JPH1156367A - Gene encoding cellulose synthesizing apparatus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject gene encoding a protein constituting a cellulose synthesizing apparatus, possibly useful for preparing wood high in cellulose content. SOLUTION: This DNA encodes a protein derived from red bean containing an amino acid sequence of the formula. An mRNA is prepared from the epicotyl of red bean, a cDNA is synthesized, a cDNA library is made, an antibody screening by an antibody to a crude purified substance of a cellulose synthesizing enzyme complex is carried out, a positive clone is subjected to second and third clonings and made into a single plaque and a base sequence is determined to give an estimated amino acid sequence.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a protein constituting cellulose synthase and its gene.

[0002]

2. Description of the Related Art Cellulose contains 2,000 to 2,000 glucose.
It is a fibrous macromolecule with about 0 bonds, and is a carbohydrate that accounts for about one-third of plants. Cellulose is used as a raw material for paper pulp, a raw material for fiber, a rough feed, and a raw material for fuel, and is a very important substance in industry.

[0003] It is considered that this cellulose is synthesized by the action of a specific enzyme in a plant cell, and isolation of a gene from a prokaryotic acetic acid bacterium has been reported for cellulose synthase. (Saxena IMet
al.Plant Mol. Biol. 15, 673-683 (1990): Wong HCet al.
Proc. Natl. Acad. Sci. USA 87, 8130-8134 (1990)). One group has created mutant strains of acetic acid bacteria that do not synthesize cellulose by mutagenesis and identified operons encoding four proteins necessary for cellulose synthesis (Wong HC et al. Proc. Natl. Acad. Sci.USA 87,8130-81
34 (1990)). These four genes have been shown by complementation tests and gene disruption experiments of mutants to be all necessary for complete cellulose synthesis. Another group reported purification of cellulose synthase from the membrane fraction of acetic acid bacteria, determination of the amino acid sequence, and isolation of the gene (Saxena IM et al. Plant Mol. Biol. 15, 673-683 (1992).
0)). It has been suggested that among these proteins, there is a protein involved in crystallization of glucan chains, in addition to a protein (Cel A) which is a center of activity for synthesizing cellulose from UDP-glucose.

In Agrobacterium, two operons required for cellulose synthesis have been identified (Matthy
sse AGet al. J. Bacteriol. 177, 1069-1075 (1995)).
This is a gene isolated from a mutant strain of Agrobacterium that does not perform cellulose synthesis, and the presence of five genes in two operons has been reported. Transposon insertion indicated that all of these genes were required for cellulose synthesis. Compared with the cellulose synthase operon of acetic acid bacteria, genes for the protein (Cel A), which is the center of the activity of synthesizing cellulose from UDP-glucose, are common, but homology was found for other genes. Not.

[0005] On the other hand, biochemical analysis of cellulose synthase of higher plants has hardly progressed. A protein having the same antigenicity as the cellulose synthase of acetic acid bacteria can be isolated from plants (Mayer R. et al. Proc. Natl. Acad. S.
ci. USA 88, 5472-54768 (1991)), and many attempts have been made to clone plant cellulose synthase using the gene of acetic acid bacteria as a probe, but no gene isolation has been reported to date. Brown and colleagues also showed that the fraction solubilized with 1% digitonin from the membrane fraction of cotton fiber had cellulose synthetic activity (Okuda K. et al. Plan
t Physiol. 101, 1131-1142 (1993)), which indicates that it contains many other components and that the yield of cellulose is very low (Delmer DP et al. Plan
t Physiol. 103, 307-308 (1993)), it would not be possible to identify the synthetic activity of cellulose.

Recently, the Cel A gene was also isolated from plants (Pear JR et al. Proc. Natl. Acad. Sci. USA 9
3,12637-12642 (1996)). This is a random sequence of about 250 cDNAs produced from cotton fibers, which have a high level of cellulose synthesis, and isolated a gene homologous to the bacterial Cel A gene. In recent years, homologues of Cel A gene have also been found in rice and arabidopsis ESTs. Delmer et al. Found that the internal region of the Cel A gene
It shows that it binds to glucose, but isolated Ce
l There is no evidence that the A gene actually does cellulose synthesis. This seems to be due to the fact that the function of the cellulose synthesis system of a plant cannot be identified simply by isolating a single gene, because of its extremely complicated structure. Thus, at present, little is known about the cellulose synthesis of plants.

[0007] Electron microscopy shows that cellulose synthesis in plants is synthesized from a device called a cellulose synthase complex in the plasma membrane (Giddings TH et al. J. Cell Biol. 84,327). -339 (198
0)). This cellulose synthase complex is commonly called a rosette and has a rosette shape composed of six granules.
The present inventors have reported the world's first isolation of a plant cellulose synthase complex using an antibody against tubulin (the meeting of the Plant Physiological Society held on March 27, 1996, collection of abstracts p67). The diameter of the granule, called the cellulose synthase complex, is about 10 nm, which is in good agreement with the size of one of the granules constituting the rosette.
However, in the report, the isolated cellulose synthase complex and the subunits constituting the complex have not been completely purified, and the gene encoding the subunit has not been isolated.

[0008]

An object of the present invention is to isolate a protein constituting the cellulose synthase complex and a gene encoding the protein.

[0009]

Means for Solving the Problems The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, a crude purified product of a cellulose synthase complex was subjected to affinity chromatography using an anti-tubulin antibody. A plurality of genes encoding proteins that bind to the isolated cellulose synthase complex were isolated by antibody screening using an antibody against the crude product. Next, by performing epitope selection using the proteins expressed in the obtained individual genes, an antibody specific to the expressed protein was purified from the antibodies against the cellulose synthase complex. Furthermore, these antibodies were reacted with the isolated granules of the cellulose synthase complex and observed with an electron microscope.As a result, the present inventors found that one of the purified antibodies actually bound to the cellulose synthase complex. I found it.

That is, the present invention relates to a protein constituting the cellulose synthesizer and a gene encoding the protein, and more specifically, (1) encodes a protein containing the amino acid sequence of SEQ ID NO: 1. DN
A, (2) DNA consisting of the nucleotide sequence of SEQ ID NO: 2
A DNA that hybridizes with the DNA encoding a protein constituting the cellulose synthase complex,
(3) a vector containing the DNA of (1) or (2), (4) a host cell containing the vector of (3),
(5) a protein comprising the amino acid sequence of SEQ ID NO: 1, (6) a protein encoded by a DNA that hybridizes with the DNA consisting of the nucleotide sequence of SEQ ID NO: 2, and comprising a cellulose synthase complex (7) The method for producing a protein according to (5) or (6), which comprises culturing the host cell according to (4). (8) The method according to (1) or (2). (9) A plant comprising the plant cell according to (8), (10) an antibody that binds to the protein according to (5) or (6).

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention relates to a protein constituting a cellulose synthase complex. Among the proteins of the present invention, an adzuki-derived protein is a subunit of a cellulose synthase complex having a molecular weight of about 33 kDa in SDS polyacrylamide gel electrophoresis. When an antibody against this protein is reacted with a cellulose synthase complex, the cellulose synthesis activity of the complex is inhibited. Therefore, it is considered that the protein is deeply involved in the cellulose synthesis activity of the cellulose synthase complex. Can be

[0012] The protein of the present invention can be prepared as a recombinant protein or a natural protein. A recombinant protein can be prepared by incorporating a DNA encoding the protein of the present invention into an appropriate expression vector, introducing the DNA into a host cell, and culturing the transformant, as described below. On the other hand, if it is a natural protein, for example, it is possible to prepare an antibody against the recombinant protein and prepare it by affinity chromatography using the antibody.

SEQ ID NO: 1 shows the amino acid sequence of a protein derived from adzuki bean. Although this amino acid sequence is not the entire sequence of the protein, compared to the deduced amino acid sequence of the full-length cDNA of the homologue isolated in tobacco and Arabidopsis, most of the protein It can be said that it is code. A person skilled in the art can isolate a full-length cDNA using a known method such as a hybridization method based on the DNA described in SEQ ID NO: 2 which encodes the protein described in SEQ ID NO: 1,
It is usually possible to isolate a complete protein based on this, and the complete protein thus isolated is also included in the protein of the present invention.

Further, those skilled in the art will modify the protein shown in SEQ ID NO: 1 by appropriately substituting the amino acid using a known method to prepare a protein having substantially the same function. It is possible to Thus, the amino acid sequence in the protein of SEQ ID NO: 1 has an amino acid sequence in which one or several amino acids have been substituted, deleted, or added, and is substantially equivalent to the protein of SEQ ID NO: 1. A protein having a function is also included in the protein of the present invention. Known methods for making such amino acid modifications include, for example, site-directed mutagenesis (eg, Zoller MJand Smith M. Methods in Enzyno
logy 100, p468 (1983)).

[0015] Further, for those skilled in the art, a hybridization technique (molecular cloning cold), which is a well-known technique, is known.
Based on the DNA sequence of SEQ ID NO: 2 (or a part thereof), a DNA highly homologous thereto is isolated from other organisms using, for example, Spring harbor laboratry Press) and the like. As in the case of the protein described in No. 1: 1, it is also possible to obtain a protein involved in cellulose synthesis. Thus, the protein of the present invention includes a protein encoded by a DNA that hybridizes with a DNA consisting of the DNA sequence of SEQ ID NO: 2, which constitutes a cellulose synthase complex. Other organisms that isolate the hybridizing DNA include, for example,
Herbaceous dicotyledonous plants such as Arabidopsis thaliana and tobacco, monocotyledonous plants such as rice and corn, and woody plants such as eucalyptus and poplar are exemplified. Note that a DNA encoding such a protein derived from another organism usually has high homology to the DNA described in SEQ ID NO: 2. High homology refers to at least 60% or more of the DNA of SEQ ID NO: 2,
It preferably refers to sequence identity of at least 70% or more, more preferably at least 80% or more. DNA like this
The conditions for the hybridization for isolating is 6 × SSC, 0.1% SDS, at 37 ° C., preferably 3 × SSC.
xSSC, 0.1% SDS, at 42 ° C, more preferably,
1xSSC, 0.1% SDS, 65 ° C.

The present invention also relates to a DNA encoding the protein of the present invention. The DNA encoding the protein of the present invention may be cDNA, genomic DNA, or synthetic.
It may be DNA. The DNA of the present invention can be used for producing a protein of the present invention as a recombinant protein, in addition to isolating a protein having substantially the same activity as the protein of SEQ ID NO: 1 described above. That is,
The DNA of the present invention is incorporated into an appropriate vector and introduced into a host cell, and the recombinant protein is expressed in the host cell.
By purifying the recombinant protein from this transformant, it is possible to produce the recombinant protein.

The host cell used for producing the recombinant protein is not particularly limited.
Yeast and the like. Further, as an expression vector used for preparation of a recombinant protein, those skilled in the art can appropriately select a suitable vector depending on the host cell,
For example, in Escherichia coli, a “pGEX-based vector” (Pharma
cia Biotech) and yeast, such as "pESP vector" (Stratagene). The introduction of the vector into the host cell is performed, for example, by the method of Hanahan (Hana
han, DJ Mol. Biol. 166, 557-580 (1983)) or an electroporation method known to those skilled in the art. In addition, general gene manipulation such as insertion of DNA into a vector is described, for example, in the literature “molecular
cloning Cold Spring harbor laboratry Press ".

The resulting transformed cells can be cultured under appropriate conditions to express the recombinant protein. Culture conditions vary depending on factors such as the type of the transformed cells to be cultured and the vector introduced therein, but if the host cell is Escherichia coli, for example, a culture medium known to those skilled in the art, such as LB medium, may be used. When the host cell is yeast, the culture is performed at about 30 ° C. using a medium known to those skilled in the art, such as a YPAD medium. In addition, those skilled in the art can appropriately select appropriate culture conditions.

The recombinant protein of the present invention can be purified from the transformant by a method known to those skilled in the art, for example, by appropriately combining various chromatography, electrophoresis, gel filtration and the like. Further, when the protein of the present invention is expressed as a fusion protein with a label such as 6X histidine or glutathione S-transferase, it is possible to easily purify the protein by affinity chromatography on the label.

Further, the DNA of the present invention can be used for producing transgenic plants. That is, a transgenic plant can be produced by introducing the DNA of the present invention into a plant cell and regenerating the plant cell into a plant. This is expected to improve the amount of cellulose synthesized in the plant and the properties of the synthesized cellulose. For example, when the DNA of the present invention is introduced into forest trees, forest trees having a high cellulose content may be able to be produced.

Plants into which the DNA of the present invention is introduced include herbaceous dicotyledonous plants such as Arabidopsis thaliana and tobacco, monocotyledonous plants such as rice and corn, and woody plants such as eucalyptus and poplar. The DNA of the present invention can be incorporated into an appropriate vector and introduced into a plant cell. In this case, pBI121 is an example of a vector to be used. DNA can be introduced into plant cells by a direct introduction method such as an electroporation method, a polyethylene glycol method, or a particle gun method. In addition, depending on the plant species, the vacuum infiltration method (Bechtold N. et al. CRAcod. Sci. Paris, Sc
ience de la vie / Life sciences 316 (1993)), a root transformation method (eg, Valvekens D.et)
Acad. Sci. USA 85, 5536-5540 (1988)) or other Agrobacterium-mediated gene transfer methods. The form of the plant cell into which the DNA is introduced varies depending on the type of the above-described gene transfer method, and includes suspension culture cells, protoplasts, callus, plant tissues such as root and leaf sections, plants, and the like. .

The regeneration of a plant from a plant cell into which the DNA of the present invention has been introduced is carried out by a method known to those skilled in the art, for example, the induction of a shoot by culturing the plant cell into which the DNA has been introduced in a shoot induction medium. Thereafter, a method such as culturing in a root induction medium to induce the root is used (for example, Damm
B. and Willmitzer L. Mol. Gen. Genet. 213, 15-20 (1988)). When DNA is introduced into a plant via Agrobacterium as in a vacuum infiltration method, for example, the method of Bechtold et al.
N.et al.CRAcod.Sci.Paris, Sciences de la vie / Lif
e sciences 316 (1993)). In addition, it was introduced by using vacuum infiltration method etc.
Since DNA is efficiently integrated into the genome of plant cells, D
It is possible to grow plants into which NA has been introduced and collect transformed seeds.

The present invention also relates to an antibody that binds to the protein of the present invention. Those skilled in the art can prepare an antibody that binds to the protein of the present invention by a conventional method. That is, in the case of a polyclonal antibody, an antibody is prepared by inoculating an antigen into an immunized animal such as a goat or a rabbit, and blood is collected and prepared from serum by salting out, ion exchange chromatography, affinity chromatography, or another method. If it is a monoclonal antibody, it is possible to inoculate a mouse with an antigen, perform cell fusion, cloning, and ascites from the immunization of the mouse, collect ascites, and perform salting out, ion exchange chromatography, It can be prepared by a method such as affinity chromatography. The antibody of the present invention can be used for, for example, controlling the amount of cellulose synthesis in plants, in addition to preparing the protein and the cellulose synthase complex of the present invention.

Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited to these Examples.

[0025]

【Example】

[Example 1] Preparation of extract solution Plant tissues or cells of adzuki bean, tobacco, and Arabidopsis thaliana were treated with 50 mM PIPES Buffer A (300 mM sucrose, 5 mM E
DTA, 5 mM EGTA, 3 mM ヂ thiothreitol, protease inhibitor (leupeptin (50 μg / ml), 0.2 mM pABS
The mixture was crushed with F (4- (2-aminoethyl) benzenesulfonyl hydrochloride) (pH 7.6) under ice cooling. 8,0 after filtration with gauze
Centrifuged at 00xg, 4 ° C for 10 minutes. 100,000xg of supernatant,
After centrifugation at 4 ° C for 20 minutes, the precipitated membrane fraction was suspended in 10 mM K-phosphate buffer containing 250 mM sucrose, and resuspended at 100,000 xg,
Washing was performed by centrifugation at 4 ° C for 20 minutes. The precipitate was suspended in a small amount of 10 mM K-phosphate buffer and subjected to a two-phase partitioning method using polyethylene glycol and dextran to separate only the cell membrane granules in the upper phase. Equivalent volume of 50 mM P
IPES B (250 mM sucrose, 0.1 mM dithiothreitol, leupeptin (50 μg / ml), 0.2 mM pABSF (fluorinated 4-
(2-aminoethyl) benzenesulfonyl hydrochloride) (pH 7.
6)), and centrifuged at 100,000xg at 4 ° C for 20 minutes.
A cell membrane precipitate was obtained. For cell membrane precipitation, 1% CHAPS (surfactant) was added, and the mixture was stirred well and kept on ice for 30 minutes. After solubilizing the cell membrane, the mixture was centrifuged at 100,000 × g at 4 ° C. for 20 minutes, and the supernatant was collected. This extract was used as a starting material for isolation of the cellulose synthase complex.

[Example 2] Preparation of tubulin antibody column Since the cellulose synthase complex contains tubulin as a main component, affinity chromatography was performed using a tubulin antibody column. Approximately 3 ml of rabbit antiserum against bean tubulin
It was mixed with 100 mM PIPES Buffer (pH 7.6) and cooled to 4 ° C. Add Sepharose 4B activated with cyanogen bromide
8 ml was added and reacted at 4 ° C. for 12 hours to bind the tubulin antibody to Sepharose. Sepharose to which the tubulin antibody was bound was thoroughly washed with a 0.2 M glycine-HCl buffer (pH 2.6) and then equilibrated with 50 mM PIPES Buffer B before use.

Example 3 Isolation of Cellulose Synthase Complex by Affinity Chromatography After flowing the extract through a tubulin antibody column at a rate of 50 microliters / min, "50 mM PIPES Buffer B + 0.2% C
HAPS + 0.3M NaCl ". The enzyme complex bound to the column was eluted with 0.2 M glycine-HCl buffer (containing 0.2% CHAPS, pH 2.6) and immediately neutralized with 2 M Tris. As a result, a large amount of granules (cellulose synthase complex) having a diameter of about 8 to 10 nm were observed. This was appropriately diluted and used as a crudely purified product of cellulose synthase.

Example 4 Preparation of Antibody and Its Effect The isolated crude product was placed in a dialysis tube, dialyzed against water to remove salts, and then concentrated by a centrifugal evaporator. Approximately 50 micrograms of concentrated protein per rabbit was mixed with adjuvant and immunized subcutaneously on the back of the rabbit. Three weeks later, approximately the same amount of protein was injected again subcutaneously. Three weeks later, the last immunization was performed in the same manner, and five days later, whole blood was collected. Thus, an antibody against the cellulose synthase complex was obtained.

Example 5 Antibody Screening mRNA was prepared from adzuki bean epicotyl and this mRNA was used as a template.
After synthesizing cDNA and inserting the cDNA into the λZAPII vector,
n vitro packaging was performed to generate a cDNA library consisting of about 5 million independent clones. This library was used for screening. Phage E. coli XL
The cells were infected with 1-Blue and grown on LB agar medium at 42 ° C. for 3.5 hours.

Next, a nitrocellulose filter previously immersed in 10 mM IPTG and dried was placed on the medium, and
Incubation was further performed at 3.5 ° C. for 3.5 hours. Remove the filter from the medium and add TBST (20mM Tris-HCL pH 7.5, 150mM NaCl, 0.05% T
Ween-20 (v / v)). Filters were shaken for 1 hour in blocking solution (1% BSA (w / v) in TBST).
The filter was reacted with the primary antibody prepared for the cellulose synthase complex for 1 hour. After washing with TBST, the filter was reacted with alkaline phosphatase-bound secondary antibody for 1 hour. After washing with TBST, the filter was washed with TBS (20 mM Tris-HCL pH 7.5, 150 mM NaCl),
Ween20 was removed. Filter the color solution (0.3mg / ml N
BT, 0.15mg / ml BCIP in 100mM Tris-HCl pH9.5,100mM Na
Cl, 5 mM MgCl ₂ ) and reacted in the dark. Wash the filter with TBS and stop solution (20 mM Tris-HCl pH2.9, 1 mM E
DTA) to stop the reaction. Filters were dried and stored in the dark. Secondary screening and tertiary screening were performed on positive clones, and single plaques were stored.

Example 6 Determination of Nucleotide Sequence The helper phage ExAssist was infected to the obtained positive clone, and in vivo excision (from λZAPII to pBluescript) was performed.
E. coli XL1-Blue having pBluescript into which cDNA was inserted was isolated. Then, E. coli XL1-Bl
The ue was cultured, and a plasmid pBluescript was prepared using "QIAprep-spin Plasmid Kit" (manufactured by QIAGEN). Using the obtained plasmid as a template, "DNA Sequencing Kit"
(Perkin Elmer) to perform a sequencing reaction. The reaction product is “Model 373S Sequencer” (PERKINE E
(LMER) and the nucleotide sequence was determined. The determined nucleotide sequence is shown in SEQ ID NO: 2, and the deduced amino acid sequence is shown in SEQ ID NO: 1.

In addition, the base sequence is analyzed by “Mac Vector”, and homology search in other organisms is performed by blast search.
And the EST database. As a result,
The DNA described in SEQ ID NO: 1 showed 55.9% homology with the Arabidopsis gene and 62.7% with the tobacco gene at the DNA level. The deduced amino acid sequence is 52.5% for Arabidopsis protein and 59.5% for tobacco protein.
Showed a homology of There is no proof that Arabidopsis and tobacco genes are involved in cellulose synthesis.

Example 7 Epitope Selection Positive clones were infected with E. coli XL1-Blue and plated on a plate.
Sprinkled to become 2000 plaques. 42 on LB agar
After growing at 3.5 ° C. for 3.5 hours, a nitrocellulose filter immersed in 10 mM IPTG and dried was placed on the medium, and incubated at 37 ° C.
Incubation was further performed at 3.5 ° C. for 3.5 hours. Remove the filter from the medium and add TBST (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% Tw
een20 (v / v)). Filters were soaked in blocking solution (1% BSA (w / v) in TBST) for 1 hour. The filter was reacted with the primary antibody prepared for the cellulose synthase complex for 1 hour. After washing with TBST, antibodies that did not bind to the positive clones were removed. The antibody bound to the positive clone was added to 2 ml of elution buffer (5 mM glycine-HCl (pH 2.3), 1
Elution was carried out with 50 mM NaCl, 0.5% Triton X-100, BSA (100 μg / ml). Tris-HCl pH7.5 was added to a final concentration of 50 mM to neutralize the antibody solution. Further elution and neutralization
Repeated twice, the antibody solution was collected. The cellulose synthase complex was subjected to SDS electrophoresis using 10% acrylamide, and the gel was placed in a transfer solution (25 mM Tris, 192 mM glycine, 2
(0% methanol, pH 8.3) for 30 minutes. Proteins are blotted electrically to a nitrocellulose filter, and the filter is washed with TBST (20 mM Tris-HCl pH 7.5, 150 mM
NaCl, 0.05% Tween20 (v / v)). Filter 1
After shaking in a blocking solution (1% BSA (w / v) in TBST) for 1 hour, the filter was reacted for 1 hour with the primary antibody prepared against the cellulose synthase complex and the above-mentioned antibody collected by elution. Was. After washing with TBST, the filter was washed with a secondary antibody to which alkaline phosphatase was bound.
Allowed to react for hours. After washing with TBST, remove the filter with TBS
(20mM Tris-HCl pH7.5, 150mM NaCl) and Tween2
0 was removed. Filter the color solution (0.3mg / ml NBT, 0.
15mg / ml BCIP in 100mM Tris-HCl pH9.5,100mM NaCl, 5m
M MgCl ₂ ) and reacted in the dark. TBS filter
And stop solution (20mM Tris-HCl pH2.9, 1mM EDTA)
To stop the reaction.

Example 8 Immune Electron Microscope A cellulose synthase complex was placed on a copper grid.
PBS containing 1% BSA (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na ₂ PO ₄
・ Blocking was performed with a 7H ₂ O, 1.4 mM KH ₂ PO _{4 (} pH 7.4) solution. The antibody obtained by the epitope selection was placed on a grid and reacted for 10 minutes. After washing with a PBS solution containing 1% BSA, a secondary antibody bound to colloidal gold was placed on a grid and reacted for 10 minutes. After washing with a PBS solution containing 1% BAS, the plate was washed with a PBS solution, and further washed with H ₂ O to remove salts. Stained with uranyl acetate. The antibodies were observed with a transmission electron microscope. As a result, the antibody actually bound to the cellulose synthase complex (FIG. 1).

Example 9 Cellulose Synthesis Activity Inhibition Experiment A cellulose synthase complex was reacted with an antibody obtained by epitope selection for 30 minutes. The substrates UDP-glucose and Mg ²⁺ were added, and the mixture was further reacted for 1 hour, and the cellulose fibers were observed with an electron microscope. As a result, it was revealed that cellulose was not synthesized from the cellulose synthase complex reacted with the antibody obtained by epitope selection, and that the antibody inhibited the cellulose synthesis activity.

[0036]

Industrial Applicability According to the present invention, a protein constituting a cellulose synthase complex, and a DN encoding the protein
A, an antibody that binds to the protein was provided. The protein and DNA of the present invention are expected to be used for improving the amount of cellulose synthesized in plants and the properties of synthesized cellulose. Further, the antibody of the present invention can be used for preparing the protein or the cellulose synthase complex of the present invention, and for regulating cellulose synthesis.

[0037]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 200 Sequence type: Amino acid Topology: Linear Sequence type: Protein sequence Lys Val Leu Pro Lys Ile Lys Lys Val Phe Glu Lys Asn Thr Ser 1 5 10 15 Lys Lys Ala Ala Ala Ala Glu Ala Thr Lys Ser Phe Asp Asp Ser Lys 20 25 30 Glu Glu Tyr Asn Lys Val Phe Glu Glu Lys Lys Thr Glu Leu Gln Thr 35 40 45 Lys Val Val Glu Ile Tyr Glu Ala Ser Pro Thr Glu Ile Lys Ser Leu 50 55 60 Val Lys Glu Arg Lys Glu Gly Gly Leu Lys Lys Asn Thr Thr Glu Val 65 70 75 His Lys Phe Leu Glu Glu Leu Val Lys Ile Asp Phe Pro Gly Ser Lys 80 85 90 95 Ala Ala Ser Glu Ala Ser Ser Lys Phe Gly Pro Thr Leu Ala Ser Ser 100 105 110 Ser Val Phe Phe Val Phe Glu Lys Val Ser Thr Phe Val Val Thr Glu 115 120 125 Glu Lys Glu Glu Lys Gly Glu Thr Lys Thr Glu Glu Thr Ser Ser Thr 130 135 140 Val Val Gln Glu Arg Glu Ile Val Val Glu Glu Glu Lys Lys Glu Asp 145 150 155 Glu Lys Ala Gln Val Ile Glu Thr Ser Gly Glu Lys Lys Val Glu Glu 160 165 170 175 Gln Pro Ala Glu Ala Ala Ala Lys Glu Glu Glu lu Lys Ser Thr Glu Ala 180 185 190 Ala Glu Lys Glu Glu Ala Ala Lys Ala 195 200 SEQ ID NO: 2 Sequence length: 902 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type : cDNA to mRNA sequence CC AAG GTT CTT CCC AAG ATC AAG AAG GTT TTC GAG AAG AAT ACC TCC 47 Lys Val Leu Pro Lys Ile Lys Lys Val Phe Glu Lys Asn Thr Ser 1 5 10 15 AAG AAA GCT GCT GCT GCT GAG GCC ACC AAG TCC TTC GAT GAC TCT AAG 95 Lys Lys Ala Ala Ala Ala Glu Ala Thr Lys Ser Phe Asp Asp Ser Lys 20 25 30 GAG GAA TAC AAC AAA GTG TTT GAA GAA AAG AAG ACT GAA CTT CAA ACT 143 Glu Glu Tyr Asn Lys Val Phe Glu Glu Lys Lys Thr Glu Leu Gln Thr 35 40 45 AAA GTG GTT GAA ATA TAT GAG GCT TCA CCA ACT GAA ATC AAG AGT TTG 191 Lys Val Val Glu Ile Tyr Glu Ala Ser Pro Thr Glu Ile Lys Ser Leu 50 55 60 GTT AAA GAA CGC AAA GAA GGA GGG CTA AAG AAG AAC ACC ACA GAA GTT 239 Val Lys Glu Arg Lys Glu Gly Gly Leu Lys Lys Asn Thr Thr Glu Val 65 70 75 CAC AAG TTC CTA GAA GAG CTT GTT AAA ATT GAT TTC CCT GGA TCA AAG 287 His Lys Phe Leu Glu Glu Leu Val Lys Ile Asp Phe Pro Gly Ser Lys 80 85 90 95 GCG GCA TCT GAA GCA TCT TCT AAG TTT GGA CCA ACC TTG GCT TCG AGT 335 Ala Ala Ser Glu Ala Ser Ser Lys Phe Gly Pro Thr Leu Ala Ser Ser 100 105 110 TCG GTT TTC TTT GTG TTT GAG AAG GTG TCA ACT TTC GTT GTT ACA GAA 383 Ser Val Phe Phe Val Phe Glu Lys Val Ser Thr Phe Val Val Thr Glu 115 120 125 GAG AAA GAA GAG AAG GGT GAA ACT AAA ACA GAA GAA ACA AGT AGT ACT 431 Glu Lys Glu Glu Lys Gly Glu Thr Lys Thr Glu Glu Thr Ser Ser Thr 130 135 140 GTT GTC CAA GAG AGA GAG ATA GTG GTT GAA GAG GAG AAA AAG GAA GAT 479 Val Val Gln Glu Arg Glu Ile Val Val Glu Glu Glu Lys Lys Glu Asp 145 150 155 GAG AAA GCA CAA GTA ATA GAG ACA AGT GGC GAG AAA AAG GTG GAA GAA 527 Glu Lys Ala Gln Val Ile Glu Thr Ser Gly Glu Lys Lys Val Glu Glu 160 165 170 175 CAA CCA GCT GAA GCT GCT GCA AAA GAG GAA GAG AAA TCT ACT GAA GCA 575 Gln Pro Ala Glu Ala Ala Ala Lys Glu Glu Glu Lys Ser Thr Glu Ala 180 185 190 GCT GAG AAA GAA GAA GCA GCA AAG GCT TGAAAATTTG GTACAGAAAA 622 Ala Glu Lys Glu Glu Ala Ala Lys Ala 195 200 AGATGTAGCT GCTTTCTTGA AGGATGCAGC AGATATACTG TACTCTTAGT TTGGATATAC 682 TGAAAATTGT GACATAGCAT TCCCTTATTC TTTTATTCCT GTGTAAAATC GGTTTTGTGG 742 TGCTTAGTTT GATTTTGCTG CAGATTTGGT GATTAGTATG CATGTGAATG TTGTATGGTG 802 AATTGTTTTG TAATTTTCAC TTTGATTTCT CATTCAAGAA ATTGTAATAA TAATTGGTTC 862 ATGGAACCCT TCCTGTAGCT AAAAAAAAAA AAAAAAAAAA 902

[Brief description of the drawings]

FIG. 1 is an electron micrograph showing the binding of an antibody against the protein of the present invention to a cellulose synthase complex.
It is observed that a black dot-like antibody is bound on the granules of the cellulose synthase complex.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12N 9/10 G01N 33/53 D G01N 33/53 33/531 A 33/531 33/566 33/566 33/573 A 33 / 573 C12N 5/00 C // (C12N 15/09 ZNA C12R 1:91) (72) Inventor Tomohiko Kato 2-1-6 Sengen, Tsukuba, Ibaraki Pref. Tsukuba Research Support Center D-21 Mitsui Co., Ltd. Plant Inside the Bio Research Laboratory (72) Inventor Shigeru Sato 2-1-6 Sengen, Tsukuba, Ibaraki Pref. Tsukuba Research Support Center D-21 Inside the Plant Bio Research Laboratory, Mitsui Co., Ltd. (72) Inventor Daisuke Shibata 2 Chien, Tsukuba, Ibaraki Pref. -1-6 Tsukuba Research Support Center D-21 Mitsui Industry Co., Ltd. Plant Biotechnology Research Institute

Claims (10)

[Claims] 1. A DNA encoding a protein comprising the amino acid sequence of SEQ ID NO: 1. 2. It consists of the nucleotide sequence of SEQ ID NO: 2.
DNA that hybridizes with DNA and encodes a protein that constitutes a cellulose synthase complex. 3. A vector comprising the DNA according to claim 1 or 2. 4. A host cell comprising the vector according to claim 3. 5. A protein comprising the amino acid sequence of SEQ ID NO: 1. 6. It consists of the nucleotide sequence of SEQ ID NO: 2.
A protein encoded by DNA that hybridizes with DNA and that constitutes a cellulose synthase complex. 7. The method for producing the protein according to claim 5, wherein the host cell according to claim 4 is cultured. 8. A plant cell into which the DNA according to claim 1 or 2 has been inserted so that it can be expressed. 9. A plant comprising the plant cell according to claim 8. 10. An antibody that binds to the protein according to claim 5 or 6.