JP2002315582A - New protein of rice phloem and its gene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To investigate the function or role of phloem protein relating to a virus transporting protein. SOLUTION: Two kinds of new phloem proteins having Ca<2+> /phospholipids bonding domain and preferably at least 50% amino acid homology to pumpkin CmPP16 protein are separated from rice.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a pump having a Ca ²⁺ / phospholipid binding domain and preferably a pumpkin Cm.
The present invention relates to a rice phloem protein having at least about 50% amino acid homology to the PP16 protein, and DNA encoding the same.

[0002]

BACKGROUND OF THE INVENTION Plants use two complementary conduits: xylem and phloem in vascular bundles for long-distance transport. For example, the vascular bundle of rice is a parallel vascular bundle and consists of a phloem and xylem. Also, for pumpkin,
The vascular bundle is a compatible vascular bundle and is composed of internal and external phloems, cambium, and xylem. The structure and physiological location of vascular bundles vary considerably between plant species, suggesting that the structure and pathway of solute transport to the leaf phloem differ (Opar
ka and Turgeon, Plant Cell, 11, 739-750 (199
9)). In angiosperms, the phloem is composed primarily of two cell types, the phloem element (SE) and its associated companion cells (CC), delivering proteins, mRNA, amino acids, and macromolecules to tissues and organs. To function as an advanced long-distance transportation system (Jorgen
sen et al., Science, 279, 1486-1487 (1998)). CC has many mitochondria and free ribosomes, which are responsible for the abnormally high density of CC cytoplasm, which distinguishes CC from other cells in the phloem. Conversely, SE is highly specialized for anabolic translocations, and most subcellular structures and organelles such as nuclei, vacuoles, ribosomes, and Golgi are degraded during phloem element development (Sjolund, Plant Cell, 9,
1137-1146 (1997)).

[0003] SE and CC are linked by a number of branched protoplasmic connections. Some of the approximately 200 soluble proteins have been identified in the phloem, and these proteins are thought to enter the SE by connecting protoplasmic connections (Xoconostle-Cazares et al., Science, 28
3, 94-98 (1999)). In addition, protoplasm communication mediates the delivery of proteins and other small and macromolecules, such as endogenous RNA molecules, to long-range translocation currents. Microinjection experiments have provided direct evidence that many phloem proteins have the ability to mediate their own transport by protoplasmic communication. A common feature of such proteins is 1 kDa to 20 kDa
Has the ability to increase the size exclusion of protoplasmic contacts to values above However, the function or role of the phloem protein in SE and the mechanism of macromolecular traffic is not at all understood.

[0004] Recently, a gene for a protein CmPP16 similar to a virus movement protein has been identified as Xoconostle-C.
Identified in pumpkins by azares et al. (Xoconostle-Cazares et al., (1999), supra). CmPP16 messenger RNA is present in the phloem tissue of the stem, but the protein is restricted to SE. It has also been shown that CmPP16 moves between cells and mediates RNA transport. This suggests that RNA molecules circulate throughout the plant through the phloem, and thus there is a superhighway for RNA-based information.

[0005]

In higher plants, the phloem plays a role in the evolved long-distance transport system.
Little is known about which types of proteins are involved in the migration of macromolecules such as proteins and polynucleotides in phloem tissue. for that reason,
It is desired to elucidate the function or role of the phloem protein related to the viral MP expressed in various plant species as described above.

[0006]

Therefore, in the present invention, phloem proteins related to the viral MP are studied diligently by using monocotyledonous rice which is different from dicotyledonous pumpkin in the tissue structure as a material. Was. As a result, a novel phloem protein gene homologous to the virus MP and a novel phloem protein gene homologous to the pumpkin CmPP16 gene were successfully isolated and expressed.

That is, the present invention provides a rice phloem protein having a Ca ²⁺ / phospholipid binding domain.

[0008] In a preferred embodiment, there is provided a rice phloem protein having at least about 50% amino acid homology to the CmPP16 protein. More preferably, the rice phloem protein has at least about 56%, more preferably at least about 58% amino acid homology with the pumpkin CmPP16 protein.

In a preferred embodiment, the rice phloem protein has the amino acid sequence of SEQ ID NO: 2.

[0010] In a preferred embodiment, the rice phloem protein has at least one amino acid substitution, insertion or deletion.

In a preferred embodiment, the rice phloem protein has the amino acid sequence of SEQ ID NO: 4.

In a preferred embodiment, the rice phloem protein has at least one amino acid substitution, insertion or deletion.

[0013] The present invention also provides a DNA encoding the rice phloem protein described in any of the above.

In a preferred embodiment, the DNA contains the nucleotide sequence of SEQ ID NO: 1.

[0015] In a preferred embodiment, the DNA comprises the nucleotide sequence of SEQ ID NO: 3.

In a preferred embodiment, the DNA contains the nucleotide sequence of SEQ ID NO: 5.

The present invention also provides a promoter comprising the nucleotide sequence of SEQ ID NO: 7.

The present invention further provides a promoter comprising the nucleotide sequence of SEQ ID NO: 8.

The promoter of the present invention also includes a promoter having a base sequence substitution, insertion, or deletion as long as the function related to gene expression is not changed.

The present invention provides a transgenic plant transformed to express any of the above rice phloem proteins in a phloem-specific manner.

[0021] In a preferred embodiment, the transgenic plant is selected from the group consisting of rice, sugarcane, corn, and melon.

[0022] The present invention also provides a method for producing any of the above rice phloem proteins using the above transgenic plant.

The present invention relates to a transgenic plant that produces a heterologous protein in a phloem-specific manner, which encodes a promoter involved in phloem-specific expression, and the heterologous protein operably linked to the promoter. D
A transgenic plant obtained by a method comprising the steps of obtaining a vector containing NA; and introducing the obtained vector into a plant.

In a preferred embodiment, the vector is
Further, it includes all or part of the DNA encoding any of the above rice phloem proteins.

In a further preferred embodiment, all or part of the DNA encoding any of the above rice phloem proteins is a DNA encoding the above heterologous protein.
And a fusion protein.

In a preferred embodiment, the promoter involved in the phloem-specific expression is a promoter comprising the nucleotide sequence of SEQ ID NO: 7 or 8.

[0027] In a preferred embodiment, the plant is selected from the group consisting of rice, sugarcane, corn, and melon.

In a preferred embodiment, the heterologous protein is secreted from the phloem into the phloem.

In another aspect of the present invention, there is provided a method for specifically producing a heterologous protein in a phloem of a plant, comprising a promoter involved in phloem-specific expression, and a promoter operably linked to the promoter. D encoding protein
Obtaining a vector comprising NA; and introducing the obtained vector into the plant to obtain a transgenic plant.

In a preferred embodiment, the vector is
Further, it includes all or part of the DNA encoding any of the above rice phloem proteins.

In a further preferred embodiment, all or a part of the DNA encoding the rice phloem protein is a DNA encoding the heterologous protein.
And a fusion protein.

In a preferred embodiment, the promoter involved in the phloem-specific expression is a promoter comprising the nucleotide sequence of SEQ ID NO: 7 or 8.

[0033] In a preferred embodiment, the plant is selected from the group consisting of rice, sugarcane, corn, and melon.

In a preferred embodiment, the heterologous protein is secreted from the phloem into the phloem.

[0035]

BEST MODE FOR CARRYING OUT THE INVENTION In the present specification, “CmPP1
"6" means Red Clover Necrosis Mosaic Virus (R
A protein originally identified from pumpkin phloem sap, having some sequence similarity to the RCNMV virus movement protein (MP), and containing a Ca ²⁺ / phospholipid binding domain. (Xoconostle-Cazares et al., (1999), supra). CmP
P16 mRNA is present in the phloem tissue of pumpkin stems, but the protein produced is restricted to SE. It is also believed that CmPP16 plays a role in migrating between cells and mediating RNA transport. As used herein, the term “virus movement protein (MP)” refers to a protein expressed by a plant virus, which has the ability to interact with protoplasm communication and mediate intercellular transport of a complex of MP and nucleic acid of the virus. Refers to a protein having

As used herein, the term “Ca ²⁺ / phospholipid-binding domain” refers to Ca ²⁺ / phospholipid-binding (dependent) C2 present in the protein kinase C family.
Domain (Kopka et al., Plant Mol. Biol., 36, 627)
-637 (1998)).

As used herein, the term “rice phloem protein” refers to a protein that is specifically expressed in the phloem of rice. In particular, the term "rice phloem protein of the present invention" refers to "RPP16 protein" or "RPP17 protein" and derivatives thereof. In addition, "derivative" means that it does not affect the function of the protein.
A protein having at least one amino acid substitution, insertion, or deletion in the amino acid sequence. As used herein, simply referring to "RPP16 protein" or "RPP17 protein" may include derivatives thereof.

As used herein, “RPP16 protein” refers to an approximately 16 kD protein having a Ca ²⁺ / phospholipid binding domain and preferably having at least about 50% amino acid homology to the pumpkin CmPP16 protein.
A rice phloem protein having the estimated molecular weight of a. Typically, a protein with a predicted molecular weight of 15.9 kDa that has 56% homology to the CmPP16 protein and has the amino acid sequence of SEQ ID NO: 2 (RP
P16-1). The estimated isoelectric point of RPP16-1 (p
I) is 4.06 and is an acidic protein. The amino acid sequence may have at least one amino acid substitution, insertion, or deletion as long as the function of the protein is not affected. Further, the RPP16 protein is expressed from a nucleotide sequence named “Rpp16-1 gene”, and this gene may be, for example, the nucleotide sequence of SEQ ID NO: 1.

As used herein, the term “RPP17 protein” refers to an approximately 17 kD protein having a Ca ²⁺ / phospholipid binding domain and preferably having at least about 50% amino acid homology to the pumpkin CmPP16 protein.
A rice phloem protein having the estimated molecular weight of a. Typically, RPP1 having the amino acid sequence of SEQ ID NO: 4
7-1 (estimated molecular weight 17.7 kDa) and SEQ ID NO: 6
RPP17-2 having an amino acid sequence of (estimated molecular weight 1
7.4 kDa), which have 58% homology with the CmPP16 protein. The estimated pIs of RPP17-1 and RPP17-2 are 6.05 and 6.26, respectively, and these proteins are neutral proteins. If it does not affect the function of the protein,
These amino acid sequences may have at least one amino acid substitution, insertion, or deletion. RPP17-2 is a protein in which three amino acids have been deleted from RPP17-1. In addition, RPP17-1 and RPP17-2 are expressed from genes named “Rpp17-1 gene” and “Rpp17-2 gene”, respectively. These genes are, for example, SEQ ID NO: 3 and SEQ ID NO: 5, respectively. It can be a base sequence.

As used herein, the terms "RPP16 promoter" and "RPP17 promoter" refer to the promoter regions located upstream of the isolated RPP16 protein and RPP17 protein, respectively. Typically, they have the nucleotide sequences represented by SEQ ID NO: 7 and SEQ ID NO: 8, respectively. "RPP16 promoter"
Strictly regulates the RPP16 protein to be specifically expressed in the branch roots in roots, in companion cells (CC) and phloem parenchyma cells in stems, and in companion cells in leaves. on the other hand,
The "RPP17 promoter" specifically expresses the RPP17 protein in branch roots in roots, in companion cells (CC) and phloem parenchyma cells in stems, and in companion cells, phloem parenchyma, and mesophyll cells in leaves. Adjust as follows. The promoter of the present invention also includes a promoter having a base sequence substitution, insertion, or deletion as long as the function related to gene expression is not changed. In addition, the promoter of the present invention not only expresses the RPP16 protein and the RPP17 protein only phloem-specifically, but also encodes a heterologous substance which is expressly bound under the control thereof.
Can also be expressed phloem-specifically.

In the present invention, in order to investigate the presence of proteins homologous to the phloem protein CmPP16 associated with the viral MP and to be expressed in various plant species, it was different from pumpkins having a compatible vascular bundle. And rice having a parallel vascular bundle was used as a material. In addition, molecular biological experiment techniques (electrophoresis of DNA, method of recovering electrophoresed DNA from gel, restriction enzyme digestion,
PCR, radiolabeling of DNA, hybridization,
Base sequence determination), and these techniques include, for example, those described in Sambrook et al., A Laboratory Manual, Second Edition.
Edition, Cold Spring Harbor Press, Cold Spring Harbor, N
Techniques commonly used by those skilled in the art as described in ew York (1989) are employed. Various techniques such as biochemistry, immunology, plant histology, and the like, unless otherwise specified, techniques commonly used by those skilled in the art are used.

The similarity search for the CmPP16 gene can be performed using an appropriate database. For example, in the case of the present invention, it can be performed using a BLAST search in a rice-expressed sequence tag (EST) database. In addition, RPP1 having the obtained deduced amino acid sequence
Computer searches for the 6 protein and the RPP17 protein can also be performed using various sequence databases currently available. In addition, phylogenetic analysis may be performed by creating a phylogenetic tree according to various currently available methods.

The protein structure can be analyzed using, for example, the free software swiss prot. CmPP 16-1, CmPP obtained by this
From the estimated isoelectric points (pI) of 16-2, RPP17-1, and RPP17-2, these proteins are putative neutral proteins, while the putative pI of RPP16-1
This indicates that this is a putative acidic protein. From the results of phylogenetic analysis along with protein characteristics,
RPP16-1 protein is a novel protein,
RPP17 protein (RPP17-1 and RPP1
7-2) is thought to be the evolutionary counterpart of the pumpkin CmPP16 protein.

The RPP16 protein and RPP17 protein can be expressed in various cells. Suitable cells include cells of bacteria, yeast, insects, plants, animals, and the like. Particularly, plants such as Escherichia coli, yeast, rice and tobacco are preferable.

When the Rpp16 gene and the Rpp17 gene are expressed in these cells, they can be expressed as a fusion protein with a detectable protein by various methods commonly used by those skilled in the art. Glutathione-S-transferase (GS
T), β-galactosidase, chloramphenicol acetyltransferase, green fluorescent protein (GFP) and the like. In particular, the Rpp16 gene and Rpp17
When the gene is expressed, a reporter gene having no intrinsic activity in the plant may be used. Examples of reporter genes for gene introduction into plants include the gusA gene encoding β-glucuronidase (GUS) and the luciferase gene.

Further, RPP16 protein and RPP
The protein that can be fused with the 17 protein is not limited to the detectable protein as described above, and may be any heterologous protein intended to be phloem-specifically expressed.

For example, in the case of a rice plant expressing the RPP16 protein or the RPP17 protein, a transgenic plant is prepared as follows. First, a genomic sequence containing the RPP16 protein or the putative promoter region of the RPP17 protein is screened by plaque hybridization.
The upstream region of the RPP16 or RPP17 open reading frame (ORF) can be obtained by screening a rice genomic library using the respective cDNA clones as probes. The putative promoter region of each flanking sequence is subcloned, for example, into the vector of pBI121, respectively. Each construct can be introduced into rice using, for example, electroporation or a vector derived from T-DNA of Agrobacterium tumefaciens to obtain transgenic rice plants that express RPP16 or RPP17 protein. The resulting transgenic rice plant is selected, for example, on a medium containing hygromycin, and the introduction of the gene into the rice genome is performed as follows.
It can be confirmed by the PCR method or the Southern method using the gusA gene as an index. RPP16 and / or RPP1
Transgenic plants expressing the seven proteins can be prepared from various plants other than rice, and for example, sugarcane, corn, and melon are preferable.

Further, the RPP16 promoter or RP
A transgenic plant that expresses a heterologous protein in a phloem-specific manner can also be prepared using DNA encoding a heterologous protein operably linked under the control of the P17 promoter. The heterologous protein can be expressed alone or as a fusion protein with all or part of the RPP16 or RPP17 protein.
Alternatively, they may be expressed separately from the RPP16 or RPP17 proteins, but may be expressed such that they interact with these proteins after expression.

The RPP16 and RPP17 proteins obtained according to the present invention have a structure similar to MP and are phloem-specifically expressed, and the expression is strictly regulated by the respective promoters. Therefore, it is necessary to develop a transgenic plant using the genes of these proteins and / or their regulatory regions, produce a heterologous substance in the phloem, and develop a technique for transporting the substance to the phloem. It is possible. For example, in addition to letting the phloem produce industrial-valued substances,
It is possible to produce a substance that confers disease or insect resistance on plants, which can be distributed throughout the plant using a long-distance transport route called the phloem. In addition, by producing a binding protein of substances (heavy metals, environmental hormones, etc.) that negatively affect the environment, it is possible to grow environmentally purified plants.

[0050]

The present invention will be described in detail with reference to the following examples, which are not intended to limit the present invention.

Example 1 New Virus M from Rice
Identification of P-like gene) Rice plant (Oryza sativa L.cvs Nip
ponbare) were grown in a greenhouse at 28 ° C. Rice cDNA was obtained from young rice leaves by Kadowaki et al. (Kadowaki et al., EMBO
J., 15, 6652-6661 (1996)).

To screen for cDNA, CmP
A similarity search for the P16 cDNA was performed using a BLAST search in a rice expressed sequence tag (EST) database, and two classes of partial sequences were picked up. Two rice EST clones R037
4 and E61187 (each with DDBJ registration number AU
031663 and C79955)
The full length was used as a probe. These hybridization probes were labeled using an enhanced chemiluminescence direct nucleic acid labeling system (Amersham Pharmacia), followed by hybridization analysis.

As a result, three clones were obtained from the cDNA library. The DNA sequence of the resulting clone was determined by the dideoxy chain termination method using a fluorescent dye primer (PE Biosystems). One of these was the nucleotide sequence shown in SEQ ID NO: 1 and contained an open reading frame (ORF) encoding 144 amino acid residues with a predicted molecular weight of 15.9 kDa. This is 16 kDa
It was named rice phloem protein (RPP16-1) (SEQ ID NO: 2). The other two clones (SEQ ID NO: 3 respectively)
And SEQ ID NO: 5) are very similar, SEQ ID NO: 3
In contrast, SEQ ID NO: 5 had only 9 nucleotide deletions in the ORF sequence. The ORFs contained in the two clones encode 159 amino acid residues with a predicted molecular weight of 17.7 kDa and 156 amino acid residues with a predicted molecular weight of 17.4 kDa, and are respectively RPP17-1 and RPP17-2 (respectively, SEQ ID NO: 4 and SEQ ID NO: 6).

Next, in order to examine the gene copy number,
Southern blot analysis was performed as follows. Rice genomic DNA (5 μg) was prepared using BamHI, EcoRI, Ec
oRV, and cut with HindIII, and 0.8
% Agarose gel for electrophoresis. These were transferred to a nylon membrane after denaturation with alkali. The hybridization probes are each Rp
p16-1 cDNA and c of Rpp17-1 or 2
DI corresponding to the entire region of cDNA for DNA
It was a G-labeled PCR fragment. Hybridization and washing were performed according to Ausubel et al. (Ausubel et al., (198
7) Current Protocols in Molecular Biology, New Yor
k: Wiley). The filters were exposed to X-ray film (Fuji Film). Hybridized bands were visualized by a chemiluminescent reaction as described in the manufacturer's instructions. As a result, a single band was observed, which strongly suggested that the Rpp16 and Rpp17 genes were single copy genes in the rice genome (data not shown). This result also indicates that Rp
9 between the p17-1 and Rpp17-2 genes
This suggests that a difference in the base sequence results from an alternative splicing event.

(Example 2: Protein RPP16-1,
Characterization of RPP17-1 and RPP17-2) The deduced amino acid sequences of pumpkin CmPP16-1 and CmPP16-2 (these are cDNA (Xoconostle-Cazares
Et al., (1999), supra)), a red clover necrosis mosaic virus movement protein (RCNMV M
P) (which is deduced from cDNA (DDDJB accession number P10838)) and rice RPP16,
The alignment with the deduced amino acid sequences of RPP17-1 and RPP17-2 (these are deduced from the cDNAs of Rpp16-1, Rpp17-1, and Rpp17-2, respectively) is shown in FIG. Identical amino acids are indicated by inversion and similar amino acids are shaded. The protein kinase C-like Ca ²⁺ / phospholipid binding (C2) domain is shown as a dark line, and the three subdomains of the C2 domain (A, B, C) are shown as white rectangles. The numbers to the right of the sequence indicate the amino acid positions of each protein.

The deduced amino acid sequences of the RPP16 and RPP17 proteins share 57% similarity with each other, and both proteins have an RCNMV M
P had local homology. The deduced amino acid sequences from the Rpp16 and Rpp17 genes are 56% and 58%, respectively, for pumpkin CmPP16.
Showed similarity. As is evident from FIG. 1, according to the motif search, the conserved regions of these proteins are:
Single Ca ²⁺ / phospholipid binding (C2) domain (Kopk
a, (1998), supra).

Next, the proteins RPP16-1, RP
A computer search for P17-1 and RPP17-2 was performed on the registered sequence data.
Similar sequences include Arabidopsis Thaliana (Arabidopsis thaliana) [DDJB accession number CAB75905, A
AF34860], chickpea (chickpea) [DDDJB
Registration number AJ012692] and Zea mays (corn) [DDJB registration numbers U64437, AF152
601] could be picked up from the database. However, these functions are not known.

Further, RPP16 protein and RP
A phylogenetic tree for the P17 protein was created with PAUP ^* 4.
0b4a (Swofford, (1998) PAUP: Phylogenetic anal
ysisusing parsimony (and other methods), version
4. Ne using Sunderland, MA: SinauerAssociates)
Created according to the ighbor-Joining Method clustering method (Saitou and Nei, Mol. Biol. Evol., 4, 406-425 (1987)), bootstrap values were obtained for 10,000 replicates. The resulting protein phylogeny is shown in FIG. The number of the branch is the percentage that supports the branch. The names of the rice proteins are shown in boxes. The results show that the RPP17 protein has a better pumping CmPP1 than the RPP16 protein in sequence alignment.
It suggests a more closely related 6 protein.

The structures of the RPP16-1, RPP17-1, and RPP17-2 proteins were
Analyzed using rot. CmPP16-1, CmP
The estimated isoelectric points (pI) of P16-2, RPP17-1, and RPP17-2 were 6.17, 5.66,
6.05 and 6.26, indicating that these proteins are putative neutral proteins. On the other hand, the estimated pI of RPP16-1 is 4.06 and RP
Shows that P16-1 is a putative acidic protein.
Not only the protein characteristics but also the results of the phylogenetic analysis indicate that the evolutionary counterpart of the pumpkin CmPP16 protein is RPP1
7 proteins.

Example 3 Immunological Examination of Protein Expression The expression of RPP16 and RPP17 proteins was
Studied by immunological detection.

First, antisera against rice RPP16 and RPP17 proteins were prepared as follows. R
Part of PP16-1 and RPP17-1 (each,
The cDNA corresponding to amino acid positions; positions 39 to 144 of SEQ ID NO: 2 and amino acid positions; positions 43 to 159 of SEQ ID NO: 4) was amplified by PCR, and a pGEX4T-3 vector containing the GST gene (Amersham Pharmacia B
iotech), and E. coli, respectively. coli
It was transformed into JM109. GST / RPP16 and GST / RPP17 fusion proteins were
Induced by the addition of PTG. Rabbits were injected with each purified fusion protein six times at two week intervals. Antiserum,
Purified by affinity purification using a CNBr activated Sepharose 4B column. RPP16-1 and RPP17-1 protein expression was detected by Western blot analysis using anti-RPP16 and anti-RPP17 antisera. The results are shown in FIGS. 3A and 3B, respectively.

In FIG. 3, the arrows indicate (a) the 30 kD peptide reacted with the anti-RPP16 antiserum and (b) the 17 kD peptide reacted with the anti-RPP17 antiserum, respectively. A peptide with a molecular weight of about 30 kDa was detected in the soluble fraction of rice total protein using anti-RPP16 antiserum (FIG. 3 (a)). Also, RPP17-1
Detection of 17 kDa in the insoluble extract
(FIG. 3 (b)). In addition, as shown below, these proteins were also found to be universally present in other plants (FIGS. 3 (c) and (d)).

Next, the leaves of each plant of rice, sugarcane, barley, adzuki beans, arabidopsis, melon and tobacco were added to 5 volumes of an extraction buffer (100 mM Tris-H).
Cl (pH 7.5); 2 mM EDTA; 5 mM 2-
(Mercaptoethanol; 10% glycerol), ground to a fine powder, centrifuged at 15,000 rpm for 30 minutes at 4 ° C., and the supernatant was collected (S1 fraction). The pellet fraction was mixed with an equal volume of buffer (50 mM Tris-H).
Cl (pH 7.5); 2 mM EDTA; 5 mM 2-
Mercaptoethanol; 10% glycerol; 50 mM
KCl; 0.2% triton X-100). Subsequently, the tubes were spun at 4 ° C for 30 minutes and centrifuged at 15,000 rpm for 30 minutes at 4 ° C, and the supernatant was collected (S2 fraction). The pellet fraction was also suspended in SDS-PAGE running buffer (insoluble fraction). Each protein was subjected to 12% SDS-PA
Separated by GE and Immobilon P
Transferred to VDF membrane (Millipore, Japan). Water-soluble soluble fraction (S1), triton X-
Leaf proteins were prepared from 100 soluble fractions (S2) and insoluble fractions (M). Further rice (lane 1); barley (lane 2); sugarcane (lane 3); arabidopsis (lane 4); melon (lane 5); adzuki (lane 6); and tobacco (lane 7).
Were also separated by SDS-PAGE, anti-RPP16 antiserum for (a) and (c) and anti-RPP1 for (b) and (d).
Western blot analysis was performed with 7 antisera.

As a result, the soluble fraction of the leaves of each plant (S1
Or S2), a protein having a structure similar to that of rice RPP16 was detected in sugarcane, barley, adzuki bean, melon, and Arabidopsis (FIG. 3).
(C)). However, the soluble 3
The 0 kDa peptide was 14 kDa larger than the 15.9 kDa mass estimated from the cDNA sequence of RPP16-1. Further, E. E. coli RPP16-1 product (amino acid position: positions 39 to 144 of SEQ ID NO: 2; As
(p: 9.52%; Glu: 12.38%) was also shown as a 11.5 kDa protein, 5.5 kDa larger than expected (data not shown). In SDS-PAGE, it has been reported that proteins with a high content of acidic amino acids migrate slower than the actual molecular weight, and thus the apparent molecular weight by SDS-PAGE is significantly increased (Takano et al., Biochemistry, 27, 1964-1972 (198
8)). Therefore, R with many acidic amino acid residues
PP16-1 protein (Asp: 8.33%; Glu: 11.11
%) Is considered to be detected as a protein larger than the actual molecular size in SDS-PAGE. These results strongly suggest that the band detected by the protein blot is a translation product of the Rpp16-1 gene.

A protein having a molecular weight similar to that of the RPP17 protein was also detected in the insoluble fraction of sugarcane, barley, tobacco, melon, and Arabidopsis leaves (FIG. 3 (d)). This result suggests that RPP17 protein is a membrane-bound protein.

These results indicate that the RPP16 and RPP17 proteins or homologs thereof are conserved throughout higher plants. Furthermore, RP
The P16 and RPP17 proteins are very different proteins in terms of intracellular localization, as they exhibit completely different protein properties in protein gel analysis.

Example 4 Production of Transgenic Plants Expressing Rpp16-gusA and Rpp17-gusA and In Situ GUS Staining
Genomic sequences containing the putative promoter regions of each of Protein 1 and RPP17-1 protein were screened by plaque hybridization. The RPP16-1 protein and the upstream region of the ORF of the RPP17-1 protein were obtained by screening a 2 × 10 ⁵ plaque rice genomic library with each cDNA clone as a probe. A 1.4 kb RPP16 promoter region (SEQ ID NO: 7) and a 2 kb RPP17 promoter region (SEQ ID NO: 8: a sequence common to RPP17-1 and RPP17-2) were added to the gus of pCAMBIA1301 respectively.
It was subcloned in-frame upstream of the A gene. These are amplified by PCR and pCAM
It was ligated upstream of the gusA gene in the VIA1301 plasmid. The resulting plasmid is Agrobacteriu
mtumefaciens T-DNA-derived vector, which is used for Agrobacterium tumefaciens strain EHA105 (ZENECA MOGE
N). Transgenic rice plant (Oryza s
ativa cvs Nipponbare) with Toki (Toki, Plant Mol. B)
iol. Rep., 15, 16-21 (1997)) by Agrobacterium-mediated transformation. Transgenic rice plants were selected on a medium containing hygromycin and the introduction of the gusA gene into the rice genome was
Confirmed by PCR. More than 40 independent transgenic lines were obtained for each construct.

Analysis of GUS gene expression was performed by fluorescence analysis of GUS activity (Jefferson, Plant Mol. Biol. Rep., 5, 387-).
405 (1987)). Transgenic Rp
GUS activity was observed in all of the roots, stems and leaves of the p16-gusA and Rpp17-gusA plants, confirming that GUS was expressed. GU
S activity was not detected in non-transformants of rice plants.

The histochemical analysis of GUS gene expression was performed by GUS staining as follows. First,
The leaf, stem and root segments were sectioned with a microslicer into 30 μm thin cross-sections, then 0.5 μm.
mM X-Gluc (5-bromo-4-chloro-3-indolyl
containing 50% β-D-glucuronic acid) and 5% methanol
2 to 2 in mM sodium phosphate buffer (pH 7.0)
Incubated for 4 hours at 37 ° C. After incubation, the samples were clarified with ethanol.

FIG. 4 shows a photograph of the result. (A),
The photographs in (b), (c) and (d) each have Rp
Fig. 3 shows a leaf cross section, an enlarged photograph thereof, a stem cross section, and a root cross section of a p16-gusA-expressing plant. (E),
The photographs of (f) and (g) show Rpp17-
1 is a cross section of leaves, stems and roots of a gusA expressing plant.
Abbreviations indicate BR: branch root; CR: crown root; Ph: phloem tissue; and TVB: transverse vascular bundle.

In the root, Rpp16-gusA and Rp
In the p17-gusA plant, GUS staining was observed in roots, but GUS staining was more pronounced in branch roots than in crown roots. To observe the expression pattern in detail, root tissue sections were prepared using a microslicer. Rpp
For both 16-gusA and Rpp17-gusA, GUS staining was observed in the vascular tissue of the branch root. On the other hand, Rpp16-gusA and Rpp17-gusA
In the cortical, epidermal and epidermal layers from plants, GU
No S staining was observed.

In the stem, Rpp16-gusA and Rp16-gusA
p17-gusA expression was detected only in small and large vascular bundles, especially phloem and xylem parenchyma.

GUS staining in the leaf blades and sheaths of Rpp16-gusA plants was examined. Leaf tissue sections were prepared from the blade blades and leaf sheath by a microslicer.
US staining was observed on longitudinal and transverse vascular bundles.
GUS staining was observed only in the large vascular CC and small vascular bundles, and not in other cell types until 8 hours after staining. GUS staining shows that C
C and in parenchyma and mesophyll cells following the vascular sheath. This result indicates that the GUS enzyme is CC
But appears to be gradually released into subsequent apoplasts, parenchyma, and mesophyll cells, i.e., the GUS staining observed in parenchyma and mesophyll cells is a consequence of the diffusion of GUS reaction products. Indicates that there is. The results also show that CC-specific expression is tightly regulated by the RPP16 promoter region.

In the leaf blades and sheaths of Rpp17-gusA plants, GUS staining is detected in CC, parenchyma, and mesophyll cells after 2 to 24 hours, whereas GUS staining is particularly preferred in CC and mesophyll cells. Was detected.

From the above results, it was found that the RPP16 promoter and the RP17 promoter controlling gusA expression exhibited similar expression patterns in roots and stems. In addition, the Rpp16 gene and Rpp17
It was suggested that the gene expression pattern was the same in roots and stems, but different in leaf blades and leaf sheaths.

[0076]

According to the present invention, two proteins having a structure similar to the virus movement protein (MP) are present in rice; one is an RPP17 protein having at least about 50% homology with the pumpkin CmPP16 protein. And the other is a pumpkin Cm like a virus MP
RPP16 protein, a novel protein having at least about 50% homology with PP16 protein, was found. These proteins are MP
Has a structure similar to that of, and is expressed phloem-specific, and its expression is strictly regulated by the respective promoters. Therefore, it is possible to develop a technique for producing a substance in the phloem and transporting the substance to the phloem using the genes of these proteins and their control regions. For example, in the phloem, it is possible to produce a useful protein or produce a substance that gives a plant disease or insect resistance, and distribute this substance throughout the plant using a long-distance transport route called a phloem. It is possible. In addition, by producing a binding protein of substances (heavy metals, environmental hormones, etc.) that negatively affect the environment, it is possible to grow environmentally purified plants.

[0077]

[Sequence List] SEQUENCE LISTING <110> Director General of National Institute of Agrobiological Science <120> Novel proteins in phloem of rice and genes there <130> P101N07031 <160> 8 <210> 1 <211> 432 <212> DNA < 213> Oryza sativa L. cvs Nipponbare <220> <221> CDS <222> (1) ... (432) <400> 1 atg gtg cag ggg acg ctc gag gtg ctg ctc gtc gga gcc aag ggc ctc 48 Met Val Gln Gly Thr Leu Glu Val Leu Leu Val Gly Ala Lys Gly Leu 1 5 10 15 gag aac acc gac tac ctg tgc aac atg gac ccg tac gcg gtt ctc aaa 96 Glu Asn Thr Asp Trp Leu Cys Asn Met Asp Pro Trp Ala Val Leu Lys 20 25 30 tgc cgc tcg cag gag cag aag agc agc gtt gcg tca ggt aaa gga tct 144 Cys Arg Ser Gln Glu Gln Lys Ser Ser Val Ala Ser Gly Lys Gly Ser 35 40 45 gac cct gaa tgg aac gaa acc ttt atg ttc agc gtc act cac aac gct 192 Asp Pro Glu Trp Asn Glu Thr Phe Met Phe Ser Val Thr His Asn Ala 50 55 60 aca gag ctc atc atc aag ttg atg gac agt gac agt ggc acg gat gat 240 Thr Glu Leu Ile Ile Lys Leu Met Asp Ser Asp Ser Gly Thr Asp Asp 65 70 75 80 g at ttt gtt gga gaa gca acg att tct ttg gaa gca atc tat aca gaa 288 Asp Phe Val Gly Glu Ala Thr Ile Ser Leu Glu Ala Ile Trp Thr Glu 85 90 95 gga agc ata ccc cca act gtt tat aat gtt gtg aaa gaa gaa tac 336 Gly Ser Ile Pro Pro Thr Val Trp Asn Val Val Lys Glu Glu Glu Trp 100 105 110 cgt gga gaa atc aaa gtg ggc ctg acg ttc act cca gag gat gat cgc 384 Arg Gly Glu Ile Lys Val Gly Leu Thr Phe Thr Pro Glu Asp Asp Arg 115 120 125 gat cgg ggt tta tct gag gaa gac att ggt gga tgg aag cag tca tct 432 Asp Arg Gly Leu Ser Glu Glu Asp Ile Gly Gly Trp Lys Gln Ser Ser 130 135 140 <210> 2 <211 > 144 <212> PRT <213> Oryza sativa L.cvs Nipponbare <400> 2 Met Val Gln Gly Thr Leu Glu Val Leu Leu Val Gly Ala Lys Gly Leu 1 5 10 15 Glu Asn Thr Asp Trp Leu Cys Asn Met Asp Pro Trp Ala Val Leu Lys 20 25 30 Cys Arg Ser Gln Glu Gln Lys Ser Ser Val Ala Ser Gly Lys Gly Ser 35 40 45 Asp Pro Glu Trp Asn Glu Thr Phe Met Phe Ser Val Thr His Asn Ala 50 55 60 Thr Glu Leu Ile Ile Lys Leu Met Asp Ser Asp Ser Gly Thr Asp Asp 65 70 75 80 Asp Phe Val Gly Glu Ala Thr Ile Ser Leu Glu Ala Ile Trp Thr Glu 85 90 95 Gly Ser Ile Pro Pro Thr Val Trp Asn Val Val Lys Glu Glu Glu Trp 100 105 110 Arg Gly Glu Ile Lys Val Gly Leu Thr Phe Thr Pro Glu Asp Asp Arg 115 120 125 Asp Arg Gly Leu Ser Glu Glu Asp Ile Gly Gly Trp Lys Gln Ser Ser 130 135 140 <210> 3 <211> 477 <212> DNA <213> Oryza sativa L.cvs Nipponbare <220> <221> CDS <222> (1) ... (477) <400> 3 atg gcg ggg agc ggt gtc ctg gag gtg cat ctc gtc gac gcc aag ggc 48 Met Ala Gly Ser Gly Val Leu Glu Val His Leu Val Asp Ala Lys Gly 1 5 10 15 ctc acc ggc aac gac ttc cta ggt gag ata ggc aag ata gac ccg tac 96 Leu Thr Gly Asn Asp Phe Leu Gly Glu Ile Gly Lys Ile Asp Pro Trp 20 25 30 gtg gtg gtg gtg cag tac cgg agc cag gag cgc aag agc agc gtc gcc aga 144 Val Val Val Gln Trp Arg Ser Gln Glu Arg Lys Ser Ser Val Ala Arg 35 40 45 gat caa ggg aag aac ccg agc tgg aac gag gtg ttc aag ttc cag atc Asp Gln Gly Lys Asn Pro Ser Trp Asn Glu Val Phe Lys Phe Gln Ile 50 55 60 aac tcc acg gcg g cg acc ggg cag cac aag ctc ttt ctg cgg ctc atg 240 Asn Ser Thr Ala Ala Thr Gly Gln His Lys Leu Phe Leu Arg Leu Met 65 70 75 80 gac cac gac acc ttc tca cgg gac gac ttc ctc ggc gaa gca acg atc 288 Asp His Asp Thr Phe Ser Arg Asp Asp Phe Leu Gly Glu Ala Thr Ile 85 90 95 aac gtg act gac ctg atc agc tta ggc atg gag cac ggc aca tgg gag 336 Asn Val Thr Asp Leu Ile Ser Leu Gly Met Glu His Gly Thr Trp Glu 100 105 110 atg agc gaa tcc aag cac cgg gtc gtc ctc gcg gac aaa aca tac cac 384 Met Ser Glu Ser Lys His Arg Val Val Leu Ala Asp Lys Thr Trp His 115 120 125 ggc gag atc aga gtc agc ctc acg ttc acg gct tct gca aag gct caa 432 Gly Glu Ile Arg Val Ser Leu Thr Phe Thr Ala Ser Ala Lys Ala Gln 130 135 140 gac cac gca gaa cag gtt gga gga tgg gcg cac agc ttt cgt cag 477 Asp His Ala Glu Gln Val Gly Gly Trp Ala His Ser Phe Arg Gln 145 150 155 <210> 4 <211> 159 <212> PRT <213> Oryza sativa L.cvs Nipponbare <400> 4 Met Ala Gly Ser Gly Val Leu Glu Val His Leu Val Asp Ala Lys Gly 1 5 10 15 Leu Thr Gly Asn As p Phe Leu Gly Glu Ile Gly Lys Ile Asp Pro Trp 20 25 30 Val Val Val Gln Trp Arg Ser Gln Glu Arg Lys Ser Ser Val Ala Arg 35 40 45 Asp Gln Gly Lys Asn Pro Ser Trp Asn Glu Val Phe Lys Phe Gln Ile 50 55 60 Asn Ser Thr Ala Ala Thr Gly Gln His Lys Leu Phe Leu Arg Leu Met 65 70 75 80 Asp His Asp Thr Phe Ser Arg Asp Asp Phe Leu Gly Glu Ala Thr Ile 85 90 95 Asn Val Thr Asp Leu Ile Ser Leu Gly Met Glu His Gly Thr Trp Glu 100 105 110 Met Ser Glu Ser Lys His Arg Val Val Leu Ala Asp Lys Thr Trp His 115 120 125 Gly Glu Ile Arg Val Ser Leu Thr Phe Thr Ala Ser Ala Lys Ala Gln 130 135 140 Asp His Ala Glu Gln Val Gly Gly Trp Ala His Ser Phe Arg Gln 145 150 155 <210> 5 <211> 468 <212> DNA <213> Oryza sativa L.cvs Nipponbare <220> <221> CDS <222> (1 ) ... (468) <400> 5 atg gcg ggg agc ggt gtc ctg gag gtg cat ctc gtc gac gcc aag ggc 48 Met Ala Gly Ser Gly Val Leu Glu Val His Leu Val Asp Ala Lys Gly 1 5 10 15 ctc acc ggc aac gac ttc cta ggc aag ata gac ccg tac gtg gtg gtg 96 Leu Thr Gly Asn Asp Phe Leu Gly Lys Ile Asp Pro Trp Val Val Val 20 25 30 cag tac cgg agc cag gag cgc aag agc agc gtc gcc aga gat caa ggg 144 Gln Trp Arg Ser Gln Glu Arg Lys Ser Ser Val Ala Arg Asp Gln Gly 35 40 45 aag aac ccg agc tgg aac gag gtg ttc aag ttc cag atc aac tcc acg 192 Lys Asn Pro Ser Trp Asn Glu Val Phe Lys Phe Gln Ile Asn Ser Thr 50 55 60 gcg gcg acc ggg cag cac aag ctc ttt ctg cgg ctc atg gac cac gac 240 Ala Ala Thr Gly Gln His Lys Leu Phe Leu Arg Leu Met Asp His Asp 65 70 75 80 acc ttc tca cgg gac gac ttc ctc ggc gaa gca acg atc aac gtg act 288 Thr Phe Ser Arg Asp Asp Phe Leu Gly Glu Ala Thr Ile Asn Val Thr 85 90 95 gac ctg atc agc tta ggc atg gag cac ggc aca tgg gag atg agc gaa 336 Asp Leu Ile Ser Leu Gly Met Glu His Gly Thr Trp Glu Met Ser Glu 100 105 110 tcc aag cac cgg gtc gtc gc gac aaa aca tac cac ggc gag atc 384 Ser Lys His Arg Val Val Leu Ala Asp Lys Thr Trp His Gly Glu Ile 115 120 125 aga gtc agc ctc acg ttc acg gct tct gca aag gct caa gac cac gca 432 Arg Val Ser Leu Thr Phe Thr Ala Ser Ala Lys Ala Gln Asp His Ala 130 135 140 gaa cag gtt gga gga tgg gcg cac agc ttt cgt cag 468 Glu Gln Val Gly Gly Trp Ala His Ser Phe Arg Gln 145 150 155 <210> 6 <211> 156 <212> PRT < 213> Oryza sativa L.cvs Nipponbare <400> 6 Met Ala Gly Ser Gly Val Leu Glu Val His Leu Val Asp Ala Lys Gly 1 5 10 15 Leu Thr Gly Asn Asp Phe Leu Gly Lys Ile Asp Pro Trp Val Val Val 20 25 30 Gln Trp Arg Ser Gln Glu Arg Lys Ser Ser Val Ala Arg Asp Gln Gly 35 40 45 Lys Asn Pro Ser Trp Asn Glu Val Phe Lys Phe Gln Ile Asn Ser Thr 50 55 60 Ala Ala Thr Gly Gln His Lys Leu Phe Leu Arg Leu Met Asp His Asp 65 70 75 80 Thr Phe Ser Arg Asp Asp Phe Leu Gly Glu Ala Thr Ile Asn Val Thr 85 90 95 Asp Leu Ile Ser Leu Gly Met Glu His Gly Thr Trp Glu Met Ser Glu 100 105 110 Ser Lys His Arg Val Val Leu Ala Asp Lys Thr Trp His Gly Glu Ile 115 120 125 Arg Val Ser Leu Thr Phe Thr Ala Ser Ala Lys Ala Gln Asp His Ala 130 135 140 Glu Gln Val Gly Gly Trp Ala His Ser Phe Arg Gln 145 150 155 <210> 7 <211> 1288 <212> DNA <213> Oryza sativa L .cvs Nipponbare <220> <221> promotor <222> (1) ... (1288) <400> 7 ggccgccgtc cgcgccaaac ccaccacggc tgcaccttct ctgtccccgt cacccgctgc 60 ctaccacttc tccccttcgc ccgtggcacc tccagcccgc ccccacgtcg cccctcctcc 120 gccgtcggct ctccacccct tcccttcccc tcccctcatc attccccttt tgaacgtaaa 180 aatgagatta caatactagt tgtctcctag tattttatat agtaaattcg tcatgcaata 240 aatttggact tttcaaaatg tttgtaaata tagccattat aaaaattaat acaaatgtat 300 ggatagttag aaaatattac taaaatatag gtggatatga tatagagggt gtaatataga 360 tgatctattg gagtgaagaa gaatatagag tgggaatctt ttttagatga ccacctaaat 420 aaggatatag atgatcaaat aaggatgctc taagcctcta actagtgtat ggctaacttt 480 tttgtactta aagcatatta gtgttcgtga cgtgtatgca atgggcacat ttttgctaaa 540 tgaaaacatc tctactatcg ccatctgact tcgaatatat gatatgtgca cctcaaatcc 600 aatggactca tatatcaatg acccacgtta gacgacaaac ttaacccttt gccaatgatc 660 acttctatca gtcaaaatgt tgaatttaca agggcttggc ccatttgata ttttttaaag 720 ttgttggccc atttgatctg gtgacaacgc cacgtgatca tcagagtgta ctattttcac 780 tattttgcg atgcc atgcc cgccg cgtcggagcc agctactgga gcgcacgccg 840 ataatgtgca cgccgacacg tccacacgca tggtgacgaa ggctgtgccc tccaattccg 900 tgcaccatcc cgacggtacc gcacggcacg gcccacggtg agatcggctc tgctttttcg 960 cccgcgaaac cggcgggaga agacaccctc tcctccctct ctccccttcc tgccgtcact 1020 catccaccag cctgggcgac gacgcaccgc ccgcggcgca ttgacttgcg cacgttacgc 1080 tgtcgtcgtt tccccctacc acgccacctc catgttccgg gggggaggcg aacgcgtccc 1140 gcgagtccca cggcccacct caactcctat aaaaactgcg ttcggattgg atctcgccgt 1200 ctcgcttcct ggtcaaattg ttcgctaata agcatcgatc gaaggcgagg ccagccagcc 1260 agccggggcg ccgggggagc tggggaag 1288 <210> 8 <211> 1572 <212> DNA <213> Oryza sativa L.cvs Nipponbare <220> <221> promotor <222> (1) ... (1572) <400 > 8 ttttcggcaa ttagaatttc ggaatctatg ctagatttgt cgagctcagt tcctcaggat 60 cttgacatgt gaattctctg tctgctttca tttttctctg tctgaggatt tcaacttttg 120 gattactgcg acactgatcc atgctttgtt ctttaattag ttatgtgttg cgcgagctat 180 cgacttcaaa ataatatgct ttttttttgt gatcgactga tcgccatata cctgtacgta 240 atgtgagacg aaaccactat tcaattttgc a gctgacgtt gtgatgttga tgcctgatgg 300 tgctctactg ctctgtttcc tgcgttcccg aaaaatactg ctctgttaat ctgtttccag 360 cgtaaattaa aacaaacggc caatgataat cgtttttaat tttttggccg agtcatcaca 420 gactcacagt atgtcaacaa ctgatgtcag tacacccgtt ctttgttatt atgacacatc 480 actgtcatga aaaccacctg ataggttaac aatcagaacc aagcagtttc tgattctgag 540 acccattctt cagaaatctg gaaattctac cgatatctat ctgcctgact atctctatct 600 cgatctcgtc caggattgtt cttgcgaact aatggttgta cgcaaattcg tctccctact 660 gcagtctcag tccaagtttc ctctttgatg attcttcgaa cttatcgcgt agattgggac 720 tcgcgcattt agccgaggaa acacgcttag cagcaagact cgatcagctc cgatcctcac 780 gttgaagcga tgaaaaaaaa tcacctcgaa ttcgatccag gcggcgagac gtgaccaaat 840 cacgtacaag tcgtcgtcga acagacctgg ttgtttaaac tactaattct aaactgcaga 900 gggtcagaga gaaagatcgt acgaaacttg tactgctaat catgcaataa gcatgtgtta 960 atgccaggac gattcactgg ggaaggaatc acaccgtggg catgctgttg actagcggaa 1020 taataccact gatttaagta gggtcatata ttgatatata gcataactgc taactgccag 1080 ttgctagtgc ctcttcggct cttccagctg ggagtcgaaa caaacaaa ag agccgtcgtt 1140 aagcacgtcc aagtaattaa attagtacgt agtagtaatc cctaagctaa aaatatggcc 1200 agttgtagct gacagctagg agtaagagat gggcaagtag agagtataga gaggaaggag 1260 agaggaagca atatgggaga acagtagtga agcctcgaac cggctcggtt cagtccaact 1320 caaccacgta gaaacgtgcg ggtttacacg gtggcgaccg cttcaaatgt gcgggagctt 1380 tgactggtcc ccagccagct cgcctataaa cgcgcgcacg ctcgagccag ctaccacggc 1440 atctgcttga gttggagcaa cacaccaaga aggccagaga gattcagaga actacaccga 1500 gaaggcgaga ttgtttgcgt ggttggtaat ttggccggcc ttgatcgatc gagcggagaa 1560 gagaagagaa ag 1572

[Brief description of the drawings]

BRIEF DESCRIPTION OF THE FIGURES FIG. 1: Migration proteins of pumpkin CmPP16 and red clover necrosis mosaic virus and rice RPP1
It is a figure which shows the alignment with 6-1 protein, RPP17-1 protein, and RPP17-2 protein.

FIG. 2. Rice RPP16-1 protein, RPP17-
FIG. 2 shows the results of phylogenetic analysis of Protein 1, RPP17-2 protein, and related sequences.

FIG. 3 is a photograph showing the results of protein blot analysis by electrophoresis of RPP16 protein and RPP17 protein.

FIG. 4. Rpp16-gusA and Rpp17-gu
It is a photograph which shows the result of GUS histochemical detection of an sA plant.

 ────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Hiroaki Shimada 3-25-4 Tsukushigaoka, Kashiwa-shi, Chiba F-term (reference) 2B030 AB03 CA15 CA17 CA19 4B024 AA08 AA11 BA79 BA80 CA04 CA07 CA09 DA01 DA05 DA06 EA04 FA02 GA11 GA27 HA11 HA13 HA14 HA20 4B065 AA11X AA26X AA88X AA88Y AB01 AC14 BA02 BA24 CA24 CA46 CA53 4H045 AA10 AA11 AA20 AA30 BA10 BA41 CA31 EA05 EA50 FA71 FA74

Claims (27)

[Claims] 1. A rice phloem protein having a Ca ²⁺ / phospholipid binding domain. 2. The rice phloem protein of claim 1, further having at least about 50% amino acid homology with the pumpkin CmPP16 protein. 3. The rice phloem protein according to claim 2, which has the amino acid sequence of SEQ ID NO: 2. 4. The rice phloem protein according to claim 2, which has at least one amino acid substitution, insertion or deletion in the protein according to claim 3. 5. The rice phloem protein according to claim 2, which has the amino acid sequence of SEQ ID NO: 4. 6. The rice phloem protein according to claim 2, which has at least one amino acid substitution, insertion or deletion in the rice phloem protein according to claim 5. A DNA encoding the rice phloem protein according to any one of claims 1 to 6. 8. The method according to claim 7, which comprises the nucleotide sequence of SEQ ID NO: 1.
DNA according to 1. 9. The method according to claim 7, which comprises the nucleotide sequence of SEQ ID NO: 3.
DNA according to 1. 10. The DNA according to claim 7, which comprises the nucleotide sequence of SEQ ID NO: 5. 11. A promoter comprising the nucleotide sequence of SEQ ID NO: 7. 12. A promoter comprising the nucleotide sequence of SEQ ID NO: 8. 13. A transgenic plant transformed so that the rice phloem protein according to any one of claims 1 to 6 is phloem-specifically expressed. 14. The transgenic plant according to claim 13, wherein said transgenic plant is selected from the group consisting of rice, sugarcane, corn, and melon. 15. A method for producing the rice phloem protein according to any one of claims 1 to 6, using the transgenic plant according to claim 13 or 14. 16. A transgenic plant producing a heterologous protein in a phloem-specific manner, comprising a promoter involved in phloem-specific expression, and a DNA encoding the heterologous protein operably linked to the promoter. A transgenic plant obtained by a method comprising the steps of: obtaining a vector containing the same; and introducing the obtained vector into a plant. 17. The transgenic plant according to claim 16, wherein the vector further comprises all or a part of the DNA according to any one of claims 7 to 10. 18. The DNA according to claim 7, wherein all or a part of the DNA is linked to DNA encoding the heterologous protein so as to express a fusion protein. 18. The transgenic plant according to 16 or 17. 19. The promoter according to claim 16, wherein the promoter is the promoter according to claim 11 or 12.
8. The transgenic plant according to any one of items 8 to 9. 20. The transgenic plant according to claim 16, wherein the plant is selected from the group consisting of rice, sugarcane, corn, and melon. 21. The transgenic plant according to claim 16, wherein the heterologous protein is secreted from the phloem into the phloem. 22. A method for specifically producing a heterologous protein in a phloem of a plant, comprising a promoter involved in phloem-specific expression, and a DNA encoding the heterologous protein operably linked to the promoter. And a step of introducing the obtained vector into the plant to obtain a transgenic plant. 23. The method according to claim 22, wherein the vector further comprises all or a part of the DNA according to any one of claims 7 to 10. 24. The DNA according to any one of claims 7 to 10, wherein all or part of the DNA is linked to DNA encoding the heterologous protein so as to express a fusion protein. 24. The transgenic plant according to 22 or 23. 25. The promoter according to claim 11, wherein the promoter is the promoter according to claim 11 or 12.
5. The method according to any one of the above items 4. 26. The method according to any of claims 22 to 25, wherein said plant is selected from the group consisting of rice, sugarcane, corn, and melon. 27. The method according to claim 22, wherein the heterologous protein is secreted from the phloem into the phloem.