JPH0779778A - Endo-type xyloglucan transferase gene - Google Patents

Abstract

PURPOSE:To provide a new endo-type xyloglucan transferase gene expressed by a specific restriction enzyme map, having important significance in the growth mechanism of plant cell wall and participating in the reconstruction, molecular weight disproportionation, etc., of xyloglucan. CONSTITUTION:This new endo-type xyloglucan transferase gene is expressed by the restriction enzyme maps of the formulas I to III and originated from corn (about 1.2 kbp), rice (about 1.1 kbp) or tobacco (about 1.2 kbp). It has important significance in the growth mechanism of plant cell wall and participates in the reconstruction, molecular weight disproportionation, etc., of xyloglucan. These genes can be produced by germinating the seeds of corn, rice, tobacco, etc., extracting mRNA from the sprout by conventional method, synthesizing a cDNA by using the mRNA as a template, preparing a cDNA library from the cDNA and cloning the library.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel endo-type xyloglucan transferase gene.

[0002]

2. Description of the Related Art Xyloglucan (hereinafter abbreviated as XG) is a main component of plant cell wall. In the plant cell wall, it binds to the surface of cellulose microfibrils to crosslink the cellulose microfibrils to form a complex network structure. There is.
It is thought that the decomposition and remodeling of XG crosslinks are required for the construction and remodeling of the cell wall, but the mechanism has not been clarified. Albersheim XG
The hypothesis that the endo-type transglycosylase acts on the mechanism of reconstitution of Escherichia coli [Plant Biochemistry, 1976 Academic Press (Academic Press), No. 225
274]. In addition, the present inventors also previously, the legume plant Vigna angularis (Vigna angularis) seedlings of the seedling epicotyl apoplast part (of the plant, all outside the plasma membrane of the cell, that is, the cell wall and the cell 20%
It has been found that the fraction from the saturated to 80% saturated ammonium sulfate precipitate disproportionates the molecular weight of XG molecules [Nishitani]
Physiologia Plantarum
tarum), Vol. 82, p. 490-497 (199
1)].

[0003]

SUMMARY OF THE INVENTION The object of the present invention is to provide an XG
The present invention provides a gene encoding an endo-type XG transferase, which is an enzyme involved in the reconstitution of the enzyme, the disproportionation of molecular weight, and the like.

[0004]

The present invention will be described in brief. The present invention relates to an endo-type XG transferase gene.
~ It is characterized by being represented by the restriction enzyme map shown in FIG. 3, respectively.

The present inventors have succeeded in isolating and purifying an enzyme involved in reconstitution of XG, disproportionation of molecular weight, etc. from Vigna angularis. Next, the enzyme has a novel plant physiologically important action of cleaving the XG molecule and recombining the resulting XG molecule fragment with another XG,
It was found that the enzyme can be used in a method for transferring XG, and the enzyme having the action was named endo-type XG transferase.

Furthermore, the present inventors conducted amino acid analysis of endo-type XG transferase from Vigna angularis,
The partial amino acid sequence is determined, then a primer for PCR is prepared from the amino acid sequence, PCR is performed using Vigna angularis cDNA as a template, and the endo-type XG
A gene encoding a transferase was amplified to prepare a probe. Subsequently, using the probe, a clone containing a gene encoding an endo-type XG transferase is searched from a cDNA library of Vigna angularis, and a gene encoding an endo-type XG transferase is isolated. The gene of interest was cloned from various plants using the gene as a probe. Further, they have found that the target gene can be cloned from another plant by using the cloned gene as a probe, thus completing the present invention.

[0007] An endo-type XG transferase can be collected from the culture by culturing an organism or cell transformed by introducing the gene. Furthermore, by introducing antisense DNA or antisense RNA of the endo-type XG transferase gene into cells, or
A DNA sequence comprising at least 15 base pairs obtained from a transferase gene is ligated to a promoter capable of acting intracellularly so that an antisense RNA of an endo-type XG transferase gene is produced by transcription. By introducing the sense RNA generation cassette into cells, the expression of endo-XG transferase in the organism can be controlled, and the morphology of the plant can be controlled by these methods. It is possible to create a plant. Further, an endo-type XG transferase is characterized in that the endo-type XG transferase gene is linked to a promoter capable of acting intracellularly so that the endo-type XG transferase is expressed.
By introducing a transferase expression cassette into an organism, an endo-type XG transferase can be expressed in the organism, the morphology of plants can be controlled by the method, and a plant body into which the expression cassette is introduced is created. can do.

The acquisition of endo-type XG transferase from Vigna angularis will be described below. The site for collecting the enzyme is not particularly limited, but for example, the apoplast part of the epicotyl of seedlings is suitable. Bigna
As a method for germinating Angularis and obtaining a crude extract solution of the apoplast portion (hereinafter referred to as apoplast solution) from this, for example, the method described in the above-mentioned Physiologia plantarum is effective. As means for purifying the enzyme from the obtained apoplast solution, for example, ammonium sulfate salting out,
It is effective to use a combination of methods such as affinity chromatography, gel filtration, ion exchange column chromatography and the like, and by these methods, a purified enzyme free of mixed glycosidase activity can be obtained.

The method for preparing the substrate used for measuring the enzyme activity will be described below. The crude purified XG derived from Tamarindus Indica L. was obtained from Dainippon Pharmaceutical Co., Ltd. (trade name: Glyloid
9S). 300 mg of the crude purified XG was added to 150 μg of Trichoderma viride cellulase in 40 ml of an aqueous solution.
Partially hydrolyzed at 45 ° C. for 2 hours. The product was autoclaved to denature the cellulase and then fractionated by ultrafiltration [Diaflo XM-300 and YM5, Amicon]. Pass the XM-300,
The fraction (40 mg or more) remaining in YM5 was dissolved in 2 ml of water and the mixture was separated on a Superose 6 prep column (16 × 5).
HPLC (Shimadzu L) equipped with 00 mm, manufactured by Pharmacia
C6A) (hereinafter this HPLC system is referred to as System A) and was eluted with 0.5 ml / min of water. Fraction (1.5
ml) was collected and lyophilized to give an XG preparation. The molecular weight of the preparation was measured by the same chromatography system as that used for measuring the enzyme activity described above.

(Method of measuring enzyme activity) The activity of the enzyme of the present invention is measured as follows. That is, 2 to 20 μg of tamarind-derived XG was added to 0.2 M sodium acetate buffer (p
Add 10 μl of enzyme solution diluted with H5.8), and add 3 at 25 ℃.
After reacting for 0 minutes, the reaction is stopped by freezing the reaction mixture at -50 ° C. Add 20 μl of reaction mixture to 5
Dissolved in 0 mM NaOH, TSK gel-3000PW column (8 × 300 mm, manufactured by Tosoh Corporation) and TSK gel-5.
A non-metal HPLC system equipped with a 000 PW column (8 × 300 mm, manufactured by Tosoh Corp.) is used and eluted with a 30 mM NaOH solution containing 15 mM sodium acetate at a flow rate of 1 ml / min. The eluate is detected by PAD equipped with a gold electrode. FIG. 4 shows an example of the detection result. That is, FIG. 4 is a diagram showing a PAD chromatograph by the above method, in which the vertical axis represents the PAD response (nA) and the horizontal axis represents the elution volume (ml). 12 (a) is an autoclave
When a purified preparation of the enzyme of the present invention denatured by treatment at 0 ° C. for 5 minutes is used, (b) is a chromatogram when an apoplast solution is used and (c) is a chromatogram when the purified enzyme is used. The arrow 1 indicates the substrate XG derived from tamarind, and the arrow 2 indicates the elution position of the monosaccharide. In (c), the width of the peak at a position 1/2 the height of the peak of 1 increases in proportion to the amount of the enzyme added to the reaction system. The increase in the width of the peak at the position is used as an indicator of the activity of the enzyme of the invention. One unit of enzyme activity when tamarind-derived XG was used as a substrate had a peak width of 2.
It is defined as the amount of enzyme that increases by 3 ml.

By analyzing the amino acid sequence of the purified endoplasmic XG transferase of Vigna angularis using, for example, a protein sequencer,
The amino acid sequence of the N-terminal portion represented by SEQ ID NO: 1 in the sequence listing is determined. A mixed primer for PCR can be prepared based on the sequence. For example, the mix primer pAZ-1 represented by SEQ ID NO: 2 in the sequence listing,
, And the mix primer pAZ-2 represented by SEQ ID NO: 3 in the sequence listing are each synthesized by a DNA synthesizer, and after purification, they can be used for searching for an endo-type XG transferase gene. For example, RNA from Vigna angularis
Was prepared, and then poly (A) ⁺ RNA was purified using Oligotex-dT30 (manufactured by Nippon Roche). Next, for example, the poly (A) ⁺ RNA and SEQ ID NO: 4 in the sequence listing were used. CDNA having a sequence of M13 primer M4 (manufactured by Takara Shuzo Co., Ltd.), which has a sequence of T's on the 3'side, and a reverse transcriptase,
To synthesize. By performing PCR using this cDNA as a template, a cDNA encoding an endo-type XG transferase
Can be amplified. Two-step PCR is effective for efficiently amplifying the target DNA. For example, the above-mentioned mix primers pAZ-1 and M are used as primers.
Perform the first step PCR using 13 primer M4,
Using this reaction product as a template, mix primer pA
Second stage P using Z-2 and M13 primer M4
By carrying out CR, a DNA fragment of about 1.1 kbp is amplified. The amplified DNA fragment is, for example, pUC119.
Can be subcloned into the Hinc II site of Next, for example, using these amplified DNAs as probes,
From a cDNA library or a genomic library prepared from Vigna angularis, a clone in which a gene encoding an endo-type XG transferase is incorporated can be searched. The cDNA library can be prepared, for example, from the above-described Vigna angularis cDNA using a cDNA synthesis kit system plus (manufactured by Amersham). By performing plaque hybridization using the above-mentioned amplified DNA fragment as a probe, for example, 5 positive plaques are obtained from 1 × 10 ⁴ plaques, and the DNA fragment inserted into the phage vector of the plaque is extracted. , For example, D with a length of about 1.1 kbp
NA fragments can be obtained. A restriction map of the fragment is shown in FIG. Further, a part of the DNA base sequence of the fragment is shown in SEQ ID NO: 5 in the sequence listing.

The fragment can be inserted into a restriction enzyme site of a suitable plasmid, and the plasmid having the fragment inserted therein can be used to transform a suitable host. The fragment is called pUC1
The plasmid integrated into the EcoRI site of 19 was pVX1.
The Escherichia coli JM109 strain, which was named 03 and transformed with pVX103, was named and displayed as Escherichia coli JM109 / pVX103, and was designated by the Institute of Microbial Science and Technology of the Institute of Industrial Science and Technology, Microtechnology Research Institute No. 4104 (FERM BP-4104). Has been deposited as.

The above-mentioned gene encoding the endo-type XG transferase derived from Vigna angularis is an example of the gene encoding the enzyme, and by using the gene or a partial sequence thereof as a probe, other plant species The endo-type XG transferase gene can be cloned. In addition, by using the primer used for cloning the gene, the gene of endo-type XG transferase can be amplified and cloned in other plant species. Examples of target plants include tomato and tobacco in the case of dicotyledonous plants, and wheat, rice and corn in the case of monocotyledonous plants.

For example, about 1.1 kbp obtained by the above operation
Was labeled with [α- ³² P] dCTP by a random primer DNA labeling kit, and this was used as a probe for hybridization.
Tomato (Lycopersicon esculentu) using ox ^TM Introductory Cloning Kit (Novagen)
m) A cDNA library prepared from RNA can be searched by the plaque hybridization method. Phage can be isolated from the positive plaque, and the endo-type XG transferase gene inserted in the phage vector can be excised as a DNA fragment of about 1.2 kbp and purified. A restriction map of the fragment is shown in FIG.
A part of the DNA base sequence of this fragment is shown in SEQ ID NO: 6 in the sequence listing.

Further, by the method described below, a plasmid containing a DNA fragment encoding the portion of base numbers 46 to 930 in the DNA base sequence shown in SEQ ID NO: 6 in the sequence listing can be constructed. The plasmid is pTX
The Escherichia coli JM109 strain, named 301, transformed with pTX301 is Escherichia coli JM109 / pTX30.
The strain is designated and designated as 1, and the strain has been deposited at the Institute of Biotechnology, Institute of Industrial Science and Technology as FERM BP-4223.

A cDNA fragment of maize and rice is screened with a cDNA fragment of about 1.1 kbp containing the endo-XG transferase gene of Vigna angularis as a probe to prepare a DNA fragment containing the endo-XG transferase gene. be able to. These DN
The restriction enzyme maps of the A fragment are shown in FIGS. 1 and 2, respectively. The plasmid into which the DNA fragment containing the endo-type XG transferase gene of maize or rice was introduced was pCX10, respectively.
E. coli JM109 strain transformed with these plasmids was named Esch.
erichia coli JM109 / pCX101, Escherichia coli JM109
Named / pRX102, Escherichia coli JM109 / pCX101
FERM P- at the Institute of Biotechnology, Institute of Biotechnology
Deposited as 13768, Escherichia coli J
M109 / pRX102 has been deposited as FERM BP-4221.

In addition, about 9 containing the endo-type XG transferase gene of tomato introduced in the plasmid pTX301.
A DNA fragment of about 1.2 bp containing the tobacco endo-type XG transferase gene can be prepared by the same method using the 30 bp DNA fragment as a probe.
About 1.2 kbp containing tobacco endo-type XG transferase gene
The plasmid introduced with the DNA fragment of the length is pNX10
Escherichia coli JM109 strain designated E. coli strain JM109 transformed with the plasmid pNX101
It is named and displayed as / pNX101 and the strain has been deposited as FERM P-13803 at the Institute of Biotechnology, Institute of Biotechnology, AIST. A restriction map of the DNA fragment having a length of about 1.2 kbp is shown in FIG. Also, the DN of this DNA fragment
A part of the A base sequence is shown in SEQ ID NO: 7 in the sequence listing.

An endo-type XG transferase gene is prepared from these strains, an appropriate expression plasmid is constructed using the gene, an appropriate host is transformed with the expression plasmid, and the transformant is transformed. The endo-type XG transferase can be produced by culturing the.

By controlling the expression of the endo-type XG transferase gene in an organism, for example, in a plant, the growth of the cell wall of the plant can be regulated. For example, by controlling the expression of these genes, the cell wall can be regulated. It can inhibit the remodeling of the plant and produce dwarf plants. As a method of controlling the expression of the endo-type XG transferase gene, for example, a method of introducing antisense DNA or antisense RNA of the endo-type XG transferase gene into an organism can be used. As the antisense DNA to be introduced, for example, the antisense DNA of the endo-type XG transferase gene obtained in the present invention or a part thereof can be used. An example of the sequence of the antisense DNA is shown in SEQ ID NO: 8 in the sequence listing. That is, SEQ ID NO: 8 in the sequence listing shows the sequence of the antisense DNA of the tobacco endo-type XG transferase gene shown in SEQ ID NO: 7 in the sequence listing. Examples of antisense DNA include
A fragment obtained by appropriately cutting a part of this antisense DNA may be used, or a DNA synthesized based on this antisense DNA sequence may be used.

The antisense RNA to be introduced is, for example, the antisense RNA of the endo-type XG transferase gene.
Alternatively, a part of it can be used. An example of the sequence of the antisense RNA is shown in SEQ ID NO: 9 in the sequence listing. That is, SEQ ID NO: 9 in the sequence listing shows the sequence of the antisense RNA of the endo-type XG transferase gene shown in SEQ ID NO: 7 in the sequence listing. For example,
A fragment obtained by appropriately cutting a part of this antisense RNA may be used, or RNA synthesized based on this antisense RNA sequence or, for example, an endo-type XG transferase gene or a part thereof may be used. , In Bidro (in v
Itro) RNA produced by reacting RNA polymerase in a transcription system may be used. Antisense DNA to be introduced
Alternatively, regarding the region and length of the antisense RNA, the introduced antisense DNA or antisense RN
A is an endogenous RNA of endo-type XG transferase in the organism
The length of the inserted fragment is preferably 15 base pairs or more, though it is not particularly limited as long as it suppresses its function by associating with. Further, the antisense DNA and the antisense RNA can be chemically modified so that they are less likely to be degraded in vivo.

As a method for introducing antisense DNA or antisense RNA into an organism, for example, microinjection method [Molecular General Genetics (Mol. Gen. Genetics), 202, 179-185 (1985)]. , Polyethylene glycol method [Nature, Vol. 296, No. 72-
74 (1982)], the Peggle cancer method [Nature, Vol. 327, pp. 70-73 (1987)], and fusion of protoplasts with antisense DNA or antisense RNA-containing small cells, cells, lysosomes and the like. Law [Proceedings of the National Academy of the Sciences of the US
A, Vol. 79, pp. 1859-1863 (198
2)], Electroporation method [Proceedings of the National Academy of the Sciences of the USA, Vol. 82, No. 5824]
Pp. 5828 (1985)] and the like.

Further, the DNA sequence obtained from the endo-type XG transferase gene is ligated to a promoter capable of acting intracellularly so that the antisense RNA of the endo-type XG transferase gene is produced by transcription. A method of constructing an RNA production cassette and introducing it into a plant is also effective. The antisense RNA generation cassette is constructed, for example, by linking the sequence of the endo-XG transferase gene or a part thereof so that the antisense RNA of the endo-XG transferase gene is generated by transcription downstream of the promoter. be able to. The gene sequence used here is an antisense RN generated by transcription.
A is an endogenous RNA of endo-type XG transferase in the organism
The length of the inserted fragment is preferably 15 base pairs or more, though it is not particularly limited as long as it suppresses its function by associating with. For example, a part of the endo-XG transferase gene is cleaved with an appropriate restriction enzyme, and ligated to an appropriate site downstream of the promoter so that the antisense RNA of the endo-XG transferase gene is generated by transcription. You can Further, for example, based on the sequence of the endo-type XG transferase gene obtained in the present invention, a primer for PCR is prepared so that an appropriate region of the endo-type XG transferase gene is amplified. Amplified DN obtained by performing PCR using the transferase gene as a template
The A fragment may be ligated downstream of the promoter region incorporated in an appropriate vector so that the antisense RNA of the endo-type XG transferase gene is generated by transcription to construct an antisense RNA generation cassette. . In this case, the amplified DNA fragment to be inserted is selected by inserting an appropriate restriction enzyme site present in the vector so that the antisense RNA of the endo-type XG transferase gene is generated by transcription. It is effective to carry out PCR using a primer having the recognition sequence of the restriction enzyme of 5) at the 5'-terminal side.

Further, for example, a cassette for expressing an endo-type XG transferase, which is linked to a promoter capable of acting intracellularly so that the endo-type XG transferase is expressed, is constructed, and the cassette is introduced into an organism such as a plant. It is possible to express the endo-type XG transferase in the plant, and thus to control the morphology of the plant. The endo-type XG transferase gene inserted into the endo-XG transferase expression cassette may include a region required for expressing the activity of the endo-XG transferase in plant cells, For example, it is desirable to include a sequence encoding a signal peptide to a stop codon. For example, a part of the endo-type XG transferase gene can be cleaved with an appropriate restriction enzyme and ligated to an appropriate site downstream of the promoter so that the endo-type XG transferase can be expressed. Further, for example, based on the sequence of the endo-type XG transferase gene obtained in the present invention, a primer for PCR is prepared so that an appropriate region of the endo-type XG transferase gene is amplified. PCR is carried out using the transferase gene as a template, and the obtained amplified DNA fragment is ligated downstream of the promoter region incorporated in an appropriate vector so that the endo-XG transferase is expressed, thereby expressing the endo-XG transferase. A cassette for use may be constructed. In this case, the amplified DNA fragment to be inserted is inserted by selecting an appropriate restriction enzyme site existing in the vector.
As in the case of the antisense RNA generation cassette, it is effective to carry out PCR using a primer having a recognition sequence for these restriction enzymes at the 5'end side.

The promoter sequence used in the cassette for producing antisense RNA or the cassette for expressing endo-type XG transferase is not particularly limited as long as it can function in the cell, and is derived from the endo-type XG transferase gene. However, it may be derived from another gene. The promoter contains a region called TATA box, which is located 20 to 30 base pairs upstream from the transcription start point (+1) and has a function of initiating transcription from RNA polymerase at an accurate position, and is used in the present invention. The promoter sequence is not necessarily limited to the region before and after the TATA box, but in addition to this region, RNA may be used for expression regulation.
It may include a region further upstream required for association with a protein other than the polymerase.

For example, when constructing an antisense RNA production cassette, if a promoter whose expression of the endo-type XG transferase gene is under the control of the natural plant body is used, the whole life cycle of the whole plant organ is used. The amount of endo-XG transferase produced throughout the stage can be reduced, resulting in dwarfing. Also, by using a non-regulatory promoter derived from another gene, it is possible to bring about a morphological change throughout the entire life cycle of the plant organ. As the non-regulatory promoter, for example, the 35S promoter of cauliflower mosaic virus can be used, which is contained in pBI121 (manufactured by Clontech). If a promoter that can be induced by stimulation is used, a plant whose morphology can be changed depending on the growth environment can be produced. For example, if a promoter that responds to light is used, the morphology of the plant can be changed depending on the conditions of light irradiation. For example, if an organ- or tissue-specific promoter is used, it is possible to cause a morphological change only in a specific organ or tissue. For example, if the promoter of a gene that is specifically transcribed in leaves is used, only the morphology of leaves can be changed. Further, for example, by using a promoter that specifically functions at a certain stage of the life cycle, the activity of endo-XG transferase can be increased or decreased only at a certain stage of the life cycle,
As a result, morphological changes can be caused only in specific organs or tissues. For example, by using a promoter capable of causing transcription only during flower organ formation, it is possible to cause morphological changes only in the flower organ. In addition, elongation of pollen tubes can be suppressed by using an antisense RNA-producing cassette constructed using a promoter that functions only during pollen tube formation, and as a result, male-sterile plants can be produced. .

In the cassette for producing antisense RNA or the cassette for expressing endo-type XG transferase, the antisense RNA of the endo-type XG transferase gene is produced downstream of the promoter by transcription, or the endo-type XG transferase is used. Efficiently terminates transcription by linking a transcription termination sequence downstream of a gene fragment (hereinafter referred to as an insertion sequence) having an endo-XG transferase gene or a partial sequence thereof linked so as to be expressed. Can be made. The transcription termination sequence may be derived from the endo-type XG transferase gene or may be derived from another gene. In addition, the efficiency of translation can be increased by linking the poly A addition sequence downstream of the insertion sequence. The poly A addition sequence may be derived from an endo-type XG transferase gene, or may be derived from another gene, for example, octopine synthetase of Agrobacterium [Diembo Journal (The
EMBOJournal), Volume 3, pages 835-846 (198).
4)] and noparin synthetase [Molecular and Applied Genetics (Mol. And Appl. Genet.)
Vol. 1, pp. 561-573 (1982)].

Various known methods can be used to introduce the antisense RNA generating cassette or the endo-type XG transferase expressing cassette into an organism. For example, these cassettes can be inserted into an appropriate vector. However, the method of introducing the vector into an organism is effective.
In this case, depending on the vector, a restriction enzyme recognition sequence, which is not present in these cassettes but is present only at the site in the vector where the cassette is to be inserted, is added to the 5'of these cassettes.
It can be added to the end and the 3'end.

The vectors into which these cassettes are inserted are
It is desirable to include a selectable marker gene so that transformed plants can be easily selected. As the selectable marker gene, for example, a gene that causes an antibiotic resistance trait (antibiotic resistance gene) can be used. Examples of such a gene include a gene that confers resistance to G418, hygromycin, bleomycin, kanamycin, gentamicin, and chloramphenicol. If an antibiotic resistance gene is included in the vector, it is easy to select transformed organisms, that is, organisms into which these cassettes have been introduced, by selecting organisms that grow in medium containing antibiotics. be able to.

The method for directly introducing the vector into which these cassettes have been inserted into an organism, such as a plant, is described in the microinjection method, polyethylene glycol method,
Examples of the method include the paringulgun method, a method of combining small cells containing cells, cells, lysosomes and the like with protoplasts, electroporation and the like.

By using a plant virus as a vector, these cassettes can be introduced into plants. As the plant virus to be used, for example, cauliflower mosaic virus (CaMV) can be used. That is, first, the viral genome is inserted into a vector derived from Escherichia coli or the like to prepare a recombinant, and then these cassettes are inserted into the viral genome. These cassettes can be inserted into a plant by excising the thus modified viral genome from the recombinant with a restriction enzyme and inoculating the plant [Hohn et al., Molecular Biology of Plant]. Tumors (Molecular Biology of Plant
Tumors), Academic Press, New York (Acad
emic Press, New York), pages 549-560 (198)
2), U.S. Pat. No. 4,407,956].

Furthermore, when a bacterium belonging to the genus Agrobacterium infects a plant, these cassettes are transferred to the plant body by utilizing the property that a part of the plasmid DNA possessed by the bacterium is transferred into the genome of the plant. Can also be introduced. Among bacteria belonging to the genus Agrobacterium, Agrobacterium tumefaciens (Agrobacteriu
m tumefaciens) infects plants and grows (crown gall)
Agrobactor rhizogenes (Agrobact
erium rhizogenes) infects plants and causes hairy roots. These are the T-DNA region (Transferred DNA) on the plasmid present in each bacterium called Ti plasmid or Ri plasmid during infection. This is because the so-called region migrates into the plant and integrates into the plant genome. Furthermore, on the Ti or Ri plasmid, there is a region called a vir region which is essential for the T-DNA region to be transferred into the plant and to be integrated into the plant genome. The vir region itself is not transferred into plants, and this vir region can function even on a plasmid different from the one in which the T-DNA region is present [Nature, vol. 303, 179- 189 pages (198
3)].

T-DNA on Ti or Ri plasmid
If the DNA desired to be integrated into the plant genome is inserted into the region, the target DNA can be integrated into the plant genome when a bacterium of the genus Agrobacterium infects the plant. Here, TD of Ti or Ri plasmid
The aneurysm in NA or the part that causes hairy roots can be removed without impairing the target transfer function, and the obtained product can be used as a vector. In the present invention, such various vectors can be used, and examples thereof include pBI121 (Clontech Co., Ltd.), pBI-H1-35S-IG (Nagoya University, provided by Dr. Kenzo Nakamura), which are called binary vectors. , A cassette for generating antisense RNA or a cassette for expressing an endo-type XG transferase can be inserted to introduce these cassettes into a plant. It should be noted that these vectors do not have the above-mentioned vir region, and the bacterium of the genus Agrobacterium used by introducing the vector is vi
It must contain another plasmid containing the r region.

Further, these vectors are shuttle vectors that can be amplified not only in bacteria of the genus Agrobacterium but also in Escherichia coli. Therefore, the recombinant operation of Ti plasmid can be carried out using Escherichia coli. Furthermore, these vectors contain an antibiotic resistance gene, and when transforming Escherichia coli, bacteria of the genus Agrobacterium, plants, etc., transformants can be easily selected. Also, these vectors contain Ca
The presence of the MV 35S promoter allows the genes inserted in these vectors to be expressed unregulated after integration into the plant genome.

Using these vectors into which an antisense RNA generating cassette or an endo-type XG transferase expressing cassette was inserted, these cassettes were transferred into the genome of the plant body via a bacterium of the genus Agrobacterium. When carrying out the transformation, any plant can be transformed as long as it is a plant that can be infected by a bacterium of the genus Agrobacterium and has a regenerated system established. Most dicotyledonous plants can be transformed using bacteria of the genus Agrobacterium, and in particular, plants that are hosts of bacteria of the genus Agrobacterium in nature are
All can be transformed in vitro. Monocotyledonous plants, including cereals, are not naturally hosts for bacteria of the genus Agrobacterium, but for example rye (nature,
325, 274-276 (1987)], corn [Science, 240, 204-207]
P. (1988)], rice [Nature, vol. 338, pp. 274-276 (1989)] and the like can be transformed in vitro.

Transformation can be carried out using (1) protoplasts or (2) tissue pieces or untreated cells. In order to use the method of (1), it is necessary to establish in advance a system for regenerating a plant from transformed protoplasts, and to use the method of (2), a bacterium of the genus Agrobacterium It is necessary to establish a system for transforming one or untreated cells and regenerating them into plants. The transformed plant can be selected by growing the plant in a medium containing a drug that can be a marker for the above-mentioned transformation.

The method of regenerating a plant differs depending on the type of plant, but in general, in the case of (1), a suspension of the transformed protoplasts, and in the case of (2), the transformation. This can be done by inducing callus from a piece of tissue or untreated cells on a plated plate and then forming shoots. Further, in general, the medium can contain hormones such as auxin and cytokinin in addition to various amino acids. Whether or not the desired antisense RNA generation cassette or the endo-XG transferase expression cassette is inserted in the genome of the transformed plant can be confirmed by a method such as Southern hybridization. Whether sense RNA or antisense RNA of the transferase gene is produced in the plant can be confirmed by a method such as Northern hybridization.

Antisense RN produced as described above
Using the plant in which the A generation cassette or the endo-type XG transferase expression cassette is inserted, the antisense strand transcription cassette or the endo-XG transferase expression cassette is transferred to the next-generation plant by mating. be able to. For example, the vector pBI-H1-35S-I
In G, an endo-type XG transferase gene obtained by the present invention or a part thereof is transferred to
To produce CaMV35S upstream, or by incorporating so that the endo-type XG transferase is expressed.
A plasmid containing an antisense RNA generation cassette or an endo-type XG transferase expression cassette in which an endo-XG transferase gene or a part thereof is linked to the promoter can be constructed. Next, using the plasmid thus constructed, a strain belonging to an appropriate Agrobacterium genus, for example, Agrobacterium tumefaciens LBA4404 [Agrobacterium tumefaciens LBA4404; Nature,
Vol. 303, pp. 179-180 (1983), available from Clontech Inc.] and infecting the target plant with the transformant, a plant can be transformed. In addition, the plasmid is digested with an appropriate restriction enzyme, the desired fragment is excised by agarose gel electrophoresis and purified, and these fragments are used as an endo-XG transferase expression cassette or an antisense RNA generation cassette to obtain another fragment. It can be integrated into a plasmid and used for plant transformation.

Based on the sequence of the tobacco endo-type XG transferase gene represented by SEQ ID NO: 7 in the sequence listing obtained by the present invention, four kinds of primers shown in SEQ ID NO: 10, 11, 12, 13 in the sequence listing , TABSSP, TABXA
P, TABXSP, TABSAP can be designed and synthesized. Primers TABSSP and TABX
SP is a sequence corresponding to bases 1 to 19 of SEQ ID NO: 7 in the sequence listing, which has a restriction enzyme SacI or XbaI cleavage site added to the 5'side corresponding to the direction 5 '→ 3', TBXAP and TABSAP. Is base number 871
This is obtained by adding a cleavage site for the restriction enzyme XbaI or SacI to the 5 ′ side of the sequence corresponding to the 3 ′ → 5 ′ direction corresponding to 888. For example, end type XG of cigarette
Using the transferase gene as a template, the gene sequence was amplified by PCR using the pair of TABSSP and TABXAP among the above primers, and the amplified DNA fragment of about 900 bp was cleaved with restriction enzymes XbaI and SacI and purified. And then Xb of pBI-H1-35S-IG
It can be inserted from the aI site to the SacI site. The thus obtained plasmid has a cauliflower mosaic virus 35S promoter sequence, and by transcribing the sequence of the portion represented by base numbers 1 to 888 of SEQ ID NO: 7 in the sequence listing at the XbaI site-SacI site downstream thereof. It is integrated so as to generate antisense RNA and has genes for kanamycin and hygromycin resistance.

Similarly, the primers TABXSP and T
A gene sequence was amplified by the PCR method using a pair of ABSAP, and the amplified DNA fragment of about 900 bp was transformed into pBI-H.
It can be inserted from the XbaI site of 1-35S-IG into the SacI site. The plasmid thus created has the same promoter and resistance gene,
It is the one in which the sequence of base numbers 1 to 888 of SEQ ID NO: 7 in the sequence listing is incorporated so that the endo-type XG transferase is expressed. Using these plasmids, suitable Agrobacterium strains such as Agrobacterium tumefaciens LBA44 can be electroporated.
Strain 04 can be transformed.

The method for transforming the target plant with the transformed Agrobacterium is not particularly limited. For example, a method can be used in which sterilized leaves are made into leaf discs of about 1 cm square and infected with transformed Agrobacterium. Transformed plants can be selected by erasing the infected leaf disk and culturing on a plate containing an antibiotic that serves as a selectable marker for the vector used. A cassette for producing antisense RNA or endo-type XG in the genome of a plant obtained on a plate containing these antibiotics
Confirmation that the cassette for transferase expression has been inserted is confirmed by D of the plant or plant formed from its seed.
NA can be extracted, and the endo-XG transferase gene or a part thereof can be used as a probe by a method such as Southern hybridization. In addition, in the transformed plant, the confirmation that the expression of the endo-type XG transferase is regulated is performed by extracting the RNA of the transformed plant or its seed and probing the endo-type XG enzyme gene. And can be carried out by a method such as Northern hybridization. For example, aseptically grown tobacco leaves are sterilized with a scalpel to make leaf discs of about 1 cm square, and transformed Agrobacterium is co-cultured with LB medium overnight culture at 28 ° C. for 3 days, After the disinfection operation, the leaves are cultivated in a solid Murashige-Skoog (MS) medium containing the surface of kanamycin-hygromycin-carbenicillin downward and 0.2 μg / ml-naphthalene acetic acid, 1 μg / ml benzyladenine. The foliage appearing after 4 weeks is cut from the disc and transferred to MS medium containing kanamycin-hygromycin-carbenicillin.
After 2 weeks, rooted plants are transferred to peat moss / vermiculite 3: 2 soil. A transformant can be obtained by sterilizing the seeds obtained after flowering and seeding them in an MS medium containing hygromycin for germination.

From this transformant, DNA is extracted according to a conventional method, and the DNA is cleaved with an appropriate restriction enzyme. As a probe, for example, primers TABSSP and TABX are used.
The presence or absence of transformation can be confirmed by carrying out Southern hybridization using a DNA sequence of about 900 bp amplified by the PCR method using the tobacco endo-type XG transferase gene as a pair of AP.

From this transformant, R
NA can be extracted to prepare a probe having a sense DNA sequence or antisense DNA sequence of about 900 bp, and Northern hybridization can be carried out using these probes to observe the expression state of endo-XG transferase.

Expression of an endo-XG transferase-encoding gene using the gene encoding the endo-XG transferase obtained in the present invention, a gene hybridizing with the gene, or a polynucleotide having a partial sequence thereof You can also observe and adjust the process. In addition, various antibodies can be prepared by using the polypeptide produced by expressing the gene encoding the endo-XG transferase obtained in the present invention.
It is useful for purification of transferase.

The endo-type XG transferase used in the present invention will be specifically described below in Reference Examples. Reference Example 1 Purification of endo-XG transferase Biguna Angularis Ovier Toucan, c
v, Takahashi (Ohwi et Ohashi, cv. Takara) seeds (manufactured by Watanabe Seed Co.), Physiologia plantarum, No. 82
Vol., Pp. 490-497 (1991), germinated to obtain an apoplast solution.

(Ammonium sulfate precipitation) A total of 600 ml of apoplast solution was mixed with 10 g of polyvinylpyrrolidone powder and 50
It contains mM dithiothreitol and 2 mM EDTA.
60 ml of 5M sodium phosphate buffer (pH 6.8) and 0
Mix at 0 ° C and hold for 5 minutes. Insoluble matter 18000 ×
After removal by centrifugation at 30 g for 30 min, ammonium sulfate powder was added to the supernatant to 80% saturation and the resulting precipitate (containing 10.5 mg protein) was collected by centrifugation. The obtained precipitate was dissolved in 2 ml of 0.2M sodium acetate buffer (pH 5.7), and 1 ml of 100% ammonium sulfate saturated solution was added to make it 33% saturated. The produced precipitate was collected by centrifugation at 18,000 xg for 30 minutes to obtain a fraction containing 1.8 mg of protein.

(Fractionation by Con-A Sepharose 6B)
The precipitate obtained by the above operation was added to 0.15 M NaCl, 1 mM MnCl ₂ , 1 mM MgCl ₂ , and 1 mM CaCl _2.
Of sodium acetate buffer a (pH 5.7) containing 2.4
It was dissolved in ml and was injected into a column of Con-A Sepharose 6B (Pharmacia) (4 ml) pre-equilibrated with the same buffer a. The column was charged with 36 ml of buffer a, 20 ml of buffer a containing 7 mM methyl-α-D-mannopyranoside, and 50
Elution was carried out successively with 40 ml of buffer a containing 0 mM methyl-α-D-mannopyranoside. The activity of the enzyme of the present invention was observed in the fraction eluted with the buffer solution a containing 500 mM of methyl-α-D-mannopyranoside. Ultra active (Ultrafree, Millipore)
Concentrate by ultrafiltration with 668 in 400 μl
A fraction containing μg of protein was obtained.

(Fractionation by TSK gel 2000SW) 200 μl of protein of the fraction obtained by the above operation was used as a eluent in 0.15M sodium acetate buffer solution of TSK gel G2000SW (7.5 × 300 mm, manufactured by Tosoh Corporation). (PH 5.
7) was subjected to column chromatography using a flow rate of 0.5 ml / min. This chromatography is repeated, the active fraction is fractionated and concentrated, and 60% in 100 μl of the above buffer solution is obtained.
A fraction containing μg of protein was obtained.

(Fractionation by Mono-S) The fraction obtained by the above operation was diluted with 500 μl of 20 mM sodium acetate solution and pre-equilibrated with 40 mM sodium acetate buffer (pH 5.7). -S, Pharmacia) column (5 x 50 mm). The column was charged with 2.5 ml of 40 mM sodium acetate buffer (pH 5.7).
20 ml of 40 mM sodium acetate buffer (pH 5.7) with a linear gradient of 0-1 M NaCl.
Was eluted at a flow rate of 0.4 ml / min to give about 30 μg of a purified preparation of the enzyme of the present invention. The results of each of the above purification steps are shown in Table 1.

[0049]

[Table 1] Table 1 Purification process of endo-type XG transferase from Vigna angularis ──────────────────────────────── ───── Purification process Total protein Total activity Specific activity Yield Purity (Unit / μg (mg) (Unit) Protein) (%) (times) ─────────────── ───────────────────── Apoplast solution 10.5 2136 0.203 100 1 33% Ammonium sulfate fraction 1.513 1766 1.17 82 5.7 Con-A Sepharose 0.668 938 1.40 43 6.9 TSK 2000SW 0.06 816 13.6 38 67 Mono-S 0.03 530 17.7 24 87 ─────────────────────────────────────

Further, in FIG. 4 described above, (b) is a chromatograph when the reaction is carried out using the apoplast solution. Arrow 2 in the figure indicates the elution position of the monomer sugar produced by the mixed glycosidase. In the purified enzyme fraction of (c), the peak of monomer sugar did not appear, showing that the glycosidase mixed in was removed by the purification operation.

(SDS-Polyacrylamide Gel Electrophoresis) A purified preparation of the enzyme of the present invention obtained in the above purification step was prepared by the method of Laemmili et al.
27, pp. 680-685 (1970)],
It was subjected to SDS-polyacrylamide gel electrophoresis. That is, a 12% polyacrylamide gel containing SDS (10 × 10 cm, 1 mm thickness) was used for electrophoresis, silver staining was performed, and the color was developed. As a result, the purified preparation of the enzyme of the present invention had a molecular weight of about 33,000. Confirmed as one band.

Reference Example 2 Cloning of Gene Encoding Endo-XG Transferase Preparation of Poly (A) ⁺ RNA Biguna Angularis Ovier Toucan, c
v. Takara seeds were germinated by the method described in Physiologia prontalum, supra. One week after germination, a lower stem of 2 to 5 cm from the tip of the bud was cut off to obtain about 3 g of plant tissue. This was immediately frozen in liquid nitrogen, ground in a mortar in the presence of liquid nitrogen to give a powder, and about 30 ml of denaturing solution [4M guanidine thiocyanate, 25 mM sodium citrate (pH 7.0), 0.1M 2-mercaptoethanol, 0 0.5% N-lauroyl sarcosine sodium]. 2m sodium acetate (pH 4.0) 3m here
l, 30 ml of water-saturated acidic phenol, 6 ml of 49: 1 chloroform / isoamyl alcohol were added sequentially, stirred well, this suspension was centrifuged to separate the aqueous layer, and isopropyl alcohol was added to this aqueous layer and centrifuged. This gave about 1 mg of RNA precipitate. The precipitate was added to an elution buffer [10 mM Tris-HCl (pH 8.0), 1 mM.
Dissolve 400 μl of EDTA (pH 8.0), 0.1% SDS, and add 1 ml of Oligotex-dT30 (Roche Japan).
Was added to adsorb only poly (A) ⁺ RNA onto the resin. Wash the resin with sterilized pure water to obtain about 10 μg
Of poly (A) ⁺ RNA were recovered.

Determination of N-Terminal Amino Acid Sequence of Endo-XG Transferase Using about 1 nmol of the purified preparation of endo-XG transferase obtained in Reference Example 1, an N-terminal amino acid sequence was analyzed by 470A protein sequencer manufactured by Applied Biosystems. The amino acid sequence of about 30 residues was determined from the end. The amino acid sequence is shown in SEQ ID NO: 1 in the sequence listing.

Based on the amino acid sequence obtained by amplification of the endo-type XG transferase cDNA by the PCR method, SEQ ID NO: 2 in the sequence listing
The primer pAZ-1 represented by SEQ ID NO: 3 and the primer pAZ-2 represented by SEQ ID NO: 3 in the sequence listing were designed, and D
It was synthesized with an NA synthesizer. Also, M13 primer M4
Similarly, a primer pTM4 having a sequence in which 20 T's were connected on the 3'side (Takara Shuzo) was synthesized. The sequence of primer pTM4 is shown in SEQ ID NO: 4 in the sequence listing.

² μg of poly (A) ⁺ RNA obtained in
5 mM MgCl ₂ , 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 0.01% gelatin, dNTPs 1 mM each, 2.5 μM primer pTM4, 20 U RNase.
CDNA was synthesized by adding the inhibitor and 50 U of reverse transcriptase RAV-2 (manufactured by Takara Shuzo Co., Ltd.) to a reaction solution having a total volume of 20 μl and performing a reverse transcription reaction at 42 ° C. for 40 minutes.

Add 25 μM MgCl ₂ 4 μ to this tube.
l, 10 × PCR buffer [500 mM KCl, 100 mM Tris-HCl (pH 8.3), 0.1% gelatin] 8 μl,
Sterile distilled water 65.5 μl, AmpuriTac ^™ DNA polymerase (manufactured by Cetus) 2.5 U, 20 pmol of primer pAZ-1, 20 pmol of M13 primer M4 (manufactured by Takara Shuzo) were added in sequence, and mineral oil was added to PCR. I served. The reaction is 94 ℃ (0.5 minutes), 45 ℃ (2 minutes), 7
The cycle of 2 ° C (1 minute) was repeated 30 times, and then the temperature was maintained at 72 ° C for 7 minutes. Next, PCR was similarly performed using 1 μl of this reaction solution as a template, pAZ-2 as a sense primer, and M13 primer M4 as an antisense primer, and the above cycle was repeated 25 times. After the reaction, the reaction solution was subjected to agarose gel electrophoresis, and it was confirmed that about 1.1 kbp cDNA was specifically amplified. This cDNA was purified and plasmid pU
It was subcloned into the restriction enzyme Hinc II site of C119.

Gene (Gene) Vol. 25, p. 263 (1983) from the poly (A) ⁺ RNA obtained in the preparation and screening of the cDNA library using the cDNA synthesis kit System Plus (manufactured by Amersham). A cDNA library using λgt10 as a vector was prepared in accordance with the method described in 1. Subcloned in 1.
A cDNA of 1 kbp was (α- ³² P) dCT using a random primer DNA labeling kit (Takara Shuzo).
It was labeled with P and used as a probe for hybridization. Using this probe, the plaque hybridization method was performed on the cDNA library prepared above. As a result of searching 1 × 10 ⁴ plaques, 5 positive plaques were obtained. Phages were isolated from these plaques and the inserted DNA was extracted. These DNAs were cleaved with restriction enzyme EcoRI, and the length of the DNA fragment was confirmed by agarose gel electrophoresis. A DNA fragment of about 1.1 kbp was purified and subcloned into the EcoRI site of plasmid pUC119,
The plasmid was named pVX103. Got about 1.
The 1 kbp DNA fragment was cleaved with several restriction enzymes and analyzed by agarose gel electrophoresis.

The results are shown in FIG. That is, FIG. 5 is a diagram showing a restriction enzyme map of a DNA fragment of about 1.1 kbp. Restriction enzymes BamHI, HindIII, KpnI, etc. do not cut the fragment.

E. coli J using the plasmid pVX103
The M109 strain was transformed and the transformant was transformed with Escherichia co
li JM109 / pVX103 was named and displayed. The strain has been deposited as Microtechnology Research Institute No. 4104 (FERM BP-4104).

Reference Example 3 Determination of nucleotide sequence of endo-type XG transferase gene 10 μg of plasmid pVX103 obtained in Reference Example 2
Was digested with the restriction enzymes XbaI and PstI. Then, the method of Henikoff et al. [Gene, Vol. 28, Vol. 3
51-359 (1984)], the DNA fragment incorporated into the vector was digested from the end on the recognition sequence side of XbaI using a deletion kit for kilosequencing (manufactured by Takara Shuzo) to obtain various sizes. A clone containing the fragment was obtained. Eight plasmids containing a fragment of an appropriate size were selected from these, and BcaBEST ^™ dideoxy sequencing according to the method of Sanger [Science, Volume 214, 1205-1210 (1981)]. About 1. using the kit (Takara Shuzo)
The nucleotide sequence of the 1 kbp DNA fragment was determined. A part of the nucleotide sequence is shown in SEQ ID NO: 5 in the sequence listing. The nucleotide numbers 57 to 872 of SEQ ID NO: 5 in the sequence listing are portions encoding the mature protein of the endotype XG transferase of Vigna angularis.

Reference Example 4 Cloning of tomato endo-type XG transferase gene and determination of nucleotide sequence Tomato (Lycopersicon esculentum) seeds (manufactured by Watanabe Shunba) were germinated, and reference examples were taken from the tissues of the upper hypocotyl part of the buds. In the same manner as in 2-, 10 μg of poly (A) ⁺ RNA was extracted, and a cDNA library was prepared using λEXlox ^™ Introductory Cloning Kit (Novagen). That is, the cDNA was synthesized according to the method described in Gene, 25, 263 (1983), and the vector λEXLX [Palazzoro (Palazzoro
olo) et al., Gene, 88, 25-36 (199).
0)], and then a cDNA library was prepared.

About 1.1 kbp cDNA obtained in Reference Example 2
Was labeled with [α- ³² P] dCTP using a random primer DNA labeling kit, and used as a probe for hybridization to perform a plaque hybridization method on the above cDNA library. About 1 × 10 ⁵ plaques resulted in about 2 positive plaques. From these plaques, according to the method of Parazolo et al.
The fragment containing A was recovered as a plasmid. This DNA
Was digested with restriction enzymes EcoRI and HindIII, and the length of the DNA fragment was confirmed by agarose gel electrophoresis. Two
The longer of the positive plaques, c with a length of approximately 1.2 kbp
You can confirm the DNA fragment, and after purifying it,
Hinc II of plasmid pUC118 was prepared by using T4 DNA polymerase (Takara Shuzo) to make the protruding end blunt.
Subcloned into the site and p
It was named TX201. The obtained DNA fragment of about 1.2 kbp was cleaved with several restriction enzymes and analyzed by agarose gel electrophoresis. The result is shown in FIG. That is, FIG. 6 is a diagram showing a restriction enzyme map of a DNA fragment of about 1.2 kbp. Plasmid pTX201 was digested with restriction enzymes BamHI and S
It was cleaved with acI, and using this DNA fragment, the nucleotide sequence of a DNA fragment of about 1.2 kbp was determined in the same manner as in Reference Example 3. A part of the sequence is shown in SEQ ID NO: 6 in the sequence listing.

Reference Example 5 (Preparation of plasmid pTX301) SEQ ID NO: 6 in Sequence Listing
Two types of primers, TOMXSP, TOM, based on the sequence of the tomato endo-XG transferase gene shown in
SAP was designed and synthesized. The respective sequences are shown in SEQ ID NOS: 14 and 15 of the sequence listing. TOMXSP is 5 '→ corresponding to base numbers 46 to 66 of SEQ ID NO: 6 in the sequence listing.
The restriction enzyme XbaI is added to the 5'side of the sequence corresponding to the 3'direction.
TOMSAP has base number 9
This is obtained by adding a cleavage site for the restriction enzyme SacI to the 5'side of the sequence corresponding to the 3 '→ 5'direction corresponding to 21 to 941.

As a template, the cDNA obtained in Reference Example 3
Approximately 1 ng (1 μl) is transferred to a 0.5 ml tube, and 10 μl of 10x PCR buffer in GeneAmp ^™ kit, 1.25
16 μl of mMdNTP mixed solution, 2.5 U of AmpliTac ^™ DNA polymerase, and 0.1 μg / μl of primer T
Sterile distilled water was added to 1 μl each of OMXSP and TOMSAP to make 100 μl, mineral oil was added, and the mixture was subjected to PCR. The reaction conditions are 94 ℃ (30 seconds), 37 ℃
The cycle of (2 minutes) and 72 ° C. (1 minute) was repeated 25 times. After completion of the reaction, the reaction solution was concentrated by ethanol precipitation, digested with XbaI and SacI, and then purified,
It was subcloned from the restriction enzyme XbaI of the binary vector pBI-H1-35S-IG to the site of the SacI site. Primer TOMXSP and TOMSAP
The plasmid into which the DNA fragment amplified by using the pair was inserted was named pTX301. The E. coli JM109 strain transformed with pTX301 was Escherichia.
It is named and displayed as coli JM109 / pTX301 and has been deposited as FERM BP-4223 at the Institute of Biotechnology, Institute of Biotechnology, AIST.

[0065]

EXAMPLES The present invention will be shown below with examples, but the present invention is not limited to the scope of these examples.

Example 1 Maize (Zea mays) seeds (manufactured by Snow Brand Seedling Co.) were germinated, and 10 μg of poly (A) was prepared in the same manner as in Reference Example 2-.
⁺ RNA was extracted and a cDNA library was prepared using the λEXlox ^™ Introductory Cloning Kit. That is, Gene, 25, 263 (1
After synthesizing cDNA according to the method described in 983), it was ligated to vector λEXLX using the EcoRI linker and HindIII linker in the kit, and then a cDNA library was prepared. The cDNA fragment of about 1.1 kbp obtained in Reference Example 2 was subjected to [α- ³² P] dCTP using a random primer DNA labeling kit.
Was labeled with and used as a probe for hybridization.
The plaque hybridization method was performed on the maize cDNA library prepared by the above method. About 1 out of 1 × 10 ⁴ plaques gave positive plaques. From these plaques, according to the method of Parazolo et al.
The fragment containing A was recovered as a plasmid. This cdn
The A fragment was cleaved with restriction enzymes EcoRI and HindIII, and the length of the cDNA insert was confirmed by agarose gel electrophoresis to confirm a cDNA fragment of about 1.2 kbp. This c
The plasmid containing the DNA fragment was named pCX101. The obtained cDNA fragment of about 1.2 kbp was cleaved with several restriction enzymes and analyzed by agarose gel electrophoresis.
The result is shown in FIG. That is, FIG. 1 is a diagram showing a restriction enzyme map of a cDNA fragment of about 1.2 kbp. Escherichia coli JM109 strain was transformed with the plasmid pCX101, and the transformant was designated and designated as Escherichia coli JM109 / pCX101. The strain has been deposited as FERM P-13768 at the Institute of Biotechnology, Institute of Biotechnology, AIST.

Example 2 Cloning of rice endo-type XG transferase gene Rice seeds (provided by Dr. Kazuhiko Nishitani, Kagoshima University) were germinated, and 10 μg of poly (A) ^{+ was} prepared in the same manner as in Reference Example 2-.
RNA was extracted and a cDNA library was prepared using the λEXlox ^™ Introductory Cloning Kit. That is, Gene, 25, 263 (19
83), after synthesizing cDNA according to the method described in
After ligation to the vector λEXLX using the EcoRI and HindIII linkers in the kit,
A cDNA library was created. The cDNA fragment of about 1.1 kbp obtained in Reference Example 2 was used as a random primer DN.
It was labeled with [α- ³² P] dCTP using the A labeling kit and used as a probe for hybridization. The plaque hybridization method was performed on the rice cDNA library prepared by the above method. 1 x 10
^{About 4} out of ⁴ plaques gave positive plaques. From these plaques, a fragment containing cDNA was recovered as a plasmid from λ phage DNA according to the method of Parazolo et al. This cDNA fragment was cleaved with restriction enzymes EcoRI and HindIII, and the length of the cDNA insert was confirmed by agarose gel electrophoresis.
A 1 kbp cDNA fragment was confirmed. The plasmid containing this cDNA fragment was designated as pRX102. Got about
The 1.1 kbp cDNA fragment was digested with several restriction enzymes and analyzed by agarose gel electrophoresis. The result is shown in Figure 2.
Shown in. That is, FIG. 2 is a diagram showing a restriction enzyme map of a cDNA fragment of about 1.1 kbp. Plasmid pRX102
Escherichia coli JM109 strain was transformed with Escherichia coli and the transformant was designated and designated as Escherichia coli JM109 / pRX102.
The strain is FER to Institute of Biotechnology
Deposited as MBP-4221.

Example 3 Cloning of tobacco endo-type XG transferase gene and determination of its nucleotide sequence Plasmid pTX301 was digested with restriction enzymes XbaI and Sac.
A DNA fragment of about 930 bp obtained by digesting with I was
It was labeled with [α- ³² P] dCTP using a random primer DNA labeling kit and used as a probe for hybridization. A plaque hybridization method was performed on a cDNA library (manufactured by Stratagene) using phage λ-ZAPII prepared from mRNA of tobacco (Nicotiana tabacum) leaves. 3.5 x
Positive plaques were obtained in a ratio of 5 to 10 ⁵ plaques. Confirm the length of the insert by PCR,
It was confirmed that a cDNA having a length of 0.7 kbp to 1.2 kbp was inserted. Of these, a fragment of about 1.2 kbp was recovered as a plasmid containing a cDNA from λ-ZAPII by the method described in the manual for the above-mentioned cDNA library, and the obtained plasmid was named pNX101. pNX101 was cleaved with several restriction enzymes and analyzed by agarose gel electrophoresis. The result is shown in FIG. That is, FIG. 3 is a diagram showing a restriction enzyme map of a DNA fragment of about 1.2 kbp. Escherichia coli JM109 strain was transformed with the plasmid pNX101, and the transformant was transformed into Escher.
It was named and displayed as ichia coli JM109 / pNX101. The strain is FERM P
It has been deposited as -13803.

The plasmid pNX101 was digested with the restriction enzyme Sac
A clone containing various sizes of fragments, which was digested with I and XbaI and digested from the end on the recognition sequence side of XbaI with the vector-dilation kit (manufactured by Takara Shuzo) to be incorporated into the vector. Got Using these clones BcaBE according to the method of Sanger
The base sequence of a DNA fragment of about 1.2 kbp was determined using ST ^™ Dideoxy Sequencing Kit (Takara Shuzo). A part of the nucleotide sequence is shown in SEQ ID NO: 7 in the sequence listing.

[0070]

INDUSTRIAL APPLICABILITY The present invention provides a gene encoding an endo-XG transferase which is involved in the transfer reaction of XG molecular weight and which has an important significance in the mechanism of plant cell wall growth.

[0071]

[Sequence list]

SEQ ID NO: 1 Sequence length: 30 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide Fragment type: N-terminal partial fragment Origin: Biological name: Vigna angularis (Vigna angularis) Sequence: Ala Asn Pro Arg Thr Pro Ile Asp Val Pro Phe Gly Arg Asn Tyr 1 5 10 15 Val Pro Thr Trp Ala Phe Asp His Ile Lys Tyr Leu Asn Gly Gly 20 25 30

SEQ ID NO: 2 Sequence Length: 29 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid (Synthetic DNA) Sequence: ATCGAYGTGC CGTTYGGGMG NAAYTAYGT 29

SEQ ID NO: 3 Sequence Length: 29 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid (Synthetic DNA) Sequence: CCGACGTGGG CGTTYGAYCA YATHAARTA 29

SEQ ID NO: 4 Sequence length: 37 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence: GTTTTCCCAG TCACGACTTT TTTTTTTTTT TTTTTTT 37

SEQ ID NO: 5 Sequence length: 1133 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Hypothetical sequence: NO Antisense: NO Origin: organism Name: Vigna angularis
is) sequence: GTTCTTCTTT GTGGACTTGT CTGATTCTGT TATCACTGGC TTCTGCTTCT TTCGCTGCCA 60 ACCCAAGAAC TCCAATTGAT GTACCATTTG GCAGAAACTA TGTGCCTACT TGGGCCTTTG 120 ATCATATCAA ATATCTCAAT GGAGGTTCTG AGATTCAGCT TCATCTCGAT AAGTACACTG 180 GTACTGGATT CCAGTCCAAA GGGTCATACT TGTTTGGTCA CTTCAGCATG TACATAAAAT 240 TGGTTCCTGG TGATTCAGCT GGCACAGTCA CTGCTTTCTA TTTATCGTCC ACAAACGCAG 300 AACATGATGA AATAGACTTC GAGTTCTTGG GAAACAGAAC TGGGCAACCA TACATTTTAC 360 AAACAAATGT GTTCACCGGA GGCAAAGGTG ACAGAGAGCA GAGAATCTAC CTCTGGTTTG 420 ACCCTACGAC TCAATACCAC AGATATTCAG TGCTATGGAA CATGTACCAG ATTGTATTCT 480 ATGTGGATGA CTACCCAATA AGGGTGTTCA AGAACAGCAA TGACTTGGGA GTGAAGTTCC 540 CCTTCAATCA ACCAATGAAA ATATACAACA GTTTGTGGAA TGCAGATGAC TGGGCTACAA 600 GGGGTGGTTT GGAGAAAACA GATTGGTCCA AAGCCCCCTT CATAGCCTCT TACAAGGGCT 660 TCCACATTGA TGGGTGTGAG GCCTCAGTGA ATGCCAAGTT CTGTGACACA CAAGGCAAGA 720 GGTGGTGGGA TCAACCAGAG TTTCGTGACC TTGATGCTGC TCAGTGGCAA AAACTGGCTT 780 GGGTACGCAA CAAATACACC ATCTACAACT ACTGCACTGA TCGCAAACGC TACTCTCAAG 840 TCCCTCC AGA GTGCACCAGA GACCGTGACA TTTAATCATT TTAACCTATA CTTTAAGGCC 900 TCATTATTTT CAATATCACA CTCCCATAAA GTTCCCACCT AAGTGCTGAT ACAGATTTCA 960 TTTGAGCTTG CTATTGCTTC CTGTATTGTT GATAATCATA TCATCATTTA TTGCTGCTAC 1020 TGTTTGGCTA TGTACCAGAT ATGGCTCTGT GCCTTAATTT GTATCTGTAA TTCTGTTTCA 1080 CTCTGAATCA ATATACTAGT AATACTACTA GTTTATACTG GTTAAAAAAA AAA 1133

SEQ ID NO: 6 Sequence length: 1128 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Hypothetical sequence: NO Antisense: NO Origin: organism name: tomato (Lycopersicon esculentum) sequence: GAAAAATACA CAAACACTGG TCTCTGATTG GATTTGTTTT TCTCACCATG GGTATCATAA 60 AAGGAGTTTT ATTTAGTATT GTTTTGATTA ATTTGTCACT TGTTGTATTT TGTGGGTATC 120 CTAGAAGGCC AGTAGATGTG CCCTTTTGGA AAAACTATGA GCCAAGTTGG GCTAGTCACC 180 ATATTAAGTT CCTCAATGGT GGTACCACTA CTGATCTTAT TCTCGACAGA TCTTCAGGAG 240 CTGGATTTCA GTCAAAGAAA TCATATCTGT TTGGGCATTT CAGTATGAAA ATGAGGCTTG 300 TTGGTGGAGA CTCAGCTGGT GTTGTCACTG CATTTTACCT GTCATCGAAT AATGCAGAGC 360 ACGATGAGAT AGATTTTGAA TTTTTGGGGA ACAGAACTGG GCAGCCATAC ATATTGCAGA 420 CAAATGTATT CACAGGAGGA AAAGGAAACA GAGAACAGAG AATATATCTT TC ATTGTCTGATCGATCGATCGATCGATCGATCGATCGATCCAGTCATCGATCCAGTCAGA TGAAGATA TACTCGAGTC TATGGGACGC AGATGATTGG GCCACAAGAG 660 GTGGGCTTGA GAAAACCAAT TGGGCCAACG CCCCATTCAC CGCGTCATAC ACATCGTTCC 720 ACGTGGATGG ATGTGAAGCT GCCACGCCAC AAGAAGTCCA AGTTTGTAAC ACTAAAGGCA 780 TGAAATGGTG GGATCAAAAG GCCTTCCAAG ATTTAGATGC ATTACAGTAT AGGAGACTTC 840 GTTGGGTTCG TCAAAAATAC ACTGTTTATA ACTATTGCAC TGATAAAGCG AGGTACCCTG 900 TTCCACCACC AGAGTGCACT AAGGACAGAG ATATTTAAAA TCATAATCAA AATTAAGAGG 960 GACTTTATGA AGAAAAAAAA CTTAATATGC TTTATGTGTG AGTATTTTAA TGATCCTTAA 1020 AACAAAGTGC TTTTAATTGA GCTGTATTTC CCTAATTCTT TTTGAGTGTA TCATTATTGG 1080 TGGAGTCATG AGGATATTAT GTATCTCATG CCAGGCCTTT CATGTCTC 1128

SEQ ID NO: 7 Sequence length: 888 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Hypothetical sequence: NO Antisense: NO Origin: organism name: tobacco (Nicotiana tabacum) sequence: ATGGGTGTAA AAGGACTTTT GTTTAGTATT GTTTTGATTA ATTTGTCATT ACTAGGACTT 60 TGTGGGTATC CCAGAAAACC AGTGGATGTA CCCTTTTGGA AAAACTATGA GCCCAGTTGG 120 GCTAGTCACC ACATCAAGTA CCTCAGTGGT GGTTCCACTG TTGATCTTGT TCTTGACAGG 180 TCTTCAGGTG CTGGATTTCA GTCAAAGAAA TCATATTTGT TTGGGCACTT TAGCATGAAA 240 CTGAAGCTTG TTGGTGGAGA CTCAGCTGGC GTTGTCACTG CATTTTACCT GTCATCGAAT 300 AATGCAGAGC ACGATGAGAT AGATTTTGAA TTCTTAGGGA ACAGGACTGG GCAACCATAC 360 ATTTTGCAGA CAAATGTGTT CACGGGAGGA AAAGGAGACA GAGAGCAGAG AATCTATCTC 420 TGGTTTGACC CAACCAAGGG TTACCATTCT TATTCTGTTC TTTGGAATG CGTCCGAGACAGACACA CCTAGATCATCAT CCAAGA ACTCAAAAGA CCTAGGTGAG GAAAACAGAC TGGTCCAATG CCCCATTTAC TGCCTCCTAC 660 ACATCATTCC ACGTGGACGG CTGTGAAGCT GCCACCCCAC AAGAAGTCCA AGTTTGTAAC 720 ACCAAAGGTAGG CATATCAAGAGATTATCATGACGAATCATGACTTAG

SEQ ID NO: 8 Sequence length: 888 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Hypothetical sequence: NO Antisense: YES Origin: organism name: tobacco (Nicotiana tabacum) sequence: TTAAATATCT CTGTCCTTAG TGCACTCTGG GGGAAGTGTA GGGTACCTCT TCCTATCAGT 60 ACAATAATTA TAAATAGTGT ATTTTTGGCG AACCCAACGA AGTCTCCTGT ATTGTAAAGC 120 ATCTAAATCT TGGAAAGCCT TTTGATCCCA CCATCTCATG CCTTTGGTGT TACAAACTTG 180 GACTTCTTGT GGGGTGGCAG CTTCACAGCC GTCCACGTGG AATGATGTGT AGGAGGCAGT 240 AAATGGGGCA TTGGACCAGT CTGTTTTCTC CAATCCACCT CTTGTGGCCC AATCATCTGC 300 ATCCCAAAGG CTTGAGTATA TTTTCATGGG CTGATTGAAT GGAAATTTCA CACCTAGGTC 360 TTTTGAGTTC TTGAATGCTC TAATTGGGAC GTCATCCACA AAGATCACAA TCTGGAAGGT 420 ATTCCAAAGA ACAGAATAAG AATGGTAACC CTTGGTTGGG TCAAACCAGA GATAGATTCT 480 CTGCTCTCGATCATCATCCACTCATCCACTCATCCATCATCACTCATCATCTCTCTCTTCTCCTCTCCCCTCTGCTAACC540TGTCCACTATCCAGCTCTCCCCCGTGAACACATTTGTC TGCAAACT C CAGCTGAGTC TCCACCAACA AGCTTCAGTT TCATGCTAAA 660 GTGCCCAAAC AAATATGATT TCTTTGACTG AAATCCAGCA CCTGAAGACC TGTCAAGAAC 720 AAGATCAACA GTGGAACCAC CACTGAGGTA CTTGATGTGG TGACTAGCCC AACTGGGCTC 780 ATAGTTTTTC CAAAAGGGTA CATCCACTGG TTTTCTGGGA TACCCACAAA GTCCTAGTAA 840 TGACAAATTA ATCAAAACAA TACTAAACAA AAGTCCTTTT ACACCCAT 888

SEQ ID NO: 9 Sequence Length: 888 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: mRNA Hypothetical Sequence: NO Antisense: YES Origin: Organism Name: tobacco (Nicotiana tabacum) sequence: UUAAAUAUCU CUGUCCUUAG UGCACUCUGG GGGAAGUGUA GGGUACCUCU UCCUAUCAGU 60 ACAAUAAUUA UAAAUAGUGU AUUUUUGGCG AACCCAACGA AGUCUCCUGU AUUGUAAAGC 120 AUCUAAAUCU UGGAAAGCCU UUUGAUCCCA CCAUCUCAUG CCUUUGGUGU UACAAACUUG 180 GACUUCUUGU GGGGUGGCAG CUUCACAGCC GUCCACGUGG AAUGAUGUGU AGGAGGCAGU 240 AAAUGGGGCA UUGGACCAGU CUGUUUUCUC CAAUCCACCU CUUGUGGCCC AAUCAUCUGC 300 AUCCCAAAGG CUUGAGUAUA UUUUCAUGGG CUGAUUGAAU GGAAAUUUCA CACCUAGGUC 360 UUUUGAGUUC UUGAAUGCUC UAAUUGGGAC GUCAUCCACA AAGAUCACAA UCUGGAAGGU 420 AUUCCAAAGA ACAGAAUAAG AAUGGUAACC CUUGGUUGGG UCAAACCAGA GAUAGAUUCU 480 CUGCUCUCUG UCUCCUUUUC CUCCCGUGAA CACAUUUGUC UGCAAAAUGU AUGGUUGCCC 540 AGUCCUGUUC CCUAAGAAUU CAAAAUCUAU CUCAUCGUGC UCUGCAUUAU UCGAUGACAG 600 GUAAAAUGCA GUGACAACGC CAGCUG AGUC UCCACCAACA AGCUUCAGUU UCAUGCUAAA 660 GUGCCCAAAC AAAUAUGAUU UCUUUGACUG AAAUCCAGCA CCUGAAGACC UGUCAAGAAC 720 AAGAUCAACA GUGGAACCAC CACUGAGGUA CUUGAUGUGG UGACUAGCCC AACUGGGCUC 780 AUAGUUUUUC CAAAAGGGUA CAUCCACUGG UUUUCUGGGA UACCCACAAA GUCCUAGUAA 840 UGACAAAUUA AUCAAAACAA UACUAAACAA AAGUCCUUUU ACACCCAU 888

SEQ ID NO: 10 Sequence Length: 30 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid (Synthetic DNA) Sequence: GAGGAGCTCC CATGGGTGTA AAAGGACTTT 30

SEQ ID NO: 11 Sequence length: 30 Sequence type: Nucleic acid Number of strands: Single-stranded Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence: TAATCTAGAT ACTTAAATAT CTCTGTCCTT 30

SEQ ID NO: 12 Sequence length: 30 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence: GAGTCTAGAC CATGGGTGTA AAAGGACTTT 30

SEQ ID NO: 13 Sequence length: 30 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence: TAAGAGCTCT ACTTAAATAT CTCTGTCCTT 30

SEQ ID NO: 14 Sequence Length: 30 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid (Synthetic DNA) Sequence: TTTTCTAGAC CATGGGTATC ATAAAAGGAG 30

SEQ ID NO: 15 Sequence length: 30 Sequence type: Nucleic acid Number of strands: Single-stranded Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence: TTTGAGCTCC CATGGGTATC ATAAAAGGAG 30

[Brief description of drawings]

FIG. 1 is a diagram showing a restriction map of an example of a gene fragment encoding the endo-type XG transferase of the present invention.

FIG. 2 is a diagram showing a restriction map of an example of a gene fragment encoding the endo-XG transferase of the present invention.

FIG. 3 is a diagram showing a restriction enzyme map of an example of a gene fragment encoding an endo-type XG transferase of the present invention.

FIG. 4 is a diagram showing a chromatograph in which a product of a reaction of endo-XG transferase of Vigna angularis is measured by PAD.

FIG. 5 is a diagram showing a restriction enzyme map of an example of a gene fragment encoding an endo-type XG transferase of Vigna angularis.

FIG. 6 is a diagram showing a restriction enzyme map of an example of a gene fragment encoding an endo-type XG transferase of tomato.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Kiyozo Asada 3-4-1 Seta, Otsu City, Shiga Prefecture, Central Research Laboratory, Mina Shuzo Co., Ltd. (72) Inventor Ikuno Kato, 3-chome Seta, Otsu City, Shiga Prefecture No. 4 No. 1 in the Central Research Laboratory, Takara Shuzo Co., Ltd.

Claims (1)

[Claims] 1. An endo-type xyloglucan transferase gene, which is represented by the restriction enzyme maps shown in FIGS. 1 to 3 of the drawings.