JPH06169778A - Structural protein gene specific to adventitious embryo of carrot with high-glycine cell wall - Google Patents

Abstract

PURPOSE:To isolate and utilize a gene specific to adventitious embryogenesis. CONSTITUTION:A gene specific to adventitious embryogenesis is obtained by isolating an RNA from carrot cells of an adventitious embryogenetic stage, establishing a cDNA library based on the RNA and hybridizing cDNA of the library with a probe specific to adventitious embryogenesis, wherein the probe is purified by subtraction method. The obtained gene or its anti-sense gene is transduced into a plant and the gene is differentiated to shift the content of the cell wall structural protein. By using a signal peptide segment of the gene, a genetic product is transferred to the cell wall.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a somatic embryogenesis-specific carrot high glycine cell wall structural protein gene, a method for inducing somatic embryogenesis using the same, and a method for modifying cell wall components.

[0002]

2. Description of the Related Art In recent years, biotechnology has been developed, and cell engineering is applied to plants to improve not only academic research but also property improvement and substance production.

It is no exaggeration to say that the basis of such plant biotechnology is the totipotency of plants, and that plant biotechnology cannot be established without this property. That is, since it has the ability to regenerate a complete plant from a single cell, it is possible to extend cell engineering technology to the plant.

Adventitious embryogenesis means that differentiated somatic cells or undifferentiated cultured cells reproduce a complete plant body through a process of embryonic development similar to that of a fertilized egg. It is a model of omnipotence. further,
Somatic embryos are the best materials available for artificial seed production. Therefore, research on somatic embryogenesis is one of the important research topics in plant biotechnology.
The isolation / analysis of the gene group related to somatic embryogenesis or the gene group that regulates the somatic embryogenesis, or the mechanism of its development has not been clarified, and the technique for inducing somatic embryos has not yet been established.

In plants, it is relatively easy to induce undifferentiated cultured cells from differentiated cells, but it is very difficult to redifferentiate them into plant bodies, and plant species that cannot induce redifferentiation are very difficult. Many. Currently, somatic embryogenesis has been reported in about 300 plants, but very few plant species are capable of adventitious embryogenesis with high reproducibility and high frequency, and a regenerative system is created with new plant species. In that case, the culture conditions such as hormone concentration must be reconsidered.

[0006] These present circumstances make it difficult to make progress in plant biotechnology (artificial seeds, mass breeding, molecular breeding, growth control, etc.) on a commercial scale. However, research on the mechanism of somatic embryogenesis and related genes, or research on the mechanism of expression regulation has not progressed so much.

Plants are an important food for us,
It is also important for global environmental protection such as maintenance of ecosystem. In recent years, production of new plants by gene manipulation techniques, that is, molecular breeding has been actively carried out, but an effective plant redifferentiation technique that can be used for industrial mass production of artificial seeds has not yet been established. Therefore, establishment of a technique for inducing differentiation into somatic embryos has become a major goal in plant biotechnology. To achieve this goal, it is necessary to isolate genes involved in somatic embryogenesis.
Therefore, the isolation of genes related to somatic embryogenesis that can be used to establish a technique for inducing differentiation of plants into somatic embryos and contribute to plant biotechnology has been awaited.

[0008]

The present invention has been made from the above viewpoints, and aims to establish a technique for inducing differentiation into somatic embryos, which is necessary to enable industrial use of plant biotechnology. It is an object of the present invention to isolate and clone a carrot somatic embryogenesis-related gene which is one of the somatic embryogenesis-related genes that can be used for

[0009]

Means for Solving the Problems As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that carrot, which is one of the somatic embryogenesis-related genes, can be obtained by using the subtraction method during cloning. The high glycine cell wall structure protein gene (hereinafter referred to as "high glycine cell wall structure protein gene") was isolated, and the present invention was accomplished.

That is, the present invention relates to 1 to 1 shown in SEQ ID NO: 1.
DNA sequence encoding the high glycine cell wall structure protein precursor having the 162nd amino acid sequence, DNA encoding the signal peptide of the high glycine cell wall structure protein having the 1st to 20th amino acid sequences shown in SEQ ID NO: 1.
It is a DNA sequence encoding the sequence and the mature form of the high glycine cell wall structure protein having the amino acid sequence of Nos. 21 to 162 shown in SEQ ID NO: 1.

The present invention also provides a method for inducing somatic embryogenesis or increasing the cell wall structure protein content by introducing a DNA sequence encoding the high glycine cell wall structure protein into plant suspension culture cells. A method for efficiently transferring the gene product to the cell wall by introducing into a plant cell a gene in which another gene is linked upstream of the DNA sequence encoding the signal peptide of the glycine cell wall structural protein, and further to a high glycine cell wall By introducing all or part of the DNA sequence encoding the structural protein into a plant cell downstream of a promoter that can be expressed in the plant cell and ligating in the forward or reverse direction with respect to the promoter, the cell wall component is introduced. A method of modifying is provided.

In the present invention, a high glycine cell wall structure protein containing a signal peptide is referred to as a "high glycine cell wall structure protein precursor", and a high glycine cell wall structure protein containing no signal peptide is referred to as a "high glycine cell wall structure protein". "Mature body". Hereinafter, the present invention will be described in detail.

<1> High Glycine Cell Wall Structure Protein Genes Most of the genes expressed in the somatic embryogenesis process are also expressed in somatic embryos and cells undergoing undifferentiated growth, and only a few genes of somatic embryos are expressed. It is expressed during formation (Sung ZR and Okimoto R., Proc. Natl. Acad. Sc
i. USA 78, 3683-3687 (1981), Sung and Okimoto
R., Proc. Natl. Acad. Sci. USA 80,2661-2665 (19
83), Fujimura T. and Komamine A., In Fujimura A. (e
d) PlantTissue & Cell Culture. Maruzen Tokyo pp.10
5-106 (1982)).

That is, since most of the genes are not specific to somatic embryogenesis, it is very difficult to clone a specific gene related to somatic embryogenesis. Therefore, as a result of diligent studies from various directions, the present inventors used the subtraction method to determine indeterminately from a cDNA library prepared from poly (A) ⁺ RNA extracted from carrot proembryo state 2 (Proembryo “State 2”). We have successfully isolated the cDNA of a high glycine cell wall structural protein associated with embryogenesis.

The isolation method will be described below.

(1) Construction of a carrot proembryostate 2 cDNA library Adventitious embryos were induced from carrot callus and m
A cDNA library for carrot proembryostate 2 was obtained by extracting RNA, synthesizing cDNA using reverse transcriptase, inserting the double-stranded product by polymerase reaction into a vector, and transforming into Escherichia coli or the like. Can be produced.

For isolation of mRNA and synthesis of cDNA, cD
Since NA cloning kits are commercially available, these may be used. In addition, a large number of vectors used for preparing the library are commercially available, and these can be used.

(2) Screening of cDNA specific to proembryostate 2 from library Obtained from the above library is a gene specific to proembryostate 2 that is specific to adventitious embryo formation but not expressed in undifferentiated state In order to do so, it is preferable to perform plaque hybridization or differential hybridization using a probe purified by the subtraction method.

The subtraction method in the present invention comprises mixing cDNA prepared from cells at the time of adventitious embryo formation with an excess amount of RNA prepared from undifferentiated cells, and separating cDNA that does not hybridize with RNA.
This is a method of isolating a specific cDNA during somatic embryogenesis.

For example, R prepared from undifferentiated cells
When NA is biotinylated and this is mixed with a cDNA solution prepared from cells at the time of somatic embryogenesis, cDNA derived from a gene that is expressed even in an undifferentiated state hybridizes with RNA. When avidin or streptavidin is added to this, it binds to biotin on biotinylated RNA. When phenol is further added, avidin or streptavidin is denatured, so that the RNA-cDNA hybrid derived from the gene expressed in an undifferentiated state is precipitated together with streptavidin. Therefore, by eliminating the precipitate, the somatic embryo-specific c
Only DNA remains in solution.

In the differential hybridization, two filters replicating from a phage plaque on a plate are hybridized with cDNA prepared from cells during somatic embryogenesis and cDNA prepared from undifferentiated cells as probes. Then, by detecting a signal that makes only the cDNA positive during somatic embryogenesis, a cDNA clone specific for somatic embryogenesis can be selected.

In the present invention, it is preferable to first carry out a primary screening by plaque hybridization using a probe purified by the subtraction method, and further confirm by differential hybridization.

The methods necessary for general gene recombination such as RNA isolation, cDNA preparation, DNA cleavage, ligation, transformation and hybridization are attached to commercially available enzymes used for each operation. Calligraphy, Molecular clonin
g (Maniatis T. et al . ColdSpring Harbor Laboratory
Press), or Current Protocols in Molecular Bio
logy (Edited by FM Ausubel et al., John Wiley & Sons, In
c., 1987).

The nucleotide sequence of the cloned cDNA is determined by the Maxam-Gilbert method or the dideoxy method (described above). The nucleotide sequence can be determined by the dideoxy method using a commercially available kit, and an autosequencer for automatically performing the sequence determination may be used.

The nucleotide sequence of the high glycine cell wall structure protein gene obtained from carrot and the amino acid sequence deduced from this sequence are shown in SEQ ID NO: 1 of the Sequence Listing. It should be noted that this gene has a sequence (SEQ ID NO: 1) encoding a signal peptide having the ability to efficiently transfer carrot high glycine cell wall structural protein to the cell wall.
Among them, a nucleotide sequence consisting of the 66th to 125th nucleotides) is also included.

<2> Utilization of high glycine cell wall structure protein gene of the present invention Since the high glycine cell wall structure protein gene was obtained by the present invention, it can be used for mass production of high glycine cell wall structure protein in carrots or plants other than carrots. Can be used. Furthermore, since DNA sequences encoding signal peptides have been obtained, it is possible to utilize these to modify cell wall components by expressing various proteins in the cell wall, and to breed new plants with disease resistance and the like. It can be applied.

For example, when the high glycine cell wall structure protein gene is connected to an appropriate promoter and introduced into carrot or other plants, the content of the high glycine cell wall structure protein can be increased. On the other hand, when at least a part of the minus strand (a sequence complementary to the coding sequence) of the gene is connected to the promoter in the reverse direction and introduced into a plant to express a so-called antisense RNA, a high glycine cell wall structure is obtained. The protein content can be reduced.

Further, DN encoding a signal peptide
When a gene to which another gene is connected to the A sequence is introduced into a plant, the gene product can be efficiently transferred to the cell wall.

Transformation of plants can be carried out by electroporation or Ti of Agrobacterium.
It can be performed by introducing DNA into protoplasts by a method using a plasmid or the like.

The following is an example of a method for preparing protoplasts in carrot, introducing DNA into protoplasts by electroporation, and regenerating transformed cells into plants.

The carrot protoplasts are prepared, for example, as follows. Cell mass State 1, which has somatic embryogenesis ability, was added to 1% Macerozyme R10, 4
% Cellulase RS (above, Kinki Yakult Co., Ltd.),
5% CaCl ₂ .2H ₂ O, 0.5% potassium dextran sulfate,
After mixing with an enzyme solution (pH 5.5) containing 0.4 M mannitol, the mixture was left standing for 3 hours, and the protoplast suspension was filtered through a 40 μm nylon mesh to obtain protoplasts.

A method for introducing DNA into the thus obtained protoplasts by electroporation (Tada, Y. et al . Theor. Appl. Genet. 80 , 475 (1
990)) is explained.

2 × 10 ⁶ protoplasts / m in electroporation buffer (70 mM KCl, 5 mM CaCl ₂ , 5 mM 2- [morpholino] ethanesulfonic acid (MES), 0.4 M mannitol)
Suspend to 1 and add 0-20 μg of DNA. As the DNA, a plasmid expressing the hygromycin resistance gene is added in addition to the introduced DNA (DNA containing the starch branching enzyme gene). In the protoplasts into which the plasmid expressing the hygromycin resistance gene has been introduced, the DNA to be introduced is also co-transfo
Since the probability of rmation is high, transformed cells can be efficiently selected by selection using hygromycin.

The protoplast / DNA mixture was allowed to stand at 0 ° C. for 20 minutes, then transferred to an ice-cooled electroporation chamber, and an electric pulse was applied thereto. The electric pulse has, for example, an initial electric field of 475 V / cm and a time attenuation coefficient T.
Give _{1/2 to} be 30.0 milliseconds. Since a device is commercially available for electroporation, this may be used. The electric pulse is, for example, the initial electric field 475.
V / cm and a time attenuation coefficient T _1/2 of 30.0 milliseconds are applied.

Next, a method for regenerating the transformed cells obtained as described above into a plant (Fujimura, T. et al . Plant
Tissue Culture Lett. 2 , 74 (1985)). The electroporated protoplasts were washed 4 times with a washing medium containing R2 inorganic salt and 0.4 M glucose by centrifugation (50 × g, 5 minutes), and then 5 × 10 ^−7.
Lin M. and M supplemented with 2,4-D, 2% sucrose.
Staba J.; Lloydia 24: 139-145 (1961) modified medium (Fuji
mura T. and Komamine A ,; PlantSci. Lett. 5: 359-364
(1975)) (hereinafter referred to as "LS modified medium"),
The cell number is adjusted to 10 ⁴ cells / ml and the cells are cultured at 25 ° C. in the dark.

On the 14th day of culture, hygromycin is added to the medium at 50 μg / ml, the culture is continued for 2 weeks, and the colonies are transferred to a fresh medium containing hygromycin at the same concentration.
When the colony grows to a diameter of 2 mm, it is transferred to LS modified medium (hormone-free) containing no hygromycin,
Cultivate under fluorescent light irradiation (3000 lux) to differentiate. The seedlings that have grown to a height of about 10 cm in a test tube are acclimated and then transferred to a pot in a greenhouse to regenerate the plant.

The selection of clones containing the high glycine cell wall structure protein gene may be carried out by collecting callus or plant cells and confirming them by a method such as Southern hybridization. After regeneration, it is preferable to check the amount of enzyme in the protein in the extract of the plant body by Western analysis or the like, while confirming that the high glycine cell wall structure protein gene has been introduced by Southern analysis or the like.

Since the high glycine cell wall structural protein gene is specifically expressed during carrot somatic embryogenesis and is involved in somatic embryogenesis, this nucleotide sequence is used as a marker for redifferentiation by somatic embryos. It enables elucidation of the mechanism and isolation of the gene that regulates it.

Therefore, the present invention establishes a technique for inducing somatic embryogenesis from undifferentiated plant suspension culture cells using a carrot high glycine cell wall structural protein which is a marker for somatic embryogenesis and is required for somatic embryogenesis. It can be used for elucidation of the mechanism of somatic embryogenesis and isolation of genes that regulate it, and is extremely useful in the technical field of regeneration for plant breeding.

Further, the constructed artificial gene is carrot,
It can be introduced into cultured cells of plants such as Arabidopsis by electroporation or particle gun method, and as a result, somatic embryogenesis is induced from transformed cultured cells obtained even in plant cultured cells that do not redifferentiate. Can be redifferentiated.

Further, the nucleotide sequence encoding a high glycine cell wall structure protein specific to somatic embryogenesis is expressed by an artificial method such as an in vitro transcription system or a microorganism such as Escherichia coli. Thus, a high-glycine cell wall structural protein associated with somatic embryogenesis can be obtained in a large amount and in a pure form. Since the obtained high glycine cell wall structural protein is a cell wall structural protein required for adventitious embryogenesis, it is effective for inducing adventitious embryogenesis in plant cells that cannot be redifferentiated or for developing a redifferentiation system. Moreover, it became possible to qualitatively modify the cell wall components related to disease resistance and the like by performing the conversion treatment of the cell wall composition using the signal peptide.

Using the nucleotide sequence of the cDNA of the present invention, a part of this nucleotide sequence is specifically mutated by using a site-specific substitution or a technique such as PCR method to improve the quality of the cell wall structure protein. Can be used for research seeking.

The carrot high glycine cell wall structure protein gene shown in SEQ ID NO: 1 is specifically expressed in the somatic embryogenesis process, and this gene can be used as a marker gene for somatic embryogenesis in plants. As a result, it becomes possible to early diagnose the presence or absence of somatic embryogenesis. In addition, the mechanism of somatic embryogenesis can be elucidated and the gene that regulates somatic embryogenesis can be isolated, which can greatly contribute to the development of a regeneration system and the development of artificial seed production technology.

The cell wall structure protein, which is a gene product of the carrot high glycine cell wall structure protein gene of the present invention, is produced in large amounts during the carrot somatic embryogenesis process and is greatly involved in somatic embryogenesis. Therefore, this gene is specifically expressed in large amounts in carrot somatic embryogenesis. Since this gene is specifically and strongly expressed in somatic embryogenesis, the promoter region of the carrot high glycine cell wall structure protein gene is very excellent as a promoter that is specifically expressed at this time. Therefore, it is considered that it becomes possible to produce an artificial gene that is specifically expressed in somatic embryogenesis (embryonic development) by binding to any gene using this promoter. That is, by binding the entire base sequence or the base sequence important for phenotype expression to an arbitrary gene, the gene can be specifically expressed in large amounts in somatic embryogenesis (embryonic development). as a result,
It is considered that it becomes possible to change the composition of the protein that constitutes the embryo and to modify the nutritional components of the plant such as increasing the contents of specific sugars and amino acids.

[0045]

EXAMPLES Examples of the present invention will be described below. Unless otherwise stated, the operation procedure is Current Protocols in M
olecular Biology (edited by FM Ausubel et al., John Wiley & So
ns, Inc., 1987).

<1> Construction of cDNA Library Derived from Proembryo State 2 (1) Culture of Carrot Callus Suspension Culture Cells Carrot cultivar "Kuroda Gosun" (Daucus carota L. c)
Callus suspension culture cells were derived from the hypocotyl of seedlings of v. "Kurodagosun"). 7 after aseptically sowing seedlings
On the day, the hypocotyl was cut into a length of 1 cm and placed in an LS modified medium containing 5 × 10 ⁻⁷ M of 2,4-D as an auxin to induce callus suspension culture cells.

The callus suspension-cultured cells thus obtained were cultivated in the dark at 27 ° C. using a reciprocal shaking culture machine (90 strokes / min. 50 mm), and 5 × 10 2,4-D was added every 7 days. ^-7 M fresh L
Subculture was maintained by transplanting to S-modified medium.

(2) Induction of pro-embryostate 2 The induction of adventitious embryos from the above callus suspension culture cells was carried out by Fujimu.
ra T. and Komamine A. Method (Plant Physiol, 64, 16
2-164 (1979)).

The carrot callus suspension culture cells on the 7th day after subculture were filtered through a 50 μm mesh fractionating funnel, and the filtrate was mixed with 3
Further filtered through a 2 μm mesh. The cells remaining on the 32 μm mesh were washed thoroughly with 2,4-D-free LS modified medium, overlaid on a 16% Ficoll 400 solution containing 2% sucrose, and the interface was disturbed, followed by centrifugation (150 g). 5 minutes).
Subsequently, it was washed several times by low-speed centrifugation using LS-modified medium containing no 2,4-D to obtain a cell mass State 1 (State 1) having about 10 cells rich in cytoplasm and having somatic embryogenesis. .

This was suspended in 200 ml of 2,4-D-free LS modified medium in a 500 ml flask at a density of 5 × 10 ² cell clusters / ml, and shake culture (90 strokes) at 27 ° C. in the dark for 3 days. / min.
50 mm) to obtain Pro Embryo State 2.

(3) Construction of cDNA Library Using the proembryostate 2 (7 g) obtained above as a material, total RNA was prepared according to the phenol-SDS method. Furthermore, poly (A) ⁺ RNA containing mRNA was isolated by oligo (dT) cellulose column chromatography, and using this as a template, cDNA was synthesized using an oligo (dT) primer and reverse transcriptase. Synthesis of cDNA using poly (A) ⁺ RNA as a template is described in Gubler U. and Hoffman B.
The method of J. (Gene, 25, 263-269 (1983)) was followed.

An EcoRI adapter (having BamHI and KpnI sites inside) was ligated to both ends of the obtained cDNA, and the lambda phage vector and λZAPII arm (Stratagene
After ligation to the EcoRI site of λZAPII / EcoRI Cloning Kit, manufactured by Incorporated, in vitro packaging kit (Stratag
Using ene, GIGAPACK Gold), packaging was performed, and a large number of recombinant λ phages were obtained by infecting E. coli XL1-Blue strain (FIG. 1, upper, middle). This was used as a carrot proembryo state 2 cDNA library. The size of this library was 1.0 × 10 ⁶ .

[0053] <2> professional Embryo state 2 specific preparation of probes (1) Poly (A) ⁺ RNA callus suspension culture cells derived from poly (A) ⁺ RNA of biotinylated callus suspension culture cells, the later subtraction Biotinylated for use in the method. For this biotinylation, subtraction kit (Invitrogen
The Subtractor ^™ I Kit manufactured by the company) was used, and the method described in the manual attached to the kit was followed.

From the carrot callus suspension culture cells 5 days after subculture, poly (A) ⁺ RNA was analyzed by the phenol-SDS method and oligo (dT) cellulose column chromatography.
(~ 10μg), and mix this poly (A) ⁺ RNA with photobiotin solution (1mg / ml) and stand in ice, 300W
Using a reflector lamp (manufactured by Toshiba), light was irradiated for 30 minutes from a distance of 10 cm above.

After irradiation, 0.1M Tris and 2-butanol were added, and the mixture was stirred and then centrifuged (15,000 × rpm for 3 minutes). After extraction with butanol four times to remove unbound photobiotin, co-precipitation with ethanol gave poly (A) ⁺ RN.
A was precipitated and dissolved in 30 μl of distilled water. Subsequently, the biotinylation treatment was repeated once again in the same manner as above to obtain biotinylated poly (A) ⁺ RNA.

(2) Preparation of probe The probe used for plaque hybridization was
Purified by the subtraction method. The probe was purified by this subtraction method according to the method of a subtraction kit (The Subtractor ^™ I Kit manufactured by Invitrogen).

[0057] The pro-embryogenic was prepared from the state 2 poly (A) ⁺ RNA as a template, ³² subjected to reverse transcription reaction with P-labeled nucleotides presence, ³² P-labeled single strand cDN was
A probe was synthesized. The biotinylated poly (A) ⁺ RNA obtained above was added to this and hybridized. After adding streptavidin to this, and further adding phenol and stirring, the aqueous phase and the phenol layer were separated, and cDNA was recovered from the aqueous layer by ethanol precipitation to obtain a pro-embryostate 2-specific probe.

<3> High Glycine Cell Wall Structural Protein cD
Screening of NA Screening of approximately 150,000 independent phage clones in the pro-embryostate 2 cDNA library was performed by plaque hybridization using a probe purified by the subtraction method.

As a result, 69 positive clones were obtained. Further, differential screening was carried out on these with cDNA derived from proembryostate 2 and cDNA derived from cell clusters 5 days after subculture as a result, and a clone designated as CEM6 was obtained.

Further, by each process of somatic embryogenesis and Northern analysis performed on cells that were proliferating undifferentiatedly, it was confirmed that the CEM6 was a clone specific to somatic embryogenesis. When Southern hybridization of chromosomal DNA was carried out in Arabidopsis (Arabidopsis thaliana), it was confirmed that a similar sequence was also present in Arabidopsis.

A plasmid clone pCEM6 having a cDNA insert was prepared from the phage DNA of CEM6 by the in vivo excision method. The in vivo excision method was according to the method of λZAPII / EcoRI cloning kit manufactured by Stratagene.

200 μl of phage solution containing CEM6, 200 μl of E. coli XL1-Blue suspension, and 1 μl of suspension of helper phage VCSM13 were mixed and allowed to stand at 37 ° C. for 15 minutes, and then 5 ml of 2 × YT medium was added to the mixture at 37 ° C. Shake culture for 70 hours
For 20 minutes and centrifuged (4000 rpm, 5 minutes) to collect the supernatant. 20 μl of 100-fold diluted supernatant and E. coli X
After 200 μl of the L1-Blue suspension was mixed and allowed to stand at 37 ° C. for 15 minutes, 1 μl of the LB medium containing 50 ppm of ampicillin was inoculated and cultured overnight. The colonized E. coli is cDNA
It contains the plasmid clone pCEM6 having an insert (FIG. 1, middle, lower row).

The nucleotide sequence of the inserted sequence in this plasmid pCEM6 was determined by the dideoxy method (Messing, Methods i).
n Enzymol., 101, 20-78 (1983)). The obtained nucleotide sequence and the amino acid sequence deduced from this sequence are shown in SEQ ID NO: 1. This sequence shows the base sequence of cDNA of carrot high glycine cell wall structure protein specific to somatic embryogenesis and the amino acid sequence of carrot high glycine cell wall structure protein. In the amino acid sequence, the sequence corresponding to the signal peptide has amino acid numbers 1 to 20, and the corresponding DNA sequence has nucleotide numbers 66 to 125.

<4> Expression of antisense gene of high glycine cell wall structure protein in carrot suspension culture cells

(1) Construction of antisense gene of high glycine cell wall structure protein pCEM6 was digested with EcoRI to prepare full-length high glycine cell wall structure protein cDNA. Next, the cleaved end was cloned into T4 DNA polymerase (DNA blunting k
It) was used for smoothing. This fragment and pBI221 whose ends were blunted with T4 DNA polymerase after digestion with SmaI
(Plasmid having 35S promoter of cauliflower mosaic virus (CaMV): Jefferson et al., EMBO J.,
6,3901 (198) fragment was ligated (FIG. 2). pB
I221 was obtained from Clontech.

From the obtained assembly of plasmids, the high glycine cell wall structural protein cD was located downstream of the 35S promoter.
A plasmid in which NA was linked in the opposite direction was selected based on the difference in restriction enzyme cleavage maps. This plasmid has the antisense high glycine cell wall structural protein gene and was named pCEM601.

(2) Introduction of antisense gene of high glycine cell wall structure protein gene into carrot suspension-cultured cells pCEM601 plasmid DNA was prepared according to the method of Tada et al.
Da et al .; Theor. Appl. Genet. (1990)), the cells were introduced into suspension-cultured cells of carrot by electroporation.

Cell mass state 1 with adventitious embryogenesis capacity: about 1
g is 1% Macerozyme R10, 4% cellulase RS (above, Kinki Yakult Co., Ltd.), 0.5% CaC
l ₂ · 2H ₂ O, potassium 0.5% dextran sulfate, 0.4 M
After mixing with 10 ml of enzyme solution (pH 5.5) containing mannitol, the mixture was allowed to stand for 3 hours, and the protoplast suspension was filtered with a nylon mesh of 40 μm to obtain protoplasts.

The obtained protoplasts were added to electroporation buffer (70 mM KCl, 5 mM CaCl ₂ , 5 mM 2- [morpholino] ethanesulfonic acid (MES), 0.4 M mannitol) at 2 × 10 5.
Suspend at ⁶ cells / ml and 0-20 μg DNA
(Plasmid expressing pCEM601 and hygromycin resistance gene) was added. After leaving this mixed solution at 0 ° C. for 20 minutes, an electric pulse (initial electric field 475 V / cm,
The cells were introduced into cells by giving a time decay coefficient T _1/2 = 30.0 ms). After the introduction, the transformed cells were selected using hygromycin to obtain carrot transformed suspension culture cells.

When DNA was extracted from the transformed cultured cells and subjected to Southern hybridization, a DNA fragment derived from pCEM601 was present in 8 strains. When these transformed cultured cells were transplanted to an LS modified medium that does not contain 2,4-D, which is a condition for forming somatic embryos, the adventitious embryo formation rate was 20% compared with the carrot suspension cultured cells used as a control. It fell to 40% and reached 28% on average. From this, it was shown that the high glycine cell wall structure protein gene is a gene having an important function in somatic embryogenesis.

<5> Introduction of Carrot High Glycine Cell Wall Structure Protein Gene into Plant Cells A fragment containing the entire region of carrot high glycine cell wall structure protein gene was prepared by subjecting pCEM6 to limited digestion with BamHI. This fragment was cloned into plasmid pBI221
Cloning into the mHI site yielded plasmid pBI6 (FIG. 3). The plasmid DNA of pBI6 was introduced into carrot protoplast cells by the electroporation method as described above. The resulting cells are 2,4-D
When cultured in an LS-modified medium containing a somatic embryo, 10% to 30%, and an average value of 21% formed an adventitious embryo despite the condition that adventitious embryo formation was not performed. When a part of the somatic embryo was regenerated into a plant, DNA was extracted, and Southern hybridization was carried out. As a result, 5 strains contained a DNA fragment that was considered to be derived from pBI6.

From these results, it was shown that by introducing the high glycine cell wall structure protein gene of the present invention, it is possible to induce somatic embryogenesis in transformed cultured cells even under non-induced somatic embryo induction conditions. .

Further, as described above, it is clear from Arabidopsis chromosomal DNA Southern hybridization that similar sequences are present in plants other than carrots. Therefore, suspension culture of plant species having no redifferentiation ability is possible. It was suggested that redifferentiation can be induced by somatic embryogenesis in cells.

<6> Isolation and Subcloning of Sequence Encoding Signal Peptide CEM6 has a cleavage site for the restriction enzyme PmlI immediately downstream of the region encoding the signal peptide. Therefore, this region can be extracted using PmlI and the EcoRI site present in the vector. Therefore, pCEM6 was digested with restriction enzymes PmlI and EcoRI (purchased from Toyobo) to obtain 0.15 kb.
And two fragments of 0.6 kb were obtained.

Next, T4 DNA polymerase (Takara Shuzo, DN
Ablunting kit) was used to blunt the ends, and these were introduced into the EcoRI site of similarly blunt-ended Bluescript II KS +. As a result, two types of plasmids were obtained.
Of these, the one containing the 0.15 kb fragment contained the signal peptide region and was designated pCEM6t (FIG. 4). This plasmid can be used to bind heterologous genes using the PstI site derived from the vector.

[0076]

INDUSTRIAL APPLICABILITY According to the present invention, a carrot high glycine cell wall structural protein gene associated with somatic embryogenesis was obtained. By expressing this gene in plants, a large amount of high glycine cell wall structure protein can be expressed. Similarly, by expressing an antisense gene of this gene, the expression level of the high glycine cell wall structure protein can be reduced.

Since the carrot high glycine cell wall structure protein gene has a DNA sequence encoding a signal peptide, it is possible to efficiently transfer other gene products to the cell wall by utilizing this signal peptide. it can.

Furthermore, since the high glycine cell wall structural protein gene is specifically expressed during carrot somatic embryogenesis and is involved in somatic embryogenesis, this nucleotide sequence is used as a marker for redifferentiation by somatic embryos. Enables the elucidation of the mechanism of and the isolation of the gene that regulates it.

Further, by introducing the high glycine cell wall structure protein gene of the present invention, it is possible to induce somatic embryogenesis of transformed cultured cells even under non-inducing conditions of somatic embryos. Furthermore, it is suggested that it is possible to induce regeneration by somatic embryogenesis in suspension-cultured cells of plant species that have no ability to redifferentiate.

[0080]

[Sequence list]

SEQ ID NO: 1 Sequence length: 747 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Origin: Organism name: Daucus carota L. cv .
“Kurodagosun”) Organization type: Proembryo State 2 (Proembryo)
"State 2") Direct origin Library name: Carrot proembryo state 2 cDNA library Clone name: CEM6 Sequence features Characteristic symbols: 5'UTR Location: 1..65 Method of determining features: P features Symbol indicating: mRNA Location: 1..747 Method by which the characteristic was determined: P Characteristic symbol: transit peptide coding region Location: 66..125 Method by which the characteristic was determined: S Characteristic symbol: mature peptide coding region Location: 126..551 Feature determining method: S Feature symbol: 3'UTR Location: 552..747 Feature determining method: P Sequence CTCAAAGCAA TATATAAATT TAGCCATTTA GGGTTTGTCT GTGTGTGTGT AAAAGTGTGT 60 GAGGA ATG GCG AAG TTG TAC GTA GTG CTT GTA CTT TCA CTA GTC CTT GTG 110 Met Ala Lys Leu Tyr Val Val Leu Val Leu Ser Leu Val Leu Val 1 5 10 15 CAT GCA ATG GCG GCA CGT GAC ATT CCT ACT GAA AAA CAT GAC ACG GAG 158 His Ala Met Ala Ala Arg Asp Ile Pro Thr Glu Lys His Asp Thr Glu 20 2 5 30 ACG GTG GTT GTC GCT ACC ACC ACT AAT GGT GGC GGC AAA GCC GTT AAG 206 Thr Val Val Val Ala Thr Thr Thr Asn Gly Gly Gly Lys Ala Val Lys 35 40 45 GAC AAG AAG TGC GCT GTT GGA TTT GTC GGC GTT GGC GGT GCG GTT GGC 254 Asp Lys Lys Cys Ala Val Gly Phe Val Gly Val Gly Gly Ala Val Gly 50 55 60 GGT GTG GCT GGT GTT GTT CCA CTT GGC GGC ATT GGC GGC GTT GGG GGA 302 Gly Val Ala Gly Val Val Pro Leu Gly Gly Ile Gly Gly Val Gly Gly 65 70 75 GCT GGT GGA GTT GGT GGT GCT GGT GGA CTT GGC GGC GCA GGT GGG CTA 350 Ala Gly Gly Val Gly Gly Ala Gly Gly Leu Gly Gly Ala Gly Gly Leu 80 85 90 95 GGA GGT CTT GGT GGA CTT GGA GGT GCA AGT GGG CTT GGT GGG CTC GGA 398 Gly Gly Leu Gly Gly Leu Gly Gly Ala Ser Gly Leu Gly Gly Leu Gly 100 105 110 GGT GCA AGT GGG CTT GGT GGA CTA GGA GGT GCA AGT GGG CTT GGG GGG 446 Gly Ala Ser Gly Leu Gly Gly Leu Gly Gly Ala Ser Gly Leu Gly Gly 115 120 125 CTC GGA GGT GCA GGT GCA GGT GGA CTA GGT GGG CTA GGT GGC GTT GGA 494 Leu Gly Gly Ala Gly Ala Gly Gly Leu Gly Gly Leu Gly Gly Val Gly 130 135 140 GGT GGC GTT GGG GGA GGT GCT GGT GGT GCC GGT GGA GTT GGT GGT GGT 542 Gly Gly Val Gly Gly Gly Ala Gly Gly Ala Gly Gly Val Gly Gly Gly 145 150 155 GTT TTA CCT TGAAATGATT AAGAACAAAG ATGCATGCAA CATTCA LeCATU TTTTTCGTAT ATTAGTACGG AGGAGCTGGT GGATCTGTTC TACTTCTAGG 651 GTTTAATTAA GTTGGCATTT GGTTTCAACA AGTATTATTG TATTATTCGA TTACGTAACT 711 ATGTTTTGTT TTCCTTAAAA AAAAAAAAAA AAAAAA 747

[Brief description of drawings]

FIG. 1 is a diagram showing a production scheme of a plasmid pCEM6.

FIG. 2 is a diagram showing a production scheme of plasmid pCEM601.

FIG. 3 is a diagram showing a production scheme of plasmid pBI6.

FIG. 4 is a diagram showing a production scheme of plasmid pCEM6t.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location // C12N 15/87

Claims (7)

[Claims] 1. A DNA sequence encoding a carrot high glycine cell wall structure protein precursor having the amino acid sequence of Nos. 1 to 162 shown in SEQ ID NO: 1. 2. A DNA sequence encoding a signal peptide of carrot high glycine cell wall structure protein having the amino acid sequence of 1 to 20 shown in SEQ ID NO: 1. 3. A DNA sequence coding for a matured carrot high glycine cell wall structure protein having the amino acid sequence of Nos. 21 to 162 shown in SEQ ID NO: 1. 4. A method for inducing somatic embryogenesis by introducing the DNA sequence according to claim 1 or 3 into plant suspension culture cells. 5. A method for increasing the cell wall structural protein content by introducing the DNA sequence according to claim 1 or 3 into a plant cell. 6. A method for efficiently transferring the gene product to the cell wall by introducing into a plant cell a DNA sequence in which another gene is linked upstream of the DNA sequence according to claim 2. 7. A plant in which all or part of the DNA sequence according to claim 1 or 3 is ligated downstream of a promoter capable of being expressed in a plant cell and in a forward or reverse direction with respect to the promoter, in a plant cell. A method of modifying a cell wall component by introducing it.