JP3513192B2 - A novel rice starch branching enzyme gene - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel rice starch branching enzyme gene and a method for changing the rice starch enzyme activity in plant cells using the gene.

[0002]

BACKGROUND OF THE INVENTION Cereals are a major source of calories for us and an important source of lipids and proteins. Therefore, qualitative and quantitative improvement of grain endosperm storage components has become a major breeding goal.

In order to achieve this goal, it is necessary to search and collect genetic resources for storage components in cereal endosperm and to evaluate their genetic and breeding properties. For example, various genetic mutations (mutations) in endosperm components have been found in maize, and they play an important role in improving the quality of maize. Furthermore, although these mutations provide valuable information when elucidating the genetic control mechanism in vivo, the gene structure and expression regulation mechanism relating to these have not been elucidated.

On the other hand, rice is mainly dietary, and the content ratio of polysaccharides in the endosperm starch and structural changes in starch greatly change the taste and cooking characteristics of rice, and the starch content in the grain is related to taste and processing. It is closely related to the characteristics. Three stages are considered as the mechanism of starch synthesis. First, the synthesis of oligosaccharide as a template for starch synthesis, its extension reaction,
Furthermore, the linear starch thus formed is synthesized in the order of branching reaction. As enzyme proteins related to starch synthesis, starch synthase bound to starch granules such as waxy protein and soluble starch synthase are known, and several types of starch branching enzymes involved in amylopectin synthesis are known to exist. There is. However, little knowledge has been obtained about these enzyme proteins.

By the way, starch is a final product of photosynthesis and is a major component of cereals, and research on its biosynthesis is one of the important subjects in plant physiology and biochemistry. Starch is composed of linear amylose in which glucose is α (1 → 4) -bonded, and amylopectin having a branched structure by α (1 → 6) bond. It is known that the content ratio of amylose and amylopectin in starch granules in plants has unique values depending on the plant species and varieties, and their higher-order structures also have unique forms.

Starch synthase, which is the main enzyme of starch biosynthesis, uses ADP glucose or UDP glucose as a substrate,
Perform the synthesis of amylose molecules. On the other hand, the amylopectin molecule has a starch synthase that forms an α (1 → 4) bond and a branching (branching) that forms an α (1 → 6) bond.
It is made by the action of enzymes in a coordinated manner. Research on the formation mechanism of the branching structure of amylopectin has been carried out with reference to the research on the branching mechanism of glycogen, and it is possible that there are enzymes with similar properties to the glycogen branching enzyme in plants. Has been suggested. However, research on the gene, or research on the genetic regulatory mechanism, has not made much progress.

[0007]

In view of the above-mentioned state of the art, the present invention focuses on a novel starch branching enzyme, which has a significant effect on amylopectin synthesis, among rice genes, and The issues are to separate, increase food production, produce rice with excellent taste, and breed plants, especially rice.

[0008]

As a result of intensive studies to solve the above-mentioned problems, the present inventor found a novel starch branching enzyme, and clarified the amino acid sequence of this enzyme protein. The gene was isolated and the present invention was achieved.

That is, the present invention encodes the cDNA of the rice starch branching enzyme gene having the base sequence shown in SEQ ID NO: 1, and the mature protein of the rice starch branching enzyme having the amino acid sequence 66 to 825 shown in SEQ ID NO: 1. Gene, a gene encoding a transit protein of a rice starch branching enzyme having the amino acid sequence of 1 to 65 shown in SEQ ID NO: 1, and a promoter of the rice starch branching enzyme gene contained in the base sequence shown in SEQ ID NO: 4 Is.

The present invention also provides a DNA in which a DNA sequence encoding all or part of the amino acid sequence shown in SEQ ID NO: 1 and a promoter expressible in plant cells are linked.
Provided is a plant cell or rice into which a sequence has been introduced, and a grain obtained by cultivating this rice.

Further, the present invention is a DN in which a DNA sequence encoding all or part of the amino acid sequence shown in SEQ ID NO: 1 and a promoter expressible in plant cells are linked.
Method for changing rice starch branching enzyme activity by introducing A sequence into plant cells, seed-specific by introducing DNA sequence combining rice starch branching enzyme gene promoter and heterologous gene into plant cells And a method for expressing said heterologous gene.

The rice starch branching enzyme gene of the present invention comprises:
Also included is a gene encoding a transit peptide that has the ability to efficiently transfer the rice starch branching enzyme to amyloplasts. In the present specification, unless otherwise specified, the “starch branching enzyme” refers to an enzyme including a transit peptide or an enzyme excluding the transit peptide, and particularly an enzyme excluding the transit peptide is referred to as a “mature enzyme”. Further, when the starch branching enzyme cDNA is used as a gene, this is referred to as "starch branching enzyme gene".

The present invention will be described in detail below. The rice starch branching enzyme according to the present invention is a novel enzyme and is named as type III. As will be described later, the present inventor fractionated rice starch branching enzyme into three types and named them type I, II and III, respectively.

Of these, starch synthesis by the type III enzyme is similar to the branching reaction in the glycogen synthesis of animals and has homology with the glycogen branching enzyme of Escherichia coli. The mechanism of this reaction is not clear, but it plays an important role in amylopectin synthesis in the early stage of seed starch formation, and the rice lacking this enzyme has a very high amylose content. Therefore, increasing the activity of this enzyme leads to increasing the content of amylopectin in the grain. Therefore, it is of great significance to enhance this enzyme activity in crop breeding.

To reduce the amylose content, there are a method of increasing the amylopectin content by suppressing the amylose synthesis system, and a method of strengthening the pathway for synthesizing amylopectin to positively increase the content. In the latter case, it also increases the amount of starch produced, so it is considered to be a very good target for rice breeding.

<1> Type III Rice Starch Branching Enzyme Gene To isolate the gene, for example, the enzyme is purified, the amino acid sequence is determined, and a synthetic oligonucleotide probe is prepared based on this sequence. There is a method of selecting from a gene library by hybridization. Therefore, a method for purifying a starch branching enzyme from rice will be exemplified below.

Grained rice ripened seeds were crushed with a juicer while cooling with ice, 50 mM Tris-HCl (pH 8.5), 5 mM EDTA, 5 m
Suspend in a buffer containing M mercaptoethanol. After removing the insoluble fraction by filtration and centrifugation, ammonium sulfate is added to precipitate the total protein. The precipitated protein fraction was dissolved in a buffer solution consisting of 50 mM Tris-HCl (pH 7.5), 5 mM EDTA and 5 mM mercaptoethanol, and after dialysis DE
Chromatography is performed using AE-cellulose (Whatman DE-52). The fraction containing the rice starch branching enzyme is Boyer
And Preiss' method (Carbohydrate Res. 61, 321-334 (197
8)) can be used for verification.

Thus, the rice starch branching enzyme was obtained by being divided into three parts, which were designated as type I, type II and type III, respectively. The active fraction was further separated and purified as a single band by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) by gel filtration column chromatography using Toyopearl HW55F, and both are novel enzymes. The type III enzyme is a protein that migrates to the 87 kDa position.

If the N-terminal amino acid sequence of the enzyme thus purified is determined, a probe can be prepared based on this sequence. III determined in the present invention
The N-terminal amino sequence of this type of starch branching enzyme is shown in SEQ ID NO: 2.

In addition, type I and type II enzymes and their genes were obtained in the same manner, but it is presumed that these enzymes are the same gene products and are divided into two types by cleavage after transcription and the like. In addition, these enzymes are completely different from the type III enzyme in terms of antigen-antibody reaction.

<2> Construction of cDNA library derived from rice ripening seeds As for the cDNA library, mRNA is extracted from rice ripening seeds, cDNA is synthesized using reverse transcriptase, and double-stranded by polymerase reaction. It can be prepared by inserting the prepared product into a vector and transforming into Escherichia coli or the like.
Since cDNA cloning kits are commercially available, these may be used.

The vector used for preparing the library is
Many types are commercially available and these can be used. The methods necessary for general gene recombination such as DNA cleavage, ligation, transformation, gene nucleotide sequence determination, and hybridization are described in the instructions attached to commercially available enzymes used for each operation, and Molecular cloning. (Maniatis T.
et al. Cold Spring Harbor Laboratory Press).

<3> Cloning of starch branching enzyme cDNA and chromosomal gene and expression in plant cells Using a probe prepared on the basis of an amino acid sequence determined from the purified enzyme, plaque hybridization or the like is carried out to obtain cDNA live. The starch branching enzyme gene can be obtained from a rally or chromosomal DNA library.

Using all or part of the obtained gene as a probe, the starch branching enzyme gene can be screened from a chromosomal DNA library by plaque hybridization or the like. Similarly,
Other starch branching enzyme genes can be selected from plant gene libraries and the genes thus obtained can also be used in the present invention.

Cloned cDNA or chromosomal DN
The base sequence of A is determined by the Maxam-Gilbert method or the dideoxy method. The nucleotide sequence can be determined by the dideoxy method using a commercially available kit, and an autosequencer for automatically performing the sequence determination may be used.

The nucleotide sequence of the thus-obtained type III starch branching enzyme cDNA and the amino acid sequence deduced from this sequence are shown in SEQ ID NO: 1. Also, chromosome D
The promoter region of the starch branching enzyme gene obtained from the NA library is shown in SEQ ID NO: 4.

An artificial starch branching enzyme expression system can be constructed by linking all or part of the gene encoding this amino acid sequence to a plant-expressible promoter such as a promoter derived from cauliflower mosaic virus. it can.

Furthermore, by transfecting all or part of the starch branching enzyme gene bound to a promoter in the reverse direction into a plant and expressing a so-called antisense RNA, translation of the starch branching enzyme gene mRNA is performed. Can be inhibited and the amount of enzyme production can be suppressed.

Furthermore, when a DNA fragment obtained by ligating a promoter of a starch branching enzyme gene as a promoter and a starch branching enzyme gene or a heterologous gene is introduced into a plant, these genes are expressed in a seed-specific manner. You can

By introducing the expression system thus constructed into plants such as rice and corn, the plants can be transformed and the starch content of the transformed plants can be arbitrarily changed. Heterologous genes can be similarly expressed in these plants.

A DNA in which the DNA sequence encoding all or part of the amino acid sequence shown in SEQ ID NO: 1 according to the present invention and a promoter that can be expressed in plant cells are linked.
The sequences include those linked in the forward direction or the reverse direction, that is, those expressing the rice starch branching enzyme gene and those expressing the antisense RNA of the same gene. This also applies to heterologous genes.

Transformation of plants is carried out by electroporation or Ti of Agrobacterium.
It can be performed by introducing DNA into protoplasts by a method using a plasmid or the like.
Hereinafter, the method of preparing protoplasts in rice, introducing DNA into protoplasts by electroporation, and regenerating transformed cells into plants will be exemplified.

Preparation of rice protoplasts is carried out, for example, as follows. Callus formed from immature embryos of rice seeds was treated with R2 inorganic salt (Ohira, K. et al. Plant Cell
Physiol. 14, 1113 (1973)), B5 vitamin (Gambor
g, O. et al. Expt. Cell Res. 50, 151 (1968)), 0.
3% casein hydrolyzate, 1 ppm 2,4-D, and 3%
Transfer to a liquid medium containing sucrose, and cultivate on a rotary shaker set at 60 rpm under irradiation with fluorescent light of 500 lux at 25 ° C.

Each week, subculture was performed in the above-mentioned fresh medium, and 1 g of cells on the 5th day of subculture was treated with 1% Macerozyme R10 and 4% cellulase RS.
(Above, made by Kinki Yakult Co., Ltd.), 0.5% CaCl ₂ · 2H ₂
Mix with 10 ml enzyme solution containing O, 0.5% potassium dextran sulfate, 0.4 M mannitol.

The pH of this solution was adjusted to 5.5 and 27 ° C.
Shake (40 rpm) for 3 hours and then leave for 3 hours.
The protoplast suspension generated by the enzyme treatment was
Debris is removed through a nylon screen of m.

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

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

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

Next, a method for regenerating a transformed cell obtained as described above into a plant (Fujimura, T. et al. Plant)
Tissue Culture Lett. 2, 74 (1985)). The electroporated protoplasts were washed 4 times with a washing medium containing R2 inorganic salt and 0.4 M glucose by centrifugation (50 × g, 5 minutes), and then 2 ppm of 2,4-D, 0. N6 medium supplemented with 5 ppm benzyladenine and 3% sucrose (Chu, C.-C. Proceedings o
f Symposium on Plant Tissue Culture (Scientific Pr
ess, Peking) p.43 (1978)) and the number of cells was 10 ⁶
Incubate in the dark at 25 ° C.

On the 14th day of culture, hygromycin is added to the medium in an amount of 50 μg / ml, the culture is continued for 2 weeks, and the colonies are transferred to a fresh medium containing hygromycin at the same concentration.
When the colony grows to a diameter of 2 mm, it is transferred to an N6 hormone-free medium containing no hygromycin, and cultured under fluorescent light irradiation (3000 lux) for differentiation. The seedlings that have grown to a height of about 10 cm in a test tube are acclimated and then transferred to a pot in a greenhouse to regenerate the plant.

The selection of the clone containing the starch branching enzyme gene may be carried out by collecting callus or plant cells and confirming it by a method such as Southern hybridization. After regeneration, it is preferable to check the amount of enzyme in the protein in the extract of plant leaves by Western analysis or the like, while confirming that the starch branching enzyme gene has been introduced by Southern analysis or the like.

<4> Utilization of Sequence Encoding Transit Peptide The starch branching enzyme has a transit peptide, which is a sequence for efficiently transferring to amyloplast, on the N-terminal side. The starch branching enzyme gene shown in SEQ ID NO: 1 also includes a portion encoding this transit peptide, and the base sequence of 128-322 corresponds to this.

When a sequence encoding an arbitrary protein is ligated to the downstream of the sequence encoding this transit peptide in frame, the protein can be efficiently converted into amyloplasts when expressed in plant cells. Can be moved to.

<Application of the Present Invention> According to the present invention, the amino acid sequences of the transit peptide of the type III rice starch branching enzyme and the protein having the enzymatic activity have been clarified.
Utilizing these, it becomes possible to mass-produce the type III rice starch branching enzyme in rice or in plants other than rice, and the present invention can be used for new breeding of rice and the like. Therefore, the present invention can be utilized in the field of plant breeding, industrial production of type III rice starch branching enzyme, and is extremely useful in the technical fields of plants, food production, enzyme industry and the like.

For the first time, the present invention succeeded in cloning and structurally analyzing a type III rice starch branching enzyme gene containing a gene encoding a transit peptide. Therefore, the present invention can exert various remarkable effects, and one example thereof will be shown below.

As already mentioned, it was obtained according to the invention.
The cDNA of type III rice starch branching enzyme encodes the entire region of this enzyme protein as shown in SEQ ID NO: 1. Therefore, it became possible to construct an expression system for an artificial type III starch branching enzyme by combining the nucleotide sequence of this cDNA with a promoter derived from cauliflower mosaic virus. It is possible to introduce the constructed artificial gene into plants such as rice and tobacco by electroporation or a method utilizing Agrobacterium, and as a result, the type III starch branch of the resulting transformed plant is obtained. It is possible to increase the production amount of dip enzyme. In addition, it was possible to increase the amylose content in seeds by using antisense technology.

CDN of type III rice starch branching enzyme of the present invention
The transit peptide encoded by A has the ability to efficiently transfer subsequent proteins to amyloplasts in rice. Therefore, when a nucleotide sequence encoding a type III starch branching enzyme is replaced with a nucleotide sequence encoding an arbitrary protein after the nucleotide sequence encoding a transit peptide, the protein is efficiently transferred to amyloplast. be able to.

Furthermore, the nucleotide sequence encoding a protein having the enzymatic activity of the type III starch branching enzyme can be used for expression of the gene by an artificial method such as an in vitro transcription system or a microorganism such as Escherichia coli. By doing II
The type I starch branching enzyme can be obtained in large quantities and in pure form. The obtained type III starch branching enzyme has an enzymatic activity for efficiently performing starch synthesis, and is therefore effective in quantitatively modifying starch. In addition, it became possible to improve the quality of starch by performing this enzyme treatment.

Using the nucleotide sequence of the cDNA of the present invention, a part of this nucleotide sequence is specifically mutated by using a method such as site-specific substitution or PCR method to form a type III starch branching enzyme. It can be used for research to improve the function of At the same time, it can be used for research on functional expression of enzyme proteins.

[0050]

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

[0051]

Example 1 Acquisition and Utilization of Type III Rice Starch Branching Enzyme cDNA <1> Purification and Characterization of Type III Rice Starch Branching Enzyme cDNA Ripe seeds of rice grown in the Koshihikari variety on ice Grind with a juicer while cooling, 50 mM Tris-HCl (pH 8.
5), 5 mM EDTA and 5 mM mercaptoethanol were suspended in a buffer solution. The insoluble fraction was removed by filtration and centrifugation, and ammonium sulfate was added to precipitate the total protein. The precipitated protein fraction was added to 50 mM Tris-HCl (pH 7.5),
It was dissolved in a buffer solution containing 5 mM EDTA and 5 mM mercaptoethanol, dialyzed and then chromatographed using DEAE-cellulose (Whatman DE-52).

The fraction containing the rice starch branching enzyme was Boyer
And Preiss' method (Carbohydrate Res. 61, 321-334 (197
8)). As a result, the rice starch branching enzyme was I
It was divided into three parts, named type II, type III, and type III. Next, the active fraction was subjected to gel filtration column chromatography using Toyopearl HW55F. Type III starch branching enzyme is SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
Was isolated and purified as a single protein band of 87 kDa.

Rabbit antiserum was prepared using the purified type III rice starch branching enzyme as an antigen, and used for subsequent Western blotting analysis and the like.

<2> Determination of N-terminal amino acid sequence of type III rice starch branching enzyme The above-mentioned type III rice starch branching enzyme solution was separated by SDS-PAGE to obtain a protein having rice starch synthase activity. The protein was taken out from the band and adsorbed on a PVDF membrane (Millipore). This is a protein analyzer (Applied Bi
Osystems Inc.) was used to examine the amino acid sequence from the N-terminal. As a result, the amino acid sequence shown in SEQ ID NO: 2 was obtained. The 15th amino acid in the sequence is Ser or C.
It was determined to be either ys.

<3> Construction of cDNA library derived from rice ripened seeds Total RNA was prepared from 20 g of rice Nipponbare ripened seeds according to the lithium chloride method. Furthermore, poly-A-RNA containing mRNA was analyzed by oligo dT cellulose column chromatography.
Was isolated, and cDNA was synthesized using the oligo dT primer and reverse transcriptase as a template. A cDNA synthesis kit manufactured by Pharmacia was used for the preparation of cDNA from total RNA. The obtained cDNA was ligated with the λgt11 arm and the cDNA, using an in vitro packaging kit (Stratagene, GIGAPACK Gold), packaging was performed, and a large number of recombinant λ phages were obtained by infecting E. coli Y1090 strain. It was This was used as a rice ripened seed cDNA library. The size of the library was 3.3x10 ⁶ .

<4> Cloning of rice starch branching enzyme cDNA From the above-mentioned N-terminal amino acid sequence of rice starch branching enzyme
The nucleotide sequence of DNA was estimated, and synthetic DNA corresponding to this amino acid sequence (corresponding to the 8th to 20th amino acid sequences in the amino acid sequence of SEQ ID NO: 2) was prepared. The sequence is shown in SEQ ID NO: 3. The other base (N) in the sequence is inosinic acid.

In the rice cDNA library described above,
300,000 independent plaques were formed on the agar medium, transferred to a nitrocellulose membrane, and
It used after ^{3 2} P labeled A as a probe was carried out screening of the cDNA of rice starch branch with enzymes. As a result, 3 positive clones were obtained. These phage DNAs were prepared and the insert was isolated by limited digestion of the phage DNA with the restriction enzyme EcoRI.
Furthermore, by inserting this fragment into the EcoRI site of plasmid pUC119, a plasmid clone (pRB33) was obtained.
I got the DNA.

<5> Analysis of Nucleotide Sequence of Starch Branching Enzyme cDNA The nucleotide sequence contained in pRB33 was determined by the dideoxy method (Messing, Methods in Enzymol., 101, 20-78 (198
3)) I went. The nucleotide sequence thus obtained and the amino acid sequence deduced from this sequence are shown in SEQ ID NO: 1.

Of this amino acid sequence, the sequence corresponding to the transit peptide is the sequence encoded by the 128th to 322nd nucleotide sequences (amino acid numbers 1 to 65).
And the sequence corresponding to the mature enzyme is 323-2602.
The sequence (amino acid number 66 to 825) encoded by the th base sequence.

When the above amino acid sequence was compared with the amino acid sequence determined from the protein of starch synthase, a matching sequence was found, and the cDNA isolated by plaque hybridization was found to be the starch branching enzyme cDNA. confirmed. Further detailed examination of the nucleotide sequence revealed that this enzyme has a transit peptide that plays an important role in translocating to amyloplast.

The type I starch branching enzyme gene obtained and determined in the same manner had almost no homology with the type III enzyme gene, and was 30% or less.

<6> Construction of antisense gene of type III rice starch branching enzyme gene and transformation of rice In the same manner as the previous section, a fragment containing the entire region of genetic information of type III rice starch synthase. After isolating T4 D
The ends were blunted using NA polymerase. Call this pBI22
Reverse cloning into the SmaI site of 1 and plasmid pBI
I got RBE3R. pBIRBE3R is located in the downstream of the 35S promoter of the cauliflower mosaic virus derived from pBI221, III
A type of starch-branching enzyme cDNA is present bound in the direction of the antisense gene. Therefore, the genetic information of the type III starch branching enzyme on this plasmid is 35S.
It is expressed by a promoter and transcribed as antisense RNA.

Transformation of rice with pBIRBE3R was performed by the method described above. As a plasmid having a hygromycin resistance gene, 10 μg of pUC19-H
Using PT, 20 μg of pBIRS3 was introduced into rice (Nipponbarre) embryo cell-derived protoplasts.

Electroporation requires an initial voltage of 47.
It was carried out by applying an electric pulse having 5 V / cm and T _1/2 of 30 milliseconds. Eight strains of transformed rice were obtained. Using a crude extract of ripened seeds obtained from the transformed strain, Western analysis using the above-mentioned antiserum was performed, and the expression level of the target type III starch branching enzyme was used as a control. Significantly lower than Nippon Barre 6
Was confirmed. The amylose content of the obtained seeds was measured by the iodine coloration method, and it was found that the amylose content was significantly higher than that of the control (30-40%).

Contrary to this example, rice with a low amylose content can be produced by ligating the type III starch branching enzyme cDNA in the forward direction to the promoter.

[0066]

Example 2 Acquisition and utilization of type III starch branching enzyme gene promoter region

<1> Preparation of Rice Genomic DNA and Construction of Genomic Library Rice nuclear DNA used as a material is the method of Rogers and Bendich (Plant
Prepared according to Mol. Biol. 5, 69 (1985). Got
DNA was digested with the restriction enzyme Sau3AI and subjected to sucrose density gradient centrifugation to obtain a fragment of 10 kb or more. These fragments were ligated to an arm of λEMBL3 phage cleaved with BamHI, followed by in vitro packaging kit (Stratagen
A large number of recombinant λ phages were obtained by packaging E. coli LE392 strain using GIGAPACK Gold manufactured by e. This was used as a rice genomic DNA library. The size of the library was 1.6 × 10 ⁶ .

<2> Isolation of Promoter Region of Type III Rice Starch Branching Enzyme Gene About 100,000 independent plaques in the above-mentioned rice genomic library were obtained in Example 1 and the type III rice starch was obtained. The promoter region of the rice branching enzyme gene was screened using the cDNA of the branching enzyme as a probe. As a result, 10 positive clones were obtained. Phage DNA was prepared from these positive clones and the size of the insert was examined. As a result, it was suggested that the obtained phage clones were derived from the same fragment. One of these phage clones was selected and named λRB3.

The insert fragment contained in the λRB3 phage genome was divided into four fragments when digested with the restriction enzyme SalI. Furthermore, Southern analysis was performed using the 5'region of the above-mentioned cDNA as a probe to identify a fragment containing the promoter region. A fragment containing the promoter region was obtained by cleaving λRB3 with restriction enzymes EcoRI and BamHI, and this was inserted into a fragment of pUC118 digested with EcoRI and BamHI, and the resulting plasmid was named pRBP1.

<3> Structural Analysis of Promoter Region of Type III Rice Starch Branching Enzyme Gene The nucleotide sequence of the pRBP1 insert fragment obtained above was determined by the dideoxy method. The structure of the promoter region was clarified by comparing the obtained base sequence with the base sequence of the above-mentioned cDNA, examining the sequences commonly found in promoters (TATA sequence, etc.), and performing Northern analysis and the like. I chose

The nucleotide sequence of the promoter region of the gene of the type III rice starch branching enzyme thus structurally analyzed is shown in SEQ ID NO: 4 in the sequence listing. In this region, the presence of nucleotide sequences corresponding to factors generally known to be present in promoters was confirmed (see Sequence Listing).

<4> Isolation of promoter of type III rice starch branching enzyme gene In order to isolate the promoter contained in pRBP1 by the PCR method, two kinds of synthetic DNA primers were synthesized. The 5'side primer has a sequence (SEQ ID NO: 5) shown in base numbers 4 to 23 in SEQ ID NO: 4, and the 3'side primer has a sequence complementary to base numbers 995 to 1115 in SEQ ID NO: 4 ( SEQ ID NO: 6). PRBP using these primers
Amplification by the PCR method of 1 was performed to amplify a fragment of about 1000 bp. The obtained fragment was inserted into the HincII site of pUC118 to obtain pRBP2. When the nucleotide sequence of this inserted fragment was determined, it was revealed that it corresponds to the promoter of the branching enzyme gene. The pRBP2 obtained here has XbaI, BamHI, and SmaI sites downstream of the promoter sequence of the type III rice branching enzyme gene. Therefore, it is possible to connect heterogeneous genes using these restriction enzyme cleavage sites.

<5> Construction of a gene in which the promoter of the type III rice branching enzyme gene and the GUS gene are linked to each other A plasmid p having a β-glucuronidase (GUS) gene
BI221 (Jefferson et al .; EMBO J. 6, 3901 (1987) pBI
221 was obtained from Clontech) was cut with BamHI and SstI to cut out the GUS gene region, and the cut ends were blunted using T4 DNA polymerase (Takara Shuzo, DNA blunting kit). This was inserted into the SmaI site of the plasmid pRBP2 having the promoter of the above-mentioned type III rice branching enzyme gene.

Thus, an artificial gene in which the GUS gene was bound to the downstream of the promoter of the type III rice branching enzyme gene was prepared. This was named pRBG1.

<6> Introduction of an artificial gene in which the promoter of the type III rice branching enzyme gene and the GUS gene are ligated into rice pRBG1 plasmid was introduced into rice (Nipponbarre) protoplast cells according to the method of Tada et al. Was introduced by electroporation. The obtained cells are
The plants were regenerated according to the method of Fujimura et al. When DNA was extracted from these cells and subjected to Southern hybridization, a DNA fragment that was considered to be derived from pRBG1 was present in 3 strains. The GUS activity of seeds of these transformed plants was determined by the method of Tada et al. (Tada et al., EMBO
J., 10, 1803-1808 (1991)), the seeds of the transformed plants showed high GUS activity.

From this, it was found that the heterologous gene can be efficiently expressed in a seed-specific manner by using the promoter of the type III rice starch branching enzyme gene.
Therefore, it was revealed that it is possible to accumulate various gene products in rice seeds by using the promoter of type III branching enzyme.

<7> Construction of a sense gene by ligating the promoter of the type III rice starch branching enzyme gene and cDNA. The plasmid pRB33 containing the cDNA of the type III rice starch branching enzyme gene was digested with EcoRI, and the digested ends were blunted using T4 DNA polymerase (Takara Shuzo, DNA blunting kit). This was ligated with the SmaI digested fragment of the plasmid pRBP2 having the promoter of the type III rice starch branching enzyme gene described above. As a result, a sense branching enzyme gene in which the type III branching enzyme gene was connected in the sense direction downstream of the promoter of the type III branching enzyme gene was created. This was named pRBPRB3.

<8> Introduction of sense type III branching enzyme gene into rice pRBPRB3 plasmid DNA was introduced into rice cells by the method described in <6>. When DNA was extracted from regenerated plant cells and subjected to Southern hybridization, 5 strains were obtained.
There was a DNA fragment derived from pRBPRB3. Seeds were taken from these transformed plants, and the activity of the branching enzyme in the endosperm was measured by the phosphorylase method (edited by Michinori Nakamura, Keiji Kainuma,
Starch, Related Glycoenzyme Experimental Method, Academic Publishing Center, 1989)
Quantified by As a result, the branching enzyme activity of the obtained 5 plant strains was 105% to 125% as compared with the control untreated plant, and the average of these increased by about 15%.

These results suggest that it is possible to increase the starch branching enzyme activity in the endosperm of transformed plants by introducing the sense type III starch branching enzyme gene of the present invention. It was It was suggested that this makes it possible to increase the amylopectin content in endosperm starch.

<9> Construction of antisense gene of type III rice starch branching enzyme gene and isolation of a fragment containing the entire region of genetic information of rice transformant rice starch synthase, and then using T4 DNA polymerase The ends were blunted. This was cloned into the SmaI site of pBI221 so that the cauliflower mosaic virus 35S promoter contained in pBI221 was in the reverse direction to the transcription direction, and the plasmid
pBIRBE3R was obtained. That is, c of type III starch branching enzyme
DNA exists by binding to the promoter in the direction of the antisense gene, and when the genetic information of this gene is expressed by the 35S promoter, antisense RNA is produced.

PBIRBE3R was introduced into rice (Nipponvalle) protoplasts by electroporation. Eight strains of transformed rice were obtained. Western analysis using the aforementioned antiserum was performed using the elementary extract of ripened seeds obtained from the transformant, and the expression level of the target type III starch branching enzyme was used as a control. It was confirmed to be significantly lower than that of Nippon Barre. In addition, the amylose content of the obtained seeds was measured by the iodine coloration method, and it was found that it was significantly higher than that of the control (30 to 40%).

[0082]

EFFECT OF THE INVENTION c of the type III rice starch branching enzyme of the present invention
An enzyme protein having an amino acid sequence defined by DNA is effectively expressed in rice and is involved in the amylose and amylopectin contents of the starch in the grain. Therefore, by introducing this base sequence into rice, the starch contained in the grain is reduced. The content of the components can be changed arbitrarily, and the taste and yield of rice can be significantly improved.

Further, by using the promoter of the rice starch branching enzyme gene obtained by the present invention, it becomes possible to specifically express the rice starch branching enzyme gene or the foreign gene in seeds.

[0084]

[Sequence list]

SEQ ID NO: 1 Array length: 2918 Sequence form: Nucleic acid Number of chains: double-stranded Topology: linear Sequence type: cDNA to mRNA origin: Organism name: Oryza sativa Tissue type: ripening seed Direct origin Library name: Rice ripening stage seed cDNA library Clone name: pRB33 Sequence features Characteristic symbol: 5'UTR Location: 1..127 Method by which the characteristics were determined: P Characteristic symbol: mRNA Location: 1..2918 Method by which the characteristics were determined: P Characteristic symbol: transit peptide Location: 128..322 How the features were determined: S Characteristic symbol: mature peptide Location: 323..2602 How the feature was determined: E Characteristic symbol: 3'UTR Location: 2603..2918 Method by which the characteristics were determined: P Array CGGCGCACAC CCACACACCG ACCACCAGGC AGCGCCTCCT CGCTTTGGCT CTCGCGTGAG 60 GAGGGTTTAG GTGGAAGCAG AGCGCGGGGG TTGCCGGGGG ATCCGATCCG GCTGCGGTGC 120 GGGCGAG ATG GCG GCG CCG GCG TCT GCG GTT CCC GGG AGC GCG GCG GGG 169         Met Ala Ala Pro Ala Ser Ala Val Pro Gly Ser Ala Ala Gly                           5 10 CTA CGG GCG GGG GCC GTG CGG TTC CCC GTG CCA GCC GGG GCC CGG AGC 217 Leu Arg Ala Gly Ala Val Arg Phe Pro Val Pro Ala Gly Ala Arg Ser  15 20 25 30 TGG CGT GCG GCG GCG GAG CTC CCGACG TCG CGG TCG CTG CTC TCC GGC 265 Trp Arg Ala Ala Ala Glu Leu ProThr Ser Arg Ser Leu Leu Ser Gly                  35 40 45 CGG AGA TTC CCC GGT GCC GTT CGCGTG GGG GGT TCC GGG GGG CGC GTG 313 Arg Arg Phe Pro Gly Ala Val ArgVal Gly Gly Ser Gly Gly Arg Val              50 55 60 GCC GTG CGC GCG GCG GGC GCG TCA GGG GAG GTG ATG ATC CCC GAG GGC 361 Ala Val Arg Ala Ala Gly Ala Ser Gly Glu Val Met Ile Pro Glu Gly          65 70 75 GAG AGC GAC GGG ATG CCG GTT TCA GCA GGT TCA GAC GAT CTG CAG TTG 409 Glu Ser Asp Gly Met Pro Val Ser Ala Gly Ser Asp Asp Leu Gln Leu      80 85 90 CCA GCC TTA GAT GAT GAA TTA AGC ACG GAG GTT GGA GCT GAA GTT GAG 457 Pro Ala Leu Asp Asp Glu Leu Ser Thr Glu Val Gly Ala Glu Val Glu  95 100 105 110 ATT GAG TCA TCT GGA GCA AGT GAC GTT GAA GGC GTG AAG AGA GTG GTT 505 Ile Glu Ser Ser Gly Ala Ser Asp Val Glu Gly Val Lys Arg Val Val                 115 120 125 GAA GAA TTA GCT GCT GAG CAG AAA CCA CGA GTT GTC CCA CCA ACA GGA 553 Glu Glu Leu Ala Ala Glu Gln Lys Pro Arg Val Val Pro Pro Thr Gly             130 135 140 GAT GGG CAA AAA ATA TTC CAG ATG GAC TCT ATG CTT AAT GGC TAT AAG 601 Asp Gly Gln Lys Ile Phe Gln Met Asp Ser Met Leu Asn Gly Tyr Lys     145 150 155 TAC CAT CTT GAA TAT CGA TAT AGC CTA TAT AGG AGA CTG CGT TCA GAC 649 Tyr His Leu Glu Tyr Arg Tyr Ser Leu Tyr Arg Arg Leu Arg Ser Asp     160 165 170 ATT GAT CAG TAT GAA GGA GGA CTG GAA ACA TTT TCT CGC GGT TAT GAG 697 Ile Asp Gln Tyr Glu Gly Gly Leu Glu Thr Phe Ser Arg Gly Tyr Glu 175 180 185 190 AAG TTT GGA TTT AAT CAC AGT GCT GAA GGT GTC ACT TAT CGA GAA TGG 745 Lys Phe Gly Phe Asn His Ser Ala Glu Gly Val Thr Tyr Arg Glu Trp                 195 200 205 GCT CCC GGG GCA CAT TCT GCA GCA TTA GTA GGT GAC TTC AAC AAT TGG 793 Ala Pro Gly Ala His Ser Ala Ala Leu Val Gly Asp Phe Asn Asn Trp             210 215 220 AAT CCA AAT GCA GAC CGC ATG AGC AAA AAT GAG TTT GGT GTT TGG GAG 841 Asn Pro Asn Ala Asp Arg Met Ser Lys Asn Glu Phe Gly Val Trp Glu         225 230 235 ATT TTT CTG CCT AAC AAT GCT GAT GGC TCA TCT CCT ATT CCA CAT GGC 889 Ile Phe Leu Pro Asn Asn Ala Asp Gly Ser Ser Pro Ile Pro His Gly     240 245 250 TCA CGT GTA AAG GTG CGA ATG GAA ACT CCA TCT GGT ATA AAG GAT TCT 937 Ser Arg Val Lys Val Arg Met Glu Thr Pro Ser Gly Ile Lys Asp Ser 255 260 265 270 ATT CCT GCC TGG ATC AAG TAC TCT GTG CAG GCC GCA GGA GAA ATC CCA 985 Ile Pro Ala Trp Ile Lys Tyr Ser Val Gln Ala Ala Gly Glu Ile Pro                 275 280 285 TAC AAT GGA ATA TAT TAT GAT CCT CCT GAA GAG GAG AAG TAC ATA TTC 1033 Tyr Asn Gly Ile Tyr Tyr Asp Pro Pro Glu Glu Glu Lys Tyr Ile Phe             290 295 300 AAG CAT CCT CAA CCT AAA AGA CCA AAG TCA TTG CGG ATA TAC GAA ACT 1081 Lys His Pro Gln Pro Lys Arg Pro Lys Ser Leu Arg Ile Tyr Glu Thr         305 310 315 CAT GTT GGA ATG AGT AGC ACG GAG CCA AAG ATC AAC ACG TAT GCA AAC 1129 His Val Gly Met Ser Ser Thr Glu Pro Lys Ile Asn Thr Tyr Ala Asn     320 325 330 TTT AGG GAT GAG GTG CTT CCA AGA ATC AAA AAG CTT GGA TAC AAT GCA 1177 Phe Arg Asp Glu Val Leu Pro Arg Ile Lys Lys Leu Gly Tyr Asn Ala 335 340 345 350 GTG CAA ATA ATG GCA ATT CAA GAG CAT GCA TAT TAT GGA AGC TTT GGG 1225 Val Gln Ile Met Ala Ile Gln Glu His Ala Tyr Tyr Gly Ser Phe Gly                 355 360 365 TAC CAT GTC ACC AAT TTC TTT GCA CCA AGT AGT CGT TTC GGG ACC CCA 1273 Tyr His Val Thr Asn Phe Phe Ala Pro Ser Ser Arg Phe Gly Thr Pro             370 375 380 GAA GAT TTA AAG TCA TTG ATT GAT AAA GCT CAT GAG CTT GGT TTA GTT 1321 Glu Asp Leu Lys Ser Leu Ile Asp Lys Ala His Glu Leu Gly Leu Val         385 390 395 GTG CTC ATG GAT GTT GTT CAC AGC CAT GCG TCA AAT AAT ACC CTA GAT 1369 Val Leu Met Asp Val Val His Ser His Ala Ser Asn Asn Thr Leu Asp     400 405 410 GGG TTG AAC GGT TTT GAT GGT ACA GAT ACG CAT TAC TTT CAT AGT GGT 1417 Gly Leu Asn Gly Phe Asp Gly Thr Asp Thr His Tyr Phe His Ser Gly 415 420 425 430 TCA CGC GGC CAT CAT TGG ATG TGG GAT TCT CGC CTT TTC AAC TAT GGG 1465 Ser Arg Gly His His Trp Met Trp Asp Ser Arg Leu Phe Asn Tyr Gly                 435 440 445 AAT TGG GAA GTT CTA AGA TTT CTA CTA TCC AAT GCA AGA TGG TGG CTC 1513 Asn Trp Glu Val Leu Arg Phe Leu Leu Ser Asn Ala Arg Trp Trp Leu             450 455 460 GAG GAG TAT AAG TTT GAT GGT TTC AGA TTT GAC GGT GTA ACC TCA ATG 1561 Glu Glu Tyr Lys Phe Asp Gly Phe Arg Phe Asp Gly Val Thr Ser Met         465 470 475 ATG TAC ACT CAT CAT GGA TTA CAA GTA GCA TTT ACG GGG AAC TAC AGT 1609 Met Tyr Thr His His Gly Leu Gln Val Ala Phe Thr Gly Asn Tyr Ser     480 485 490 GAA TAC TTT GGA TTT GCC ACT GAT GCT GAT GCA GTA GTT TAC TTG ATG 1657 Glu Tyr Phe Gly Phe Ala Thr Asp Ala Asp Ala Val Val Tyr Leu Met 495 500 505 510 CTG GTA AAT GAT TTA ATT CAT GGA CTT TAT CCT GAG GCC ATA ACC ATC 1705 Leu Val Asn Asp Leu Ile His Gly Leu Tyr Pro Glu Ala Ile Thr Ile                 515 520 525 GGT GAA GAT GTC AGT GGA ATG CCT ACA TTT GCC CTT CCT GTT CAA GAT 1753 Gly Glu Asp Val Ser Gly Met Pro Thr Phe Ala Leu Pro Val Gln Asp             530 535 540 GGT GGG GTT GGT TTT GAT TAT CGC CTT CAT ATG GCT GTT CCT GAC AAA 1801 Gly Gly Val Gly Phe Asp Tyr Arg Leu His Met Ala Val Pro Asp Lys         545 550 555 TGG ATT GAA CTC CTC AAG CAA AGT GAT GAA TCT TGG AAG ATG GGT GAT 1849 Trp Ile Glu Leu Leu Lys Gln Ser Asp Glu Ser Trp Lys Met Gly Asp     560 565 570 ATT GTG CAC ACA CTG ACT AAC AGA AGG TGG TCA GAG AAG TGT GTT ACT 1897 Ile Val His Thr Leu Thr Asn Arg Arg Trp Ser Glu Lys Cys Val Thr 575 580 585 590 TAT GCT GAA AGT CAT GAT CAA GCA CTA GTT GGT GAC AAA ACT ATT GCA 1945 Tyr Ala Glu Ser His Asp Gln Ala Leu Val Gly Asp Lys Thr Ile Ala                 595 600 605 TTC TGG TTG ATG GAC AAG GAT ATG TAT GAT TTT ATG GCT CTG GAC AGA 1993 Phe Trp Leu Met Asp Lys Asp Met Tyr Asp Phe Met Ala Leu Asp Arg             610 615 620 CCG GCA ACA CCT AGC ATT GAT CGT GGA ATA GCA TTG CAT AAA ATG ATT 2041 Pro Ala Thr Pro Ser Ile Asp Arg Gly Ile Ala Leu His Lys Met Ile         625 630 635 AGA CTT ATC ACA ATG GGG TTA GGA GGA GAA GGC TAT CTT AAC TTT ATG 2089 Arg Leu Ile Thr Met Gly Leu Gly Gly Glu Gly Tyr Leu Asn Phe Met     640 645 650 GGA AAT GAG TTC GGA CAT CCT GAA TGG ATT GAT TTT CCA AGA GCT CCA 2137 Gly Asn Glu Phe Gly His Pro Glu Trp Ile Asp Phe Pro Arg Ala Pro 655 660 665 670 CAA GTA CTT CCA AAT GGT AAA TTC ATC CCA GGG AAT AAC AAC AGT TAT 2185 Gln Val Leu Pro Asn Gly Lys Phe Ile Pro Gly Asn Asn Asn Ser Tyr                 675 680 685 GAT AAA TGC CGT CGA AGA TTT GAC CTG GGT GAT GCG GAC TAT CTT AGG 2233 Asp Lys Cys Arg Arg Arg Phe Asp Leu Gly Asp Ala Asp Tyr Leu Arg             690 695 700 TAT CGT GGC ATG CTA GAG TTT GAC CGC GCG ATG CAG TCT CTC GAG GAA 2281 Tyr Arg Gly Met Leu Glu Phe Asp Arg Ala Met Gln Ser Leu Glu Glu         705 710 715 AAA TAT GGG TTC ATG ACA TCA GAC CAC CAG TAC ATA TCT CGA AAG CAT 2329 Lys Tyr Gly Phe Met Thr Ser Asp His Gln Tyr Ile Ser Arg Lys His     720 725 730 GAA GAG GAT AAG ATG ATT ATA TTT GAG AAG GGA GAT CTG GTA TTT GTG 2377 Glu Glu Asp Lys Met Ile Ile Phe Glu Lys Gly Asp Leu Val Phe Val 735 740 745 750 TTC AAC TTC CAT TGG AGT AAC AGC TAT TTT GAC TAC CGT GTT GGT TGT 2425 Phe Asn Phe His Trp Ser Asn Ser Tyr Phe Asp Tyr Arg Val Gly Cys                 755 760 765 TTA AAG CCA GGG AAA TAT AAG GTG GTC TTG GAC TCA GAT GCT GGA CTC 2473 Leu Lys Pro Gly Lys Tyr Lys Val Val Leu Asp Ser Asp Ala Gly Leu             770 775 780 TTT GGT GGA TTT GGC AGG ATC CAT CAC ACT GCA GAG CAC TTC ACT GCC 2521 Phe Gly Gly Phe Gly Arg Ile His His Thr Ala Glu His Phe Thr Ala         785 790 795 GAT TGT TCA CAT GAC AAC AGG CCC TAC TCG TTC TCA GTT TAT TCT CCT 2569 Asp Cys Ser His Asp Asn Arg Pro Tyr Ser Phe Ser Val Tyr Ser Pro     800 805 810 AGC AGA ACC TGC GTT GTC TAT GCT CCA GCG GAA TGAGAACACC AAGAGGCAGC 2622 Ser Arg Thr Cys Val Val Tyr Ala Pro Ala Glu 815 820 825 ATGCAAGTGT GTGCGGCTGG CTAGTGCGAA GGAGCAAGAA AAACTAGTTG CCAGCAATCT 2682 GTGAACGGCT TTCCTAGGTT CTGCTTCGAT GAATGCCGGA TAGACTAGAC AGCTTGCTTT 2742 TGTGCTTTGC GCTCCCAATT TGTAGTTTTA GTTTGTGAGG GAAAGAAACG TTTATTTGTA 2802 ATTATCTATG GCTGTCGAAC GGCGACGAAA CCATGAACCC CGTATATTTG TTGGTACCGT 2862 TCGAACTGCC AGTTATACAT AGTTCTGCAC TTCTGTACAT CTTGTGATGC TTGAATC 2919

SEQ ID NO: 2 Array length: 20 Sequence shape: Amino acid Topology: linear Sequence type: Origin: Organism name: Oryza sativa Tissue type: ripening seed Array Ala Ala Gly Ala Ser Gly Glu Val Met Ile Pro Glu Gly Glu Xaa Asp                   5 10 15 Gly Met Pro Val              20

SEQ ID NO: 3 Array length: 38 Sequence form: Nucleic acid Number of chains: single strand Topology: linear Sequence type: Other nucleic acids Synthetic DNA Array ACNGGCATNC CRTCRCAYTC NCCYTCNGGN ATCATNAC 38

SEQ ID NO: 4 Array length: 1110 Sequence form: Nucleic acid Number of chains: double-stranded Topology: linear Sequence Type: Genomic DNA origin: Organism name: Oryza sativa Tissue type: ripening seed Direct origin Clone name: pRBP1 Sequence features Characteristic symbol: promoter Location: 1..996 Method by which the characteristics were determined: P Characteristic symbol: TATA signal Location: 953..958 How the features were determined: S Characteristic symbol: mRNA Location: 997..1110 How the features were determined: S Array GAATTCTTAA GTACTCAACT CACACGTTTG GTGTGCTTGG TTTCATGGGC TGGCCCAACA 60 GAAAAATCGC TCTGCCCAAT CCGCTGGACG CACGCGGAGT CGCCAGTCTG CTAGAGGATG 120 TGCTCCATTT TGGCAGCCCA TTGTTCACCG CGAGAAAGGA TGATGGACCA CAGGCTACTA 180 GGCTAGTCAG GTCACGCTTC GCACGAGTGC TTCTCCCTCA CGTGCTCCTC AACGTTTACA 240 CAGAGGTGAC AGAGCAGAAC AGAAAAATGA AACAACAGAG CACCGGTGTT GCGTTCTTCC 300 ATTCACTGAG AATTTGTTTC GTGTAAGCCT CCTGGGTCCA GCCCAACTCC CATCTTCCCT 360 TTTGTTGTTT TCCCTGGGTA ACAAAAAGCG CGACCAAAGA GGACAAGGAA AAACAAAAAG 420 AAGCACAGGA GTAGCAAGTA GCAGGGAGGG AGACGCGACG CAAGCGGCGT TACGGGTCGA 480 GAGGGAAAGC CTCCCCGGCG TTGGTTTCCT TTTCCTCTCC TCTTCCTCCC CAACCCGCGC 540 CGTGCCGCAC GCCGCGCCAC GCCAACTCGA TCGGTGGTGA CTCCGCCTGC CTCCACCCGC 600 GGCGCACGCC CGCTGCCCTC CACCTTCCAC CTGGCAGCAG CGGCTGGCAT GGCAGTGGAG 660 ACGGCGCGCT CCTCTCCTCT CCCTTCCCCC GGCTAACGCA AACGTGACGC CCGGGCGCGC 720 ACGGGAAAGC GGACGCTTTC ATCGCGAGGG GGGAGGGGAA AAGCCTCGCC CTCCGCCCTC 780 GCCGCCTGAC TCCGTGCGCC GCGGATTGGA CCGACGCGCA ACCGCCTCTT CCGCCCCCGT 840 CCCCCCAGTG CACGCATCGC ACACCCACCC TCGGCGGCCT CGATGCCACT CTCGCGTGAG 900 GGGTTCGCTC CATCAGGACC AGAGCTCCTC CTCCTCGTCC TCTATATAGC GCGGCGCCCT 960 CCGCTCCTCC TAGCTTCAGC ACCAGTGCAC CCGCACGCGC GCACACCCAC ACACCGACCA 1020 CCAGGCAGCG CCTCCTCGCT TTGGCTCTCG CGTGAGGAGG GTTTAGGTGG AAGCAGAGCG 1080 CGGGGGTTGC CGGGGGATCC 1100

SEQ ID NO: 5 Array length: 20 Sequence form: Nucleic acid Number of chains: single strand Topology: linear Sequence type: Other nucleic acids Synthetic DNA Array TTCTTAAGTA CTCAACTCAC 20

SEQ ID NO: 6 Array length: 21 Sequence form: Nucleic acid Number of chains: single strand Topology: linear Sequence type: Other nucleic acids Synthetic DNA Array GGTGTGTGGG TGTGCGCGCGT 21

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁷ identification code FI C12N 9/10 C12N 5/00 C (58) Fields investigated (Int.Cl. ⁷ , DB name) C12N 15/54 A01H 1 / 00 A01H 5/00 SwissProt / PIR / GeneS eq GenBank / EMBL / DDBJ / GeneSeq BIOSIS / MEDLINE / WPID S (STN)

Claims (10)

(57) [Claims] 1. A rice starch branching enzyme gene having the base sequence shown in SEQ ID NO: 1. 2. A gene encoding the protein shown in (A) or (B) below: (A) A mature protein of a rice starch branching enzyme (B) consisting of the amino acid sequence from the 66th to 825th amino acid sequence shown in SEQ ID NO: 1. A protein comprising an amino acid sequence in which one or several amino acids are substituted / deleted or inserted in the 66th to 825th amino acid sequence shown in SEQ ID NO: 1 and having starch branching enzyme activity. 3. A gene encoding a transit protein of a rice starch branching enzyme, which is composed of the 1st to 65th amino acid sequences shown in SEQ ID NO: 1. 4. A promoter having the 1st to 996th nucleotide sequence shown in SEQ ID NO: 4. 5. By introducing a DNA sequence in which a DNA encoding the protein shown in (C) or (D) below and a promoter that can be expressed in plant cells are linked .
Modified to enhance starch branching enzyme activity.
N : (C) Rice starch branching enzyme protein consisting of the amino acid sequence shown in SEQ ID NO: 1, or (D) the amino acid sequence shown in SEQ ID NO: 1 in which one or several amino acids have been substituted / deleted or inserted A protein consisting of a sequence and having starch branching enzyme activity. 6. The rice according to claim 5, wherein the promoter is the promoter according to claim 4. 7. A grain having an increased starch content, which is obtained by cultivating the rice according to claim 5 . 8. A starch branching enzyme by introducing into a rice a DNA sequence in which a DNA sequence encoding a protein shown in (C) or (D) below and a promoter expressible in plant cells are linked. Method of changing the activity: (C) a rice starch branching enzyme protein consisting of the amino acid sequence shown in SEQ ID NO: 1 or (D) an amino acid sequence shown in SEQ ID NO: 1 in which one or several amino acids are substituted / deleted or A protein consisting of an inserted amino acid sequence and having starch branching enzyme activity. 9. The method for changing the starch branching enzyme activity according to claim 8, wherein the promoter is the promoter according to claim 4. 10. A method for introducing the DNA sequence in which the promoter according to claim 4 is combined with a heterologous gene into rice to express the heterologous gene in a seed-specific manner.