JP2007151435A - Transformed plant having high starch-accumulating ability and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a plant suiting to environmental stress becoming aggravated, and having high starch/energy-accumulating ability even in the very bad environment; and to provide a method for producing the plant. <P>SOLUTION: The transformed plant having the high starch-accumulating ability is obtained by transporting/localizing a functional protein through an endoplasmic reticulum-Golgi body system to a plasmid by plasmid-transportation/localization construct including a specific peptide region of rice α-amylase I-1 and a vesicle membrane-permeable signal peptide region to modify the function of the plasmid. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a transformed plant having a high starch accumulation ability and a method for producing the same.

In recent years, deterioration of the natural environment on a global scale such as global warming has become a major problem. In Japan, it is already known that environmental stress has had a great influence on the growth and physiology of important crops such as rice, especially carbohydrate metabolism, leading to a decrease in sales and quality.
The accumulation of photosynthetic products in starch is the most important energy storage process not only for plants but for all life on earth. This accumulation in starch is carried out by amyloplasts with differentiated chloroplasts that have photosynthetic functions. Therefore, in order to breed environmental stress-resistant crops, this chloroplast / amyloplast (plastid) function modification method is used. Research is underway. The differentiation and functional expression of plastids is governed by the genes of this organelle itself and the genes encoded in the genome of the cell nucleus. As a method for modifying the function of plastids, two methods have been known. One is a method of directly introducing a gene into a plastid, and the other is a method in which a chloroplast transition signal gene called a transit peptide is fused to a functional protein gene to be introduced into a chloroplast, and then the cell nucleus. It is a method of introducing into the genome. The introduced gene is translated in the cytoplasm and transported and localized to plastids by transit peptides. However, with the former method, it is difficult to modify the plastids of the whole plant, and with the latter method, the recombinant protein is translated in the cytoplasm, which may cause a burden on the cells.
By the way, α-amylase (EC 3.2.1.1) is a starch hydrolyzing enzyme, secreted from the scutellum tissue and the aleurone layer to the endosperm which is the starch storage tissue, and decomposes the starch granules to germinate And it plays an important role of supplying the carbon source and energy source necessary for growth growth. It has been clarified that there are at least 11 kinds of rice α-amylase genes. Among them, α-amylase I-1 (AmyI-1) having a typical N-linked sugar chain is a germinated rice seed. Has been reported to play an important role in the degradation of endosperm storage starches (Non-patent Documents 1 and 2).
Asatsuma, S., Sawada, C., Itoh, K., Okito, M., Kitajima, A., Mitsui, T. (2005): Involvement of a-amylase I-1 in starch degradation in rice chloroplasts. Physiol. 46: 858-869 Nanjo, Y., Asatsuma, S., Itoh, K., Hori, H. and Mitsui T. (2004) Proteomic identification of a-amylase isoforms encoded by RAmy3B / 3C from germinating rice seeds. Biosci. Biotech. Biochem. 68 : 112-118

  However, little is known about the details of the targeting mechanism and genetic system for chloroplasts and amyloplasts (plastids) of rice α-amylase. Breeding crops with high chloroplast / amyloplast photosynthetic and starch accumulation ability plays an important role in accumulating the most important energy not only for plants but also for all life on earth. Research is progressing from the direction, and the results are expected.

  Therefore, the present invention has been made under the background described above, and provides a plant that adapts to a severe environmental stress and has a high starch energy accumulation ability efficiently even in a poor environment, and a method for producing the same. The purpose is to do.

  In view of the above problems, the present inventors have examined the targeting mechanism of enzyme proteins encoded by the nuclear genome in cereal cells to plastids and clarified the existence of the targeting mechanism of plastid constituent proteins (see FIG. 1). . Specifically, the targeting of rice α-amylase I-1 to plastids was inhibited by AtArf1 involved in the transport from the endoplasmic reticulum to the Golgi apparatus or the dominant negative mutant of AtSar1, and therefore, from the endoplasmic reticulum to the Golgi apparatus. Clarified that the transportation of is indispensable. That is, plastid transport / localization via the endoplasmic reticulum-Golgi system is transit peptide-independent plastid localization, and plastid transport / localization of proteins that are not compatible with protein permeation channels in the plastid envelope May be possible. Moreover, it discovered that (alpha) -amylase I-1 (AmyI-1) which does not have the typical transit peptide in connection with plastid localization localizes to the chloroplast and cell wall compartment of a rice green leaf cell.

  In addition, rice α-amylase was known as a secretory enzyme, but as a result of analyzing the rice rice α-amylase suppressed expression and overexpressing transformed rice plant, It has been found that α-amylase is involved. Furthermore, the role of α-amylase I-1 on starch accumulation in living tissue cells was examined using α-amylase I-1 expression-suppressed and overexpressed rice plants. In rice lines, α-amylase I-1 expression was almost completely suppressed, and a marked increase in starch accumulation was observed in the leaf tissue compared to the wild type.

  The present invention has been completed based on the above research results and knowledge, and provides the following components.

The polypeptide according to claim 1 of the present invention is characterized by comprising the following amino acid sequence (a) or (b).
(A) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1.
(B) A polypeptide comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence set forth in SEQ ID NO: 1 and having a targeting effect on plastids.

  The gene according to claim 2 of the present invention encodes the polypeptide according to claim 1.

The polypeptide according to claim 3 in the present invention is characterized by comprising the following amino acid sequence (c) or (d).
(C) A polypeptide having the amino acid sequence represented by SEQ ID NO: 2.
(D) A polypeptide comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence set forth in SEQ ID NO: 2, and having an endoplasmic reticulum membrane permeation effect.

  The gene according to claim 4 in the present invention encodes the polypeptide according to claim 3.

  The vector according to claim 5 of the present invention comprises the gene according to claim 2.

  The vector according to claim 6 of the present invention comprises the gene according to claims 2 and 4.

  The transformed plant according to claim 7 of the present invention is characterized in that the gene according to claim 2 is introduced.

  The transformed plant according to claim 8 of the present invention is characterized in that the gene according to claims 2 and 4 is introduced.

  The transformed plant according to claim 9 of the present invention is characterized in that, in claim 7 or 8, the transformed plant is a monocotyledonous plant.

  In the transformed plant according to claim 10 of the present invention, the vector according to claim 5 or 6 is introduced into a callus derived from a monocotyledonous plant, and after the callus is propagated, the plant body is redifferentiated from the callus. It is characterized by being obtained.

  In the method for producing a transformed plant according to claim 11 of the present invention, the vector according to claim 5 or 6 is introduced into a callus derived from a monocotyledonous plant using Agrobacterium, and the callus is proliferated. The plant body is redifferentiated from callus.

  ADVANTAGE OF THE INVENTION According to this invention, it becomes possible to produce the transformed plant which adapts to the environmental stress which will become more serious from now on, and can accumulate starch efficiently in a bad environment. Moreover, the photosynthetic ability and starch accumulation ability of plastids can be enhanced, and the supply of starch, which is a carbon neutral industrial material, can be increased. Moreover, plant starch is a very important nutrient for human beings, and it is possible to expand the use of starch by modifying the structure of starch granules, and is a useful material in industrial production such as biodegradable resins.

(vector)
In the present invention, as a vector used for introducing a gene into a plant, a transgene can be introduced into a target cell (host cell) and the transgene can be expressed in the target cell. If it is, it will not specifically limit, According to the objective, a suitable vector (a plasmid, a bacteriophage, a virus, etc.) will be utilized. Many vectors are commercially available, and in the present invention, an appropriate one can be selected and used according to the purpose. The method for producing the vector of the present invention is not particularly limited, and any method known to those skilled in the art may be used.

An example of a preferred plastid transport / localization construct in the present invention is shown in FIG. AmyI-1SB shown in FIG. 2 indicates the peptide region (SEQ ID NO: 1) of 301-369 of α-amylase I-1, and is essential for plastid targeting. In general, even when a part of an amino acid having a specific function is modified, the function may be maintained. In view of this, the AmyI-1SB polypeptide is characterized by comprising the amino acid sequence described in the following (a) or (b): (a) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1 A peptide; (b) a polypeptide comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence set forth in SEQ ID NO: 1 and having a targeting effect on plastids; peptide.
Moreover, SP shows ER membrane permeation | transduction signal peptide, for example, AmyI-1SP (sequence number 2) is mentioned, However, It is not limited to this, You may use SP of other plant secretion protein. The AmyI-1SP polypeptide is characterized by comprising the following amino acid sequence (c) or (d): (c) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2; (d) A polypeptide comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence set forth in SEQ ID NO: 2, and having an endoplasmic reticulum membrane permeation effect. The functional protein may be inserted either on the N-terminal side or on the C-terminal side of AmyI-1SB.

(Transformation method)
For introducing a vector into a plant, various methods known to those skilled in the art such as polyethylene glycol method, electroporation method (electroporation method), Agrobacterium-mediated method, particle gun method and the like can be used. Preparation of a vector suitable for each method and regeneration of a plant from transformed plant cells can be performed by methods known to those skilled in the art depending on the type of plant cell.

(Target plant)
The target plant in the present invention is a monocotyledonous plant. Examples of monocotyledonous plants include Gramineae (genus Gramineae, Wheat genus, Barley, Rye, Gramineae, Corn genus, Sugar cane genus), Lily family (eggaceae, garlic genus, etc.).

  The present invention will be described in detail below with reference to examples of the present invention, but the present invention is not limited to these examples.

  The biological materials and reagents used in the following Examples 1 to 3 are as follows.

(Test plant)
Onion (Allium cepa) epidermal cells were cultured as a plant to be used for transformation (target plant) on a 2,4-D-free MS medium supplemented with 0.6% gellite in the dark at 25 ° C. Later used.

(Plasmid construct)
Total RNA was isolated from scutellum tissue of rice seeds on day 6 of germination. The cDNA library was prepared using pBluescriptIISK (+/−) (Takara Bio Inc.) according to the manufacturer's protocol. The cDNA library was selected using specific probes of α-amylase II-3 (RAmy3E), II-5 (RAmy3B), and II-6 (RAmy3C) (Sheu, J.-J., Yu, T .-S., Tong, W.-F., Yu, S.-M. (1996) Carbohydrate starvation stimulates differential expression of rice a-amylase genes that is modulated through complicated transcriptional and posttranscriptional processes.J. Biol. Chem. 271: 26998-27004.) The resulting full-length cDNAs (referred to as AmyII-3, AmyII-5, and AmyII-6) were cloned into pBluescript SK (+ • −) to create pAmyII-3, pAmyII-5, and pAmyII-6. (Figure 3). pAmyI-1 and pAmyII-4 are Asatsuma S, Sawada C, Itoh K, Okito M, Kitajima A, Mitsui T (2005) Involvement of α-amylase I-1 in starch degradation in rice chloroplasts. Plant & Cell Physiology 46: Prepared according to the method described in 858-869. (FIG. 4).

  Full-length α-amylase I-1 cDNA and various fragments partially cleaved at the C-terminus were amplified by PCR using pAmyI-1 and a primer sequence set (Table 1) as template DNA.

  Full-length α-amylase II-3, II-4, II-5, II-6 cDNA, and their N-terminal signal sequence regions were used as template DNAs for pAmyII-3, pAmyII-4, pAmyII-5 and pAmyII-6. And it amplified by PCR using a primer sequence set (Table 2).

  A BamHI PCR amplified fragment was inserted into the BamHI site of pGFP and pAmyI-1-GFP, pAmyI-1 (SP) -GFP, pAmyI-1 (Δ101-428) -GFP, pAmyI-1 (Δ201-428) -GFP, pAmyI-1 (Δ301-428) -GFP, pAmyI-1 (Δ370-428) -GFP, pAmyII-3-GFP, pAmyII-3 (SP) -GFP, pAmyII-4-GFP, pAmyII-4 (SP)- GFP, pAmyII-5-GFP, and pAmyII-6-GFP (FIG. 5) were made.

  Using PCR, one amino acid in which tryptophan 302 was substituted with alanine 302 was introduced to prepare AmyI-1 (W302A). Mutagenic forward primers (F-ATG + 1 and F-W302A) and reverse primers (R-end and R-W302A) were synthesized (Table 3).

  A first PCR was performed using a mutagenic primer set and pAmyI-1 as template DNA. A second PCR was performed using F-ATG + 1 and R-end primers and the first PCR product as template DNA. The second PCR product was cleaved with BamHI and cloned into the BamHI site of pGFP to create pAmyI-1 (W302A) (FIG. 6).

  For DsRed2 expression in onion epidermal cells, a BamHI-NotI PCR amplified fragment from pDsRed2 (Clonetech) was cloned into the BamHI-NotI digested site of BamHI-NotI digested pGFP to produce pDsRed (FIG. 7). Rice waxy gene (Itoh, K., Ozaki, H., Okada, K., Hori, H., Takeda, Y., Mitsui, T. (2003) Introduction of Wx transgene into rice wx mutants leads to both high- and low-amylose rice. Plant Cell Physiol., 44: 473-480) using the first 1-111 amino acids of the transit peptide sequence with two primers 5'-ctggatccatgtcggctctcaccacg-3 'and 5'-tatggatccggcaggggggagggccaccgag-3' PCR amplification was performed, and then the PCR product was digested with BamHI to prepare pWxTP-DsRed. The BamHI digested fragment was inserted into the BamHI digested site of pDsRed (FIG. 8). Furthermore, the amplified WxTP fragment of BamHI PCR was cloned into the BamHI digestion site of pGFP to construct pWxTP-GFP (FIG. 9).

  PPTS2-GFP (Mano, S., Nakamori, C., Hayashi, M., Kato, A., Kondo, M., Nishimura, M. (2002) Distribution and characterization of peroxisomes in Arabidopsis by visualization Plant Cell Physiol. 43: 331-41.) and pmt-GFP (Niwa, Y., Hirano, T., Yoshimoto, K., Shimizu, M., Kobayashi, H) with GFP: dynamic morphology and actin-dependent movement. (1999) Non-invasive quantitative detection and applications of non-toxic, S65T-type green fluorescent protein in living plants. Plant J. 18, 455-463. 10).

(Gene transfer method)
Plasmid DNA was introduced into onion epidermal cells using a particle delivery system (PDS-1000 / He, BIO-RAD). Specifically, 3 μg of plasmid DNA dissolved in 10 μl of sterilized water, 10 μl of a gold particle suspension (60 μg / ml of gold particles with a diameter of 1.0 μm), 10 μl of 2.5 mM CaCl ₂ and 4 μl Of 0.1 M spermidine was added and suspended, and allowed to stand at room temperature for 30 minutes. Gold particles coated with plasmid DNA were washed with cold ethanol and then gently suspended in 10 μl of ethanol. A 1100 psi rupture disk was used and introduced twice per sample.

(Observation method)
GFP, DsRed and mRFP fluorescence images were observed and photographed using an electric fluorescence microscope (Olympus BX-61) and a CCD camera (Cool SNAP fx, Roper Scientific), and image analysis software (Lumina Vision, Mitani Corp.) ). GFP was excited at 470-490 nm and detected at 510-550 nm. DsRed and mRFP were excited at 520-550 nm and detected ≧ 580 nm. 20-30 images were taken at 10 μm intervals per cell, and after deconvolution processing, one image was obtained.

  A rice α-amylase gene and a green fluorescent protein (GFP) gene were fused, and the fusion product was introduced into an onion cell to examine transport and localization of GFP.

  AmyI-1-GFP and WxTP-DsRed were simultaneously introduced into onion epidermal cells with a particle gun and allowed to express transiently (FIG. 11). WxTP-DsRed is a marker indicating localization to plastids. As shown in FIGS. 11A and 11D, the fluorescence of AmyI-1-GFP was localized not only on the cell surface but also in some organelle in the cell. In B and E, the fluorescence of WxTP-DsRed showed plastids, and strumule, a structure peculiar to plastids, was also confirmed (FIG. 11E). C is an image in which A and B are superimposed, F is an image in which D and E are superimposed, and the fluorescence of AmyI-1-GFP partially coincides with the fluorescence of WxTP-DsRed, and AmyI-1 is localized in plastid Showed that to do. From this result, it was shown that the rice α-amylase gene and the green fluorescent protein (GFP) gene were fused, and this fusion product was introduced into the onion cell to transport and localize GFP in the plastid of the onion cell. It was. In addition, it was revealed that α-amylase I-1 contains a plastid localization signal common to both rice and onion cells.

  In order to determine a region indispensable for plastid targeting of α-amylase I-1, a fusion protein of α-amylase I-1 and GFP lacking a part of the C-terminal end was transiently expressed. Localization was investigated.

FIG. 12 shows α-amylase I-1 (AmyI-1Δ34-428, AmyI-1Δ101-428, AmyI-1Δ201-428, AmyI-1Δ301-428, AmyI-1Δ370-428) in which the C-terminal is partially cleaved. Show. AmyI-1Δ34-428 contained only the N-terminal signal peptide of α-amylase I-1. The predicted signal peptidase cleavage site is on the N-terminal side of Gln ²⁶ . AmyI-1Δ301-428 contains an N-linked sugar chain binding site (Asn ²⁶⁵ , -Gly ²⁶⁶ , -Thr ²⁶⁷ ). AmyI-1Δ370-428 has a complete A and B domain containing a putative starch binding site ( ³⁰¹ Typ- ³⁰² Typ) (Robert, X., Haser, R., Mori, H., Svensson, B. , Aghajari, N. (2005) Oligosaccharide binding to barley a-amylase 1. J. Biol. Chem. 280: 32968-32978). The amino acid sequence of 370 to 428 residues corresponds to the C-domain that made up the five antiparallel β-sheets. As shown in FIG. 12 and Table 4, the AmyI-1-GFP fusion protein lacking many C-terminals was localized in the endoplasmic reticulum (ER) -like network structure, and was hardly localized in the plastid. However, the AmyI-1Δ370-428-GFP fusion protein was localized to plastids in the onion cells transfected with the full-length AmyI-1-GFP. From these results, it is clear that the peptide region of 301-369 amino acid residues (α-AmyI-1SB (SEQ ID NO: 1)) is essential for plastid targeting of α-amylase I-1 in onion epidermal cells. became.

  Whether α-amylase isoforms (AmyII-3, AmyII-4, AmyII-5, and AmyII-6) are localized in the plastid of onion epidermis cells is determined by a fusion protein of α-amylase isoform and GFP ( AmyII-3-GFP, AmyII-4-GFP, AmyII-5-GFP and AmyII-6-GFP).

  There are many isoforms of rice α-amylase, and these isoforms are known to be encoded by many genes (O'Neill, SD, Kumagai, MH, Majumdar, A., Huang , N., Sutliff, TD and Rodriguez, RL (1990) The alpha-amylase genes in Oryza sativa: characterization of cDNAclones and mRNA expression during seed germination. Mol. Gen. Genet. 221: 235-244; Huang, N., Koizumi, N., Reinl, S., Rodriguez, RL (1990a) Structural organization and differential expression of rice a-amylase genes. Nucl. Acid Res. 18: 7007-7013; Huang, N., Sutliff, TD, Litts, JC, Rodriguez, RL (1990b) Classification and characterization of the rice a-amylase multigene family. Plant Mol. Biol. 14: 655-668; Kim, J.-K. and Wu, R. (1992) Nucleotide sequence of a high-pI rice (Oryza sativa) a-amylase gene. Plant Mol. Biol. 18: 399-402). In addition, in the previous study, the present inventors have reported that natural expression products of RAmy1A, RAmy3B, RAmy3C, RAmy3D, and RAmy3E were detected in rice plants and tissue culture (Mitsui, T., Yamaguchi , J. and Akazawa, T. (1996) Physicochemical and serological characterization of rice a-amylase isoforms and identification of their corresponding genes.Plant Physiol. 110: 1395-1404; Nanjo, Y., Asatsuma, S., Itoh, K , Hori, H. and Mitsui T. (2004) Proteomic identification of a-amylase isoforms encoded by RAmy3B / 3C from germinating rice seeds. Biosci. Biotech. Biochem. 68: 112-118).

  Compared to the localization of AmyI-1-GFP in plastids, AmyII-3-GFP, AmyII-4-GFP, AmyII-5-GFP, and AmyII-6-GFP are hardly localized in plastids (Table 4).

  Furthermore, the targeting properties of α-amylase I-1-GFP fusion protein with mutated starch binding sites in onion cells were examined and compared.

  FIG. 13 shows rice α-amylase isoforms (amylase I-1 (M24286); amylase II-3 (M59352); amylase II-4 (M24287); amylase II-5 (X56337); amylase II-6 (X56338). )) Sequence comparison of amino acid sequences. The amino acid sequence between α-amylase I-1 (RAmy1A), II-3 (RAmy3E), II-4 (RAmy3D) and II-5 / 6 (RAmy3B / 3C) shown in FIG. 13 is about 83 to 90%. Have homology.

³⁰¹ Typ- ³⁰² Typ was a unique amino acid residue in the polypeptide of α-amylase I-1. Other α-amylase isoforms (α-amylase II-3, II-4, II-5 and II-6) did not have tryptophan dipeptides in the amino acid residues of peptide regions 301 to 369 (FIG. 13). When Reptophan 302 was replaced with Alanine 302, the targeting of AmyI-1-GFP to plastids was strongly inhibited, and AmyI-1 (W302A) -GFP was retained in an ER-like network (FIG. 14, Table 4). ). These results revealed that the ³⁰² Typ residue is essential for plastid targeting of α-amylase I-1 in onion epidermal cells. FIG. 15 shows the signal region of α-amylase I-1 for plastid targeting. “SP” shown in FIG. 15 means an ER (endoplasmic reticulum) membrane permeation signal peptide, and “TSR” means a signal region for plastid targeting (ie, 301-369 peptide region of α-amylase I-1). (Starch binding region)). In addition, α-amylase is composed of three domains, domain A folds into (β / α) 8 barrels and contains the catalytic residue of the enzyme, and domain B contains the third β chain of domain A and 3 A large loop protruding from between the first α-helix, domain C is located at the C-terminus of the (β / α) ₈ barrel and consists of the β chain.

  The biological materials, reagents, experimental methods, etc. used in Example 4 below are as follows. Note that materials and the like that are not particularly described are the same as those used in the above examples.

(Test plant)
The rice cultivar “Nihonbare” (Oryza sativa L. cv. Nipponbare) was used as a test plant. The rice seeds were provided by the Niigata Agricultural Research Institute.

(Analysis method)
Starch content is Matsukura C, Saitoh T, Hirose T, Ohsugi R, Perata P, Yamaguchi J (2000) Sugar uptake and transport in rice embryo.Expression of companion cell-specific sucrose transporter (OsSUT1) induced by sugar and light. Plant Physiology 124: 85-93, and Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding .. Anal. Biochem. 72: 248-254 Determined according to. The analysis method of α-amylase was performed according to the method described in Mitsui T, Yamaguchi J, Akazawa T (1996) Physicochemical and serological characterization of rice α-amylase isoforms and identification of their corresponding genes.Plant Physiology 110: 1395-1404. .

(Construction of vector)
Construction of pZH2B-35S-AmyI-1 and pTN1-35S-AmyII-4 was carried out by Asatsuma S, Sawada C, Itoh K, Okito M, Kitajima A, Mitsui T (2005) Involvement of α-amylase I-1 in starch degradation in rice chloroplasts. Plant & Cell Physiology 46: 858-869 and Hajdukiewicz P, Svab Z, Maliga P (1994) The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation.Plant Molecular Biology 25: 989-994 According to the method (FIG. 16). The fragment obtained by digesting pAmyII-4 with BamHI-EcoRI was cloned into the corresponding restriction site obtained by digesting pTN1-35S-AmyI-1 with BamHI-EcoRI to create pTN1-35S-AmyII-4 ( FIG. 17).

(Preparation of α-amylase overexpressing plant)
The vector prepared as described above is introduced into competent cells of Agrobacterium tumefaciens EHA101 strain treated with 20 mM CaCl ₂ , and Agrobacterium introduced by plate culture in the dark is introduced. Selected. Agrobacterium introduced with the plasmid was infected with callus derived from rice embryonic tissue in the presence of acetosyringone. After infection, callus from which the plasmid was induced was selected on a plate medium containing antibiotics and redifferentiated into plant bodies by plant hormones (NAA) (Hiei Y, Ohta S, Komari T, Kumashiro T (1994) Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA.Plant Journal 6: 271-282 and Asatsuma S, Sawada C, Itoh K, Okito M, Kitajima A, Mitsui T (2005) Involvement of α-amylase I-1 in starch degradation in rice chloroplasts. Plant & Cell Physiology 46: 858-869).

(Plant growth)
The regenerated plant body was transferred to a pot and allowed to grow until it reached about 20 cm in a gloss chamber at 27 ° C. for 12 hours in the light and 8 hours in the dark. Thereafter, it was transferred to a greenhouse and grown for about 6 months until seeds were collected. After flowering, seeds were cut in about 40 days.

(Southern blot analysis, Northern blot analysis, Western blot analysis)
Genomic DNA (5 μg) isolated from young leaves of wild type and transformed rice was digested with EcoRI, fractionated by 1% agarose gel electrophoresis, and then transferred and fixed to Hybond-N + nylon membrane (Amersham). Northern blot analysis was performed by Mitsui T, Loboda T, Kamimura I, Hori H, Itoh K, Mitsunaga S, (1999) Sucrose-controlled transport and turnover of α-amylase in rice (Oryza sativa L.) cells..Plant Cell Physiol 40 884-893 and Kashem MA, Itoh K, Iwabuchi S, Hori H, Mitsui T (2000) Possible involvement of phosphoinositide-Ca2 + signaling in the regulation of alpha-amylase expression and germination of rice seed (Oryza sativa L.). The procedure was described in Plant & Cell Physiology 41: 399-407. For Southern blot analysis, specific DNA of the α-amylase I-1 gene and α-amylase II-4 gene (Kashem et al. 2000, supra) was used as a radioactive probe. Autoradiography was performed using an image analysis apparatus BAS5000 (Fuji Film). Western blot analysis, methods for preparing anti-α-amylase I-1 antibody and anti-α-amylase II-4 antibody, etc. are described in Mitsui T, Yamaguchi J, Akazawa T (1996) Physicochemical and serological characterization of rice α-amylase isoforms and identification. It was performed according to the method described in Plant Physiology 110: 1395-1404. A peroxidase-labeled anti-rabbit IgG antibody was used as the secondary antibody. The protein band was visualized using ECL (chemiluminescence, Amersham) and quantified using LAS3000 (Fujifilm). The amount of each protein of α-amylase I-1 and II-4 expressed in a wild type rice plant was defined as 1 unit.

  Western blot analysis was performed using specific antibodies (eg, anti-α-amylases I-1, II-3, II-4, II-5 and II-6), and α- in rice ripening seeds during the ripening period. The expression of amylase isoforms was examined. Expression of α-amylase I-1 and II-4 was detected in ripened seeds on the 10th day after heading (FIG. 18). Interestingly, it was suggested that α-amylase II-4 is the most abundant isoform in ripened seeds.

  PZH2B-35S-AmyI-1 and pTN1-35S-AmyII containing α-amylase I-1 (AmyI-1) and α-amylase II-4 cDNA (AmyII-4), respectively, under the control of the cauliflower mosaic virus (CaMV) 35S promoter -4 was used to produce transgenic rice plants by Agrobacterium-mediated transformation. The presence of AmyI-1 and AmyII-4 was confirmed in transformed plants regenerated using Southern hybridization. The resulting transformed rice plant had 1-8 copies of the transgene (not shown). In addition, α-amylase gene expression in these gene-introduced lines transformed with pZH2B-35S-AmyI-1 and pTN1-35S-AmyII-4 was examined using Northern and Western blot analysis. The results are shown in FIG. As shown in FIG. 19, both of these transformed rice lines showed a marked increase in α-amylase mRNA and protein in adult leaves and T1 generation shoot tissues. In addition, the starch content in leaves overexpressing α-amylase I-1 (A3-1) is significantly reduced compared to that of the wild type (WT), and the starch content is about 42% of the wild type. Met. (FIG. 20). On the other hand, the starch content in leaves overexpressing α-amylase II-4 (D1-4) was slightly reduced, about 82% of the wild-type starch content.

  In the ripening period of the T3-1 generation of A3-1 and D1-4, the higher activity of α-amylase compared to the wild type is the ripening of both transformed plants expressing α-amylase I-1 and II-4. Detected in seeds (Figure 21). The vertical axis in FIG. 21 indicates α-amylase activity (unit / seed), and the horizontal axis indicates the number of days after heading (daf). The level of α-amylase activity contained in the ripened seeds of the D1-4 line was about twice that of the A3-1 line.

  Furthermore, the characteristics of ripening seed phenotypes of A3-1 and D1-4 lines were analyzed. When Western blot analysis was carried out using specific antibodies against α-amylase I-1 and α-amylase II-4, α-amylase I- One molecule showed an increase of 36 times, and α-amylase II-4 molecule showed an increase of 44 times in mature seeds of the D1-4 line (FIG. 22-A). An increase in enzyme activity was shown in both mature seeds of lines A3-1 and D1-4, but a more significant increase was shown in line D1-4 (FIG. 22-B). The dry weights of ripening seeds of A3-1 and D1-4 lines were 4% lower in A3-1 line and 11% lower in D1-4 line than wild type seeds (FIG. 22-). C).

  Further, the quality of the mature seeds of the transgenic rice lines (A3-1 and D1-4 lines) overexpressing α-amylase I-1 and α-amylase II-4 was evaluated by both reflected light image and transmitted light image. Evaluation was made (FIG. 23). As shown in Table 5, in the seeds obtained from the transformed rice lines, abnormal starch accumulation was observed, and the percentages of normal grains of the wild type, A3-1 line, and D1-4 line were 90%, respectively. %, 54.4%, and 36.5%. The characteristic morphological phenotypes of the abnormal seeds of lines A3-1 and D1-4 were white core and whole opaque white, respectively (FIG. 23, Table 5). These results indicated that abnormal expression of α-amylase activity affected starch accumulation and rice grain structure. In addition, α-amylase I-1 and α-amylase II-4 have the ability to localize to amyloplasts in the endosperm of ripened seeds and are considered to have a function of regulating starch accumulation (degradation).

It is a figure which shows the plastid targeting mechanism of (alpha) -amylase I-1 in a higher plant cell. It is a figure which shows the example which constructed | assembled the plastid construct. It is a figure which shows the preparation process of pAmyII-3, pAmyII-5, and pAmyII-6. It is a map of pAmyI-1 and pAmyII-4. It is a figure which shows the preparation process of various pAmy-GFP. It is a figure which shows the preparation process of pAmyI-1 (W302A) -GFP. It is a figure which shows the preparation process of pDsRed. It is a figure which shows the preparation process of pWxTP-DsRed. It is a figure which shows the preparation process of pWxTP-GFP. It is a figure which shows the example which constructed | assembled pPTS2-GFP and pmt-GFP. It is a figure which shows the result of simultaneous expression in the onion epidermal cell of AmyI-1-GFP and WxTP-DsRed. A and D show fluorescent images of AmyI-1-GFP, B and E show fluorescent images of WxTP-DsRed, and C and F show superposed images of GFP and DsRed. The lower figures (D, E, F) are enlarged views of the upper figures (A, B, C). It is a figure which shows the localization of the partially cut | disconnected alpha-amylase I-1-GFP in the onion epidermal cell into which the gene was introduced. The left side, the center, and the right side of A to E show a WxTP-DsRed image, an AmyI-1-GFP image, and a superimposed image of WxTP-DsRed and AmyI-1-GFP, respectively. Amino acids of rice α-amylase isoforms (Amylase I-1 (M24286); Amylase II-3 (M59352); Amylase II-4 (M24287); Amylase II-5 (X56337); Amylase II-6 (X56338)) It is a figure which shows the arrangement | sequence comparison of an arrangement | sequence. The arrowhead indicates the cleavage site of the N-terminal signal peptide. The part marked with ** (asterisk 2) indicates a putative starch binding site. Underlined points indicate glycosylation sites. It is a figure which shows the simultaneous expression in the onion epidermal cell of AmyI-1 (W302A) -GFP and WxTP-DsRed. It is a figure which shows the signal area | region of (alpha) -amylase I-1 regarding the targeting to plastid. It is a figure which shows the preparation process of pZH2B-35S-AmyI-1. It is a figure which shows the preparation process of pTN1-35S-AmyII-4. It is a figure which shows the result of having performed the Western blot analysis using anti-alpha-amylase I-1, II-3, II-4, II-5, and II-6. It is a figure which shows the result of having performed the northern blot analysis and the Western blot analysis of the rice plant which overexpressed alpha-amylase I-1 (A) and alpha-amylase II-4 (B). (A) shows the results of starch content in leaves overexpressing wild type (WT), α-amylase I-1 (A3-1) and α-amylase II-4 (D1-4); ) Is a graph showing the results of α-amylase expression. It is a figure which shows the change of (alpha)-amylase activity in the matured seed of a transformed plant (A3-1 and D1-4) and a wild type (WT). (A) shows the results of expression of α-amylase in mature seeds overexpressing wild-type (WT), α-amylase I-1 (A3-1) and α-amylase II-4 (D1-4), (B) is a graph showing the results of α-amylase activity, and (C) is a graph showing the results of dry weight of ripe seeds. (A) is a reflected light image of fully-ripened seeds of a transgenic rice line (A3-1 and D1-4 lines) overexpressing wild-type (WT), α-amylase I-1 and α-amylase II-4. (B) is a figure which shows a transmitted light image.

Claims (11)

A polypeptide comprising the amino acid sequence described in (a) or (b) below.
(A) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1.
(B) A polypeptide comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence set forth in SEQ ID NO: 1 and having a targeting effect on plastids. A gene encoding the polypeptide according to claim 1. A polypeptide comprising an amino acid sequence described in the following (c) or (d):
(C) A polypeptide having the amino acid sequence represented by SEQ ID NO: 2.
(D) A polypeptide comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence set forth in SEQ ID NO: 2, and having an endoplasmic reticulum membrane permeation effect. A gene encoding the polypeptide according to claim 3. A vector comprising the gene according to claim 2. A vector comprising the gene according to claim 2 or 4. A transformed plant, wherein the gene according to claim 2 is introduced. A transformed plant, wherein the gene according to claim 2 or 4 is introduced. The transformed plant according to claim 7 or 8, wherein the transformed plant is a monocotyledonous plant. A transformed plant obtained by introducing the vector of claim 5 or 6 into a callus derived from a monocotyledonous plant, growing the callus, and then redifferentiating the plant body from the callus. A transformed plant, wherein the vector according to claim 5 or 6 is introduced into a callus derived from a monocotyledonous plant using Agrobacterium, and after the callus is grown, the plant body is redifferentiated from the callus. Manufacturing method.