JP2003143988A - Salt resistant eucalypt and method for preparing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain salt resistant eucalypt strong against environmental stress by imparting betaine-producing ability to the eucalypt. SOLUTION: Choline oxidase gene which catalyzes one step oxidation from choline to betaine is introduced in chromosome DNA of eucalypt. A shoot having at least one end with a growing point and a base without the growing point is used as a tissue for introducing the gene and the gene is introduced into the base of the shoot to initiate adventitious buds. Thus, an individual in which the choline oxidase gene is introduced into the chromosome DNA is prepared independent of species even in the genus Eucalyptus having many species to which the gene is difficultly introduced.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a plant having a novel property, and more particularly to a salt-tolerant eucalyptus resistant to environmental stress.

[0002]

2. Description of the Related Art Afforestation on uncultivated soil such as salt-accumulated soil plays a major role in preventing desert expansion, vegetation and global warming.

However, the growth of plants in salt-accumulated soil is
(1) The accumulated salt reduces the water potential in the soil, and the plant cannot absorb water. (2) The salt absorbed (invaded) in the plant disturbs metabolism. 3) Salt prevents the absorption of other ions required for growth (Fumihiko Sato, Plant Cell Engineering Supplement, "Environmental Problems and Plant Biotechnology", p.33-39, 199).
4) Caused by factors such as Above all, when water cannot be absorbed, plant cells lose turgor pressure and close stomata, and photosynthesis is reduced, and growth is largely inhibited.

In order to cope with such salt damage, salt-tolerant plants have a compatible solute (co) for maintaining intracellular osmotic pressure.
Adaptable by biosynthesizing the compatible solution in response to changes in external osmotic pressure. Such compatible solutes are all low-molecular-weight, highly water-soluble, difficult to metabolize, and substances that do not affect metabolism, for example,
Glycine betaine (betaine), amphoteric compounds such as proline, and polyols such as vinylitol and mannitol are known.

Among these compatible solutes, betaine is
Plants and bacteria that are adaptable to high salt concentration and / or water-deficient environments, such as higher plants such as the family Rubiaceae, Gramineae, Solanaceae, etc. (Eg, D. Rhodes and
A. D. Hanson, Annu. Rev. PlatP
hysiol. Plant Mol. Biol. Four
4: 357-3584, 1993). In these plants and / or bacteria, betaine maintains intracellular osmotic pressure (SP Robinson and GPJ).
one, Aust. J. Plant Physio
l. , 13: 659-668, 1986), but also prevents the dissociation of soluble enzyme under high salt concentration (Gabbay-Azaria et al., Arc.
h. Bichem. Biophys. 264: 333
-339, 1988), protecting photosystem II complexes (eg, Murata t al., FEBS Le.
tt. 296: 187-189, 1992).

There are mainly two biosynthetic pathways for betaine. That is, choline is oxidized in two steps to produce betaine, and Escherichia coli and spinach ( Spincia ole) are produced.
racea ) and the like, and a route in soil fungus Arthrobacter, etc., in which choline is oxidized in one step to produce betaine.

In spinach, for example, ferredoxin-dependent choline monooxygenase causes the first-stage oxidation (R. Brouquisse et al., P.
ant Physiol. , 90: 322-329,
1989), NAD-dependent betaine aldehyde dehydrogenase (EA Weretilnyk et al, P.
lanta. 178: 342-352, 1989) catalyzes the second stage oxidation (FIG. 1). It has been observed that exposure of such plants to salt stress increases the activity of both enzymes and increases the amount of betaine (eg, AD Hanson et al., Pro.
c. Natl. Acad. Sci. U. S. A. , 8
2: 3678-3682, 1985). On the other hand, the soil fungus Arthrobacter globiforms ( Arthroba
In C. tergulobiformis , choline oxidase catalyzes the one-step oxidation of choline to betaine (S. Ikuta et al., J. B.
iochem. , 82: 1741-1749, 197.
7, FIG. 2).

[0008]

Therefore, it is considered that if the genes encoding these betaine synthases can be incorporated into a plant and constitutively expressed, salt tolerance can be imparted to the plant. , And research for that purpose is being actively conducted (for example, M. Nomura e.
t al. , Plant Physiol. , 107:
703-708, 1995). However, eucalyptus, which grows quickly and is useful as a greening tree, a pulp material, etc., has not yet succeeded in incorporating such a gene and stably expressing it. This is partly because it is generally difficult to introduce genes into Eucalyptus plants.

In view of the above problems, the present invention introduces a betaine synthase gene into a chromosomal DNA of a eucalyptus plant and stably expresses it, and is excellent in environmental stress, particularly salt tolerance. The purpose was to produce eucalyptus that can grow healthy even in salt-accumulated soil that was not used as the ground.

[0010]

Means for Solving the Problems As a result of intensive studies, the inventors of the present invention have used a shoot having at least one end having a growth point and a base having no growth point as a tissue for gene transfer, and The choline oxidase-encoding gene is introduced into Eucalyptus plants by allowing the adventitious bud to differentiate by introducing the choline oxidase-encoding gene into the base that does not have the growth point while maintaining the growth point at one end. Then, the above object was achieved.

That is, the present invention provides a Eucalyptus plant in which a gene encoding choline oxidase is introduced into chromosomal DNA. Furthermore, the present invention, as a tissue for gene transfer, at least one end having a growth point,
In addition, a shoot having a base without a growth point was collected, and a gene encoding choline oxidase was introduced into the base without a growth point while keeping the growth point at least at one end to differentiate adventitious buds. A method for producing a Eucalyptus plant into which a gene encoding choline oxidase is introduced is also provided.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION The gene encoding choline oxidase used in the present invention (hereinafter simply referred to as choline oxidase gene) is a protein having a function capable of converting choline into betaine in a one-step reaction shown in FIG. Is a gene that encodes. Such genes include
For example, a gene consisting of a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 1, or one or more amino acid sequences added, deleted and / or other than the amino acid sequence set forth in SEQ ID NO: 1 A gene having a nucleotide sequence encoding an amino acid sequence modified by substitution with an amino acid and encoding a protein having choline oxidase activity can be used.

[0013] These genes, belonging to the genus Arthrobacter is a soil bacteria of the gram-positive, preferably Arthrobacter Gurobiforumusu (Arthrobacter gl
obiformis , hereafter simply referred to as A. It is called Globiforms. ) And Arthrobacter pasence ( Arthr
(Obactor pascens ), particularly preferably A. Although it can be obtained by cloning of Globiformus genomic DNA or cDNA, it may be derived from other organisms. Further, those chemically synthesized by a standard method can also be used as the choline oxidase gene of the present invention.

Generally, the target gene (choline oxidase gene in the case of the present invention) is present in the tissue for gene transfer.
In introducing, the selectable marker gene is introduced together with the target gene, and the expression of this selectable marker gene is used as an index for the target gene introduction. In the present invention, as a selectable marker gene, genes widely involved in antibiotic resistance such as a widely used kanamycin resistance gene (NPTII gene) and hygromycin resistance gene (HPT gene), and nopaline synthase gene (NOS) are used. Gene), a gene involved in amino acid synthesis such as octopine synthase gene (OCS gene), a gene involved in pesticide resistance such as sulfonylurea resistance gene (ALS gene), etc. it can.

Further, as proposed by the applicant in Japanese Patent Laid-Open No. 7-313432, genes involved in plant morphological differentiation (morphological abnormality induction genes), for example, ipt gene (A.
C. Smigocki, L .; D. Owens, Pro
c. Natl. Acad. Sci. USA, 85:51
31, 1988), β-glucuronidase gene (Morten Joersbo and Fin).
n T. Okkels, Plant Cell Rep
orts, 16: 219-221, 1996), CKI.
1 gene (T. Kakimoto, Science, 2
74: 982, 1996) and its mutants (CKI2 gene), iaa M (tryptophanmonooxy).
gene) gene (H. J. Klee et a.
l. , GENES & DEVELOPMENT, 1:
86, 1987), gene 5 gene (H. koerb
er et al. , EMBO Journal, 1
0: 3983, 1991), gene 6b gene (PJJ Hoyaas et al., Pla.
nt Mol. Biol. , 11: 791, 198
8), and the rol gene group of rolA to D (FFW).
hite et al. J. Bacteriol. ,
164: 33, 1985) and the like may be used as a selectable marker gene. In this case, these selectable marker genes are combined with a detachable DNA element such as a transposon or site-specific recombination system so that the selectable marker gene is eliminated together with the detachable DNA element. Finally, the effect of the selectable marker gene used for selection is eliminated and the effect of cells or tissues in which only the target gene remains in the chromosomal DNA, that is, the plant body, that is, the selectable marker gene is eliminated. The eucalyptus into which the choline oxidase gene has been introduced can be efficiently obtained.

By the way, in order to produce a protein such as an enzyme from a gene, a promoter sequence (expression initiation sequence), a terminator sequence (expression termination sequence), etc., in addition to the gene encoding the information of these polypeptides, can be produced. A regulatory sequence called is required. Therefore, also in the present invention, in order to express the choline oxidase gene, the selectable marker gene and the like in Eucalyptus cells and to produce the proteins encoded by these genes, the promoter sequence on the 5 ′ side of the choline oxidase gene described above is used. Is ligated to the 3'side with a terminator sequence and introduced into eucalyptus chromosomal DNA. Therefore, in the following, if necessary, the gene encoding the information of the polypeptide is a structural gene, and the structural gene having a regulatory sequence such as a promoter sequence and a terminator sequence is simply a gene to distinguish them.

Currently, as a promoter sequence that functions in plants, the 35S promoter of cauliflower mosaic virus (JT Odell et al., Natu) is used.
re (London), 313: 810, 1985),
Promoter of nopaline synthase (WHRR Lan
gridd et al. , Plant CellR
ep. 4: 355, 1985), a promoter of ribulose diphosphate carboxylase / oxygenase small subunit (R. Fluhr et al., Pro.
c. Natl. Acad. Sci. USA, 83:23.
58, 1986) and the like, as the terminator sequence,
Nopaline synthase polyadenylation signal (A.De
picker et al. J. Mol. Appl.
Gen. , 1: 561, 1982), polyadenylation signal of octopine synthase (J. Gielen et.
al. , EMBO J .; 3: 835, 1984) and the like are known. Therefore, also in the present invention, these promoters or terminators can be used as promoters or terminators of the choline oxidase gene, the selectable marker gene, etc. introduced into the eucalyptus chromosomal DNA.

The above-mentioned gene, its regulatory sequence, other factors and the like can be introduced into a plant cell in a form of being integrated into a vector, and can be integrated into a plant chromosome DNA by the function of this vector. There is no problem as long as they are introduced into the same cell of the tissue for gene introduction, even if they are introduced into the same vector or introduced into different vectors. However, when these genes and the like are integrated on the same vector, it is necessary to arrange them so that the presence of one gene or the like does not inhibit the expression of the other gene or the like.

In the present invention, as such a vector, a known vector for introducing a gene into plants can be appropriately modified and used. In addition, the introduction of the vector into plant cells, cauliflower mosaic virus,
Gemini virus, tobacco mosaic virus, brom mosaic virus, Agrobacterium tumefaciens (hereinafter abbreviated as A. tumefaciens), Agrobacterium rhizogenes, etc., indirectly or via a microinjection method, or a microinjection method, It can be directly carried out by a physical / chemical method such as an electroporation method, a polyethylene glycol method, a fusion method, a particle gun method (I. Potrykus,
Annu. Rev. Plant Physiol. Pl
antol. Biol. 42: 205, 1991).

In the present invention, the tissue for gene transfer is a tissue obtained from a plant of the genus Eucalyptus, in which a system for introducing a foreign gene into chromosomal DNA and a system for regenerating a plant from the tissue have been established. Anything will do as long as it exists. However, from the viewpoint of gene transfer efficiency, it is preferable to use a shoot having at least one end having a growth point and a base having no growth point as such a tissue. More specifically, using such a shoot as a tissue for gene transfer, the choline oxidase gene and other necessary genes (selection marker gene, etc.) are added to the base that does not have the growth point while maintaining the growth point.
And factors are introduced to differentiate adventitious buds. Then, since adventitious buds into which these genes have been introduced will be re-differentiated with a high probability, even in the genus Eucalyptus, where many genes are difficult to introduce, the choline oxidase gene is present in the chromosomal DNA regardless of the species. The introduced individual can be obtained.

Hereinafter, a method for producing a Eucalyptus plant into which a choline oxidase gene has been introduced by this method will be described.

The term “shoot” as used herein refers to a shoot in which fixed buds or adventitious buds are elongated. In order to further improve the gene transfer efficiency, a tissue or site having a high redifferentiation ability, for example, a juvenile tissue having a low content of polyphenols causing browning of the tissue is used as the gene transfer tissue. Is desirable. From such a viewpoint, in the present invention, hypocotyls obtained by germination of seeds, and those obtained by extending apical buds and lateral buds of seedlings cultivated in a culture vessel are selected as suitable gene transfer tissues. To be done.

However, these shoots must have at least one end having a growth point and a base having no growth point. Such shoots, for example,
When the hypocotyl is used as the tissue for gene transfer of the present invention, after germination of the seed, the root and the part having the root disc of the root should be cut off from the hypocotyl, leaving the apical bud and the apical bud primitive. Can be obtained by In this case, one end with a growing point refers to the shoot end where the apical shoots are present, and the base without a growing point refers to the part where the end face with the roots cut off is present. Becomes Further, when the apical buds or lateral buds of seedlings are used as the tissue for gene transfer of the present invention, they can be obtained by simply cutting them off from the seedlings. In this case, the one end with the growing point is
As in the case of the hypocotyl, it refers to the shoot end with the apical bud, etc., and the base without the growing point refers to the part with the cut surface from the seedling.

In the case of gene transfer to the shoot base, for example, by the gene transfer method using Agrobacterium (Agrobacterium method), a suitable vector incorporating the choline oxidase gene or the like is introduced into Agrobacterium. It may be introduced in advance and the above-mentioned shoot having a wound at the base may be dipped in a liquid in which this Agrobacterium is suspended. Since infection with Agrobacterium occurs at a wound on a plant tissue, the choline oxidase gene or the like is introduced into the shoot base by immersing it in the suspension of Agrobacterium. In addition, when using a hypocotyl from which roots are cut off as a tissue for gene transfer, or when cutting out an expanded apical shoot from a seedling and using it, the end surface of the shoot base generated during the cutting The (cut surface) becomes the infection site of Agrobacterium. That is, when the gene is introduced into these tissues, it is not necessary to intentionally scratch the shoot base. Genes are introduced into the shoot base by simply immersing these tissues in the Agrobacterium bacterium solution.

To infect a plant tissue with Agrobacterium, after immersing the tissue for gene transfer in a bacterial solution, transfer this tissue onto a solid medium and co-cultivate with the Agrobacterium for several days. By doing so, it becomes certain. Examples of the medium for co-culture include MS (Murashig).
e and Skoog, Physiol. Plan
t, 1962, 15: 473-497) and WPM (Lo
yd and McCown, Prop. Int. Pl
ant Prop. Soc. , 1980, 30: 421
-427) and other well-known basal media, or those modified for eucalyptus, with a carbon source and a solidifying agent for the media, and if necessary, plant hormones such as auxins and cytokinins. It can be used by appropriately adding. At this time, sucrose 1 as a carbon source
0-30 g / l, agar 5-10 as a solidifying agent for the medium
g / l or 1 to 4 g / l of gellan gum, 0.01 as zeatin or benzyladenine as cytokinins
˜5.0 mg / l, and as auxins, naphthalene acetic acid (NAA), indole butyric acid (IBA), indole acetic acid, etc. are often used in a concentration range of 0.01 to 2.0 mg / l. In addition, acetosyringon 10-200m
In some cases, the infectivity of the bacterium may be increased by adding g / l to the medium for coculture.

On the other hand, when the choline oxidase gene is introduced by a particle gun, the above coculture medium can be used as a medium for gene introduction treatment. In this case, the tissue for gene transfer may be placed on this medium with the site to which the target gene is to be transferred, that is, the shoot base facing upward, and the gene gun may be operated by operating a particle gun according to a standard method.

In the above-mentioned gene transfer method, the use of a cytokinin-related gene as the selectable marker gene can further improve the gene transfer efficiency. Here, the cytokinin-related gene is a gene that acts in the direction of increasing the influence of cytokinin in the introduced plant cell and increases the adventitious bud differentiation ability of the cell. Examples of such genes include A. Ip, which is a cytokinin synthesis gene derived from Tumefaciens
The t gene, the β-glucuronidase gene derived from Escherichia coli, which is a gene that activates inactive cytokinin, and the CKI1 gene, which is derived from Arabidopsis thaliana and is considered to be a cytokinin receptor gene, are typical . Among them, the ipt gene used in the examples of the present application is the most well known gene whose function has been elucidated.

In order to differentiate adventitious buds from shoots into which the choline oxidase gene or the like has been introduced at the base, the tissue may be cultured in an appropriate medium. At this time, as the adventitious bud differentiation medium, for example, an MS medium in which the concentration ratio of ammonia nitrogen and nitrate nitrogen is 1: 3 (hereinafter simply referred to as modified MS medium) is diluted 1 to 4 times. , Sucrose 10-30g / l, gellan gum 1-
4 g / l or agar 5-10 g / l, zeatin 0.2-5.0 mg / l as plant hormone and NAA or IBA
What added 0.01-1.0 mg / l can be used. However, when a cytokinin-related gene is used as a selection marker gene, plant hormones may be added (hormone-free) or auxin alone may be added. When the gene is introduced by the above-mentioned Agrobacterium method, antibiotics such as carbenicillin, ticarcillin, cefotaxime and the like are added to this medium in an amount of 10 to 10000 mg / l in order to eradicate Agrobacterium. Temperature is 15 to 30 ° C, light intensity is 0 to 200 μmo
1 / m ² / s is preferred. The shoot growth point retained at the time of gene transfer is not particularly required in the subsequent process, and therefore it may be excised at an appropriate time.

The shoots cultured in the adventitious shoot differentiation medium normally differentiate adventitious shoots within a few weeks after the start of culture. At this time, callus may slightly grow before adventitious bud differentiation. The adventitious bud that differentiates here has the target gene introduced with higher efficiency than the case where it is obtained by a gene transfer operation into eucalyptus based on the conventional method. However, when a gene is introduced using a morphological abnormality-inducing gene such as a cytokinin-related gene as a selection marker gene, a morphological abnormality is caused by this gene, and an adventitious bud into which a target gene has been introduced is a multiblast. May show abnormal morphology. However, even in such a case, as shown in the above-mentioned JP-A-9-154580, if this morphological abnormality-inducing gene is used in combination with a DNA factor having elimination ability, the influence of the morphological abnormality-inducing gene is finally obtained. It can be eliminated to obtain adventitious buds with normal morphology.

The individual into which the target gene has been introduced is cut off from the adventitious buds thus obtained, and, for example, auxins are
If transplanted to a rooting medium containing 1.0 mg / l and rooted,
Can be played.

[0031]

In the present invention, the choline oxidase gene is introduced into the chromosomal DNA of Eucalyptus plants. As a result, choline oxidase or a protein having choline oxidase activity is produced in the plant body, and betaine is produced from choline existing in the body in the first place by a one-step oxidation reaction catalyzed by these proteins. . Since betaine is an excellent compatible solute for maintaining intracellular osmotic pressure, the eucalyptus from which betaine has been produced in this way acquires traits such as salt tolerance.

For introducing the choline oxidase gene into eucalyptus, a shoot having at least one end having a growth point and a base having no growth point is used as a tissue for gene introduction, and the growth point is used. While holding it,
It can be efficiently carried out by introducing a choline oxidase gene or the like into the base having no growth point to differentiate adventitious buds.

The reason for this is not always clear. However, it is considered that the growth point existing at least at one end of the same shoot contributes in some way. When a gene is introduced, plant tissues and cells are always damaged to some extent, but the resilience from this damage is enhanced by the presence of the growth point, so that the cells to which the gene is introduced maintain active growth. This may have favored the introduction of the choline oxidase gene and / or the redifferentiation of adventitious shoots into which the choline oxidase gene was introduced.

In general, the shoot base having no growth point is suitable for rooting. Therefore, when a cytokinin-related gene was used as a selectable marker gene, and a choline oxidase gene was introduced into this portion, the difference in the capacity for adventitious bud differentiation between cells into which the gene had been introduced and cells into which it had not been introduced. It is considered that the cell size is significantly increased, and as a result, cells into which the choline oxidase gene has been introduced selectively differentiate adventitious buds. Therefore, the use of such a gene as a selectable marker gene is more advantageous for the differentiation of adventitious shoots into which the choline oxidase gene has been introduced.

[0035]

EXAMPLES The present invention will be described below based on examples, but the present invention is not limited to the following examples. In the following examples, more detailed experimental operations are performed using the molecular cloning second method unless otherwise specified.
Plate (Sambrook et al., 1989) or according to the manufacturer's instructions.

Example 1 I.D. Agrobacterium of plasmid pMATCODA
Introduction to A. Tumefaciens 4404 strain, 10 ml of YEB
Liquid medium (beef extract 5 g / l, yeast extract 1 g /
1, peptone 1 g / l, sucrose 5 g / l, 2 mM MgS
O ₄ , pH 7.2 at 22 ° C. (hereinafter, unless otherwise indicated, the pH at 22 ° C.) was inoculated, and OD630.
Was cultured at 28 ° C until the range of 0.4 to 0.6. This culture solution was centrifuged at 6900 xg at 4 ° C for 10 minutes to collect the cells, and the obtained cells were mixed with 20 ml of 10 mM HE.
Suspend in PES (pH 8.0) and then resuspend at 6900 x
The cells were collected by centrifugation at 4 ° C for 10 minutes. Then, the cells were suspended in 200 μl of YEB liquid medium, which was used as a vector pMATCO containing the choline oxidase gene.
It was used as a bacterial solution for introducing DA (FIG. 3).

FIG. 3 is a schematic diagram mainly showing a portion of the pMATCODA structure which is to be incorporated into the chromosomal DNA of a plant. In the figure, 35s-P is 35S
Promoter, tr is a gene encoding a transit peptide which is a chloroplast translocation signal derived from tobacco ribulose 1,5-bisphosphate carboxylase-oxygenase small subunit, and codA is a base encoding the amino acid sequence of SEQ ID NO: 1. A. A. Choline oxidase structural gene (co
dA gene) and ter are polyadenylation signals of nopaline synthase. The black triangles surrounded by rectangles are the recombinant sequences Rs of the site-specific recombination system (R / Rs system) obtained from Zygosaccharomyces rouxii and its sequence direction, and R is the same site-specific recombination system recombinase. gene, ipt is ipt gene, parB
-P indicates the parB promoter. Although not shown in the figure, this plasmid also has a kanamycin resistance gene. However, this kanamycin resistance gene is not integrated into the chromosomal DNA of plants. As the promoter and terminator of the ipt gene, the original promoter and terminator of this gene are used.

When this plasmid is used as a vector for gene transfer, the chromosomal DNA of the plant has a co-oxidase gene having 35s-P as a promoter.
fusion gene dA gene and transit peptide, also, ipt gene as the selectable marker gene, together with the R gene and the recombinant sequence Rs of R / RS system site-specific recombination system is a DNA factor having a leaving Hanareno, once Be incorporated. However, when the R gene is subsequently expressed, the produced recombinant enzyme binds to Rs, and the region sandwiched by Rs (corresponding to a DNA element having a releasing ability in the present application),
That is, since the R gene itself and the ipt gene are eliminated,
After all, only the choline oxidase gene remains in the chromosomal DNA.

PMATCODA on Agrobacterium
Was introduced by electroporation. That is, 50 μl of the plasmid-introducing bacterial solution and 2 μg of pMATCODA were mixed in a 0.5 ml tube, and the mixed solution was adjusted to 2.5 kV and 25
By applying electric pulse of μF, 200Ω, p
MATCODA is A. Introduced into Tumefaciens 4404 strain (GENE PULSER manufactured by Bio-Rad Co., Ltd.
Use II. ). The cells after electroporation were added to 200 μl of YEB liquid medium and cultured by shaking at 25 ° C. for 1 hour, and then cultured on YEB agar medium containing 50 mg / l kanamycin (agar 1.5 w / v%, Other composition was the same as above.) And pMATCODA-introduced strain was selected by culturing at 28 ° C. for 2 days. pMA
The final confirmation of the introduction of TCODA was carried out by cutting the plasmid extracted by the alkaline method from the strain thus selected with a restriction enzyme and comparing the electrophoretic patterns of the cut fragments.

II. From Agrobacterium to Eucalyptus
The introduction of pMATCODA Eucalyptus camaldulensis (Eucalypt
us camaldulensis , hereinafter E. Abbreviated as Kamaldrensis. ) Seeds in 1% 70% ethanol
Soak for 2 minutes, then soak in 2% aqueous sodium hypochlorite solution for about 2 hours with stirring to sterilize, wash well with sterile water, seed on a germination medium, and in a 4 ° C refrigerator to promote germination. After storing it for 2 days or more at 25 ℃, light intensity of about 40μ
It was cultivated under all-light condition of mol / m ² / s and germinated. As the germination medium, a modified MS medium in which the concentration ratio of ammonia nitrogen and nitrate nitrogen was 1: 3 was diluted 2-fold (hereinafter referred to as Eucalyptus basic medium), and 10 g / sucrose was added. 1 and 8 g / l of agar were added and used.

On the other hand, the Agrobacterium 4404 strain into which pMATCODA had been introduced in I was cultured overnight in YEB liquid medium at 25 ° C. and 180 rpm, and then OD600.
Was diluted to 0.5 to give an Agrobacterium suspension for infection.

1-2 weeks after the seeds were sown in the germination medium, the roots and cotyledons of the germinated seedlings were cut off, and the hypocotyls were collected leaving only the apical buds at one end thereof, and the Agrobacterium suspension for infection was suspended. It was immersed in the liquid. Then, after removing excess bacterial solution, this hypocotyl was added to eucalyptus basal medium with 40 mg of acetosyringone.
/ L, sucrose 10 g / l, and agar 8 g / l were used to infect Agrobacterium by co-culturing with Agrobacterium in the dark at 25 ° C for 2 days.

After co-cultivation, the hypocotyls were treated with eucalyptus basal medium containing 250 mg / l of ticarcillin and 10 g of sucrose.
1 / l, agar 8g / l
At a temperature of ℃ and light intensity of about 40 μmol / m ² / s
The cells were cultured while being subcultured in a medium having the same composition every two weeks.
Then, three months after the infection with Agrobacterium, among the adventitious buds that had differentiated and extended, those that appeared to show normal morphology by visual observation were selected and rooted in a rooting medium. Then, a redifferentiated individual was obtained. As a rooting medium, IBA 0.05 mg / l, cefotaxime 100 mg / l, sucrose 10 g / l, and gellan gum 2.5 g / l were added to a eucalyptus basic medium.

At this time, 435 hypocotyls were used for Agrobacterium infection, and 85 redifferentiated individuals were obtained from 19 hypocotyls.

III. Introduction of pMATCODA Eucalyptus
In analysis II, the regenerated plants 85 individuals obtained from hypocotyl after Agrobacterium infection, chromosomal DNA was extracted by a conventional method, with these DNA, elimination of primer and ipt gene present in the codA gene can be detected PCR was carried out using each of the primers capable of detecting the codA gene and the presence of ip
Detachment of the t gene was confirmed. In these 33 redifferentiated individuals, the ipt gene was once introduced into the chromosomal DNA together with the choline oxidase gene, but thereafter, the ipt gene together with the detachable DNA factor,
It is considered that the choline oxidase gene is eliminated from the introduced DNA and only the choline oxidase gene remains in the chromosomal DNA.

Presence of codA gene and ip by PCR
Western blotting analysis was performed on 16 out of the 33 redifferentiated individuals in which elimination of the t gene was confirmed, and production of choline oxidase protein was confirmed. That is, 500 mg of leaves were collected from 16 of these redifferentiated individuals and 2 controls to obtain a buffer solution (100 mM phosphate buffer solution (pH 7.0), 150 mM sodium chloride, 10 mM).
EDTA, 10 mM thiourea, 0.3% Tween
20, 0.05% Triton X-100, 10 mM
750 μl of DTT) was added, and the mixture was crushed on ice in a microcentrifuge tube to give a homogenate.
SDS-PAGE was performed on the soluble protein obtained by centrifugation at 000 × g for 10 minutes. In addition, here, for the control, E. The seedlings obtained by clonally propagating Camaldrensis seedlings were grown to the same degree as the redifferentiated individuals to be compared, and used.

The separation pattern after SDS-PAGE was a nylon membrane (Immobilon PVD manufactured by Millipore).
F.), and the nylon membrane was treated with an antibody against choline oxidase (P. Deshniumu et al.,
Plant Mol. Biol. , 29: 897-90
Created by the method described in 7, 1995. ), And after the incubation, the presence or absence of production of choline oxidase was examined by ECL Western blottin.
g analysis system (Amersham
(Pharmacia Biotech).

As a result, the presence of an immunoreactive protein was confirmed at the position of 64 kDa corresponding to choline oxidase in all individuals subjected to Western blot analysis. This is because not only the choline oxidase gene was introduced into the chromosomal DNA but also choline oxidase was expressed to produce choline oxidase in the 16 redifferentiated individuals, that is, the choline oxidase gene was chromosomal DNA. It is shown that the precursor which is integrated into the correct position of the, and the resulting precursor is correctly processed and converted into a mature protein.

FIG. 4 shows a part of the result of the Western blot analysis.

IV. Introduction of pMATCODA Eucalyptus resistance
Out of 85 redifferentiated individuals obtained in the saltiness test II, 43 (including choline oxidase transgenic and non-transgenic individuals) derived from 8 hypocotyls were cut into shoots containing nodes, Sodium chloride 200m in Eucalyptus basic medium
M, cefotaxime (100 mg / l) and agar (8 g / l) were used for culturing, and then fixed buds that had developed from the node were collected.
It was cultured in a medium supplemented with 0 mM, and its salt tolerance was tested. In addition, both this node culture and the culture for the salt tolerance test are 2
It was carried out at 7 ° C. under a totally bright condition with a light intensity of 51 μmol / m ² / s.

As a result, rooting was observed from two lines of buds (buds obtained from two different redifferentiated individuals),
It was confirmed that these are also resistant to 200 mM sodium chloride. On the other hand, in the control in which salt tolerance was tested in the same manner, all buds of 39 strains derived from 7 seeds died, and none of them could grow in the presence of 200 mM sodium chloride.

[0052]

INDUSTRIAL APPLICABILITY According to the present invention, betaine, which is an excellent compatible solute for maintaining the intracellular osmotic pressure, is produced from choline, which was originally present in the cells of eucalyptus, by a one-step oxidation reaction. Become so.

Therefore, according to the present invention, salt-tolerant eucalyptus can be obtained. Since such eucalyptus grows even if it is planted in a salt-accumulated soil or the like, it can play a major role in preventing desert expansion, greening, and global warming.

Further, in the present invention, when the choline oxidase gene is introduced into a Eucalyptus plant,
As a tissue for gene transfer, using a shoot having at least one end having a growth point, and a base portion having no growth point, while maintaining the growth point, the gene was added to the base portion having no growth point. By introducing the choline oxidase gene, adventitious buds into which the choline oxidase gene has been introduced can be redifferentiated with high probability.

Therefore, according to the method for producing a Eucalyptus plant according to the present invention, the choline oxidase gene was introduced into the chromosomal DNA regardless of the species, even in Eucalyptus, which has many species for which gene transfer is difficult. Individuals can be obtained.

[Sequence list]

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[Procedure amendment]

[Submission date] February 19, 2002 (2002.2.1
9)

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] Brief description of the drawing

[Correction method] Added

[Correction content]

[Brief description of drawings]

FIG. 1 is a diagram showing a two-step oxidation process of choline to betaine.

FIG. 2 is a diagram showing a one-step oxidation process of choline to betaine.

FIG. 3 is a schematic diagram showing the structure of a part of the plasmid pMATCODA that integrates into plant chromosomal DNA.

FIG. 4 shows that E. coli in which the codA gene has been introduced. It is a figure which shows a part of the result of the western blot analysis performed about the camaldrensis.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Etsuko Matsunaga             5-21-1 Oji, Kita-ku, Tokyo Nippon Paper Industries             Technical Research Institute Co., Ltd. (72) Inventor Hiroyasu Ebinuma             5-21-1 Oji, Kita-ku, Tokyo Nippon Paper Industries             Technical Research Institute Co., Ltd. (72) Inventor Norio Murata             1464, 64, Fubuki-cho, Okazaki-shi, Aichi F-term (reference) 2B030 AB02 AD04 CA15 CA17 CA19                 4B024 AA08 AA17 BA08 CA03 DA01                       EA04 GA14 GA17                 4B065 AA13Y AA89X AB01 AC06                       AC14 BA02 CA28 CA53

Claims (5)

[Claims] 1. A Eucalyptus plant in which a gene encoding choline oxidase is introduced into chromosomal DNA. 2. The gene encoding choline oxidase is derived from the soil fungus Arthrobacter.
The Eucalyptus plant described. 3. A choline oxidase-encoding gene has a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 1 of the Sequence Listing, or one or more amino acids relative to the amino acid sequence set forth in SEQ ID NO: 1. A base sequence encoding an amino acid sequence, wherein the sequence is modified by addition, deletion and / or substitution with another amino acid, and
The Eucalyptus plant according to claim 1 or 2, which comprises a nucleotide sequence encoding a protein having choline oxidase activity. 4. A shoot having at least one end having a growth point and a base having no growth point as a tissue for gene transfer is collected, and the growth point is retained while the growth point at at least one end is retained. The plant of the genus Eucalyptus according to claim 1, 2 or 3, which is obtained by introducing a gene encoding choline oxidase into a base which does not have it and differentiating adventitious buds. 5. A shoot having at least one end having a growth point and a base having no growth point as a tissue for gene transfer is collected, and the growth point is maintained while the growth point of at least one end is retained. A method for producing a Eucalyptus plant into which a gene encoding choline oxidase has been introduced, which comprises introducing a gene encoding choline oxidase into a base not having the gene to differentiate adventitious buds.