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CA 02600560 2007-09-11
DESCRIPTION
Method for producing plant forming nodules with high nitrogen-fixing activity
Technical Field
The present invention relates to a method for producing a nodulating plant
that
forms nodules with high nitrogen-fixing activity.
Background Art
Leguminous crops such as Glycine max (soybean), Phaseolus angularis (azuki
bean), and Phaseolus vulgaris (kidney bean) form nodules (nodulation), which
are
symbiotic organs, through infection with root nodule bacteria. Bacteroids of
symbiotic
root nodule bacteria within such nodules fix atmospheric nitrogen, so that the
plants can
grow well even in soil with low nitrogen content. Hence, when a leguminous
crop is
cultivated, the seeds of the crop are often treated with pre-cultured root
nodule bacteria
in addition to an application of a general fertilizer. To further enhance the
nitrogen-
fixing ability of the nodules of leguminous crops, root nodule bacteria with
enhanced
nitrogen-fixing ability have also been developed (JP Patent Publication
(Kokai) No.
2003-33174 A).
In addition to leguminous crops, it is known that symbiotic nitrogen-fixing
bacteria can invade and proliferate in the roots of leguminous trees such as
the genus
Acacia and Albizia julibrissin and nonleguminous trees such as Alnus japonica
and
Alnus firma, form nodules, efficiently fix atmospheric nitrogen, and thus
supply nitrogen
to the host trees. These nodulating trees have high nitrogen concentrations in
their
living leaves and thus have high nitrogen concentrations in fallen leaves.
Accordingly,
nodulating trees are effective in increasing microbial levels in soil and
improving the
quality of the soil so as to fertile the soil. Thus, such trees are also used
as the so-
called soil-improving trees for greening waste land.
Enhancement of the nitrogen-fixing ability of nodules of such plants is
thought
to be very useful in terms not only of agriculture but also of environmental
conservation.
Meanwhile, it is known that in the nodule cells of leguminous plants,
symbiotic
globin genes that are unique in leguminous plants are very strongly expressed.
Symbiotic hemoglobin (also referred to as leghemoglobin) is composed of a
globin that
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is a gene product of such a symbiotic globin gene and heme, and it is said to
account for
20% to 30% of all the soluble proteins in the nodules. But the symbiotic
hemoglobin is
completely absent in tissues other than the nodules. Symbiotic hemoglobin has
strong
affinity for oxygen and the like, as with hemoglobin in animal blood. The
symbiotic
hemoglobin is believed to serve a function of regulating the oxygen partial
pressure
within nodule cells at a level that is sufficient for respiration of root
nodule bacteria
within nodules and that prevents nitrogenase from being inactivated since the
nitrogenase is required for the nitrogen-fixing ability of root nodule
bacteria, but can be
inactivated by oxygen.
With the recent development of gene analysis technology, it has been revealed
that plants other than leguminous plants also have globin genes. Such globin
genes
discovered in plants other than leguminous plants differ from the symbiotic
globin genes
of leguminous plants. Thus such globin genes have been termed "nonsymbiotic
globin
genes" (also referred to as nonsymbiotic hemoglobin genes). It is currently
believed
that all plants have nonsymbiotic globin genes. In other words, leguminous
plants have
both symbiotic globin genes and nonsymbiotic globin genes, but non-leguminous
plants
have nonsymbiotic globin genes alone. A nonsymbiotic globin gene of Lotus
japonicus
that is a model plant belonging to the family Leguminoseae has also been
reported
(Uchiumi et al., Plant Cell Physiol. (2002) 43(11): pp. 1351-1358).
Unlike symbiotic globin genes, nonsymbiotic globin genes are known to be
expressed in all plant tissues. It has been reported that when a plant is
exposed to a
low temperature (4 C) or low-oxygen partial pressure (at oxygen concentration
of 5% or
less), the expression level of its nonsymbiotic globin gene is increased. It
has also
been reported that resistance against low oxygen stress has been enhanced in
Arabidopsis thaliana into which a nonsymbiotic globin gene has been introduced
and
overexpressed (Hunt, P.W., et al., Proc. Natl. Acad. Sci. (2002) U.S.A. 99:
pp. 17197-
17202).
Disclosure of the Invention
An object of the present invention is to provide a method for producing a
nodulating plant that forms nodules having enhanced nitrogen-fixing activity,
and a
nodulating plant that exhibits high nitrogen-fixing activity of its nodules.
As a result of intensive studies to achieve the above object, the present
inventors
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have discovered that Lotus japonicus into which a Lotus japonicus nonsymbiotic
globin gene has been introduced and overexpressed, and Lotus japonicus into
which
an Alnus firma nonsymbiotic globin gene has been introduced and overexpressed
can form nodules having high nitrogen-fixing activity. The present inventors
have
completed the present invention based on such findings.
The present invention encompasses the following.
[1] A method for producing a nodulating plant capable of forming nodules
having
enhanced nitrogen-fixing activity, comprising causing overexpression of a
nonsymbiotic globin gene in a nodulating plant.
More preferably, the nonsymbiotic globin gene in this method comprises
a DNA selected from the group consisting of the following (a) to (e):
(a) a DNA comprising the nucleotide sequence shown in SEQ ID NO: 1
01 8;
(b) a DNA which hybridizes under stringent conditions to a DNA
comprising a nucleotide sequence complementary to the nucleotide sequence
shown
in SEQ ID NO: 1 or 8 and encodes a protein having nonsymbiotic globin
activity;
(c) a DNA encoding a protein comprising the amino acid sequence
shown in SEQ ID NO: 2 or 9;
(d) a DNA encoding a protein that comprises an amino acid sequence
derived from the amino acid sequence shown in SEQ ID NO: 2 or 9 by deletion,
substitution, or addition of 1 to 50 amino acids and has nonsymbiotic globin
activity;
and
(e) a DNA comprising the nucleotide sequence shown in SEQ ID NO: 3
or 10.
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In this method, preferably, the overexpression of the nonsymbiotic
globin gene is caused by introducing the nonsymbiotic globin gene ligated to
an
overexpression promoter into a nodulating plant.
In a particular embodiment, the invention relates to a method for
producing a nodulating plant capable of forming nodules having enhanced
nitrogen-
fixing activity, comprising: causing overexpression of a nonsymbiotic globin
gene in a
nodulating plant by introducing the nonsymbiotic globin gene ligated to an
overexpression promoter into the nodulating plant; inoculating the nodulating
plant
which has overexpressed the nonsymbiotic globin gene with a symbiotic nitrogen-
fixing bacterium to form nodules; and screening for a plant having the
enhanced
nitrogen-fixing activity in the nodules formed, wherein said nonsymbiotic
globin gene
comprises a DNA selected from the group consisting of the following (a) to
(d): (a) a
DNA comprising the nucleotide sequence shown in SEQ ID NO: 1 or 8; (b) a DNA
encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 2 or
9;
(c) a DNA encoding a protein that comprises an amino acid sequence derived
from
the amino acid sequence shown in SEQ ID NO: 2 or 9 by deletion, substitution,
or
addition of 1 to 10 amino acids and that has nonsymbiotic globin activity; and
(d) a
DNA comprising the nucleotide sequence shown in SEQ ID NO: 3 or 10.
According to this method, preferably, a nodulating plant capable of
forming nodules having an enhanced nitrogen-fixing activity at least 3 times
greater
than that of a wild-type strain can be produced. Such nodulating plant that is
produced according to this method can form nodules having enhanced nitrogen-
fixing
activity, preferably nodules that exhibit nitrogen-fixing activity of 10
nM/min/g to
100 nM/min/g.
The nodulating plant in which the overexpression of the nonsymbiotic
globin gene is caused in this method is preferably a leguminous plant. More
preferably, according to this method, the nodulating plant which has
overexpressed
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the nonsymbiotic globin gene is further inoculated with a symbiotic nitrogen-
fixing
bacterium.
[2] A nodulating plant which is produced according to the method of
[1] above, wherein the nodulating plant is capable of forming nodules having
enhanced nitrogen-fixing activity. Such nodulating plant more preferably has
nodules
on its roots.
[3] A vector for enhancing the nitrogen-fixing activity of nodules,
comprising
a nonsymbiotic globin gene ligated to an overexpression promoter.
The nonsymbiotic globin gene to be contained in the vector is preferably
derived from a leguminous plant or a nonleguminous nodulating plant. The
nonsymbiotic globin gene comprises more preferably the DNA described in (a) to
(e)
of [1] above.
[4] A method for enhancing nitrogen-fixing efficiency upon plant
cultivation,
comprising cultivating the nodulating plant of [2] above.
[5] Use of a nonsymbiotic globin gene for producing a transformed
nodulating plant capable of forming nodules having enhanced nitrogen-fixing
activity,
wherein said nonsymbiotic globin gene comprises a DNA selected from the group
consisting of the following (a) to (d): (a) a DNA comprising the nucleotide
sequence
shown in SEQ ID NO: 1 or 8; (b) a DNA encoding a protein comprising the amino
acid sequence shown in SEQ ID NO: 2 or 9; (c) a DNA encoding a protein that
comprises an amino acid sequence derived from the amino acid sequence shown in
SEQ ID NO: 2 or 9 by deletion, substitution, or addition of 1 to 10 amino
acids and
that has nonsymbiotic globin activity; and (d) a DNA comprising the nucleotide
sequence shown in SEQ ID NO: 3 or 10.
According to the method for producing a nodulating plant of the present
invention, the nitrogen-fixing activity of nodules of a nodulating plant of
interest can
be markedly improved. The vector of the present invention for enhancing the
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nitrogen-fixing activity of nodules can, when it is used in such production
method,
contribute to significant enhancement of the nitrogen-fixing activity of the
nodules.
Furthermore, a nodulating plant obtainable according to the present invention
is
capable of forming nodules having high nitrogen-fixing activity, resulting in
promoted
growth of the plant and increased nitrogen content. The present invention also
relates to a method for increasing the nitrogen fixation level upon plant
cultivation
through cultivation of the nodulating plant, and the method makes it possible
not only
to increase the amount of nitrogen to be fixed from atmosphere in a given
environment and to increase the yield or the growth amount of the relevant
nodulating
plant under such environment, but also to increase the amount of nitrogen in
soil and
thus fertilize the soil.
This description includes the disclosure of the description and drawings
of Japanese Patent Application No. 2005-071677, from which the present
application
claims priority.
Brief Description of the Drawings
Fig. 1 shows the genomic structure and the amino acid sequence of
Lotus
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CA 02600560 2007-09-11
japonicus nonsymbiotic globin gene.
Fig. 2 shows expression levels of a nonsymbiotic globin gene in wild-type
Lotus
japonicus. Fig. 2A shows the expression levels in different tissues. Fig. 2B
shows the
expression levels under stress conditions.
Fig. 3 shows the structure of a transformation vector used for transformation
of
hairy roots.
Fig. 4 shows photographs showing experimental results of confirming the
introduction of nonsymbiotic globin gene in transformants. Fig. 4A shows hairy
root
induction in transformants.
Fig. 4B shows GFP fluorescence as observed in
transformant roots. Fig. 4C shows the expression of the transgenes as
confirmed by
RT-PCR.
Fig. 5 shows photographs showing nodules formed on transformed hairy roots.
Fig. 5A shows Lotus japonicus hairy roots (control roots) obtained as a result
of
introduction of a vector not containing an LjHbl gene. Fig. 5B shows hairy
roots into
which the LjHbl gene has been introduced and overexpressed. White arrowhead
marks
indicate nodules formed on transformed hairy roots. Mesh arrowhead marks
indicate
nodules formed on non-transformed hairy roots. It is shown that only the
transformed
nodules emitted fluorescence.
Fig. 6 shows ethylene amount data measured by gas chromatography, showing
the nitrogen-fixing activity (ARA activity) levels of nodules formed on
transformed
hairy roots.
Fig. 7 is a schematic diagram showing a vector used for expression of Lotus
japonicus globin proteins in Escherichia coli.
Fig. 8 shows absorbance spectra showing affinity of Lotus japonicus globin
proteins (symbiotic [left graph, Fig. 8A] and nonsymbiotic [right graph, Fig.
8B])
expressed in Escherichia coil, for nitrogen monoxide. Each line denotes data
obtained
via measurement at different times for mixing of the globin proteins with
nitrogen
monoxide.
Fig. 9 shows photographs showing the expression of AfHbl transgene in Lotus
japonicus transformants as confirmed by RT-PCR.
Fig. 10 shows nitrogen-fixing activity (ARA activity) levels in whole
transformed plants (Lotus japonicus) into which AfHbl has been introduced and
the
same in the thus formed nodules.
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Best Mode for Carrying Out the Invention
Hereinafter, the present invention will be described in detail.
1. Nodulating plant
According to the present invention, a nodulating plant capable of forming
nodules having enhanced nitrogen-fixing activity is produced by causing
overexpression
of a nonsymbiotic globin gene in a nodulating plant. In the Description,
"nodulating
plant" means a plant of a species that is capable of nodulating or forming
nodules.
Nodules are symbiotic organs with granular structures formed due to the
inhabitation of
symbiotic nitrogen-fixing bacteria in the roots of nodulating plants. Examples
of such
nodulating plants include not only leguminous plants (including leguminous
crops and
leguminous trees), but also some nonleguminous plants (mainly nonleguminous
trees)
such as members of the family Betulaceae or the family Alnus, which are
generically
named actinorhizal plants. Root nodule bacteria live symbiotically in the
roots of a
leguminous plant as symbiotic nitrogen-fixing bacteria, forming nodules. In
the case
of some nonleguminous plants mentioned above, Actinomycetes live symbiotically
as
symbiotic nitrogen-fixing bacteria in the roots, forming nodules. Examples of
nodulating plants useful in the present invention include, when they are
leguminous
plants, Lotus japonicus, Glycine max (soybean), Phaseolus angularis (azuki
bean),
Phaseolus vulgaris (kidney bean), Pisum sativum (pea), Vicia faba, Arachis
hypogaea,
Medicago sativa (alfalfa), Medicago truncatula, the genus Trifolium (clover),
Vigna
sinensis, Lens esculenta (lentil), Rob ma pseudoacacia, Cytisus scoparius, the
genus
Lespedeza, Sophora japonica, and Paraserianthes Falcataria. Examples of
nodulating
plants useful in the present invention include, when they are non-leguminous
plants,
Alnus firma, Alnus japonica, Myrica rubra, Casuarina stricta, Coriaria
japonica, and
the genus Elaeagnus.
In the present invention, nodulating plants may be either plant bodies with
nodules or plant bodies with no nodules at a time point at which a
nonsymbiotic globin
gene is introduced, at which the plant body is regenerated, or any other time
points, as
long as they are plants of biological species capable of nodulating or forming
nodules.
In the Description, the term "nodulating plant" refers not only to nodulating
plant bodies (whole plants), but also plant organs (e.g., leaves, petals,
stems, roots,
seeds, hypocotyls, and cotyledons), plant tissues (e.g., epidermis, phloem,
parenchyma,
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CA 02600560 2007-09-11
xylem, vascular bundles, palisade tissues, and spongy tissues), and any
portions of plant
bodies such as cultured plant cells (e.g., calli), for example.
2. Nonsymbiotic globin gene and obtainment thereof
The nonsymbiotic globin gene according to the present invention encodes a
globin protein that becomes associated with heme (a complex salt of porphyrin
and
ferrous ion) to form nonsymbiotic hemoglobin. As with animal hemoglobin,
nonsymbiotic hemoglobin is a protein having strong affinity for oxygen, carbon
dioxide,
carbon monoxide, or the like. The nonsymbiotic hemoglobin has stronger
affinity for
oxygen, nitrogen monoxide, or the like than symbiotic hemoglobin. In the
Description,
the activity of globin encoded by such nonsymbiotic globin gene is referred to
as
nonsymbiotic globin activity.
The nonsymbiotic globin gene according to the present invention can be
isolated
from any plant. The nonsymbiotic globin gene of the present invention is more
preferably derived from a nodulating plant. The nonsymbiotic globin gene may
be
isolated from a leguminous plant such as Lotus japonicus or Glycine max
(soybean) or
may be isolated from a nonleguminous nodulating plant such as Alnus firma.
Such
nonsymbiotic globin gene to be used in the present invention may also be
isolated from
a plant other than a nodulating plant, such as a monocotyledon (e.g., Hordeum
vulgare
(barley), Oryza sativa (rice), or Zea mays (corn)). Examples of a known
nonsymbiotic
globin gene that can be used in the present invention are as follows: Lotus
japonicus
(Uchiumi et al., Plant Cell Physiol. (2002) 43(11): pp. 1351-1358), Alnus
firma
(DDBREMBL/GenBank accession No. AB221344), Oryza sativa (rice)
(DDBREMBL/GenBank accession No. U76030), Medicago sativa (alfalfa)
(DDBREMBL/GenBank accession No. AF172172; Serogelyes et al., FEBS Lett. (2000)
482, pp. 125-130), Glycine max (soybean) (DDBREMBL/GenBank accession No.
U47143; Anderson, et al., Proc. Natl. Acad. Sci. U.S.A. (1996) 93(12), pp.
5682-5687),
and Arabidopsis thaliana (DDBREMBL/GenBank accession No. U94998; Trevaskis et
al., PNAS (1997) 94 pp. 12230-12234).
The nonsymbiotic globin gene to be used in the present invention may be a
cDNA or a genomic DNA containing exons and introns. Examples of a "gene" used
herein include a DNA and an RNA. Examples of such DNA include at least a
genomic
DNA, a cDNA, and a synthetic DNA. Examples of such RNA include an mRNA and
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the like. The "gene" used herein may also contain an untranslated region (UTR)
sequence in addition to a coding sequence.
In a non-limiting preferred embodiment, a Lotus japonicus nonsymbiotic globin
gene can be used as the nonsymbiotic globin gene derived from a leguminous
plant
according to the present invention. For example, a DNA comprising the
nucleotide
sequence of SEQ ID NO: 1 isolated from Lotus japonicus or a DNA of a genomic
fragment comprising the nucleotide sequence of SEQ ID NO: 3 isolated from
Lotus
japonicus can be appropriately used as the nonsymbiotic globin gene of the
present
invention. Furthermore, a DNA encoding a Lotus japonicus nonsymbiotic globin
protein that comprises the amino acid sequence of SEQ ID NO: 2 can also be
advantageously used.
As the nonsymbiotic globin gene of the present invention, a DNA encoding a
protein that comprises an amino acid sequence derived from the amino acid
sequence
shown in SEQ ID NO: 2 by deletion, substitution, or addition of 1 to 50,
preferably 1 to
35, more preferably 1 or several (e.g., 2 to 10) amino acids may also be used,
as long as
it has nonsymbiotic globin activity. Furthermore, such nonsymbiotic globin
gene to be
used in the present invention may also be a DNA that hybridizes under
stringent
conditions to a DNA comprising a nucleotide sequence complementary to the
nucleotide
sequence shown in SEQ ID NO: 1 or to a DNA comprising a nucleotide sequence
complementary to the nucleotide sequence of a DNA encoding the nonsymbiotic
globin
protein comprising the amino acid sequence of SEQ ID NO: 2 and that encodes a
protein
that has nonsymbiotic globin activity. The nonsymbiotic globin gene of the
present
invention may also be a DNA encoding a protein that comprises an amino acid
sequence
having at least 80% identity with the amino acid sequence of SEQ ID NO: 2 and
that has
nonsymbiotic globin activity.
In another embodiment, an Alnus firma nonsymbiotic globin gene can be
appropriately used as the nonsymbiotic globin gene derived from a
nonleguminous
nodulating plant according to the present invention, but examples are not
limited thereto.
For example, as the nonsymbiotic globin gene of the present invention, a DNA
comprising the nucleotide sequence of SEQ ID NO: 8 isolated from Alnus firma
or a
DNA of a genomic fragment comprising the nucleotide sequence of SEQ ID NO: 10
isolated from Alnus firma can be appropriately used. Moreover, a DNA encoding
an
Alnus firma nonsymbiotic globin protein comprising the amino acid sequence of
SEQ ID
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NO: 9 can also be advantageously used.
As the nonsymbiotic globin gene of the present invention, a DNA encoding a
protein that comprises an amino acid sequence derived from the amino acid
sequence
shown in SEQ ID NO: 9 by deletion, substitution, or addition of 1 to 50,
preferably 1 to
35, and more preferably 1 or several (e.g., 2 to 10) amino acids may also be
used, as
long as it has nonsymbiotic globin activity. Such nonsymbiotic globin gene to
be used
in the present invention may be a DNA that hybridizes under stringent
conditions to a
DNA comprising a nucleotide sequence complementary to the nucleotide sequence
shown in SEQ ID NO: 8 or to a DNA comprising a nucleotide sequence
complementary
to the nucleotide sequence of a DNA encoding the nonsymbiotic globin protein
comprising the amino acid sequence of SEQ ID NO: 9 and that encodes a protein
that
has nonsymbiotic globin activity. The nonsymbiotic globin gene of the present
invention may also be a DNA encoding a protein that comprises an amino acid
sequence
having at least 80% identity with the amino acid sequence of SEQ ID NO: 9 and
that has
nonsymbiotic globin activity.
In the present invention, the term "stringent conditions" refers to conditions
under which namely a specific hybrid is formed. Under examples of such
conditions,
nucleic acids sharing high homology, and specifically, DNAs having 90% or more
and
preferably 95% or more homology, hybridize to each other, but nucleic acids
sharing
homology lower than such homology do not hybridize to each other. More
specifically,
such stringent conditions refer to conditions in which the sodium salt
concentration
ranges from 15 mM to 750 mM, preferably 50 mM to 750 mM, and more preferably
300
mM to 750 mM, the temperature ranges from 25 C to 70 C, and preferably 50 C to
70 C, and more preferably 55 C to 65 C, and the formamide concentration ranges
from
0% to 50%, preferably 20% to 50%, and more preferably 35% to 45%. Furthermore,
under stringent conditions, post-hybridization filter-washing conditions
generally are
conditions in which the sodium salt concentration ranges from 15 mM to 600 mM,
preferably 50 mM to 600 mM, and more preferably 300 mM to 600 mM, and the
temperature ranges from 50 C to 70 C, preferably 55 C to 70 C, and more
preferably
60 C to 65 C.
In a non-limiting embodiment of the present invention, the nonsymbiotic globin
activity of a globin protein encoded by a nonsymbiotic globin gene is
represented by the
affinity of the nonsymbiotic hemoglobin comprising such globin protein for
oxygen,
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carbon dioxide, nitrogen monoxide, or the like. The nonsymbiotic globin
activity of
the present invention is determined as described below, for example. A globin
protein
is recombinantly produced using an expression vector containing the
nonsymbiotic
globin gene encoding the relevant nonsymbiotic globin and then nonsymbiotic
hemoglobin is reconstructed using the thus obtained globin protein and heme.
The
affinity of the nonsymbiotic hemoglobin for oxygen, carbon dioxide, nitrogen
monoxide,
or the like is measured by a conventional technique and thus the measured
value can be
used as an indicator of the nonsymbiotic globin activity.
The affinity of the
nonsymbiotic hemoglobin for oxygen, carbon dioxide, or nitrogen monoxide can
be
measured by methods known by persons skilled in the art.
In an example, the affinity of such nonsymbiotic hemoglobin for nitrogen
monoxide can be determined based on the absorbance spectrum obtained by
measuring
absorbance at a wavelength between 500 nm and 600 nm in the presence of
nitrogen
monoxide. In general, when an absorbance curve is plotted for hemoglobin using
light
with a wavelength between 500 nm and 600 nm, two characteristic peaks are
observed at
540 nm and 575 nm (see Fig. 8). These 2 peaks disappear in the case of
hemoglobin
observed after the completion of the reaction with nitrogen monoxide. Hence,
the
affinity of a hemoglobin protein for nitrogen monoxide can be measured by
mixing the
hemoglobin protein with nitrogen monoxide and then analyzing the absorbance
spectrum
according to a conventional technique. Generally, isolated and/or purified
hemoglobin
is present in a form that is bound with oxygen (referred to as oxyhemoglobin).
When
recombinantly produced for example, nonsymbiotic hemoglobin is also isolated
in a
form that is bound with oxygen. The nonsymbiotic hemoglobin to be used herein
has
higher affinity for nitrogen monoxide than for oxygen. Therefore, when
nitrogen
monoxide is added to isolated nonsymbiotic hemoglobin, two peaks as observed
in the
absorbance spectrum disappear and then a shift to the spectrum obtained after
completion of the reaction with nitrogen monoxide is observed. Regarding such
method for measuring the affinity of hemoglobin for nitrogen monoxide, Michele
Perazzoli et al., The Plant Cell (2004) 16, pp. 2785-2794 can be referred to.
Through comparison analysis of the absorbance spectrum measured as described
above between nonsymbiotic hemoglobin and symbiotic hemoglobin, their
affinities for
nitrogen monoxide can be relatively compared. Comparison of the absorbance
spectra
reveals that nonsymbiotic hemoglobin can bind to nitrogen monoxide more
rapidly than
CA 02600560 2007-09-11
symbiotic hemoglobin.
In the present invention, the affinity of nonsymbiotic
hemoglobin for nitrogen monoxide can be determined using as an indicator the
time
required for mixing with nitrogen monoxide until the binding of nonsymbiotic
hemoglobin with nitrogen monoxide is observed in the absorbance spectrum.
In the present invention, nonsymbiotic globin may be recombinantly produced
in Escherichia coli using a vector containing a gene encoding such
nonsymbiotic globin
(SEQ ID NO: 2 or 9), and then nonsymbiotic hemoglobin that is reconstructed
with the
nonsymbiotic globin and heme in the Escherichia colt may be collected. The
affinity
of the nonsymbiotic hemoglobin for nitrogen monoxide can then be measured by
the
above-described method, for example. Preferably, the nonsymbiotic globin of
the
present invention has the same degree of affinity for nitrogen monoxide as
that of
nonsymbiotic hemoglobin comprising such nonsymbiotic globin (SEQ ID NO: 2 or
9),
but the examples are not limited thereto.
The nonsymbiotic globin gene according to the present invention as described
above can be obtained as a nucleic acid fragment by performing PCR
amplification
using primers designed based on the sequences of SEQ ID NOs: 1 to 3 and 8 to
10, for
example, and a nucleic acid derived from any plant (e.g., a nodulating plant
such as
Lotus japonicus or Alnus firma) as a template. Furthermore, the nonsymbiotic
globin
gene of the present invention can be obtained as a nucleic acid fragment by
performing
hybridization using a nucleic acid derived from any plant (e.g., a nodulating
plant such
as Lotus japonicus or Alnus firma) as a template and a DNA fragment that is a
portion of
the nonsymbiotic globin gene as a probe. A nucleic acid to be used as a
template in
these methods may be a genomic DNA extracted from any plant by a conventional
technique or a cDNA synthesized via reverse transcription from a mRNA
extracted by a
conventional technique, for example. A nucleic acid to be used as a template
may be a
purified genomic DNA, a purified cDNA, a cDNA library, a genomic DNA library,
or
the like. Alternatively, such nonsymbiotic globin gene to be used in the
present
invention may also be synthesized as a nucleic acid fragment by various
nucleic acid
sequence synthesis methods known in the art, such as a chemical synthesis
method.
Furthermore, desired mutation may be introduced into the nucleotide sequence
(ultimately, into the amino acid sequence encoded by the nucleotide sequence)
of the
thus obtained nonsymbiotic globin gene by site-specific mutagenesis or the
like. For
introduction of a mutation into a gene, known techniques such as the Kunkel
method,
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CA 02600560 2007-09-11
the gapped duplex method, or any techniques based on those methods can be
employed.
For example, mutation may be introduced using a mutagenesis kit (e.g., Mutan-K
(TAKARA) or Mutan-G (TAKARA)) utilizing site-specific mutagenesis or the LA
PCR
in vitro Mutagenesis Series Kit (TAKARA).
In addition, experimental protocols employed in the present invention, such as
mRNA preparation, cDNA preparation, PCR, RT-PCR, library construction,
ligation into
vectors, transformation of cells, DNA sequencing, chemical synthesis of
nucleic acids,
determination of the amino acid sequence on the N-terminal side of a protein,
mutagenesis, and protein extraction can be performed according to methods as
described
in ordinary laboratory manuals. An example of such a laboratory manual is
Sambrook
et al., Molecular Cloning, A Laboratory Manual, 2001, Eds., Sambrook, J. &
Russell, D.
W., Cold Spring Harbor Laboratory Press.
3. Overexpression of nonsymbiotic globin genes in nodulating plants
According to the present invention, overexpression of a nonsymbiotic globin
gene is caused in a nodulating plant by genetic engineering techniques. The
wording
"causing overexpression" used herein means that: genetically engineering a
gene in a
host organism by any of genetic engineering techniques (e.g., gene transfer)
such that
the gene is expressed at a level above the normal expression level of the gene
in the host
organism (e.g., higher by 10% or more); or introducing a gene into a host
organism that
does not have the gene such that the gene is expressed therein. Specific means
for
causing overexpression of a nonsymbiotic globin gene are not limited and any
techniques known by persons skilled in the art can be employed. An example of
a
general method is a technique that involves ligating a nonsymbiotic globin
gene
downstream of an overexpression promoter so as to enable the expression of the
gene in
a correct reading frame and then introducing the thus constructed
transformation vector
into a nodulating plant, but the examples are not limited thereto. In this
case, a
nonsymbiotic globin gene is incorporated into a vector by, for example,
excising a DNA
fragment containing the nonsymbiotic globin gene using an appropriate
restriction
enzyme and then inserting in-frame and ligating the DNA fragment into an
appropriate
restriction enzyme site located downstream of an overexpression promoter in an
expression vector containing the overexpression promoter. Alternatively, a DNA
fragment previously prepared by ligating a nonsymbiotic globin gene downstream
of an
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CA 02600560 2007-09-11
overexpression promoter may be incorporated into a vector. A genomic fragment
of a
nonsymbiotic globin gene ligated to an overexpression promoter may also be
inserted
into the genomic DNA of a nodulating plant via a process such as homologous
recombination. As nodulating plants in which overexpression of nonsymbiotic
globin
genes is caused, any of the above nodulating plants (e.g., Lotus japonicus)
can be
appropriately used.
In the present invention, "overexpression promoter" means a promoter capable
of causing strong expression (large amount expression) of a gene that has been
ligated
thereto in host plant cells. The overexpression promoter of the present
invention may
particularly be a nodule-specific promoter that enables nodule-specific
expression. The
overexpression promoter of the present invention may be either an inducible
promoter or
a constitutive promoter. A promoter is a DNA comprising an expression control
region
generally located on the 5' upstream of a structural gene or a modified
sequence thereof.
In the present invention, any promoters appropriate for foreign gene
expression in plant
cells can be used as overexpression promoters. Preferred examples of such
overexpression promoters to be used in the present invention include, but are
not limited
to, a cauliflower mosaic virus (CaMV) 35S promoter, a rice actin promoter, a
modified
355 promoter, a tobacco PR 1 a promoter, and an Arabidopsis thaliana PR-1
promoter.
As a transformation vector for introduction of a nonsymbiotic globin gene, any
gene introduction vector for plant cells can be used. For example, when an
Agrobacterium method is employed, an Agrobacterium-derived plasmid vector
(e.g., a Ti
plasmid) or a binary vector is preferably used. Such transformation vector to
be used
in the present invention contain a nonsymbiotic globin gene and optionally an
overexpression vector, and may further contain a selectable marker gene for
facilitating
selection of transformants, a reporter gene, a replication origin (e.g., Ti or
Ri plasmid-
derived replication origin) for use with a binary vector system, and the like.
Examples
of a selectable marker gene include drug resistance genes such as a Cefotax
gene, a
hygromycin resistance gene, a dihydrofolate reductase gene, an ampicillin
resistance
gene, a neomycin resistance gene, and a kanamycin resistance gene. Examples of
a
reporter gene include a green fluorescence protein gene (GFP) and luciferase
genes
(LUC and LUX).
In the present invention, a "vector" encompasses so-called
expression cassette. An "expression cassette" means a DNA fragment containing
a
promoter DNA sequence and the DNA sequence of a gene to be expressed, in which
the
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CA 02600560 2007-09-11
DNA sequence of the gene is ligated to the promoter DNA sequence such that the
gene
can be expressed in plant cells. Such expression cassette may lack the ability
of
autonomous replication.
Furthermore, examples of a vector that contains an overexpression promoter in
advance include pBI-based binary vectors (e.g., pKANNIBAL, 1G121-Hm, pBI121,
pBI101, pBI101.2, pBI101.3, and pCAMBIA1301).
The present invention also provides a vector comprising the above-mentioned
nonsymbiotic globin gene ligated to an overexpression promoter. Such
transformation
vector can be very conveniently used because the vector can be introduced into
any
nodulating plants to enhance the nitrogen-fixing activity of nodules.
Any plant transformation methods that are broadly used for plants can be used
as a method for introducing a transformation vector into a nodulating plant,
including,
but are not limited to, an Agrobacterium method, a particle gun method, an
electroporation method, a polyethylene glycol (PEG) method, a microinjection
method,
and a protoplast fusion method. These plant transformation methods are
described in
ordinary textbooks such as "New Edition, Experimental Protocols for Model
Plants,
Genetic Techniques to Genomic Analysis" (under the supervision of Isao
Shimamoto
and Kiyotaka Okada, (2001) Shujunsha Co., Ltd.). Preferably, plant cells which
have
been subjected to the introduction of the transformation vector are selected
by a method
utilizing a selectable marker such as kanamycin resistance, and confirmed for
the
expression of the transgene through detection of a reporter protein or
expression analysis
of the transgene. Preferably, in addition to that, the plant bodies are
regenerated by a
conventional technique.
More specifically, in the case of the Agrobacterium method, for example, the
method of Nagel et al is employed. First, a vector is introduced into
Agrobacterium by
electroporation. Next, plants are infected with the thus transformed
Agrobacterium to
introduce the gene of interest into the plants according to the method
described in Plant
Molecular Biology Manual (S. B. Gelvin et al., Academic Publishers); Thykaer,
T. et al.,
Cell Biology, 2nd ed. (1998) pp. 518-525; Stiller, J., et al., J. Exp. Bot.
(1997) 48, pp.
1357-1365; Ogar, P. et al., Plant Science (1996) 116 159-168; or Hiei Y. et
al., Plant J.
(1994) 6, 271-282. Plant bodies are then regenerated.
When the polyethylene glycol method is employed, first, cell walls may be
removed by digesting them with enzymes to obtain protoplasts. A nonsymbiotic
globin
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CA 02600560 2007-09-11
gene may be then introduced into the cells using polyethylene glycol and then
the cells
may be regenerated into plant bodies (Datta SK: In Gene Transfer To Plants
(Potrykus I
and Spangenberg, Eds) pp. 66-74 (1995)).
When the electroporation method is employed, first, cell walls may be removed
by digesting them with an enzyme to obtain protoplasts. Electric pulses may be
applied to the protoplasts to introduce a nonsymbiotic globin gene into cells
and then the
cells may be regenerated into plant bodies (Toki S, et al., Plant Physiol.,
100: 1503
(1992)).
When the particle gun method is employed, plant bodies, plant organs, and
plant
tissues may be used intact. Alternatively, sections or protoplasts may be
prepared
therefrom and then used (Christou P, et al., Biotechnology 9: 957 (1991)). The
prepared sample is bombarded with microparticles (each with a diameter between
approximately 1 m and 2 pm) of gold, tungsten, or the like that have been
coated with a
nonsymbiotic globin gene with high pressure gas using a gene transfer
apparatus (e.g.,
PDS-1000 (BIO-RAD)) to introduce the nonsymbiotic globin gene into plant
cells.
Treatment conditions may vary depending on plant and sample used, and but in
general,
the bombardment is performed at a pressure between approximately 450 psi and
2000
psi and at a distance approximately between 4 cm and 12 cm from the target.
Once the
nonsymbiotic globin gene is introduced into the cells, the cells may be
regenerated into
plant bodies as described above.
In the thus transformed nodulating plant of the present invention, the
nonsymbiotic globin gene may be expressed in an inserted form in the genomic
DNA of
the plant or may be expressed as an extragenomic DNA (e.g., in a form that is
retained
in a vector).
Expression of a nonsymbiotic globin gene in transformed plant cells or plant
tissues (e.g., hairy roots, leaves, stems, and nodules) in which the
overexpression of the
nonsymbiotic globin gene has been caused as described above or in the
regenerated plant
bodies thereof is preferably confirmed by a conventional technique such as a
Northern
blotting method, a Southern blotting method, or a method on the expression of
a reporter
gene.
4. Formation of nodules in plant bodies and determination of nitrogen-fixing
activity of
nodules
CA 02600560 2007-09-11
Transformed nodulating plants in which overexpression of a nonsymbiotic
globin gene has been caused according to the present invention can form
nodules when
they are grown under a given environment in which a symbiotic nitrogen-fixing
bacterium is present. However, it is also possible to artificially cause
nodulation
through inoculation of such nodulating plant with a symbiotic nitrogen-fixing
bacterium.
Such nodulating plant can be inoculated with a symbiotic nitrogen-fixing
bacterium
according to a method well known by persons skilled in the art (e.g., see
Higashi, S.,
Katahira, S. and Abe, M. Plant and Soil 81, pp. 91-99 (1984)).
The term "symbiotic nitrogen-fixing bacteria" used herein means
microorganisms that are capable of living symbiotically with plants, forming
nodules,
fixing nitrogen into ammonia and supplying it to the host plants (that is,
have nitrogen-
fixing ability). Such symbiotic nitrogen-fixing bacteria include, when
they are
eubacteria, root nodule bacteria of the genera Rhizobium, Bradyrhizobium,
Azorhizobium, Sinorhizobium, Mesorhizobium, and Allorhizobium, and when they
are
Actinomycetes, bacteria of the genus Frankia. Root nodule bacteria are known
to
infect leguminous plants and plants of the genus Parasponia of the family
Ulmaceae.
Bacteria of the genus Frankia are known to infect mainly trees of the family
Betulaceae,
Myricaceae, or the like. Some exceptions are also known for such symbiotic
nitrogen-
fixing bacterium-host plant relationship. Hence, a transformed nodulating
plant may
be inoculated with a symbiotic nitrogen-fixing bacterium capable of infecting
the plant
species to which the target plant belongs. For example, Lotus japonicus may be
inoculated with Lotus japonicus root nodule bacteria (Mesorhizobiwn loti).
When a nodulating plant that has overexpressed a nonsymbiotic globin gene is
inoculated with a symbiotic nitrogen-fixing bacterium to form nodules
according to the
present invention, nitrogen-fixing activity in the nodules is significantly
enhanced.
The nitrogen-fixing activity of such nodules is enhanced at least 2 times and
preferably
at least 3 times (e.g., 3 to 6 times) per unit weight of the nodules, compared
with the
level of such activity in a control plant (wild-type strain) of the same
species that has
not overexpressed the nonsymbiotic globin gene.
The nitrogen-fixing activity of nodules may be determined using any method for
determining nitrogen-fixing activity that is known by persons skilled in the
art. In the
present invention, the nitrogen-fixing activity of nodules is preferably
determined as the
activity of reducing acetylene to ethylene (acetylene reduction activity: ARA
activity) in
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CA 02600560 2007-09-11
an extract of the nodules. ARA activity can be expressed as the amount of
ethylene
generated per unit weight (e.g., 1 gram) of nodules and per unit time (e.g., 1
hour or 1
minute). Such ARA activity may be determined according to the Examples
described
below, for example. The nodulating plant that has overexpressed a nonsymbiotic
globin gene according to the present invention exhibits ARA activity of
between 10
nM/min/g and 100 nM/min/g and preferably of between 11 nM/min/g and 40
nM/min/g
in nodules, but examples are not limited thereto.
The present invention further relates to a nodulating plant obtained as
described
above, which has overexpressed a nonsymbiotic globin gene and has nodules on
its
roots.
5. Other embodiments
A nodulating plant that is produced by the method of the present invention
forms nodules exhibiting high nitrogen-fixing activity. Hence, through
cultivation of
such plant, a large amount of nitrogen in atmosphere can be fixed under an
environment
for cultivation. Therefore, the present invention also provides a method for
enhancing
nitrogen-fixing efficiency upon plant cultivation. The expression "enhancing
nitrogen-
fixing efficiency upon plant cultivation" means to increase the nitrogen
fixation level
per predetermined cultivation area or per plant body under cultivation within
a
predetermined time period, compared with the nitrogen fixation level achieved
by a non-
transformed corresponding plant under the same environment. In the present
invention,
"cultivation" means to intentionally grow a relevant plant in a specific place
or under
specific environment.
In the present invention, the expression "cultivation"
encompasses agricultural cultivation, but agricultural work (tilling, seeding,
planting
seedlings, thinning out, disinfection, pruning, thinning, and harvest) is not
always
required. Examples of cultivation in the present invention include, but are
not limited
thereto, cultivation of crops or plants for gardening, landscaping, gardening,
planting for
greening degraded lands or seashores, planting for fertilization of
oligotrophic soil, and
planting for improving the quality of soil such as saline soil or dry soil.
Through enhancement of nitrogen-fixing efficiency upon plant cultivation, a
nodulating plant that is produced by the method of the present invention can
increase the
amount of nitrogen to be fixed from atmosphere under a given environment, can
increase the concentration of fixed nitrogen in the relevant plant tissues,
and thus can
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CA 02600560 2007-09-11
increase the yield or the growth amount of the nodulating plant. Furthermore,
in the
long term, cultivation of the nodulating plant of the present invention
enables
fertilization of soil through enhancement of the amount of nitrogen in soil
and increase
of the yields of other plants using such soil. Moreover, cultivation of the
nodulating
plant of the present invention enables effective greening of oligotrophic
soil, saline soil,
dry soil, and the like.
Examples
The present invention is further illustrated with reference to the following
examples. However, these examples do not limit the technical scope of the
present
invention.
[Example 1] Isolation and identification of Lotus japonicus nonsymbiotic
globin gene
(LjHbl)
A nonsymbiotic globin gene (LjHbl) of Lotus japonicus was isolated through
screening of a Lotus japonicus genomic library (Sato et al. (2000) DNA Res. 8:
pp. 311-
318) by means of the PCR method. Primers used for screening were designed
based on
the sequence of a nonsymbiotic globin gene homolog (clone name: AV413959,
DDBREMBL/GenBank accession No. AB238220) that is present in the EST library of
Lotus japonicus. The primer sequences are shown as follows:
LjHblF 1 : 5'-TTCTCACTTCACTTCCATCGC-3' (SEQ ID NO: 4, forward primer);
LjHb1F2: 5'-TTGGTCAAGTCATGGAGCG-3' (SEQ ID NO: 5, forward primer);
LjHb1R1: 5'-TCACAGTGACTTTTCCAGCG-3' (SEQ ID NO: 6, reverse primer);
and
LjHb1R2: 5'-AGACAGACATGGCATGAGGC-3' (SEQ ID NO: 7, reverse primer).
PCR for amplification of the LjHbl gene was performed under the following
reaction conditions using GeneAmp(R) PCR System 9700 (Applied Biosystems): 30
cycles of 94 C for 30 seconds, 55 C for 30 seconds, and 72 C for 30 seconds.
As a result of the screening of Lotus japonicus genomic library, the LjHbl
gene
present in a TAC clone (LjT0I01) was identified. The LjHbl gene had a full
length of
1012 bp spanning from the initiation codon to the termination codon and was a
gene
encoding 161 amino acid residues, as revealed by sequence analysis of the
LjHbl gene
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CA 02600560 2007-09-11
in the Lotus japonicus genome. The genomic structure of the Lotus japonicus
nonsymbiotic globin gene (LjHbl) and the amino acid sequence encoded by the
gene are
shown in Fig. 1. The LjHbl gene had a structure containing four exons and
three
introns, which is a common structure among plant globin genes, and was present
on
chromosome 3 among the six chromosomes of Lotus japonicus.
[Example 2] Expression analysis of the nonsymbiotic globin gene (LjHbl)
The expression analysis of LjHbl was performed by means of RT-PCR in two
experimental series in which the expression in different tissues and the
expression under
stress conditions were examined, respectively. In the experimental series
involving the
examination of the expression in different tissues, four types of tissue
sample of Lotus
japonicus (grown individual plants on week 6 after germination) were used: 1)
leaves; 2)
stems; 3) roots; and 4) nodules. In the experimental series involving the
examination
of the expression under stress conditions, the four types of sample used
herein were: 1)
untreated (control) sample; 2) sucrose-added sample; 3) low temperature-
treated sample;
and 4) low oxygen-treated sample. Primers LjHb1F1 and LjHb1R2 that had been
used
in Example 1 were used for the RT-PCR. A one-step RT-PCR kit (QIAGEN) was used
for the reverse transcription and the amplification of transcription products
thereof.
The expression levels of the LjHbl gene were determined by electrophoresis.
Electrophoretic photographs were subjected to imaging. LjHbl expression levels
were
expressed as relative values based on the band intensities.
Fig. 2A shows the experimental results of examining the expression in
different
tissues. It was demonstrated that LjHbl is expressed in various organs of
grown
individual plants. Expression levels (relative values) were: 0.4 in the
leaves; 0.4 in the
stems; 1.0 in the roots; and 36.0 in the nodules. Thus, LjHbl was particularly
strongly
expressed in the nodule tissues. Furthermore, Fig. 2B shows the experimental
results
of examining the expression under stress conditions. It was demonstrated that
LjHbl is
also strongly expressed via stress treatments such as low temperature
treatment (see,
Fig. 2B; expression level: approximately 250) and low oxygen treatment (Fig.
2B;
expression level: approximately 450), in terms of relative expression level
compared
with an untreated sample.
[Example 3] Construction of transformation vectors
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For the purpose of producing transformed Lotus japonicus plant bodies and
transformed hairy roots which overexpress LjHbl, a gene construct comprising
LjHbl
cDNA ligated to a strong promoter was prepared. Further, for construction of
vectors
to be used for transformation, plasmids pKANNIBAL (Wesley et al. 2001 the
plant
Journal 27, 581-590), pHKN29 (Kumagai and Kouchi, 2003 MPMI 16(8), 663-668)
and
pIG121-Hm (Ohta et al., 1990, Plant Cell Physiol., 31, 805-813) were used.
Total RNA was extracted according to a conventional technique from Lotus
japonicus nodules, and then cDNA was synthesized by RT-PCR. Full-length LjHbl
cDNA was cloned through PCR using the resulting cDNA as a template. In
particular,
the obtained full-length LjHbl cDNA (SEQ ID NO: 1) was ligated downstream of
the
cauliflower mosaic virus 35S promoter in pKANNIBAL. Further, 35S-LjHbl cDNA
fragment was excised from the resulting vector and then ligated downstream of
the GFP
region in pHKN29 to produce plasmid vector pR35SLjHb1 (Fig. 3). This
pR35SLjHb1
was used for induction of transformed hairy roots as described below.
Furthermore, full-length LjHbl cDNA was ligated downstream of the 35S
promoter in pIG12I-Hm to produce a plasmid vector pT35S1Abl. This pT35S1Abl
was used for producing transformed Lotus japonicus plant bodies as described
below.
[Example 4] Production of transformed hairy roots and confirmation of gene
introduction and gene expression
Transformed Lotus japonicus hairy roots into which LjHbl gene had been
introduced were produced by a hairy-root-inducing transformation system
mediated by
Agrobacterium rhizogenes using the plasmid vectors constructed in Example 3.
First, the vector pR35SLjHb1 that enables overexpression of LjHbl constructed
in Example 3 was directly introduced into Agrobacterium rhizogenes LBA1334
(provided by Dr. Clara Diaz (Institute Molecular Plant Science, Leiden
University)) by
electroporation. Lotus japonicus seedlings on day 5 after seeding, excised at
the
hypocotyl portions, were inoculated with a bacterial cell suspension of the
Agrobacterium rhizogenes harboring pR35SLjHb 1. Subsequently, the seedlings
were
placed on sterilized filter papers and then co-cultivated in the co-
cultivation medium
(1/10 B5, 0.5 1.tg/m1 BAP, 0.05
NAA, 5 mM MES (pH 5.2), and 20 jig/m1
acetosyringone) for 5 days. After the completion of co-cultivation, the
seedlings were
placed in GamborgB5 medium (produced by Nihon Pharmaceutical Co., Ltd., and
CA 02600560 2007-09-11
marketed by Wako Pure Chemical Industries, Ltd.) supplemented with antibiotic
cefotax
(200 g/m1; Chugai Pharmaceutical Co., Ltd.) so that hairy roots were induced.
Fig.
4A shows the thus induced hairy roots. In addition, Fig. 4A shows various
samples
differing in terms of growth stage or period of hairy root development.
The presence or the absence of the transgene LjHbl in hairy roots was
confirmed through GFP fluorescence detection and detection of a fusion gene of
35S
promoter and LjHbl via PCR amplification. In hairy roots into which the LjHbl
gene
had been introduced, GFP was produced and thereby green fluorescence was
observed
(Fig. 4B). In Fig. 4B, the left (upper and lower) photographs are bright-field
images
observed and the right (upper and lower) photographs are dark-field images
observed.
GFP fluorescence was clearly observed in roots as a result of overexpressing
the
symbiotic globin gene.
Furthermore, total RNA was extracted according to a conventional technique
from the transformed hairy roots, and then RT-PCR was performed so that the
expression of the transgene LjHbl was confirmed. In addition, Lotus japonicus
hairy
roots derived from a wild-type strain were used as controls. Fig. 4C shows the
results.
As shown in Fig. 4C, the LjeIF-4A gene which is known to be expressed at the
same
level in any Lotus japonicus tissues and at any stages was used as an
indicator for
demonstrating that the RNA amount used for the causing of the gene expression
was the
same in both test and control samples.
As demonstrated by such results, in hairy roots into which the LjHbl gene had
been introduced, LjHbl gene expression was induced in an amount approximately
more
than 100 times greater than that in the hairy roots of the wild-type strain
into which
LjHbl gene had not been introduced.
[Example 5] Nodulation of transformed hairy roots and measurement of nitrogen-
fixing
activity of the nodules
The plants were induced to develop hairy roots and LjHbl gene introduction
therein was confirmed in Example 4. The plants were transplanted to culture
soil
(vermiculite : pearlite = 4 : 1). Fahraeus medium (Fahraeus, (1957) J. Gen.
Microbiol.
16(2) 374-381) supplemented with KNO3 up to a final concentration of 1 mM was
given
to the plants, and the plants were grown for 1 week. Subsequently, the plants
were
transplanted to a fresh culture soil and then the hairy roots were inoculated
with Lotus
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japonicus root nodule bacteria (Mesorhizobium loti MAFF 303099) at a
concentration of
1 x 107 cells/ml. After inoculation with the root nodule bacteria, medium
containing
no nitrogen source was given thereto, and then the plants were further grown
for 4
weeks. The transformed plants were kept under the growth conditions as in a
plant
growth chamber, with a 16 hours light/8 hours dark cycle, at 25 C to 26 C. 4
weeks
later, nodules were formed on the hairy roots (Fig. 5).
Next, the nitrogen-fixing activity of the nodules formed on the hairy roots
was
determined as activity of reducing acetylene into ethylene (Acetylene
Reduction
Activity; ARA). Specifically, first, nodules collected from the hairy roots
were put into
a 15-cm test tube and then the test tube was sealed with a rubber cap. Air
within the
test tube was sufficiently aspirated off using an aspirator and then the test
tube was
filled with an acetylene gas. After 2 hours of incubation at room temperature,
the gas
within the test tube was collected and then the amount of generated ethylene
was
measured by gas chromatography. Fig. 6 shows the measurement results.
As in the measurement results shown in Fig. 6, ARA activity per unit weight of
nodules (i.e., per gram weight of nodules) formed on the hairy roots into
which LjHbl
had been introduced and overexpressed was calculated to be 11.15 nM/min/g
[1.45 x
21.5 - 0.5 = 30.67 (nM/g); 30.67 (nM) / 2.75 minutes = approximately 11.15
nM/min/g].
On the other hand, as a control, ARA activity per unit weight of nodules (per
gram
weight of nodules) formed on the hairy roots into which no LjHbl had been
introduced
was calculated to be 2.12 nM/min/g [1.45 x 4 - 0.5 = 5.3 (nM/g); 5.3 (nM) /
2.5 minutes
= 2.12 nM/min/g]. It was shown that the nitrogen-fixing activity of nodules
formed on
the hairy roots (transformants) into which LjHbl had been introduced and
overexpressed
was 5 times or more greater than that of the control.
[Example 6] Production of transformed plant bodies
1) Infection of Lotus iaponicus with Agrobacterium tumefaciens (A.
tumefaciens)
Each hypocotyl was excised from a Lotus japonicus seedling on day 5 after
seeding by cutting at a site immediately below the cotyledon and at a boundary
of the
root. Meanwhile, pT35S1jLb 1 constructed in Example 3 was directly introduced
into
Agrobacterium tumefaciens (A. tumefaciens) EHA105 (provided by Dr. Kenzo
Nakamura at the Nagoya University) by electroporation. Acetosyringone was
added at
a final concentration of 100 AM to a bacterial cell suspension (1.0 x 107
cells/ml, 0D600
22
CA 02600560 2007-09-11
= 0.10 x 0.15) of the resulting Agrobacteriwn tumefaciens EHA105 harboring
pT35S1jLb 1. The excised hypocotyl was immersed in the suspension solution.
The
hypocotyl was sliced into sections with a thickness of approximately 5 mm in
the
solution. The sections were continuously immersed in the bacterial cell
suspension for
30 minutes, so that the sections were infected with Agrobacterium. After
infection, the
sections were placed on sterilized filter paper and then co-cultivated at 25 C
for 3 to 5
days in the co-cultivation medium (1/10 B5, 0.5 ii,g/m1 BAP, 0.05 pg/ml NAA, 5
mM
MES (pH 5.2), and 20 jig/m1 acetosyringone).
2) Callus induction
Co-cultivated sections were transferred onto a callus medium (1 x B5, 2%
sucrose, 0.5 jig/ml BAP, 0.05 jig/m1 NAA, 10 mM NH4, and 0.3% phytagel)
supplemented with antibiotic cefotax (250 gimp for sterilization and
antibiotic
hygromycin B for selection of the transformant. The sections were cultured at
25 C
with a 14 hours light/10 hours dark cycle for 5 weeks. Transplantation of
sections was
performed every 1 to 2 weeks.
3) Induction of shoots from calli
After 5 weeks of culture in the above callus medium, the sections were
transferred onto a shoot induction medium (1 x B5, 2% sucrose, 0.5 g/m1 BAP,
0.05
jig/ml NAA, 10 mM NH4, and 0.3% phytagel). The sections were cultured at 25 C
with a 14 hours light (6100 lux)/10 hours dark cycle for 2 weeks.
Subsequently, the
calli formed from the sections were transplanted in shoot induction medium not
supplemented with hygromycin B. The calli were cultured under culture
conditions
that were the same as those described above for 3 weeks. Transplantation of
calli was
performed every 1 to 2 weeks.
4) Shoot elongation
CaIli were transferred onto a shoot elongation medium (1 x B5, 2% sucrose, 0.2
jig/ml BAP, and 0.3% phytagel) and then cultured at 25 C with a 14 hours light
(6100
lux)/10 hours dark cycle for 3 weeks. Transplantation of calli was performed
every 1
to 2 weeks. Subsequently, calli were transplanted onto shoot elongation medium
containing no plant hormone. The calli were then cultured under culture
conditions
similar to those described above for 2 to 3 weeks, thereby promoting shoot
elongation.
5) Induction and elongation of roots
5 mm or longer shoots generated from the calli placed on the shoot elongation
23
= CA 02600560 2007-09-11
medium were cut from each shoot base portion using a razor. The shoot base
portions
inserted lengthwise in a root induction medium (1/2 B5, 1% sucrose, 0.5 tg/m1
NAA,
and 0.4% phytagel), and the shoots were cultured with a 14 hours light (6100
lux)/10
hours dark cycle for 1 weeks or more. Subsequently, the shoots with bloating
cut areas
were inserted into a root elongation medium (1/2 B5 and 1% sucrose). The
shoots were
then cultured under culture conditions similar to those described above for 2
to 3 weeks,
thereby promoting root elongation.
6) Cultivation of transformed plant bodies
Plant bodies were obtained through root elongation as described above. The
plant bodies were extracted from the medium and then gel that had adhered to
the roots
was thoroughly washed off in water. The plant bodies were transplanted onto
vermiculite impregnated with commercial B5 medium (Wako Pure Chemical
Industries,
Ltd.) diluted 1:10. The plant bodies were then cultivated with a 14 hours
light (6100
lux)/10 hours dark cycle. The thus cultivated Lotus japonicus plant bodies
produced
seeds, and the seeds were harvested. Subsequently, the seeds were seeded in
Power
soil (culture soil for gardening; Kureha Chemical Co.) for cultivation.
Whether or not the LjHbl gene had been successfully introduced into the thus
grown plant bodies was confirmed by detecting the fusion gene of a 35S
promoter and
LjHbl through PCR amplification thereof. Furthermore, increases in the
expression
levels of the LjHbl gene were confirmed by RT-PCR.
[Example 7] Nodulation and nitrogen-fixing activity of transformed Lotus
japonicus
plant bodies
Transformed Lotus japonicus plant bodies were produced and the introduction
and expression of the LjHbl gene into the plant bodies were confirmed as in
Example 6
described above. The transformed plant bodies were transplanted into culture
soil
(vermiculite : pearlite = 4 : 1) for cultivation. The plant bodies were
inoculated with
Lotus japonicus root nodule bacteria (Mesorhizobium loti MAFF 303099) at a
concentration of 1 x 107 cells/ml. After inoculation with the root nodule
bacteria, the
transformed plant bodies were cultivated in a plant growth chamber, with a 16
hours
light (6100 lux)/8 hours dark cycle at 25 C to 26 C for 4 weeks. After 4 weeks
of
cultivation, the formed nodules were collected from the plant bodies and then
ARA
activity was determined in a manner similar to that in Example 5. As a result
of
24
CA 02600560 2007-09-11
determination, the ARA activity per unit weight of nodules (per gram weight of
nodules)
formed by transformed Lotus japonicus plant bodies overexpressing LjHbl was
calculated to be 17.21 nM/min/g. As a control, the ARA activity per unit
weight (per
gram weight of nodules) of nodules formed on Lotus japonicus (general Lotus
japonicus,
wild-type) into which LjHbl had not been introduced was calculated to be 5.05
nM/min/g.
In addition, the average number of nodules formed on each plant body obtained
in this example was 7 in both transformants and non-transformants. No
particular
differences were observed in the appearance of nodules, such as size and
color.
[Example 8] Comparison of the affinity for nitrogen monoxide of Lotus
japonicus
nonsymbiotic globin with that of Lotus japonicus symbiotic globin
The full-length LjHbl cDNA (the sequence spanning from the initiation codon
to the termination codon thereof is shown in SEQ ID NO: 1, which encodes the
amino
acid sequence of SEQ ID NO: 2) obtained in Example 3 was cloned into a protein
expression vector pGEX4T-3 (Amersham Pharmacia Biotech) according to a
conventional technique (Fig. 7). The vector was then introduced into
Escherichia coil
to obtain transformants. For preparation of a control sample, a Lotus
japonicus
symbiotic globin gene was similarly cloned into an expression vector pGEX4T-3
(Fig.
7), and the vector was then introduced into Escherichia coil to obtain
transformants.
The thus obtained Escherichia coil transformants were cultured for inducing
gene
expression. Hence, soluble active nonsymbiotic globin could be
recombinantly
produced within bacterial cells of Escherichia coil in large amounts.
Subsequently,
Escherichia coli cells that had expressed the nonsymbiotic globin gene were
collected
according to a conventional technique. After disruption of E. coli cells,
protein
purification was performed, so that active nonsymbiotic hemoglobin could be
obtained.
The globin to be obtained through disruption of Escherichia coil cells was
associated
with heme from Escherichia coil and thus isolated as a form having hemoglobin
activity,
because heme is also supplied in Escherichia coil into which the nonsymbiotic
globin
gene had been introduced.
Subsequently, nitrogen monoxide was mixed with the thus obtained symbiotic
hemoglobin and then absorbance was measured over time. Fig. 8 shows absorbance
spectra obtained at wavelengths between 500 nm and 600 nm at 0 minutes, 5
minutes, 15
CA 02600560 2007-09-11
minutes, and 30 minutes after the initiation of mixing with nitrogen monoxide.
As
shown in Fig. 8, as the time of mixing with nitrogen monoxide lengthens, two
peaks at
540 nm and 575 nm progressively disappeared.
Particularly in the case of
nonsymbiotic hemoglobin, the 575-nm peak disappeared at a faster rate and
almost
completely disappeared at 15 minutes after the initiation of mixing. On the
other hand,
in the case of symbiotic hemoglobin, the 540-nm peak was not dramatically
decreased.
Even at 30 minutes after the initiation of mixing, the 575-nm peak remained,
although it
was weak, so that two peaks were still observed.
[Example 9] Isolation and identification of Alnus firma nonsymbiotic globin
gene
(AfHb 1)
A nonsymbiotic globin gene (AfHbl) of Alnus firma was isolated by screening
of an Alnus firma nodule cDNA library (Sasakura, F. et al., "A class 1
hemoglobin gene
from Alnus firma functions in symbiotic and nonsymbiotic tissues to detoxify
nitric
oxide." Mol. Plant Microbe. Interact. (2006) 19(4) in press) using a DNA
fragment of
the Lotus japonicus nonsymbiotic globin gene LjHbl as a probe.
The probe for screening was designed based on the sequence of the Lotus
japonicus nonsymbiotic globin gene Lj Hb 1 (clone name: AV413959,
DDRI/EMBL/GenBank accession No. AB238220). The probe was obtained by PCR
amplification from the genomic DNA of Lotus japonicus using the primers
LjHb1F1 (5'-
TTCTCACTTCACTTCCATCGC-3': SEQ ID NO: 4) and LjHb1R1 (5'-
TCACAGTGACTTTTCCAGCG-3': SEQ ID NO: 6) used in Example 1. PCR for
amplifying a LjHbl fragment to be used as a probe was performed using
GeneAmp(R)
PCR System 9700 (Applied Biosystems) under conditions consisting of 30 cycles
of
94 C for 30 seconds, 55 C for 30 seconds, and 72 C for 30 seconds.
As a result of such screening of the Alnus firma nodule cDNA library, the
AfHbl
gene was identified. As a result of nucleotide sequence analysis, the AfHbl
gene was
found to be a gene comprising a sequence of 483 bp in length spanning from the
initiation codon to the termination codon in the cDNA and encoding 160 amino
acids.
The nucleotide sequence spanning from the initiation codon to the termination
codon of
cDNA sequence of AfHbl (DDBJ/EMBL/GenBank accession No. AB221344) is shown
in SEQ ID NO: 8. The amino acid sequence encoded by the nucleotide sequence is
shown in SEQ ID NO: 9. Moreover, the genomic DNA sequence (from the initiation
26
CA 02600560 2007-09-11
codon to the termination codon) of the Alnus firma AfHbl gene is shown in SEQ
ID NO:
10.
[Example 10] Expression analysis of Alnus firma nonsymbiotic globin gene
(AfHbl)
The expression analysis of AfHbl was performed by RT-PCR using mRNAs
extracted according to a conventional technique from the tissues of various
organs of
Alnus firma as samples. The following AfHb1F1 and AfHb1R3 primers were used
for
the RT-PCR. A One-Step RT-PCR kit (QIAGEN) was used for the reverse
transcription
and the amplification of transcription products thereof. The following AfHb1F1
and
AfHb1R3 primers were also used for amplification of the reverse transcription
products.
The primer sequences used herein are shown as follows:
Affib1F1: 5'-GCTGCTATCAAATCTGCAAT-3' (SEQ ID NO: 11, forward primer)
and
AfHb1R3: 5'-GGGGGGCTGTGATTTTAG-3' (SEQ ID NO: 12, reverse primer).
The thus obtained amplified products were electrophoresed and then
electrophoretic photographs obtained were subjected to imaging. The expression
level
of the AfHbl gene in each tissue was determined based on the band intensity.
The results of the expression analysis by means of RT-PCR revealed that the
AfHbl gene had been expressed in various organs of the grown plants of Alnus
firma.
In particular, the AfHbl gene had been strongly expressed in nodule tissues.
As with
the Lotus japonicus LjHbl gene, it was revealed that the expression of the
AlHbl gene
was also strongly induced by stress treatment such as low temperature
treatment.
[Example 11] Construction of a transformation vector
Production of transformed Lotus japonicus overexpressing the AfHbl gene was
attempted to elucidate the functions of the AfHbl gene. For vector
construction for
transformation, plasmids pKANNIBAL (Wesley et al., 2001, The Plant Journal 27,
581-
590) and pHKN29 (Kumagai and Kouchi. 2003 MPMI 16(8), 663-668) were used.
First, cDNA was synthesized by reverse transcription from total RNA extracted
from Alnus firma nodules. Full-length AfHbl cDNA was cloned by means of PCR
using the synthesized cDNA as a template. The thus obtained AfHbl cDNA was
ligated
downstream of the cauliflower mosaic virus 35S promoter in pKANNIBAL.
Moreover,
the 35S-AfHbl cDNA fragment was excised from that and then ligated downstream
of
27
CA 02600560 2007-09-11
GFP region in pHKN29, thereby finally preparing a transformation vector
pAfHb1S.
[Example 121 Production of transformed hairy roots
A hairy-root-inducing transformation system mediated by Agrobacterium
rhizogenes was employed for transformation of Lotus japonicus with AfHbl. The
transformation method is based on the principle of co-transfection, by which
hairy roots
are induced and transformed by Agrobacterium rhizogenes. Transformed hairy
roots in
this example were produced according to the method of Example 3.
The transformation vector pAfHb1S that enables overexpression of the AfHbl
gene constructed in Example 11 was directly introduced into Agrobacterium
rhizogenes
LBA1334 by electroporation. Lotus japonicus seedlings on day 5 after seeding,
which
had been excised at the hypocotyl portions, were infected through inoculation
with a
bacterial cell suspension of the Agrobacterium rhizogenes LBA1334 in which the
vector
pAfHb1S had been introduced. Subsequently, the seedlings were placed on
sterilized
filter paper and then co-cultivated in co-cultivation medium for 5 days. After
the
completion of co-cultivation, the seedlings were placed on agar medium of
Gamborg B5
medium (produced by Nihon Pharmaceutical Co., Ltd. and marketed by Wako Pure
Chemical Industries, Ltd.) supplemented with antibiotic cefotax (200 ,g/m1;
Chugai
Pharmaceutical Co., Ltd.), so that hairy roots were induced.
The presence or the absence of transgene AfHbl in hairy roots was confirmed
through GFP fluorescence detection and RT-PCR, in a manner similar to that in
Example
4.
As a result, in all the individual Lotus japonicus plants that had been
infected with
Agrobacterium rhizogenes into which AfHbl had been introduced, hairy roots
emitting
GFP fluorescence were induced. On the other hand, as a result of confirmation
by RT-
PCR, AfHb 1 gene expression was induced in hairy roots into which OA 1 had
been
introduced (transformants) at an expression level of approximately more than
100 times
greater than that in wild-type strain hairy roots into which AfHbl had not
been
introduced. Fig. 9 shows the results of confirming AfHbl gene expression using
RT-
PCR. In Fig. 9, LjeIF-4A is a positive control.
[Example 13] Analysis of the phenotypes of individual transformed plants
It was confirmed in Example 12 that hairy roots had undergone transformation
with the Aflibl gene. Plants having such hairy roots were transplanted to
culture soil
28
CA 02600560 2007-09-11
(vermiculite : pearlite = 4 : 1). Fahraeus medium (Fahraeus, (1957) J. Gen.
Microbiol.
16(2) 374-381) supplemented with KNO3 up to a final concentration of 1 mM was
given
to the plants and then the plants were grown for 1 week. Subsequently, the
plants were
transplanted to a fresh culture soil, and the hairy roots were inoculated with
Lotus
japonicus root nodule bacteria (Mesorhizobium loti MAFF 303099) at a
concentration of
1 x 107 cells/ml. After inoculation with the root nodule bacteria, Fahraeus
medium
containing no nitrogen source was given thereto, and then the plants were
further grown
for 4 weeks. The transformed plants were kept under the growth conditions as
in a
plant growth chamber, with a 16 hours light/8 hours dark cycle, at 25 C to 26
C.
Nodules were formed on the hairy roots of the grown plant bodies. The average
number of nodules formed per each plant body was 9 in both transformants and
non-
transformants. No particular differences were observed among transformants and
non-
transformants in terms of appearance of nodules, such as size and color.
Next, the nitrogen-fixing activity of the nodules formed on the hairy roots
was
determined as one of phenotypes of the transformants. The nitrogen-fixing
activity of
nodules was determined as activity of reducing acetylene into ethylene
(Acetylene
Reduction Activity; ARA) according to the method described in Example 5. The
results are shown in Fig. 10. In Fig. 10, "control" indicates the results
obtained using,
as a sample, Lotus japonicus into which the plasmid pHKN29 that does not
contain the
AfHbl gene had been introduced.
As a result of determination, nodules formed on hairy roots into which AfHbl
had been introduced and overexpressed, exhibited nitrogen-fixing activity 3 to
5 times
greater per unit weight of nodules compared with that of nodules formed on
hairy roots
of the wild-type strain Lotus japonicus into which AfHbl had not been
introduced. As
shown in Fig. 10, nodules formed on hairy roots into which the overexpression
of AfHbl
had been caused exhibited ARA activity (average) of 7.2 nM/min/g per unit
weight.
Determination was also performed for the whole Lotus japonicus plant bodies
into
which Aft-1bl had been introduced, and the ARA activity (average) per unit
weight was
shown to be 13 nM/min/g. On the other hand, as a control, nodules formed on
wild-
type strain hairy roots into which AfHbl had not been introduced exhibited ARA
activity (average) of 2.6 nM/min/g per unit weight. The whole wild-type strain
plant
bodies into which AfHbl had not been introduced exhibited ARA activity
(average) of 5
nM/min/g per unit weight.
29
CA 02600560 2007-09-11
Based on the above results, it was demonstrated that the Lotus japonicus into
which the Alnus firma nonsymbiotic globin gene, AfHbl, had been introduced
also
exhibits a significantly improved nitrogen-fixing activity of nodules formed
on the hairy
roots.
Industrial Applicability
According to the method for producing a nodulating plant of the present
invention, a nodulating plant with nodules having markedly improved nitrogen-
fixing
activity can be obtained. Further, the method for enhancing nitrogen fixation
levels
upon plant cultivation through cultivation of the nodulating plant of the
present
invention can be used for increasing the yield or the growth amount of such
nodulating
plant or fertilizing soil by increasing the nitrogen level in soil and
greening degraded
lands and the like.
All publications, patents, and patent applications cited herein are
incorporated
herein by reference in their entirety.
Sequence listing free text
The sequences of SEQ ID NOs: 4 to 7, 11, and 12 represent primers.
30
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