U.S. patent application number 09/834624 was filed with the patent office on 2001-11-08 for oxidative stress resistance gene.
This patent application is currently assigned to BTG INTERNATIONAL LIMITED.. Invention is credited to Balazs, Barna, Deak, Maria, Dudits, Denes, Kiraly, Zoltan, Sass, Laszlo, Torok, Karolyne.
Application Number | 20010039670 09/834624 |
Document ID | / |
Family ID | 10989471 |
Filed Date | 2001-11-08 |
United States Patent
Application |
20010039670 |
Kind Code |
A1 |
Deak, Maria ; et
al. |
November 8, 2001 |
Oxidative stress resistance gene
Abstract
The invention relates to plants, especially transgenic plants,
plant parts and plant cells overproducing an iron binding protein
(e.g. ferritin) and having an enhanced resistance against a wide
range of abiotic and biotic oxidative stress conditions (e.g.
against treatment with paraquat or fusaric acid and against viral,
bacterial and fungal infections). The invention also comprises
nucleic acid sequences encoding an alfalfa ferritin or functional
variants thereof and the use of said sequences for rendering plants
resistant against oxidative stress conditions. The invention is
useful for reducing environmental damages of crops caused by a wide
variety of stress conditions.
Inventors: |
Deak, Maria; (Szeged,
HU) ; Dudits, Denes; (Szeged, HU) ; Torok,
Karolyne; (Szeged-Tape, HU) ; Sass, Laszlo;
(Szeged, HU) ; Balazs, Barna; (Budapest, HU)
; Kiraly, Zoltan; (Budapest, HU) |
Correspondence
Address: |
Nixon & Vanderhye P.C.
8th Floor
1100 N. Glebe Rd.
Arlington
VA
22201
US
|
Assignee: |
BTG INTERNATIONAL LIMITED.
|
Family ID: |
10989471 |
Appl. No.: |
09/834624 |
Filed: |
April 16, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09834624 |
Apr 16, 2001 |
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09418830 |
Oct 15, 1999 |
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09418830 |
Oct 15, 1999 |
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PCT/GB98/01108 |
Apr 16, 1998 |
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Current U.S.
Class: |
800/279 ;
435/419; 435/69.8; 536/23.6; 800/301 |
Current CPC
Class: |
C07K 14/415 20130101;
C12N 15/8282 20130101; C12N 15/8274 20130101; C12N 15/8281
20130101; C12N 15/8271 20130101; C12N 15/8283 20130101 |
Class at
Publication: |
800/279 ;
800/301; 435/419; 536/23.6; 435/69.8 |
International
Class: |
C12N 015/82; C12N
005/14; C12N 015/29; A01H 005/00 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 16, 1997 |
HU |
P9700762 |
Sep 22, 1997 |
HU |
P9700762 |
Mar 9, 1998 |
HU |
P9700762 |
Claims
1. A plant cell which overproduces an iron binding protein
characterised in that the protein is at least 90% homologous to
that of SEQ ID No 2.
2. A plant cell of claim 1 characterised in that the iron binding
protein has at least 50% sequence identity to the protein having
the amino acid sequence of SEQ ID No 2.
3. A plant cell according to claims 1 or 2 characterised in that it
has been transformed by the introduction of a nucleic acid coding
for the expression of an iron binding protein.
4. A plant cell of any one of claims 1 to 3 characterised in that
the iron binding protein is a ferritin.
5. A plant or a plant part comprising a cell according to anyone of
claims 1 to 4.
6. A plant or plant part according to claim 5 in the leaves of
which the intensity of the photosynthetic reactions is not
decreased by more than 10% following, a 70 hours treatment with 10
.mu.M paraquat.
7. A plant or plant part as claimed in claim 6 characterised in
that the intensity is not decreased following the treatment.
8. A plant or a plant part according to claims 5, 6 or 7
characterised in that it has an enhanced resistance against fusaric
acid treatment and/or infections of viral and/or bacterial and/or
fungal origin.
9. A plant or plant part as claimed in any one of claims 5 to 8
characterised in that the iron binding protein is ectopically
expressed in vegetative tissues.
10. A plant or plant part as claimed in any one of claims 1 to 9
characterised in that the protein is expressed under control of a
promoter comprising the ribulose-1,5-biphosphate carboxylase small
subunit gene promoter.
11. A plant as claimed in any one of claims 1 to 10 characterised
in that the protein is truncated at the N-terminal transit
region.
12. Use of a nucleotide sequence coding for a ferritin having the
sequence of SEQ ID No 1 or a sequence having at least 90% homology
thereto for the preparation of a plant cell, plant or plant part as
claimed in any one of claims 1 to 11.
13. Use of a nucleotide sequence encoding for an iron binding
protein as a molecular biological agent for transforming a plant
cell, plant or plant part for the purpose of making the plant cell,
plant or part resistant to oxidative stress.
14. Use as claimed in claim 13, characterised in that it is for
making the plant cell, plant or part resistant to the damaging
effects of abiotic and biotic stresses.
15. Use as claimed in claim 14 characterised in that the stresses
include one or more of oxidatively induced free radicals, viral,
bacterial and fungal infections, low and high temperatures and
drought.
16. Use as claimed in claim 14 characterised in that the stresses
include all of oxidatively induced free radicals, and viral,
bacterial and fungal infections
17. Isolated, enriched, cell free and/or recombinant nucleic acid
comprising the sequence SEQ ID No 1 or having at least 90% homology
therewith or complementary sequences to such sequences.
18. Nucleic acid as claimed in claim 17 having at least 95%
homology therewith or a complementary sequence thereto.
19. Nucleic acid as claimed in any one of claims 16 to 18 not
including a ferritin leader sequence from Medicago saliva L.
20. A method of providing crops in an environment subject to
oxidative stress whereby the crop is able to resist at least 20%
increased oxidative stress as compared to a naturally provided
counterpart crops characterised in that it comprises transforming a
natural counterpart crop plant cell or plant with a nucleotide
sequence encoding for an iron binding protein and growing said
plant in that environment.
21. A method or use as claimed in any one of claims 13 to 16 and 20
characterised in that the nucleotide sequence encodes for a
ferritin of at least 90% homology to that of SEQ ID No 2.
Description
[0001] The present invention relates to plants, especially
transgenic plants, plant parts and plant cells overproducing an
iron binding protein (e.g. ferritin) and having an enhanced
resistance against a wide range of abiotic and biotic oxidative
stress conditions (e.g. against treatment with paraquat or fusaric
acid and against viral, bacterial and fungal infections). The
invention also comprises nucleic acid sequences encoding an alfalfa
ferritin or functional variants thereof and the use of said
sequences for rendering plants resistant against oxidative stress
conditions.
[0002] The invention is useful for reducing environmental damages
of crops caused by a wide variety of stress conditions.
[0003] With respect to the present specification and claims, we
will use the following technical terms in accordance with the given
definitions. With regard to the interpretation of the present
invention, it shall be understood that the below defined terms are
used in accordance with the given definitions even if said
definitions might not be in perfect harmony with the usual
interpretation of said technical term.
[0004] A "functional variant" of a protein is a polypeptide the
amino acid sequence of which can be derived from the amino acid
sequence of the original protein by the substitution, deletion
and/or addition of one or more amino acid residue in a way that, in
spite of the change in the amino acid sequence, the functional
variant retains at least a part of at least one of the biological
activities of the original protein that is detectable for a person
skilled in the art. A functional variant is generally at least 50%
homologous (i.e. the amino acid sequence of it is 50% identical),
advantageously at least 70% homologous and even more advantageously
at least 90% homologous to the protein from which it can be
derived. Any functional part of a protein or a variant thereof is
also termed functional variant.
[0005] The term "overproducing" is used herein in the most general
sense possible. A special type of molecule (usually a polypeptide
or an RNA) is said to be "overproduced" in a cell if it is produced
at a level significantly and detectably higher (e.g. 20% higher)
than natural level. Overproduction of a molecule in a cell can be
achieved via both traditional mutation and selection techniques and
genetic manipulation methods. The term "ectopic expression" is used
herein to designate a special realisation of overproduction in the
sense that, for example, an ectopically expressed protein is
produced at a spatial point of a plant where it is naturally not at
all (or not detectably) expressed, that is, said protein is
overproduced at said point.
[0006] A plant, plant part, a plant tissue or a plant cell is said
to have an enhanced resistance against a damaging effect, eg.
damaging agent, if it can tolerate a significantly and detectably
(e.g. at least 20%) stronger damaging effect, eg. dose or intensity
of damaging agent, of the same type, without suffering any
detectable damage, than its natural counterpart would do.
[0007] Within the framework of the present description a "ferritin
protein" is defined, as it is usual in the art, as a protein
capable binding iron ions (Theil E. C., 1987, Ann. Rev. Biochem.
56:289-315). The members of the eucariotic ferritin family are
highly conserved both in their amino acid sequence and three
dimensional structure (Lobraux, S. et al., 1992, Biochem. J.
288:931-939).
[0008] The term "oxidative stress" is again used in very general
sense comprising all kind of abiotic (e.g. treatment with different
chemical agents or exposure to extreme weather conditions like high
or low temperature or drought) and biotic (infection by different
infectious agents) stress conditions in the manifestation of
damaging effects of which oxidatively induced active radicals play
a detectable role.
[0009] During their different developmental stages plants are
exposed to an extremely wide range of both biotic and abiotic
stress conditions. It is, thus, a very important task of high
economic significance to develop new breeding stocks of enhanced
general stress resistance.
[0010] Under stress conditions such as high light intensity, UV-B
irradiation, heavy metal contamination, high or low temperature,
water deficiency, flooding, wounding, infection by viruses,
bacteria, fungi, damage caused by insects and the like, oxygen
toxicity can significantly contribute to the damage of crop plants.
Reactive oxygen species as singlet oxygen, superoxide radical
(O.sub.2), hydroxyl radical (OH.sup.+) and hydrogen peroxide
(H.sub.2O.sub.2) play a key role in injury of stressed plants.
There is good evidence that the biological damage attributed to
superoxide and hydrogen peroxide is dependent on the presence of
iron. The intracellular pool of free iron can react with
H.sub.2O.sub.2 or O.sub.2.sup.- giving rise to the very reactive
hydroxyl radical via Haber-Wiess or Fenton reaction (Halliwell and
Gutteridge, 1984, Biochem. J. 219:1-14). Intracellularly, most of
the non-metabolised iron is sequestered in ferritin; therefore
ferritin is able to restrict the availability of iron and so the
generation of the very reactive hydroxyl radicals. cDNAs encoding
ferritin have been isolated from variety of plant species. These
proteins are highly conserved both in amino acids sequence and
three dimension structure (Lobreoux S. et al. 1992, Biochem. J.
288:931-939.). Ferritins are localised in chloroplasts and iron can
activate their synthesis (Seckbach, J. 1982, J. Plant. Nutr.
5:369-394; Lobraux et al. 1992, Plant Mol. Biol. 19:563-575). Under
normal growth conditions ferritin is synthesised only in embryo and
not in vegetative organs, like roots and leaves (Lobraux and Briat:
Biochem. J. 1991, 274:601-606).
[0011] Significant antioxidant effect of ferritin molecules can be
expected in systems where ferritin synthesis and degradation is
released from the normal metabolic regulation. It has been
demonstrated that during oxidative stress conditions, degradation
of ferritin molecules occurs and the so released iron ions highly
accelerate the production rate of the damaging radical species
(Cairo et al. 1995, Journal of Biochemical Chemistry
270:700-703).
[0012] Numerous traditional plant breeding and genetic manipulation
approaches are known in the art for improving the resistance of
specific crops against preselected desired stress conditions (e.g.
against cold, drought, UV light or pathogens). These known methods,
however, will not be individually detailed herein as they are all
highly different from the approach of the invention. The common
feature of all these previously disclosed approaches is that they
enhance the resistance of different plants against a single
preselected stress condition (or against a limited groups of stress
conditions of the same origin) e.g. by expressing a specific
resistance gene. However, no specific approach providing plants
with resistance against a wide range of both abiotic and biotic
stress conditions is known in the art.
[0013] It is, thus, an object of the present invention to provide a
novel and general method suitable to provide crops, especially
transgenic crops, with enhanced resistance against a wide range of
both abiotic and biotic stress conditions.
[0014] According to another aspect, it is also an object of the
invention to provide crops and breeding material, advantageously
transgenic, having increased resistance against a wide range of
both abiotic and biotic stress conditions.
[0015] On the basis of the foregoing disclosure it has, thus,
became clear to the inventors that a substantially new genetic
manipulation approach is to be developed so as to achieve the above
defined objects, possibly targeting a common step in the damaging
mechanism of the different abiotic and biotic stress
conditions.
[0016] The approach of the present invention is, thus, based on the
novel theoretical concept that overproducing or ectopically
expressing ferritin or other iron binding proteins, e.g.
transferrins, in different organs of plants will lower the
intracellular iron concentration and, therefore, reduce the
damaging effects of oxygen induced free radicals. It is important
to emphasise hereby that the approach of the invention is
absolutely novel, as there is no method disclosed in the art that
would provide plants with resistance against a wide range of both
abiotic and biotic stress conditions. Furthermore, there is no
approach disclosed so far according to which a ferritin protein is
overproduced in plants for any reason in any manner.
[0017] Therefore, to achieve the above-defined objects of the
invention we have cloned a ferritin cDNA gene from alfalfa
(Medicago saliva L.) and overproduced it in vegetative tissues of
tobacco plants.
[0018] It was found, that in accordance with the basic concept of
the invention, the transgenic tobacco plants expressing the alfalfa
ferritin ectopically in their vegetative tissues show significantly
higher resistance towards both abiotic (treatment with different
chemicals) and biotic (viral, bacterial and fungal infections)
stress conditions than the starting tobacco plants and
transformants show improved general adaptation and regeneration
characteristics, as well.
[0019] It should be also emphasised that according to the basic
concept of the invention it is probable that overproduced ferritin
molecules express their general protective effect via binding free
iron ions in the plant cells, it can be assumed that by
overproducing other iron binding proteins, such as transferring, a
similar protective effect could be achieved.
[0020] The present invention, therefore, provides plant cells
overproducing an iron binding protein and having an enhanced
resistance against oxidative stress. The plants of the invention
are advantageously produced by genetic manipulation methods known
per se but can also be produced via traditional mutation and
selection techniques. The transformed plants of the invention show
significantly higher resistance to oxidative stress conditions than
their unmodified counterparts not expressing elevated levels of an
iron binding protein.
[0021] The plant cells according to the invention are
advantageously transgenic cells transformed by the introduction of
a nucleic acid, eg in the form of vector, coding for the expression
of an iron binding protein, advantageously ferritin.
[0022] According to preferred embodiments of the invention there
are provided plant cells of the invention are overproducing a
ferritin having the amino acid sequence of SEQ ID No 2 as shown in
the attached sequence listing, or a functional variant thereof,
said functional variant being advantageously at least 50%, more
advantageously at least 70% and even more advantageously at least
90% homologous to said ferritin polypeptide.
[0023] The invention further provides plants, advantageously
transgenic plants, and parts thereof comprising cells according to
the invention.
[0024] According to a preferred embodiment of the invention, the
intensity of the photosynthetic reactions is not decreased
following a 70 hours treatment with 10 .mu.M paraquat in the leaves
of the plants of the invention.
[0025] Plant, plant parts or plant cells according to the invention
advantageously have an enhanced resistance against fusaric acid
treatment and/or infections of viral and/or bacterial and/or fungal
origin.
[0026] The invention also provides isolated, enriched, cell free
and/or recombinant nucleic acids of sequence comprising or
consisting of a nucleotide sequence coding for an alfalfa ferritin
having the sequence of SEQ ID No: 2, as shown in the sequence
listing included herewith, and functional variants thereof.
Preferably the nucleic acids have homology of at least 90% with
that of SEQ ID No 1 (the DNA sequence in FIG. 1), more preferably
at least 95% and most preferably at least 98%. Preferably these
nucleic acids are cDNAs, recombinant vectors or other recombinant
constructs.
[0027] The invention also comprises the use of these nucleic acids
for the preparation of plant cells, plant parts and plants
according to the invention.
[0028] The present disclosure and examples below demonstrate that
synthesis of the iron binding protein ferritin in vegetative
tissues of plants provides resistance against paraquat generated
free radicals. In agreement with this observation we have also
demonstrated a significant reduction of the symptoms after
infection of the transgenic plants of the invention with a wide
range of unrelated pathogens. This novel technology according to
the present invention based on ectopic expression of an iron
binding protein, therefore, potentially has high agronomic
significance in that it may reduce primary oxidative damage in
crops caused by either biotic or abiotic stress conditions.
[0029] Though not wishing to be bound to any theoretical
interpretation, we think that the above findings support the
assumption that iron ions are more effectively bound in the plants
according to the invention as a consequence of the overproduction
of an iron binding protein (ferritin) and, therefore, damage caused
by oxygen induced free radicals are reduced and general adaptation
and regenerative properties of the plants of the invention are
improved. Plants according to the invention will also be able to
show resistance against other stress conditions not tested so far,
e.g. against extremely low or high temperatures and drought, where
such iron mediated degeneration is implicated.
[0030] Though it is considered to be quite clear from the above
disclosure for a person skilled in the art, we also wish to
emphasise here the universality of the approach according to the
invention. We have demonstrated that ectopical expression of an
iron binding protein of alfalfa origin in vegetative tissues
confers resistance to transgenic tobacco plants against a wide
variety of stress conditions. As the pioneering approach of the
invention is based on the absolutely general concept of reducing
the concentration of iron ions in the targeted tissues of plants, a
person skilled in the art will understand that the advantages of
the invention can not be restricted to the specific embodiments
shown but this novel approach for ensuring general stress
resistance to plants can be used in the case of any other crops of
agronomic or horticultural significance.
[0031] It is also demonstrated that the ectopic overexpression of
the embryo specific alfalfa ferritin in vegetative tissues of
tobacco plants, using two different types of promoters (one of
plant and the one of viral origin) is not at all damaging to the
targeted tissue. It should, thus, be contemplated that any type of
plant specific promoters (either constitutive or spatially and/or
developmentally regulated) can be used in the invention for the
overexpression of an iron binding protein in the desired plant part
or tissue.
[0032] As the concentration of harmful radicals is usually the
highest in the photosynthesising green tissues, these tissues are
the first useful targets of the approach according to the
invention. It should be understood, however, that, because of its
above demonstrated highly universal basic concept, the invention is
not at all limited to conferring stress resistance to green
tissues, but it is also useful for making resistant all other plant
parts of interest (e.g. root, stem, flower, fruit or specific parts
of the foregoing) supposedly exposed to any kind of oxidative
stress conditions (e.g. to infection by any type of pathogens).
FIGURES
[0033] FIG. 1.
[0034] The nucleotide sequence of ferritin cDNA isolated from
alfalfa (Medicago sativa) and the deduced amino acid sequence. The
underlined part corresponds to the transit peptide.
[0035] FIG. 2.
[0036] Comparison of ferritin amino acid sequences from different
species. HuHfer: human H subunit (Boyd et al. 1985, J. Biol. Chem.
260:11755) HoLfer: horse spleen L-chain (Heuterspreute and
Chrichton 1981, FEBS Lett. 129:322) Peafer: pea seed ferritin
(Lobreoux S. et al. 1992, J. Biochem. 288:931) Msfer1: alfalfa
ferritin (unpublished)
[0037] FIG. 3
[0038] Accumulation of ferritin mRNAs in vegetative tissues of
transgenic tobacco plants according to the invention.
[0039] FIG. 4.
[0040] Detection of ferritin in cell extract from leaves of
transgenic tobacco plants according to the invention after
SDS-PAGE.
[0041] FIG. 5.
[0042] Detection of ferritin in cell extract from leaves of
transgenic tobacco plants according to the invention by using FLAG
antibody in western blot analysis.
[0043] FIG. 6
[0044] Changes in fluorescence intensity (Fv/Fm) in leaf discs from
SR1 control and C3 transformed plants of the invention during
treatment with 10 .mu.M paraquat.
[0045] FIG. 7.
[0046] Changes in fluorescence intensity (Fv/Fm) in leaf discs from
SR1 control and various transformed plants according to the
invention after treatment with 10 and 20 .mu.M paraquat.
[0047] FIG. 8.
[0048] The chlorophyll content in leaves of control SR1 and
transformed plants of the invention after 3 day-treatment with 10
and 20 .mu.M paraquat.
[0049] Some preferred embodiments and the concept of the invention
will be further illustrated by way of the below experimental
examples. It shall be understood, however, that the examples below
are presented only for more comprehensive understanding of the
spirit of the invention and are no way illustrative or limiting as
to the scope of the invention, which is, in turn, defined in the
attached claims.
[0050] In the below examples, we demonstrate numerous advantages of
the of the invention as reduced to practice. We have isolated a
full length cDNA clone (MsFER1) encoding a novel alfalfa ferritin
polypeptide and used this cDNA for the expression of ferritin in
vegetative tissues of transgenic tobacco plants under the
transcriptional control of different promoters. After biochemically
characterising the ferritin expressing transgenic tobacco plants,
we have demonstrated that most of them have a significantly
enhanced resistance against different abiotic (treatment with
damaging chemical agents) and biotic (infections with viral,
bacterial and fungal pathogens) stress conditions of very different
origin.
EXAMPLE 1
[0051] Identifying a full length alfalfa ferritin cDNA (MsFER1)
clone and determining the nucleic acid and deduced amino acid
sequence thereof
[0052] For constructing a cDNA library and isolating the cDNA clone
coding for ferritin, the usual methods of recombinant DNA
technology were used as described in "Molecular cloning, A
Laboratory Manual" (2. edition, Cold Spring Harbour N.Y, 1989) by
Sambrook J., Fritsch, F. F. and Maniatis T.
[0053] Specifically, the alfalfa ferritin cDNA clone was isolated
as follows.
[0054] Total cellular RNA was isolated from in vitro cultured
alfalfa tissues primarily consisting of somatic embryos. Small
amounts of callus tissues were also present in the samples (Cathala
et al. 1983, DNA, 2:329-335). The formation of somatic embryos was
induced by applying auxin (2,4 dichlorophenoxyacetic acid) shock as
described by Dudits et al. (1991, J. Cell Science,
99:473-482.).
[0055] Then, mRNA was purified from the total cellular RNA isolate
by oligo-dT cellulose chomatography (Aviv H. and Leder P. 1972,
Proc. Natl. Acad. Sci. USA 69:1408-1412.).
[0056] First strand cDNA was then synthesised with AMV reverse
transcriptase in the presence of oligo dT primer. The second strand
was produced by DNA polymerase I. After RNase H treatment, cDNA was
annealed into a PstI digested pGEM2 vector (Promega) after dC and
dG homolinker addition with terminal transferase. The products were
transformed into E. coli MC 1061 strain and selected by 100 mg/l
ampicillin. 2.5.times.10.sup.5 primary transformants were produced
by using 25 .mu.g cDNA. Ninety-six percent of the clones comprised
a cDNA insert of significant size. The procedure is described in
detail by De Loose et al. (1988, Gene 70:13-23).
[0057] Fifty ESTs were then identified by randomly sequencing
preselected cDNA clones. Pharmacia T7 Sequencing Kit or USB
Sequenase 2.0 kit were used for sequencing according to the
instructions of the manufacturer. Preselection was done by Northern
hybridising the cDNA clones with an RNA probe prepared from somatic
embryos. Strongly hybridising clones were selected.
[0058] Sequence alignment based on GenBank and EMBL data bank
revealed one cDNA clone (designated as MsFER1) with high homology
to the known ferritins at the deduced amino acid sequence. FIG. 1
presents both nucleotide (SEQ ID NO: 1) and amino acid sequence
(SEQ ID NO: 2) of the MsFER1 clone. The cDNA insert comprised in
this clone is 1036 bp long and the coding region is found between
positions 40 and 789. The encoded protein is composed by 251 amino
acids and it shares high amino acid sequence identity with
ferritins of different origin. As shown in FIG. 2, plant ferritins
share 39-49% amino acid identity with the human H and the horse L
ferritins, while the sequences of the pea and the alfalfa ferritins
are very similar to each other (89% of amino acid identity) in the
mature protein. The two plant ferritins, however, significantly
differ in their regions corresponding to the chloroplasts leader
sequence. Here the identity is only 47% in the amino acid sequence.
Therefore, the newly described alfalfa ferritin can be considered
as a novel variant in the ferritin family.
EXAMPLE 2
[0059] Introduction of the alfalfa ferritin cDNA into tobacco
plants for ectopic expression of this protein in vegetative
tissues
[0060] The applied transformation technology is based on the
Agrobacterium gene delivery system reviewed by Hinchee et al.
"Plant Cell and Tissue Culture" pp. 231-270, eds. I. K. Vasil T. A
Thorpe, Kluwer Academic Publisher 1994. In the present Examples we
have used the system with vectors described by Pellegrineschi et
al. (1995, Biochemical Society Transitions 23:247-250).
[0061] The MsFER1 cDNA was cloned into the BamHI/KpnI sites of the
transformation vector where it was functionally attached to the
promoter of the small subunit gene of ribulose 1.5-bisphosphate
carboxylase. This promoter is originally described by Mauer and
Chui (1985, Nucleic Acid Res. 7:2373-2387) and is especially useful
for expression in the green tissues of plants. The transgenic
clones obtained after transforming tobacco plants named as A2, C3
and C8 carry this construct. Alternatively, the MsFER1 cDNA was
also linked to the viral promoter CaMV35S described by Benfey et
al. (1989, The EMBO J. 8:2195-2202). For cloning the fall length
construct we used the following PCR primer synthesised in a Per
Septives automated DNS synthesiser.
CCG AAT TCC ATG GCT CTT TCA GCT TCC (SEQ ID NO: 3)
[0062] Eco RI site coding region of the ferritin transit
peptide
[0063] We have also generated a truncated version of MsFER1 cDNA
that lacks a significant part of the targeting leader sequence (see
FIG. 1). In this cloning procedure we used the following PCR
primer:
GGG AAT TCC ATG GAT GGT GAT AAG AGG (SEQ ID NO: 4)
[0064] Eco RI site coding region within the transit peptide
[0065] The above mentioned oligonucleotides and T7 sequencing
primer (Boehringer Mannheim) were used for PCR amplification of the
defined DNA fragments (Mullis and Faloona 1987, Meth. Enzymol.
155:335).
[0066] The EcoRI/KpnI digested PCR products (the KpnI restriction
site originates from the T7 primer sequence) were cloned into the
pFLAG-ATS vector (Scientific Imaging System Kodak, USA) digested
with the same restriction enzymes. The presence of the FLAG
sequence in these constructs enables the immunological tracking of
the protein synthesis by using the Anti Flag M2 antibody
(Scientific Imaging System Kodak, USA).
[0067] For generating full FLAG FERR and partial (pt) FLAG FERR
constructs we synthesised the following Bam-Flag
oligonucleotide:
CGG ATC CAT GGA CTA CAA GGA CGA GGA (SEQ ID NO: 5)
[0068] BamHI site MET FLAG coding sequence
[0069] Using this primer and the C24 sequencing primer of the pFLAG
vector PCR products were produced and cloned into the
transformation vector at BamHI/KpnI sites.
[0070] The so constructed binary vectors were mobilised into an
Agrobacterium strain. Tobacco leaf discs were then co-cultured with
the Agrobacterium cells and transformants were selected on
kanamycin containing medium as described by Claes et al. (1991. The
Plant Journal 1:15-26). Primary transformants were self-pollinated
and T2 plants were further characterised.
EXAMPLE 3
[0071] Molecular evidence for ectopic synthesis of ferritin in
transgenic tobacco plants
[0072] Northern blot hybridisation was firstly used to show the
functional activity of the introduced constructs. Northern
hybridisation was done according to the widely used protocols
(Sambrook J., Fritsch F. F. and Maniatis T.: "Molecular cloning, A
Laboratory Manual" 2. edition, Cold Spring Harbour N.Y., 1989).
First, total RNA samples were isolated from the vegetative tissues
of the transformants (see Cathala et al. 1983, DNA 2:329-335).
After agarose gel electrophoresis in formaldehyde, RNA was
transferred onto Hybond-N filters (Amersham, Inc.). Radiolabelled
probes were generated by random-primed .sup.32P-labelling
(Freinberg A. P. and Vogelstein B. 1983. Anal. Biochem.
137:266-267). After 4-12 hours of prehybridisation in the presence
of formaldehyde, the hybridisation was carried out at 42.degree. C.
for 16-24 hours. Washing conditions were as follows: 65-72.degree.
C., 0.1.times.SSC, 0.1% SDS. As shown on FIG. 3, significant
amounts of ferritin mRNA accumulated in the transformed plants.
[0073] Synthesis of ferritin was analysed by biochemical methods
after partial purification as described by Laulhere and Laulhere
(1989, J. Biol. Chem. 264:3629-3635). After SDS-PAGE, the protein
profiles of the control SRI plants and the transformants (C3, A2,
C8) differed significantly. The later ones accumulated variable
amounts of ferritin having a molecular mass of 26 kDa (FIG. 4).
Using the FLAG detection system we could also show the synthesis of
ferritin by chemiluminescence (Super Sigfnal.sup.RM-CL-HRP System,
Pierce) in several transgenic tobacco plants (FIG. 5). The
presented molecular data convincingly show the lack of ferritin in
the control plants and accumulation of this protein in vegetative
tissues of the transformants.
EXAMPLE 4
[0074] Ectopic synthesis of ferritin provides oxidative stress
resistance for transgenic plants
[0075] To test the basic concept of this invention we have analysed
the paraquat (Pq) resistance of control and transformed tobacco
plants. It is well documented that electrons produced during
photosynthetic electron transport reduce Pq and free radicals are
formed (Ashton F. M. and Crafts A. S. 1981, Mode of action of
herbicides, Wiley, New York). Leaf discs from control and
transformed plants were exposed to 10 or 20 .mu.M Pq.
[0076] Functional damage was monitored by measuring the light
activated fluorescence with PAM fluorimeter as described by Vass I.
et al. (1996. Biochemistry, 35:8964-8973). As an example, FIG. 6
shows the loss of photosynthetic function in the control SR1 leaves
after 60 hours of 10 .mu.M Pq treatment, while the C3 transformant
retained its photosynthetic activity during the period analysed.
Similar responses can be observed after Pq treatment of two other
transformants (Full9, Pt2) in which the ferritin gene is expressed
under the control of the CaMV35S promoter. These experiments were
repeated several times (see also FIG. 7) and the functional
characterisation convincingly proved the paraquat-resistance in the
case of the transformants. This resistance can also be seen when
the changes in chlorophyll contents are monitored (FIG. 8).
[0077] Using Pq as the inducer of free radicals, the presented
results support the conclusion that transformed plants synthesising
ferritin in their vegetative tissues express elevated tolerance
against damage caused by free radicals. Here we have to mention
that the analysed transformed plants lack any visible alterations
during their growth under greenhouse conditions. The photosynthetic
function of transformants is similar to the control SR1 plants .
Under control conditions these plants exhibit normal chlorophyll
content (FIG. 8).
EXAMPLE 5
[0078] Transformed plants overproducing ferritin have enhanced
resistance against fusaric acid treatment
[0079] The involvement of free radicals in pathogenicity has been
proposed by K. E. Hammond-Kosack and J. D. G. Jones (1996, The
Plant Cell 8:1773-1791). Therefore, we have tested the development
of symptoms on transformants expressing the alfalfa ferritin gene
in green tissues. First, we analysed the effects of fusaric acid as
a non-specific fungal toxin. Fusaric acid in different
concentrations was injected into control (SR1) and transgenic (A2,
C3 and C8) tobacco leaves. 2 days after injection we analysed the
degree of necrotization on leaf tissues. Table 1 summarises the
results of 3 experiments.
1TABLE 1 Reduced necrotization in transformed plants expressing
ferritin after fusaric acid treatment 2.5 .times. 10.sup.-3 M 1.25
.times. 10.sup.-3 M 2.5 10.sup.-3 M SR1 XXX (100%) XX(X) (73%) XXX
(100%) A2 X (28%) -(X) (16.5%) X(X) (50%) C3 X (65%) -(X) (16.5%)
X(X) (50%) C8 X (70%) X(X) (50%) XX (73.5%) -no necrotization; XXX
= severe necrotization (or % of necrotization of the injected leaf
area)
[0080] The presented data show considerable reduction of
necrotization on the leaves of the transformed plants. These
results also promise enhanced resistance against damages caused by
fungal infections.
EXAMPLE 6
[0081] Transformed plants overproducing ferritin have enhanced
resistance against Pseudomonas infection
[0082] Along the same concept, we have injected Pseudomonas
syringae bacterium suspension (10.sup.7/ml) into the leaves of
tobacco plants. After 20 hours, the degree of necrotization (HR)
were analysed. Table 2. summarises the results indicating reduced
necrosis in two transgenic lines (A2, C8).
2TABLE 2 The degree of necrotization after bacterial infection Exp.
A Exp. B SR1 100% 100% full necrosis A2 20% 30% C3 100% 100% C8 30%
35%
[0083] The above data demonstrates that at least two of the
transformants (A2 and C8) have a significantly enhanced resistance
against the damages caused by bacterial infection.
EXAMPLE 7
[0084] Transformed plants overproducing ferritin have enhanced
resistance against Botrytis and Alternaria fungal infection
[0085] We have tested the response of transgenic lines according to
Ex. 2 against two fungal pathogens such as Alternaria alternata and
Botrytis cinerea. Leaves were detached and inoculated with agar
block (5 mm diam) from the fungal culture. Table 3 summarises the
sizes of necrotised leaf areas of the treated control and
transformant plants.
3TABLE 3 Effect of fungal pathogens on transformed plants
Alternaria alternata Botrytis cinerea Tobacco mm.sup.2 % mm.sup.2 %
SR1 723.4 .+-. 224.0 100.0 516.9 .+-. 118.4 100.0 A2 204.1 .+-.
50.2 28.2 278.3 .+-. 101.6 53.8 C3 286.5 .+-. 59.6 39.6 56.9 .+-.
6.7 11.0 C8 198.2 .+-. 85.2 27.4 290.5 .+-. 89.5 56.2 PT1 543.2
.+-. 163.2 75.1 497.7 .+-. 117.7 96.3 PT2 407.0 .+-. 123.6 56.3
334.8 .+-. 80.4 64.8 PT5 520.1 .+-. 140.1 71.9 257.5 .+-. 56.5 49.8
Full4 268.9 .+-. 14.5 37.2 317.1 .+-. 62.8 61.3 Full5 435.0 .+-.
55.3 60.1 476.1 .+-. 96.1 92.1 Full9 113.4 .+-. 63.0 15.7 278.3
.+-. 101.6 53.8
[0086] From the above data, one can conclude that the transformed
tobacco plants--comprising either the full or the truncated version
of the ferritin gene--expressing ferritin in their leaves show
significantly reduced symptoms (though in variable degree) in
comparison with control plants.
EXAMPLE 8
[0087] Transformed plants overproducing ferritin have enhanced
resistance against tobacco necrotic virus (TNV) infection
[0088] In the infection of plants with viruses oxygen generation
plays an essential role in pathogenicity. Therefore we have tested
the responses of the transformed and control (SR1) plant against
tobacco necrotic virus (TNV) infection (Table 4).
4TABLE 4 Reduction of number and area of lesion in transformed
tobacco plants after TNV infection Experiment 1 SR1 (control) 17.4
.+-. 2.5 (total lesion, cm.sup.2) C3 (transformant) 6.4 .+-. 1.4 C8
(transformant) 6.5 .+-. 1.2 A2 (transformant) 6.1 .+-. 1.7
Experiment 2 SR1 (control) 50.0 .+-. 10.0 (number of lesions) 35.8
.+-. 21.7 Pt1 (transformant) 25.8 .+-. 7.3 Pt2 (transformant) 12.5
.+-. 8.4 Pt5 (transformant) 18.7 .+-. 12.3 Pt9 (transformant) 38.3
.+-. 8.6 Full4 (transformant) 45.0 .+-. 19.1 Full5 (transformant)
32.0 .+-. 16.8 Full6 (transformant) 46.2 .+-. 6.1 Full9
(transformant) 21.7 .+-. 13.7
[0089] As shown in Table 4, significant reduction in number of
lesions can be recognised in transformed tobacco lines carrying the
alfalfa ferritin gene under the control of promoter from small
subunit of RUBISCO (C3, C8 and A2). It might have a functional
significance that among the transformed lines expressing the
ferritin gene under the control of the CaMV35S promoter, two lines
expressing the truncated ferritin gene showed the most significant
reduction in the symptoms.
Sequence CWU 0
0
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