sequence
occurring uniquely in the genome, which mediates a strong ex-
pression of an arbitrary heterologous gene primarily in the
cotyledons and in the endosperm dependency on seed
development.
The most important component part of the cassette is the SBP
promoter, the sequence of which is shown in Figure 1. Com-
pared with analog promoters in this field, this promoter has
the benefit of great strength and seed-specificity. Its use
for the expression of foreign genes, even without the DNA se-
quence of a signal peptide, is also part of the scope of the
invention.
Together with the transcriptionally regulatory sequences, the
expression cassette also, if need be, contains a signal pep-
tide, which enables the transport of the required gene prod-
uct into the protein bodies, thus preventing decomposition of
the gene products to a great extent. The optional use of the
authentic signal peptide enables the transport of the synthe-
sised foreign proteins to and storage in the protein bodies.
The genes to be expressed can be integrated either as tran-
scription or as translation fusions, they can be varied to a
great extent, for example genes can be used for the produc-
tion of enzymes (e. g. amylase, xylanase), pharmaceutical
products or for the over-expression of proteins with a high
share of essential amino-acids (e. g. 2S globulin of the bra-
zil nut rich in methionine) or of other proteins influencing
the properties of the seeds. Further possibilities can be
found in the reduction or elimination of gene products
through the integration of genes in an anti-sense orienta-
tion. By inserting regulatory genes under the control of this
seed-specific promoter, metabolic processes in the seeds can
3
CA 02348888 2001-05-O1
also be influenced. The cassette can also be used in order to
express the SBP gene inherent to the promoter from field
beans into other species. The use of other terminators, for
example the termination sequence of the gene to be expressed,
is a further possibility of optimal use of the cassette. As a
concrete example, the gene of f3-glucuronidase (GUS) was used
to show the specificity of the promoter (Fig. 2b, c)..
The nucleotide sequence of the expression cassette contains
transcriptionally regulatory areas, guaranteeing a strong
specific expression of an arbitrary gene into the seed of
plants. The Northern (Fig. 2a) shows the high seed-specific
expression in the various tissues of Vicia faba. The GUS data
in Figs. 2b and 2c show on the one hand the distribution of
the f3-glucuronidase in the sections through ripe tobacco
seeds and, on the other, the accumulation of the i3-glucuroni-
dase in the transgenic tobacco seeds as a function of devel-
opment.
The plasmids containing the expression cassette, preferably
the plasmids pSBPROCS and pPTVSBPRGUS, are also to be placed
under protection.
The scope of the invention also includes the use of the ex-
pression cassette according to claims 12-16, which is done by
transformation into bacteria strains and subsequent transfer
of the resulting recombinant clones into preferably dicotyl
plants. The plants expressing the required gene product in
the seed are selected and bred as genetically stable lines.
After harvesting, the required gene products are extracted
from the transgenic seeds in a way basically already known.
This invention is also interesting for applications in which
the required gene product is expressed under the control of
various promoters, in order to increase the total of the ex-
pression rates, in order to make better use of the develop-
ment period of the seeds and to avoid effects by co-suppres-
sion. This expression cassette is also suited for co- and
4
CA 02348888 2001-05-O1
multiple transformations with the objective of expressing
various gene products. A variety of new expression cassettes
is needed for these strategies in order to be able to select
the correct ones.
The entire method for the alteration of a plant cell is por-
trayed in an example (pSBPOCS).
The invention is to be explained in more detail below with
examples of embodiments.
Methods
1. Cloning method
For cloning, the vectors pUCl8 /7/, pBK-CMV (Stratagene) and
pOCSl (Plant Genetic Systems, Gent, Belgium) and for plant
transformation the vectors BIN19 /8/, and, after deletion of
the GUS gene, pGPTV-BAR /9/ were used.
2. Bacteria strains
For the transformation to E. coli, strain DHSa /10/ was used.
The binary plasmids were inserted into the agro-bacteria
strain EHA105 /11/ by conjugation.
3. Plant transformation
The transformation of Nicotiana tabacum was done by the
leafdisk method /12/ and the transformation of Vicia narbo-
nensis with the help of the method described by Pickardt in
1991 /13/ by agrobacterium mediated gene transfer.
4. Analysis of genomic DNA from transgenic plants
The genomic DNA of the transgenic tobaco and V. narbonensis
plants was isolated with the help of the DNA isolation kit of
the firm of Macherey & Nagel. In a first step, the transgenic
lines were identified via PCR with gene-specific primers. The
integration of foreign DNA was examined by means of "Southern
5
CA 02348888 2001-05-O1
blot" analyses of 20~cg of DNA following suitable restriction
digestion.
5. i3-glucuronidase activity test (GUS assay)
The reporter gene f3-glucuronidase is a bacterial enzyme ac-
cessible to both quantitative /14/ and also histo-chemical
activity assays. Tissue samples were incubated over night at
37°C in 1 mM X-Gluc, 50mM Na phosphate (pH 7.0) and 0.1%
Tween 20. For sections, the tissues were fixed, embedded in
paraffin and cut to a section thickness of 15 - 30 ~,m on a
microtome.
Examples of embodiments
The invention, which contains the production of a new, seed-
specific expression cassette as well as the plasmids and
transgenic plants derived from them, is explained below -
partly with the help of the figures - using an example of an
embodiment.
1.) Cloning and structure analysis of an SBP seed protein
gene from Vicia faba
Primers (5'-GAAGACCCTGAGCTCGTAACTTGCAA-ACAC- 3' and 5'
AGTACTCATAGATCTCTGGGTGATGTTGGT-3') were derived from the se
quence of a cDNA clone which codes for the sucrose binding
protein of the soybean /15/. The gene-specific probe was then
amplified, cloned and sequenced by means of RT - PCR on mRNA,
isolated from immature cotyledons of V. faba. The PCR product
was identified as the gene fragment homolagous to the sucrose
binding protein and was used as a probe for the isolation of
the complete cDNA from a cotyledon-specific ~, Zap Express
cDNA Bank of V. faba L. var. minor. One of the isolated
clones (VfSBP20), which has a homology of 68% on the
nucleotide level, codes for the complete SBP-homologous gene
from the field bean. But it differs from the gene isolated
from the soybean in both the expression (F'ig. 2a) and also in
the function (no sucrose binding).
6
CA 02348888 2001-05-O1
2) Isolation of the regulatory sequences by means of PCR
The regulatory sequences were isolated with the help of the
"Universal GenomeWalkerT'"Kit" of the firm of CLONTECH and the
gene-specific primers PSBP1, position 159 (5'-AATCCTCA
CACTTCTCCATGCATATCCGTTTGTCC-3'), PSBP2, position 118 (5'-
GCCCTGCAGAT-CGCATTTGTCTTTGCA-3') and PSBP3, position 85 (5'-
CTGGGTCCTTTTCTTTTCTGG- C-3'). Following prior digestion of
the genomic DNA of V.faba with ScaI (a) and StuI (b) and
ligation of the adapters, a two-step PCR was done in accor-
dance with the description of the kit with the following pa-
rameters: 7 cycles of 94°C, 2s, 72°C, 3 min and 32 cycles of
94°C, 2s, 67°C, 3 min and 4 min 67°C. The PCR
preparations
were diluted 1:50 and 1~,1 of each were amplified in a second
PCR (5 cycles of 94°C, 2s, 72°C, 3 min and 20 cycles of
94°C,
2s, 67°C and 4 min at 67°C. In the Agarosegel, bands of 1.7 kb
from (a) and 1.9 kb from (b) were verified via a Southern
blot. These bands were then cloned into the pUCl8 and se-
quenced. The clones SBPR7 and SBPR15 were then identified by
a sequence comparison as the promoters matching gene VfSBP20.
They represent allelic variants of gene VfSBP20, with both
clones having 100% sequence identity with clone VfSBP20 in
the corresponding area . On the 5 ' side of the ATG of the SBP
gene, 1539 by were isolated with clone SBPR7 and 1750 by with
clone SBPR15. They differ by 23 base pair substitutions and
two insertions. The restriction maps of clone pSBPR7 and
pSBPRI5 are shown in Fig. 3, the sequence of clone pSBPRIS in
Fig. 1.
3a) Proof of the seed-specific expression in tobacco
With the help of the reporter gene of ~3-glucuronidase, the
seed-specific expression of the isolated regulatory sequences
SBPR7 and SBPR15 was to be tested. For this, the binary plas-
mid pBI101 /14/, which contains the promoter-free glucuroni-
dase gene behind a poly-linker, was cut with SmaI and dephos-
phorylated. The promoters were isolated from the plasmids
pSBPR7 and pSBPRI5 respectively by means of an SalI/NcoI
7
CA 02348888 2001-05-O1
digestion and the ends smoothed. The fragments were then
cloned into the SmaI site of binary plasmids pBI101 in front
of the reporter gene, with plasmids pBISBPR7GUS and
pBISBPR15GUS resulting. These plasmids were then transferred
to the agro-bacteria strain EHA105 and the chimerical agro-
bacteria containing SBP promoter/glucuronidase gene were used
for the transformation of tobacco. The results are shown in
Figures 2b and 2c. The analysis of the transgenic tobacco
seeds shows a strong blue coloration and thus a strong activ-
ity of the glucuronidase in the endosperm and in the
cotyledons of the tobacco seeds, also according to the seed
development. No glucuronidase activity was detected in other
tissues. The two slightly different nucleotide sequences
SBPR7 and SBPR15 also do not differ in their expression be-
haviour. These data show that the isolated regulatory se-
quences fused with the ~-glucuronidase gene result in a
strong and strictly seed-specific expression in the tobacco.
3b) Proof of the seed-specific expression in peas
In order to show that a seed-specific expression is also to
be expected in legumes, the SalI/NcoI fragment of plasmid
pSBPRI5 was cloned into the SalI/NcoI cut plasmid pGUSl
(Plant Genetic Systems, Gent). From the resulting plasmid
pSBPGUS, the fusion of the SBPR15 promoter/GUS/ocs-terminator
was cut out with SalI/SmaI, smoothed and ligated into the bi-
nary plasmid pGPTV-Bar, EcoRI/SmaI cut (Fig. 4). pGPTV-Bar
/9/ is a binary plasmid mediating phosphinothricin resistance
which is successfully used for the transformation of peas.
This plasmid has been called pPTVSBPRGUS (Fig. 4). The embry-
os of the transgenic pea lines generated with this plasmid
show a strong blue coloration after a histo-chemical analy-
sis.
3c) Proof of the transient expression in embryos of Vicia
faba, Vicia narbonensis, Pisum sativum and Brassica napus
With plasmid pSBPGUS, isolated embryos of Vicia faba, Vicia
narbonensis, Pisum sativum and Brassica napus were shot by
CA 02348888 2001-05-O1
means of the Biolistics PDS-1000/He Particle Delivery System
under the following conditions. The coating preparation com-
prised 501 of gold (Hereaus, 0.6-3~,m, 50 mg/ml), 10.1 of
Qiagen-cleaned plasmid-DNA (l~,g/~l), 501 of 2.5M CaCl2 and
101 of O.1M spermidine. At 1800 Psi and a vacuum of 27 inch
Hg, the embryos lying on an agar panel were then shot and
subsequently cultivated in MS-2% sucrose liquid medium for 2
days. There was then a reaction over night at 37°C with X-
Gluc (1mM) in 50mM Na phosphate (pH 7.0) and 0.1°s Tween 20.
Unlike the negative control (promoter-free pGUSl), a number
of blue dots were registered in the above mentioned embryos,
showing that the SBP promoter functions in the seeds.
4.) Production of the expression cassette for over-expression
of heterologous genes in the seed
In order to make the regulatory sequences available for the
over-expression of foreign genes, the SalI fragment of the
longer clone SBPR15 was isolated and smoothed and cloned into
the SmaI location of plasmid pOCSl (Plant Genetic Systems,
Gent, Belgium). This cassette thus contains the promoter re-
gion, the complete 5' untranslated region, the complete sig-
nal peptide, the first five triplets of the ripe protein
(Fig. 1) and the 3' untranslated area with the polyadenyla-
tion signals of the octopine synthase gene''(Fig. 5). The NcoI
location can be used for transcription fusions with foreign
genes, the BamHI location for translation fusions. After the
insertion of the foreign gene, the sequence containing the
promoter, regulatory sequences, the foreign gene and the 3'
termination sequences is cut out with restriction enzymes and
cloned into a binary vector with the herbicide resistance
suitable for the plant transformation.
As an example of this, the BamHI fragment. of the gene of Xy-
lanaseZ of Clostridium thermocellum was cloned into the BamHI
location of plasmid pSBPOCS as a translation fusion. From the
resulting plasmid pSBPRXYNZ (Fig. 6), the smoothed
Asp718/SphI fragment was ligated with the binary vector
9
CA 02348888 2001-05-O1
pGPTV-Bar, which was cut with the enzymes EcoRI/SmaI and
smoothed. After transformation into the agro-bacteria strain
EHA105, N. Tabacum was transformed. The strong expression of
the Xylanase Z was shown in the ripe transgenic seeds in a
Western blot (Fig. 7).
Literature:
1. Herrera-Estrella,L., Depicker,A., Van Montagu,M. and
Schell,J. (1983) Nature, 303,No.5914, 209-213.
2. Velten,J., Velten,L., Hani,R. and Schull,J. (1984) EMBO
J. 3, 2723-2730.
3. Koziel,M.G., Adams,T.L., Hazlet,M.A., Damm,D.,
Miller, J., Dahlbeck,D., Jayne,S. and Staskawicz,B.J.
(1984) Journ. of Molec. and Appl. Genet. 2, 549-562.
4. Goldberg,R.B. (1986) Phil. Trans. R. Soc. Lond. B314,
343-353.
5. Hall, T. C. et al (1996) US Patent 5,504,200
6. Conrad,U. et al. (19--) German Patent DE 196 04 588.6
7. Yanisch-Perron,C., Vieira,J. and Messing, J. (1985) Gene,
33, 103-119.
8. Bevan,M. (1984) Nucl. Acids Res. 12, 8711-8720.
9. Becker,D., Kemper,E., Schell,J. and Masterson,R. (1992)
Plant Mol. Biol. 20, 1195-1197.
10. Hanahan,D. (1983) J. Mol. Biol. 166, 557-580.
11. Hood,E.E., Gelvin,S.B., Melchers,L.S. and Hoekema,A.
(1993) Transgenic. Res. 2, 208-218.
12. Baumlein,H., Boerjan,W., Nagy,I., Bassuner,R., Van
Montagu,M., Inze,D. and Wobus,U. (1991) Mol Gen. Genet,
225, 459-467.
13. Pickardt,T., Meixner,M., Schade,V. and Schieder,0.
(1991) Plant Cell Report, 9, 535-538.
14. Jefferson,R.A. (1987) Plant Molec. Biol. Rep. 5, 387-
405.
CA 02348888 2001-05-O1
15. Grimes,H.D., Overvoorde,P.J., Ripp,K., Franceschi,V.R.
and Hitz,W.D. (1992; The Plant Cell, 4, 1561-1574.
11
