JP2013116047A - Gene capable of forming er body, and use thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To elucidate the mechanism of forming an ER body which is one construct originating from an endoplasmic reticulum, and to develop a technology for imparting a new trait to plant bodies.SOLUTION: There is provided a DNA construct which comprises DNA comprising a polynucleotide sequence represented by SEQ ID NO: 2, 4, 6 or 8, for generating an ER body in plant bodies and a DNA comprising a polynucleotide sequence represented by SEQ ID NO: 12, 14 or 16, or a composition containing the DNA construct; or a composition which comprises a first DNA construct comprising a first gene encoding a protein comprising an amino acid sequence represented by SEQ ID NO: 1, 3, 5 or 7, and a second DNA construct comprising a second gene encoding a protein comprising an amino acid sequence represented by SEQ ID NO: 11, 13 or 15.

Description

  The present invention relates to a gene having an ER body-forming ability and use thereof, and more particularly to a gene capable of forming an ER body, which is an organelle present only in Brassicaceae plants, in non-Brassic plants and use thereof. .

  Plant cells, unlike animal cells, produce various structures derived from the endoplasmic reticulum (ER). The ER body, which is one of such ER-derived structures, is an ER-derived structure found in Arabidopsis thaliana (hereinafter referred to as Arabidopsis thaliana), and is an organelle that accumulates glucosidase in plants belonging to the order of Brassica. Yes (see Non-Patent Document 1). Unlike other structures from ER, the ER body has a characteristic shape and size that is very long and large.

  In Arabidopsis thaliana, ER bodies are observed in epidermal cells of seedlings (see Non-Patent Document 2). In addition, a plant lacking the ER body exhibits overinfection with the fungus Piriformospora indica (see Non-Patent Document 3). These suggest that the ER body is involved in the defense system against fungal infection. Furthermore, the ER body is induced by wounds and jasmonic acid, which suggests that the ER body is involved in pest resistance (see Non-Patent Documents 4 and 5).

  Plants are known to use secondary metabolites as repellents, and some of them are also known to accumulate in vacuoles as glycoside precursors. As described above, the ER body is a unique organelle that is observed only in the plants of the Brassicaceae, and is considered to be involved in a defense system against fungal infection and resistance to pest damage. In Arabidopsis, the present inventors have previously identified NAI2, an ER body protein, that this protein is required for ER body formation and is responsible for the accumulation of β-glucosidase in the ER body. (See Non-Patent Document 2). From these findings, it is considered that when plant cells are destroyed by insects or the like, β-glucosidase contained in the ER body removes sugar from the glycoside and produces an active repellent substance.

Y. Hayashi et al., Plant Cell Physiol. 42: 894-899 (2001) K. Yamada et al., Plant Cell 20: 2529 (2008) I. Sherameti et al., Plant J. 54: 428-439 (2008) R. Matsushima et al., Plant Physiol. 130: 1807-1814 (2002) I. Hara-Nishimura and R. Matsushima, Curr. Opin. Plant Biol. 6: 583-588 (2003)

  Thus, the ER body is a very important organelle. However, as described above, the ER body that is considered to be involved in disease resistance and the like is a structure that is formed only in a very limited aspect, and specifically, only the seedlings of the cruciferous plants. It is a unique organelle that is observed only when it has been observed and grown and has been damaged by insects. NAI2 and its counterpart necessary for the formation of such an ER body do not exist in other plants. Furthermore, it is completely unknown how NAI2, a non-membrane protein, regulates ER body formation. If the formation mechanism of the ER body can be elucidated, disease resistance and the like can be imparted to the grown Brassicaceae plant.

  The present invention has been made in view of the above-described problems, and an object thereof is to provide a technique for elucidating the formation mechanism of an ER body and imparting a new character to a plant body.

  NAI2 is required for ER body formation and is responsible for the accumulation of PYK10 having β-glucosidase activity in the ER body. PYK10, like NAI2, is a protein specific to Brassica and is a soluble protein having an ER retention signal. NAI2 deficiency does not affect PYK10 mRNA levels, but reduces PYK10 protein levels, and PYK10 protein is uniformly distributed throughout the ER (see Non-Patent Document 2). According to these findings, the presence of NAI2 forms an ER body in the order of Brassica, accumulates PYK10 as an ER body protein in the ER body, and produces an active repellent substance with cell destruction. Conceivable.

  In order for the ER body to be formed as a structure in the cell, the presence of a membrane protein is strongly suggested. In particular, the localization of NAI2 that does not have an ER retention signal (KDEL) in the ER body strongly suggests the presence of a membrane protein as a binding partner. Therefore, the present inventors performed a transcriptome analysis using a mutant in which an ER body is not formed (nai1 mutant). As a result, NAI1 encoding a bHLH type transcriptional regulatory factor is found to control the expression of PYK10 and NAI2, and also controls the expression of MEB (membrane protein of ER body) 1 and 2 encoding a transmembrane protein. I found. Moreover, when NAI2 was depleted, the localization of MEB1 and MEB2 was changed and distributed within the ER network. These findings suggest that the ER body membrane has specific properties and that NAI2 regulates the formation of this ER body membrane by controlling the accumulation of MEB1 and MEB2. . Therefore, the present inventors tried to form an ER body using NAI2 and MEB. However, ER bodies could not be formed using these proteins.

  While repeating such trial and error, the present inventors have found that an ER body can be successfully formed by using PYK10 together with NAI2 based on a unique viewpoint, and have completed the present invention. Although PYK10 was known to be a soluble protein that accumulates in the formed ER body, PYK10, which was thought to have only such ER body accumulation ability, is essential for ER body formation. That is, a person skilled in the art cannot predict or expect.

  That is, the DNA construct according to the present invention includes a gene encoding a protein having an ER body-forming ability and a gene encoding a protein having an ER body-accumulating ability.

  In the present invention, the protein having the ability to form an ER body (also referred to as a first polypeptide) is (i) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1, 3, 5 or 7, or (ii) SEQ ID NO: It is preferably a protein consisting of an amino acid sequence in which one or several amino acids of the amino acid sequence shown in 1, 3, 5 or 7 are substituted, added or deleted, and having an ER body-forming ability, A protein having a body accumulation ability (also referred to as a second polypeptide) is (iii) a protein consisting of the amino acid sequence shown in SEQ ID NO: 11, 13 or 15, or (iv) shown in SEQ ID NO: 11, 13 or 15. An amino acid sequence in which one or several amino acids are substituted, added or deleted, and ER body accumulation ability Is preferably a protein having.

  The DNA construct according to the present invention includes a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 9 as a gene encoding a further protein (also referred to as a third polypeptide) having an ER body accumulation ability, or a sequence It may further comprise a gene consisting of an amino acid sequence in which one or several amino acids of the amino acid sequence shown in No. 9 are substituted, added or deleted, and encoding a protein having an ER body accumulation ability. In this case, a large amount of membrane protein can be accumulated in the ER body.

  In the present invention, a gene encoding a protein having an ER body-forming ability (also referred to as a first gene) is (A) DNA comprising a polynucleotide sequence shown in SEQ ID NO: 2, 4, or 6, (B) A DNA comprising a polynucleotide sequence in which one or several nucleotides of the polynucleotide sequence shown in SEQ ID NO: 2, 4 or 6 are deleted, substituted or added, and encodes a protein having an ER body-forming ability (C) a DNA that hybridizes under stringent conditions to a polynucleotide comprising a complementary sequence of the polynucleotide sequence shown in SEQ ID NO: 2, 4 or 6, and encodes a protein having an ER body-forming ability, Or (D) a polynucleotide sequence shown in SEQ ID NO: 2, 4 or 6 And a DNA encoding a protein having an ER body-forming ability, and a gene encoding a protein having an ER body-accumulating ability (the first (Also referred to as 2 genes)) (a) DNA consisting of the polynucleotide sequence shown in SEQ ID NO: 12, 14 or 16; (b) 1 or several of the polynucleotide sequence shown in SEQ ID NO: 12, 14 or 16 A DNA comprising a polynucleotide sequence from which nucleotides of the above are deleted, substituted or added, and encoding a protein having an ER body accumulation ability, (c) a polynucleotide sequence represented by SEQ ID NO: 12, 14 or 16 Under conditions that are stringent to a polynucleotide comprising the complementary sequence of DNA encoding a protein that is hybridized and has the ability to accumulate ER bodies, or (d) DNA having a homology of 80% or more with a polynucleotide consisting of the polynucleotide sequence shown in SEQ ID NO: 12, 14, or 16 And DNA encoding a protein having an ER body accumulation ability.

  The DNA construct according to the present invention is a DNA comprising a polynucleotide sequence represented by SEQ ID NO: 10 as a gene (also referred to as a third gene) encoding a further protein having ER body accumulation ability; DNA consisting of a polynucleotide sequence in which one or several nucleotides of the nucleotide sequence are deleted, substituted or added, and encoding a protein having an ER body accumulation ability; the polynucleotide shown in SEQ ID NO: 10 DNA that hybridizes under stringent conditions to a polynucleotide consisting of a complementary sequence of the sequence and encodes a protein having the ability to accumulate ER bodies; or 80% of the polynucleotide consisting of the polynucleotide sequence shown in SEQ ID NO: 10 The above homology To a DNA, and a DNA encoding a protein having an ER body integrated ability, it may further include a gene that contains a. In this case, a large amount of membrane protein can be accumulated in the ER body.

  The DNA construct according to the present invention preferably further includes a promoter. When the DNA construct includes a single promoter, the promoter is operably linked to the first gene and the second gene, and a plurality of promoters are included. The first promoter is operably linked to the first gene and the second promoter is operably linked to the second gene. Here, the first gene is a gene encoding a protein having an ER body forming ability, and the second gene is a gene encoding a protein having an ER body accumulating ability. The second promoter may be the same as or different from the first promoter.

  When the DNA construct according to the present invention includes a third gene, the third gene may be operably linked to a single promoter together with the first gene and the second gene. It may be connected as possible. The third promoter may be the same as or different from the first promoter and the second promoter.

  The DNA construct according to the present invention preferably further includes a gene (also referred to as a target gene) encoding a target protein for accumulation in the ER body. In this case, the second gene and the target gene are preferably linked in frame so as to generate a fusion protein of the second polypeptide and the target protein. When a gene encoding an additional protein having an ER body accumulation ability is further included in addition to the second gene, the additional gene and the target gene are generated so as to generate a fusion protein of the additional protein and the target protein. They may be connected in-frame.

  The composition according to the present invention is characterized by containing the above-described DNA construct in order to produce a ER body in a plant body. The kit according to the present invention is characterized by comprising the above-described DNA construct in order to generate a ER body in a plant body. In addition, when said DNA construct further contains the target gene, the composition concerning this invention can be used for the objective of storing the target protein in a plant body.

  Furthermore, the composition according to the present invention encodes a first DNA construct containing a gene encoding a protein having an ER body-forming ability and a protein having an ER body accumulation ability in order to generate an ER body in a plant. And a second DNA construct containing the gene to be treated. In addition, the kit according to the present invention encodes a first DNA construct containing a gene encoding a protein having an ER body-forming ability and a protein having an ER body accumulation ability in order to generate an ER body in a plant. And a second DNA construct containing the gene.

  In the composition or kit according to the present invention, the protein having an ER body-forming ability is (i) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1, 3, 5 or 7, or (ii) SEQ ID NO: 1, 3 It is preferably a protein having an ER body-forming ability, consisting of an amino acid sequence in which one or several amino acids of the amino acid sequence shown in 5 or 7 are substituted, added or deleted, and having an ER body-forming ability. (Iii) a protein comprising the amino acid sequence shown in SEQ ID NO: 11, 13 or 15, or (iv) one or several amino acids of the amino acid sequence shown in SEQ ID NO: 11, 13 or 15 It is preferably a protein consisting of a substituted, added or deleted amino acid sequence and having the ability to accumulate ER bodies. There.

  The composition according to the present invention may further contain a third DNA construct containing an additional gene encoding an additional protein (also referred to as a third polypeptide) having an ER body accumulation ability. The kit according to the present invention may further include a third DNA construct containing an additional gene encoding an additional protein having an ER body accumulation ability. The gene includes a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 9, or an amino acid in which one or several amino acids of the amino acid sequence shown in SEQ ID NO: 9 are substituted, added or deleted A gene encoding a protein consisting of a sequence and having the ability to accumulate ER bodies can be mentioned.

  In the composition or kit according to the present invention, the gene encoding the protein having ER body-forming ability is (A) DNA consisting of the polynucleotide sequence shown in SEQ ID NO: 2, 4 or 6, and (B) SEQ ID NO: DNA comprising a polynucleotide sequence in which one or several nucleotides of the polynucleotide sequence shown in 2, 4 or 6 are deleted, substituted or added, and encoding a protein having an ER body-forming ability (C) DNA encoding a protein that hybridizes under stringent conditions to a polynucleotide consisting of a complementary sequence of the polynucleotide sequence shown in SEQ ID NO: 2, 4 or 6 and has the ability to form an ER body, or (D) the polynucleotide sequence shown in SEQ ID NO: 2, 4 or 6 And a DNA encoding a protein having an ER body-forming ability, and the gene encoding the protein having an ER body-accumulating ability is preferably a DNA encoding a protein having an ER body-forming ability. (A) DNA comprising the polynucleotide sequence shown in SEQ ID NO: 12, 14 or 16; (b) one or several nucleotides of the polynucleotide sequence shown in SEQ ID NO: 12, 14 or 16 are deleted or substituted Or a DNA consisting of an added polynucleotide sequence and a DNA encoding a protein having the ability to accumulate ER bodies, (c) a polynucleotide consisting of a complementary sequence of the polynucleotide sequence shown in SEQ ID NO: 12, 14 or 16 Hybridize under stringent conditions, and DNA encoding a protein having R body accumulation ability, or (d) DNA having 80% or more homology with a polynucleotide comprising the polynucleotide sequence shown in SEQ ID NO: 12, 14, or 16, and ER body It preferably contains DNA encoding a protein having an accumulation ability.

  The composition concerning this invention may further contain the 3rd DNA construct containing the further gene (it is also mentioned 3rd gene) which codes the further protein which has ER body integration | stacking ability. The kit according to the present invention may further include a third DNA construct containing an additional gene encoding an additional protein having an ER body accumulation ability. The gene includes a DNA comprising the polynucleotide sequence shown in SEQ ID NO: 10; a polynucleotide sequence in which one or several nucleotides of the polynucleotide sequence shown in SEQ ID NO: 10 are deleted, substituted or added. DNA which is a DNA and encodes a protein having ER body accumulation ability; hybridizes under stringent conditions to a polynucleotide comprising a complementary sequence of the polynucleotide sequence shown in SEQ ID NO: 10, and has ER body accumulation ability Or a DNA encoding a protein having a ER body accumulation ability, or a DNA having a homology of 80% or more with a polynucleotide comprising the polynucleotide sequence shown in SEQ ID NO: 10 The genes that are That.

  In the composition or kit according to the present invention, each of the first DNA construct and the second DNA construct preferably further contains a promoter, and the first promoter is operably linked to the first gene in the first DNA construct, In the second DNA construct, the second promoter is operably linked to the second gene. Here, the first gene is a gene encoding a protein having an ER body forming ability, and the second gene is a gene encoding a protein having an ER body accumulating ability. The second promoter may be the same as or different from the first promoter. When a third DNA construct is further used, the third promoter is operably linked to the third gene in the third DNA construct. The third promoter may be the same as or different from the first promoter and the second promoter.

  In the composition or kit according to the present invention, the second DNA construct preferably further includes a target gene encoding a target protein to be accumulated in the ER body. In this case, the second gene and the target gene are linked in frame so as to generate a fusion protein of the second polypeptide and the target protein. The composition or kit having such a configuration can be used for the purpose of storing the target protein in a plant.

  In addition, when the 3rd DNA construct containing the further gene which codes the further protein which has ER body integration | stacking ability is further used, the target gene may be contained in the 3rd DNA construct. In this case, the additional gene and the target gene are linked in-frame so as to generate a fusion protein of the additional protein and the target protein. In this case, a large amount of membrane protein can be accumulated in the ER body. The composition or kit having such a configuration can be used for the purpose of storing the target protein in a plant.

  The plant body to which the present invention is applied may be a monocotyledonous plant or a dicotyledonous plant.

  ER is an organelle for the synthesis of secreted proteins, and the synthesized protein is rapidly delivered from the ER to the target site. It has become clear from many examples that such ER is used as a place for protein accumulation in plants. This is expected to use the ER body as a storage place.

  However, as described above, the ER body is a unique organelle that is observed only in plants of the Brassicaceae, and NAI2 and its counterpart necessary for ER body formation do not exist in other plants. These items suggest that it is not easy to form ER bodies in plants other than the Brassicaceae. Moreover, it is particularly remarkable that the formation of ER bodies in monocotyledonous plants (rice, onions, etc.) that have no technical correlation with Brassicaceae exceeds the range that can be predicted by those skilled in the art. Is.

  The plant according to the present invention is characterized by expressing a protein having an ER body-forming ability and a protein having an ER body accumulation ability. It should be noted that natural cruciferous plants (for example, Arabidopsis thaliana) expressing the endogenous first polypeptide and second polypeptide are not included in the plant of the present invention. Further, the above-described additional protein having the ability to accumulate ER bodies may be further expressed. The method for producing a plant according to the present invention is characterized by including the step of introducing the above-described DNA construct into the plant. The DNA construct to be introduced into the plant body may be provided as the above-described composition or as the above-described kit.

  In addition, the method for producing a plant according to the present invention may be a method for generating an ER body in a plant. The plant according to the present invention may express a protein having an ER body-forming ability and a fusion protein of a protein having an ER body accumulation ability and a target protein. Alternatively, the plant according to the present invention may express a protein having an ER body forming ability, a protein having an ER body accumulation ability, and a fusion protein of a target protein and a further protein having an ER body accumulation ability. In the present invention, the protein having the ER body forming ability, the protein having the ER body accumulating ability, and the additional protein having the ER body accumulating ability may have the above-described configuration. In addition, a natural Brassica plant (for example, Arabidopsis thaliana) expressing a fusion protein of an endogenous first polypeptide and a second polypeptide with any polypeptide is included in the plant of the present invention. Absent.

  The method for producing a plant according to the present invention includes a step of introducing a DNA construct containing the target gene described above into the plant. The DNA construct to be introduced into the plant body may be provided as the composition or the kit. The method for producing a plant according to the present invention may be a method for storing a target protein in the plant.

  By using the present invention, it is possible to elucidate the formation mechanism of an ER body, which is one of structures derived from the endoplasmic reticulum (ER), and provide a technique for imparting a new character to a plant body. Specifically, ER bodies that exist only in Brassicaceae plants can be formed in other plants, particularly monocotyledonous plants. Thereby, the storage site | part of the protein which could not be expected until now can be formed in various plant bodies.

It is a figure which shows that simultaneous expression of NAI2 and PYK10 is enough for formation of ER body. The fluorescent image shown shows a green fluorescent protein targeted to ER in onion epithelial cells. It is a figure which shows the high magnification image (left) of the endogenous ER body of Arabidopsis thaliana, and the high magnification image (right) of the ER body artificially induced | guided | derived to the onion. It is a figure which shows that MEB2 was integrated | stacked on ER body induced | guided | derived with the onion epithelial cell. NAI2 gene and PYK10 gene were introduced together into onion epithelial cells to induce ER body formation (lower panel). The bar is 5 μm. It is a figure which shows that ER body formation was induced | guided | derived in tobacco callus by introduce | transducing NAI2 gene and PYK10 gene together.

[ER body]
The ER body that is considered to be involved in pest resistance and the like is a structure that is formed only in a very limited aspect in the Brassicaceae plant. Specifically, it is observed only in the seedlings of the Brassicaceae plant. It is a unique organelle that is observed only when it has been damaged by insects.

  NAI2, which is an ER body protein, is important for ER body formation, and ER body is not formed by nai2 mutant (see Non-Patent Document 2). However, the present inventors have found that expression of NAI2 alone in onion epithelial cells is not sufficient for the formation of ER bodies. However, ER bodies were induced in onion epithelial cells by simultaneously introducing NAI2 and PYK10. This suggests that NAI2 and PYK10 proteins can interact to form a concentrated matrix of the ER body, thereby controlling the shape of the ER body.

  In onion epithelial cells, NAI2 and PYK10 induced an ER body of the same shape and size as the endogenous ER body of Arabidopsis thaliana. Furthermore, expression of NAI2 and PYK10 in onion epithelial cells not only induces the formation of ER bodies, but also the membrane protein of ER bodies, MEB2, localizes to ER bodies induced in the cells. . Thus, NAI2 and PYK10 are thought to control the shape of the ER body and accumulate the ER body membrane proteins to establish the original properties of the ER body membrane. In the present specification, the endogenous ER body is described using Arabidopsis thaliana, but the native structure of the ER body is a plant of the order of the Brassicaceae other than Arabidopsis (for example, Arabis alpina, Brassica oleracea, Raphanus). sativus, Capparis spinosa, Cleome spinosa, etc.).

  The NAI2 protein consists of 772 amino acids and has a signal peptide at its amino terminus, so it is considered that the NAI2 protein is a secreted protein. The region of the N-terminal half of the NAI2 protein has a Glu-Phe-Glu (EFE) motif that repeats about 40 amino acid sequences including the characteristic Glu-Phe-Glu sequence 10 times. The EFE motif region of NAI2 protein is hydrophilic. The region of the C-terminal half of the NAI2 protein is unique and we have called the NAI2 domain. The NAI2 protein having such a structure can be referred to as a protein having an ER body-forming ability because an ER body is not formed in the nai2 mutant.

  NAI2 (SEQ ID NOs: 1 and 2) having such a unique structure forms a family. Examples of proteins structurally related to NAI2 in Arabidopsis include TSK binding protein 1 (TSA1 / At1g52410 (SEQ ID NOs: 3 and 4)) and At3g15960 (SEQ ID NOs: 5 and 6). TSA1 has a signal peptide, 10 EFE repeats, and a NAI2 domain. At3g15960 is a smaller protein than NAI2 or TSA1, has a signal peptide and a NAI2 domain, but does not have an EFE repeat. A protein having a signal peptide, 9 EFE repeats, and a NAI2 domain (TC74287 (SEQ ID NOs: 7 and 8)) in B. napus is also exemplified as a protein structurally related to NAI2. In addition, NAI2 homologues were identified in Brassicaceae plants (for example, B. rapa (AC189268, AC189514), B. oleracea (BZ479594, BZ006167), etc.). However, NAI2 homologs were not found not only in animals and fungi but also in rice and poplar.

  Similar to NAI2, PYK10 (SEQ ID NO: 11 and 12), which is a protein specific to Brassica and has a ER retention signal (KDEL (SEQ ID NO: 17)), is distributed throughout the ER in the nai2 mutant. Is accumulated in the ER body in the wild type, it can be referred to as a protein having ER body accumulation ability. PYK10 also forms a family of structurally related proteins. Examples of PYK10 homologs include BGLU18 (SEQ ID NOs: 13 and 14) and BGLU21 (SEQ ID NOs: 15 and 16), which also have the function of β-glucosidase. BGLU18 is also a soluble protein with a similar sequence (REEL (SEQ ID NO: 18)) and, like PYK10, accumulates in the ER body induced by wounds (data not shown).

[DNA construct]
The present invention provides a DNA construct for forming an ER body, which is an organelle present only in Brassicaceae plants, in non-Brassic plants. The DNA construct according to the present invention includes a gene encoding a protein having an ER body-forming ability and a gene encoding a protein having an ER body-accumulating ability.

  As used herein, the “NAI2 family molecule” described above is intended as the “protein having the ability to form an ER body”. The term “ER body-forming ability” intends the ability (activity) capable of forming an ER body when expressed in a Brassicaceae plant in which formation of the ER body is inhibited.

  As used herein, the “protein having the ability to accumulate ER bodies” is intended to include the aforementioned PYK10 family molecules. The term “ER body accumulation ability” intends the ability (activity) that can be accumulated in the formed ER body when an ER body is formed in a Brassicaceae plant in which formation of the ER body is inhibited.

  In one embodiment, the protein having an ER body-forming ability (also referred to as a first polypeptide) is (i) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1, 3, 5 or 7, or (ii) a sequence It may be a protein having an ER body-forming ability, consisting of an amino acid sequence in which one or several amino acids of the amino acid sequence shown in Nos. 1, 3, 5 or 7 are substituted, added or deleted. In addition, a protein having an ER body accumulation ability (also referred to as a second polypeptide) is (iii) a protein consisting of the amino acid sequence shown in SEQ ID NO: 11, 13 or 15, or (iv) SEQ ID NO: 11, 13 or 15 may be a protein having an ER body accumulation ability, consisting of an amino acid sequence in which one or several amino acids are substituted, added, or deleted.

  When used in terms of a polypeptide (amino acid), “one or several” is preferably in the range of 1-30, more preferably in the range of 1-20, and more preferably 1-10. More preferably, it is in the range of 1 to 5, and still more preferably in the range of 1 to 5. A person skilled in the art can easily understand the extent of the number of amino acids indicated by the term “one or several” depending on the length of the target polypeptide.

  As used herein, the term “polypeptide” is used interchangeably with “peptide” or “protein” and is intended to be a polymer of amino acids. A polypeptide “fragment” is intended to be a partial fragment of the polypeptide. The polypeptides according to the invention may also be produced recombinantly, chemically synthesized or isolated from natural sources.

  In another embodiment, a gene encoding a protein having an ER body-forming ability (also referred to as a first gene) is (A) DNA comprising a polynucleotide sequence shown in SEQ ID NO: 2, 4 or 6, (B) A DNA comprising a polynucleotide sequence in which one or several nucleotides of the polynucleotide sequence shown in SEQ ID NO: 2, 4 or 6 are deleted, substituted or added, and encodes a protein having an ER body-forming ability (C) a DNA that hybridizes under stringent conditions to a polynucleotide comprising a complementary sequence of the polynucleotide sequence shown in SEQ ID NO: 2, 4 or 6, and encodes a protein having an ER body-forming ability, Or (D) a polynucleotide sequence shown in SEQ ID NO: 2, 4 or 6 Comprising polynucleotide is DNA having a homology of 80% or more, and contains a DNA, which encodes a protein having an ER body forming ability. In addition, a gene encoding a protein having an ER body accumulation ability (also referred to as a second gene) is (a) DNA consisting of a polynucleotide sequence shown in SEQ ID NO: 12, 14 or 16, (b) SEQ ID NO: 12, A DNA consisting of a polynucleotide sequence in which one or several nucleotides of the polynucleotide sequence shown in 14 or 16 are deleted, substituted or added, and encoding a protein having an ER body accumulation ability; c) DNA that hybridizes under stringent conditions to a polynucleotide comprising a complementary sequence of the polynucleotide sequence shown in SEQ ID NO: 12, 14, or 16 and encodes a protein having the ability to accumulate ER bodies, or (d ) Polynucleotide shown in SEQ ID NO: 12, 14 or 16 A DNA having a polynucleotide homology of 80% or more consisting of sequence and DNA encoding a protein having an ER body integrated ability contains.

  When used in terms of polynucleotide (base), “one or several” is preferably in the range of 1 to 100, more preferably in the range of 1 to 50, and 1 to 30. More preferably, it is in the range of 1 to 15, and still more preferably in the range of 1 to 15. A person skilled in the art can easily understand the extent of the range of the number of bases indicated by the term “one or several” depending on the length of the target polynucleotide. For example, in the case of oligonucleotides, the term “one or several” is preferably in the range of 1-10, more preferably in the range of 1-7, and in the range of 1-5. More preferably, it is still more preferably in the range of 1 to 3.

  As used herein, the term “polynucleotide” is used interchangeably with “gene”, “nucleic acid” or “nucleic acid molecule” and is intended to be a polymer of nucleotides. A “fragment” of a polynucleotide is intended to be a partial fragment of the polynucleotide. As used herein, the term “nucleotide sequence” is used interchangeably with “nucleic acid sequence” or “base sequence”.

  The polynucleotide according to the present invention may exist in the form of RNA (eg, mRNA) or in the form of DNA (eg, cDNA or genomic DNA). DNA can be double-stranded or single-stranded. Single-stranded DNA or RNA can be the coding strand (also known as the sense strand) or it can be the non-coding strand (also known as the antisense strand).

  As used herein, the term “oligonucleotide” is intended to be a combination of several to several tens of nucleotides, and is used interchangeably with “polynucleotide”.

  As used herein, the term “stringent (hybridization) conditions” refers to a hybridization solution (50% formamide, 5 × SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM phosphorous. After overnight incubation at 42 ° C. in sodium acid (pH 7.6), 5 × Denhardt's solution, 10% dextran sulfate, and 20 μg / ml denatured sheared salmon sperm DNA, Although it is intended to wash the filter in 1 × SSC, the washing conditions at high stringency are appropriately changed depending on the polynucleotide to be hybridized. For example, when using mammalian-derived DNA, 0.1% SDS Washing at 65 ° C. in 0.5 × SSC containing (preferably 15 minutes × 2 times), and more preferably, E. When using E. coli-derived DNA, washing at 68 ° C. in 0.1 × SSC containing 0.1% SDS (preferably 15 minutes × 2 times) is preferable, and when using RNA, 0.1% Washing at 68 ° C. in 0.1 × SSC containing SDS (preferably twice for 15 minutes) is preferable. When oligonucleotides are used, in 0.1 × SSC containing 0.1% SDS Washing at the hybridization temperature (preferably 15 minutes × 2 times) is preferred.

  As used herein, the homology for the polypeptide or polynucleotide of interest is preferably 80% or more, more preferably 85% or more, and more preferably 90% or more. Preferably, it is more preferably 95% or more.

  The DNA construct according to the present invention preferably further includes a promoter. When the DNA construct includes a single promoter, the promoter is operably linked to the first gene and the second gene, and a plurality of promoters are included. The first promoter is operably linked to the first gene and the second promoter is operably linked to the second gene. Here, the first gene is a gene encoding a protein having an ER body forming ability, and the second gene is a gene encoding a protein having an ER body accumulating ability. The second promoter may be the same as or different from the first promoter.

  The promoter used in the present invention is not particularly limited, and those skilled in the art can appropriately select a promoter that is well-known as a preferred promoter for protein expression in plants.

  The DNA construct according to the present invention may further include a gene (also referred to as a target gene) encoding a target protein to be accumulated in the ER body. In this case, the gene encoding the protein having the ER body accumulation ability and the gene encoding the target protein are linked in frame so as to generate a fusion protein of the protein having the ER body accumulation ability and the target protein. .

  If a DNA construct having such a structure is used, a protein storage site that could not be expected so far can be formed in various plants. That is, the DNA construct of the present invention can be used for the purpose of storing a target protein in a plant body.

  When used for the purpose of storing a target protein in a plant body, the DNA construct according to the present invention may contain two or more genes encoding a protein having an ER body accumulation ability, at least one of which contains a PYK10 family molecule. Any gene may be used as long as it encodes, and the other gene may encode a MEB2 family molecule, for example. From the viewpoint that MEB2, which is a membrane protein of the ER body, is localized in the ER body induced in cells, MEB2 can also be said to be a protein having an ER body accumulation ability. When a target protein is accumulated in an ER body using a DNA construct having such a configuration, at least one gene encoding a protein having an ER body accumulation ability and a gene encoding the target protein are linked in frame. Thus, a fusion protein of at least one protein having an ER body accumulation ability and a target protein may be generated.

  As used herein, MEB2 protein is a protein consisting of the amino acid sequence shown in SEQ ID NO: 9, or one or several amino acids in the amino acid sequence shown in SEQ ID NO: 9 are substituted. It is a protein consisting of an added or deleted amino acid sequence and having ER body accumulation ability. The MEB2 gene is a DNA comprising the polynucleotide sequence shown in SEQ ID NO: 10; from a polynucleotide sequence in which one or several nucleotides of the polynucleotide sequence shown in SEQ ID NO: 10 are deleted, substituted or added. A DNA encoding a protein having the ability to accumulate ER bodies; hybridizing under stringent conditions to a polynucleotide comprising a complementary sequence of the polynucleotide sequence shown in SEQ ID NO: 10, and capable of accumulating ER bodies Or a DNA encoding a protein having a homology of 80% or more with a polynucleotide comprising the polynucleotide sequence shown in SEQ ID NO: 10 and having an ER body accumulation ability, Containing genes That.

[Composition and kit]
As used herein, a “composition” is intended to be a form in which various components are contained in one substance. In addition, as used herein, a “kit” is intended to be a form in which at least one of various components is contained in another substance.

  In general, a composition is intended to be “one or more components that are homogeneously present as a whole and grasped as a single substance”. For example, a single composition containing substance A as a main component, as a main component The single composition containing the substance A and the substance B can be a single composition. These compositions may contain other components other than the substance A and the substance B within a range not inhibiting the function of the main component. The composition according to the present invention may be used alone or in combination with other substances or compositions. In this case, other substances or compositions to be used together may or may not be provided in the composition according to the present invention. In the latter case, these cannot be recognized as a composition as a whole, but in this case, those skilled in the art can easily fall into the category of “kits” described later and be provided as a kit rather than as a composition. To understand.

  As used herein, the term “kit” intends a package with a container (eg, bottle, plate, tube, dish, etc.) containing a particular material. Preferably, instructions for using each material are provided. As used herein, in the aspect of a kit, “comprising” is intended to mean being contained in any of the individual containers that make up the kit. In addition, the kit according to the present invention may be a package in which a plurality of different compositions are packed together, where the form of the composition may be a form as described above, or in the case of a solution form in a container. It may be included. The kit according to the present invention may comprise substance A and substance B mixed in the same container or in separate containers. The “instructions” may be written or printed on paper or other media, or may be affixed to electronic media such as magnetic tape, computer readable disk or tape, CD-ROM, etc. .

  The present invention provides a composition for forming ER bodies in non-brassic plants. In one embodiment, the composition according to the invention contains a single DNA construct as described above. The present invention also provides a kit for forming ER bodies in non-brassic plants. In one embodiment, the composition according to the invention comprises a single DNA construct as described above.

  In another embodiment of the invention, the configuration of the single DNA construct described above may be included in a separate DNA construct. That is, in one embodiment, the composition according to the present invention includes a first DNA construct containing a gene encoding a protein having an ER body-forming ability and a gene encoding a protein having an ER body-accumulating ability. And a second DNA construct. The composition concerning this embodiment may further contain the 3rd DNA construct containing the additional gene which codes the additional protein which has ER body integration | stacking ability. In one embodiment, the kit according to the present invention includes a first DNA construct containing a gene encoding a protein having ER body-forming ability and a gene encoding a protein having ER body-accumulating ability. 2 DNA construct. The kit according to the present embodiment may further include a third DNA construct containing an additional gene encoding an additional protein having an ER body accumulation ability.

  In the composition and kit according to the present invention, the second DNA construct or the third DNA construct may further comprise a target gene encoding a target protein to be accumulated in the ER body, as described above. The composition or kit having such a configuration can be used for the purpose of storing the target protein in a plant.

[Plant body and production method thereof]
By using the DNA construct, composition and kit as described above, a plant having an ER body can be produced. That is, this invention provides the plant body which has ER body, and its production method.

  The plant according to the present invention only needs to express the protein encoded by the gene (polynucleotide) contained in each DNA construct as a result of introducing the above-described DNA construct into the plant. That is, the method for producing a plant according to the present invention may include a step of introducing the above-described DNA construct into the plant. For the gene introduction procedure in the present invention, a technique well known in the art may be used, and a so-called “transformant plant” production method may be employed. In addition, the said DNA construct may be provided as a composition or kit mentioned above.

  As used herein, the term “transformant” intends not only a cell, tissue or organ, but also an individual organism. In addition, it can be said that the transformant according to the present invention is only required to express at least the gene contained in the DNA construct according to the present invention and express the protein encoded by this gene. That is, it should be noted that transformants generated by means other than expression vectors are also included in the technical scope of the present invention. Moreover, it is preferable that the desired protein is stably expressed in the plant body (transformant) according to the present invention.

  As used herein, “gene introduced” or “gene introduced” is expressed in a target cell (host cell) by a known genetic engineering technique (gene manipulation technique). It is intended to be introduced (ie, transformant). When the present invention is applied to the industrial field using plants, various crops (plants and crops produced in the agriculture, forestry and fisheries industry) can be cited as application targets. Specific examples include cereals (rice, wheat, corn, etc.), timbers (pine, cedar, cypress, etc.), various vegetables, and flower buds.

  Plants to be transformed in the present invention include whole plants, plant organs (eg leaves, petals, stems, roots, seeds, etc.), plant tissues (eg epidermis, phloem, soft tissue, xylem, vascular bundle, It means any of a palisade tissue, a spongy tissue, etc.) or a plant culture cell, or various forms of plant cells (eg, suspension culture cells), protoplasts, leaf sections, callus, and the like. The plant used for transformation is not particularly limited, and may be any plant belonging to the monocotyledonous plant class or the dicotyledonous plant class. In one embodiment, the plant according to the present invention may be a grown plant individual, a plant cell, a plant tissue, a callus, or a seed.

  For the introduction of genes into plants, transformation methods known to those skilled in the art (for example, the Agrobacterium method, gene gun (particle bombardment), PEG method, electroporation method, etc.) are used. And the method of directly introducing into plant cells. When the Agrobacterium method is used, the constructed plant expression vector is introduced into an appropriate Agrobacterium, and this strain is infected with a sterile cultured leaf piece according to a method well known in the art (for example, the leaf disc method). Good. Electroporation and gene gun methods are known as methods for directly introducing genes into plant cells or plant tissues. When using a gene gun, a plant body, a plant organ, and a plant tissue itself may be used as they are, or may be used after preparing a section, or a protoplast may be prepared and used. It should be noted that regeneration of a plant body from a cell or plant tissue into which a gene has been introduced can be performed by methods known to those skilled in the art depending on the type of plant cell.

  When plant cultured cells are used as a host, the recombinant vector can be obtained by, for example, microinjection, electroporation (electroporation), polyethylene glycol, gene gun (particle gun), protoplast fusion, calcium phosphate, etc. Introduced into cultured cells. Callus and the like obtained as a result of transformation can be used as they are for cell culture, tissue culture or organ culture as they are, and a plant hormone culture method (auxin, Cytokinin, gibberellin, abscisic acid, ethylene, brassinolide, etc.) can be regenerated into plants.

  Whether or not the gene has been introduced into the plant can be confirmed by detecting the target gene using a PCR method, Southern hybridization method, Northern hybridization method or the like. Detection of the target gene can be a step of hybridizing the nucleic acid probe to the target gene. As used herein, the “nucleic acid probe” is not particularly limited as long as it can hybridize to a target nucleic acid, and may be a so-called hybridization probe or a PCR primer. Hybridization techniques and PCR techniques are well known in the art and can be easily performed by those skilled in the art.

  The nucleic acid probe is not particularly limited as long as it is an oligonucleotide containing a partial sequence of the target gene (or its complementary strand). In the case of in situ hybridization, the nucleic acid probe is preferably an oligonucleotide composed of 15 to 50 consecutive bases of the nucleotide sequence described in the sequence listing or its complementary sequence, more preferably 20 to 50, and still more preferably 20 An oligonucleotide consisting of ˜45, most preferably 25 to 40 consecutive bases. In the case of PCR (including in situ PCR), the nucleic acid probe is preferably an oligonucleotide consisting of 15 to 50 consecutive bases of the nucleotide sequence described in the sequence listing or its complementary sequence, more preferably 15 to An oligonucleotide consisting of 40, more preferably 15 to 35, most preferably 25 to 35 consecutive bases. Of course, the oligonucleotide used in the Example mentioned later may be sufficient.

  Once a transformed plant in which the polynucleotide of the present invention is integrated into the genome is obtained, offspring can be obtained by sexual reproduction or asexual reproduction of the plant. Further, for example, seeds, fruits, cuttings, tubers, tuberous roots, strains, callus, protoplasts, and the like can be obtained from the plant or its progeny or clones thereof, and the plant can be mass-produced based on them it can. Accordingly, the present invention also includes a plant into which a desired protein is introduced so that it can be expressed, or a progeny of the plant having the same properties as the plant, or a tissue derived therefrom.

  Those skilled in the art who read this specification will easily understand that such a method for producing a plant can be a method for generating an ER body in a plant. That is, the present invention also provides a method for generating an ER body in a plant body.

  When the DNA construct described above contains a gene encoding a protein intended for storage, a plant body in which the target protein is stored in the ER body can be produced by using the present invention. That is, this invention provides the plant body which stored the target protein in ER body, and its production method. Similarly, the present invention provides a method for storing a target protein in a plant.

  In addition, when purifying the stored target protein, after preparing a cell extract from cells or tissues by a well-known method (for example, a method in which cells or tissues are disrupted and then centrifuged to recover a soluble fraction) From this cell extract, well-known methods (for example, affinity purification using an antibody against the protein of interest, ammonium sulfate precipitation or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction Chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography) are preferred, but not limited thereto. Most preferably, high performance liquid chromatography (“HPLC”) is used for purification.

  The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope shown in the claims, and embodiments obtained by appropriately combining technical means disclosed in different embodiments. Is also included in the technical scope of the present invention.

  Moreover, all the academic literatures and patent literatures described in this specification are incorporated herein by reference.

  In accordance with the procedure described in Non-Patent Document 2, using the primers and template EST clones (U21078 for MEB1, U23495 for MEB2, U16255 for NAI2) or mRNA as described in Table 1, or cDNA containing the gene of interest The fragments were PCR amplified and inserted into the expression vector pUGW2, respectively. The EST clone used is a Salk / Stanford / PGEC Consortium full-length cDNA / ORF clone obtained from the Arabidopsis Biological Resource Center.

  The obtained expression vector was introduced into onion epithelial cells or tobacco callus by particle bombardment according to the procedure described in Non-Patent Document 2. In order to visualize the endoplasmic reticulum and the ER body with green fluorescent protein, an expression vector, pBI221 • SP-GFP-HDEL was simultaneously introduced. 6 to 12 hours after gene introduction, observation was performed with a confocal laser microscope according to the procedure described in Non-Patent Document 2.

  Endogenous ER bodies are only observed in Brassicaceae plants. It is of great interest to form organelles unique to Brassicaceae plants in non-Brassic plants. NAI2 is a protein having an ER body-forming ability that is essential for the formation of endogenous ER bodies. It was confirmed whether such NAI2 could induce ER body formation in non-brassic plants. However, no ER body was observed when NAI2 was introduced into onion epithelial cells (upper right of FIG. 1). Similarly, when PYK10 having ER body accumulation ability was introduced into onion epithelial cells, no ER body was observed (upper left in FIG. 1).

  It was confirmed whether it is possible to induce ER body formation in onion epithelial cells using the membrane proteins MEB1 and MEB2 whose expression is controlled by the transcription factor NAI1, which controls the expression of NAI2 and PYK10. However, even when MEB1 and MEB2 were introduced, the ER body was not induced (the middle left in FIG. 1). The possibility that ER body formation is induced based on the interaction between MEB1 and MEB2 which are membrane proteins of ER body and PYK10 which is soluble ER body protein was examined. Was not induced (middle right of FIG. 1). Furthermore, the possibility of ER body formation being induced based on the interaction between membrane proteins MEB1 and MEB2 and NAI2 having the ability to form ER bodies was examined. Was not done (lower left of FIG. 1).

  Various conditions were also examined for the gene introduction procedure and the observation period, but favorable results could not be obtained. This suggests that the ER body formation mechanism in Brassicaceae plants is very complicated, and it is very difficult to form organelles unique to Brassicaceae plants in non-Brassic plants.

  Therefore, these genes were combined and introduced into onion epithelial cells for the purpose of examining in more detail how the localization of MEB1, MEB2, and PYK10 changes with or without NAI2. Then, when NAI2 and PYK10 were expressed at the same time, an elongated structure that was not normally observed in onion epithelial cells was observed (lower right in FIG. 1). Although its shape is similar to that of the ER body, it was completely unexpected that a unique organelle called the ER body could be constructed only by expressing two proteins, NAI2 and PYK10, which are non-membrane proteins. The elongated structures formed in onion epithelial cells were compared morphologically with the endogenous ER body in Arabidopsis thaliana. As shown in FIG. 2, this artificially derived structure was the same size and shape as the endogenous ER body in Arabidopsis (FIG. 2). An ER body was formed only by expressing NAI2 and PYK10 proteins not only in onion epithelial cells but also in tobacco cultured cells (FIG. 4).

  In order to further investigate whether this artificially derived structure is an ER body, it was examined whether the localization of MEB2, which is a membrane protein of the ER body, changes depending on the presence or absence of NAI2 and PYK10. In order to produce a fusion gene of GFP and MEB2, a cDNA fragment containing the MEB2 gene was PCR amplified and inserted into an expression vector pUGW6 containing the GFP gene. The obtained vector was introduced into an onion epithelial cell together with a vector containing the NAI2 gene or PYK10 gene. In order to visualize the endoplasmic reticulum and ER body with a red fluorescent protein, the expression vector pBI221 • SP-tdTOM-KDEL was simultaneously introduced. As a result, in the absence of NAI2 and PYK10 (ie, the structure was not induced), MEB2 was dispersed throughout the ER and its signal was very low (top in FIG. 3). However, in the presence of NAI2 and PYK10 (that is, the structure is induced), MEB2 was attracted to the structure and localized in the film form of the structure (bottom of FIG. 3).

  From these results, it was found that it is necessary and sufficient to express both NAI2 and PYK10 in order to form ER bodies, which are organelles unique to Brassicaceae plants, in non-Brassic plants.

  In Arabidopsis, NAI2 is an ER body component for ER body formation. The inventors have found that mutations in PYK10, the major ER body component, change the morphology of the ER body (not shown). This suggests that PYK10 also regulates ER body formation, but is consistent with the above results. In Arabidopsis, PYK10 protein is easily detected by CBB staining, but NAI2 protein is not detected. This indicates that PYK10 is more abundant than NAI2. This suggests that NAI2 functions as a chaperone of PYK10, facilitating PYK10 accumulation and enrichment.

  By using the present invention, it is possible to stock up the target protein in a plant used as a protein production factory with resource saving and energy saving.

Claims (16)

A DNA construct comprising a first gene encoding a first polypeptide and a second gene encoding a second polypeptide,
The first polypeptide is
(I) a protein comprising the amino acid sequence shown in SEQ ID NO: 1, 3, 5 or 7, or (ii) one or several amino acids of the amino acid sequence shown in SEQ ID NO: 1, 3, 5 or 7 are substituted, A protein consisting of an added or deleted amino acid sequence and having the ability to form an ER body,
The second polypeptide is
(Iii) a protein comprising the amino acid sequence shown in SEQ ID NO: 11, 13 or 15, or (iv) one or several amino acids of the amino acid sequence shown in SEQ ID NO: 11, 13 or 15 are substituted, added or deleted A DNA construct or a first gene, which is a protein comprising an amino acid sequence and having an ER body accumulation ability,
(A) DNA comprising the polynucleotide sequence shown in SEQ ID NO: 2, 4, 6 or 8;
(B) DNA comprising a polynucleotide sequence in which one or several nucleotides of the polynucleotide sequence shown in SEQ ID NO: 2, 4, 6 or 8 have been deleted, substituted or added, and ER body-forming ability DNA encoding a protein having
(C) DNA encoding a protein that hybridizes under stringent conditions to a polynucleotide consisting of a complementary sequence of the polynucleotide sequence shown in SEQ ID NO: 2, 4, 6 or 8, and has an ER body-forming ability, or (D) DNA having a homology of 80% or more with a polynucleotide comprising the polynucleotide sequence shown in SEQ ID NO: 2, 4, 6 or 8 and encoding a protein having an ER body-forming ability
Contains
The second gene is
(A) DNA comprising the polynucleotide sequence shown in SEQ ID NO: 12, 14 or 16,
(B) DNA consisting of a polynucleotide sequence in which one or several nucleotides of the polynucleotide sequence shown in SEQ ID NO: 12, 14 or 16 are deleted, substituted or added, and has the ability to accumulate ER bodies DNA encoding a protein,
(C) DNA that hybridizes under stringent conditions to a polynucleotide comprising a complementary sequence of the polynucleotide sequence shown in SEQ ID NO: 12, 14, or 16 and encodes a protein having an ER body accumulation ability, or (d ) DNA having a homology of 80% or more with a polynucleotide comprising the polynucleotide sequence shown in SEQ ID NO: 12, 14, or 16 and encoding a protein having an ER body accumulation ability
A DNA construct comprising: Further comprising a third gene encoding a third polypeptide;
A third polypeptide is
(I) a protein consisting of the amino acid sequence shown in SEQ ID NO: 9, or (ii) an amino acid sequence in which one or several amino acids of the amino acid sequence shown in SEQ ID NO: 9 are substituted, added or deleted, And a protein having an ER body accumulation ability,
The third gene is
(A) DNA consisting of the polynucleotide sequence shown in SEQ ID NO: 10,
(B) a DNA comprising a polynucleotide sequence in which one or several nucleotides of the polynucleotide sequence shown in SEQ ID NO: 10 are deleted, substituted or added, and encodes a protein having an ER body accumulation ability DNA,
(C) DNA that hybridizes under stringent conditions to a polynucleotide comprising a complementary sequence of the polynucleotide sequence shown in SEQ ID NO: 10 and encodes a protein having ER body accumulation ability, or (d) SEQ ID NO: 10 A DNA having a homology of 80% or more with a polynucleotide comprising the polynucleotide sequence shown in FIG. 5 and encoding a protein having an ER body accumulation ability
The DNA construct according to claim 1, comprising:   The target gene encoding the target protein is further included, and the second gene or the third gene and the target gene are in frame so as to generate a fusion protein of the second polypeptide or the third polypeptide and the target protein. The DNA construct according to claim 1 or 2, which is ligated together.   A composition for generating an ER body in a plant, comprising the DNA construct according to any one of claims 1 to 3.   The composition for storing the target protein in a plant body containing the DNA construct of Claim 3. Generating an ER body in a plant containing a first DNA construct comprising a first gene encoding a first polypeptide and a second DNA construct comprising a second gene encoding a second polypeptide A composition for causing
The first polypeptide is
(I) a protein comprising the amino acid sequence shown in SEQ ID NO: 1, 3, 5 or 7, or (ii) one or several amino acids of the amino acid sequence shown in SEQ ID NO: 1, 3, 5 or 7 are substituted, A protein consisting of an added or deleted amino acid sequence and having the ability to form an ER body,
The second polypeptide is
(Iii) a protein comprising the amino acid sequence shown in SEQ ID NO: 11, 13 or 15, or (iv) one or several amino acids of the amino acid sequence shown in SEQ ID NO: 11, 13 or 15 are substituted, added or deleted A composition comprising the amino acid sequence and a protein having an ER body accumulation ability, or a first gene,
(A) DNA comprising the polynucleotide sequence shown in SEQ ID NO: 2, 4, 6 or 8;
(B) DNA comprising a polynucleotide sequence in which one or several nucleotides of the polynucleotide sequence shown in SEQ ID NO: 2, 4, 6 or 8 have been deleted, substituted or added, and ER body-forming ability DNA encoding a protein having
(C) DNA encoding a protein that hybridizes under stringent conditions to a polynucleotide consisting of a complementary sequence of the polynucleotide sequence shown in SEQ ID NO: 2, 4, 6 or 8, and has an ER body-forming ability, or (D) DNA having a homology of 80% or more with a polynucleotide comprising the polynucleotide sequence shown in SEQ ID NO: 2, 4, 6 or 8 and encoding a protein having an ER body-forming ability
Contains
The second gene is
(A) DNA comprising the polynucleotide sequence shown in SEQ ID NO: 12, 14 or 16,
(B) DNA consisting of a polynucleotide sequence in which one or several nucleotides of the polynucleotide sequence shown in SEQ ID NO: 12, 14 or 16 are deleted, substituted or added, and has the ability to accumulate ER bodies DNA encoding a protein,
(C) DNA that hybridizes under stringent conditions to a polynucleotide comprising a complementary sequence of the polynucleotide sequence shown in SEQ ID NO: 12, 14, or 16 and encodes a protein having an ER body accumulation ability, or (d ) DNA having a homology of 80% or more with a polynucleotide comprising the polynucleotide sequence shown in SEQ ID NO: 12, 14, or 16 and encoding a protein having an ER body accumulation ability
A composition comprising: 6. The composition of claim 5, further comprising a third DNA construct comprising a third gene encoding a third polypeptide,
A third polypeptide is
(I) a protein consisting of the amino acid sequence shown in SEQ ID NO: 9, or (ii) an amino acid sequence in which one or several amino acids of the amino acid sequence shown in SEQ ID NO: 9 are substituted, added or deleted, And a protein having an ER body accumulation ability,
The third gene is
(A) DNA consisting of the polynucleotide sequence shown in SEQ ID NO: 10,
(B) a DNA comprising a polynucleotide sequence in which one or several nucleotides of the polynucleotide sequence shown in SEQ ID NO: 10 are deleted, substituted or added, and encodes a protein having an ER body accumulation ability DNA,
(C) DNA that hybridizes under stringent conditions to a polynucleotide comprising a complementary sequence of the polynucleotide sequence shown in SEQ ID NO: 10 and encodes a protein having ER body accumulation ability, or (d) SEQ ID NO: 10 A DNA having a homology of 80% or more with a polynucleotide comprising the polynucleotide sequence shown in FIG. 5 and encoding a protein having an ER body accumulation ability
A composition comprising:   The second DNA construct further includes a target gene encoding the target protein, and the second gene or the third gene and the target gene so as to generate a fusion protein of the second polypeptide or the third polypeptide and the target protein. The composition according to claim 6 or 7, wherein and are linked in-frame.   The composition according to claim 8, which is used for storing a target protein in a plant body.   The composition according to any one of claims 4 to 9, wherein the plant is a monocotyledonous plant.   A plant body having an ER body comprising the step of introducing the DNA construct according to any one of claims 1 to 3 or the composition according to any one of claims 4 to 10 into the plant body. Manufacturing method. Expressing a first polypeptide and a second polypeptide;
The first polypeptide is
(I) a protein comprising the amino acid sequence shown in SEQ ID NO: 1, 3, 5 or 7, or (ii) one or several amino acids of the amino acid sequence shown in SEQ ID NO: 1, 3, 5 or 7 are substituted, A protein consisting of an added or deleted amino acid sequence and having the ability to form an ER body,
The second polypeptide is
(Iii) a protein comprising the amino acid sequence shown in SEQ ID NO: 11, 13 or 15, or (iv) one or several amino acids of the amino acid sequence shown in SEQ ID NO: 11, 13 or 15 are substituted, added or deleted A plant comprising a protein having an amino acid sequence and having an ER body accumulation ability.   An ER body is produced in a plant body, comprising the step of introducing the DNA construct according to any one of claims 1 to 3 or the composition according to any one of claims 4 to 10 into the plant body. How to make.   A method for producing a plant body in which a protein of interest is stored in an ER body, comprising the step of introducing the DNA construct according to claim 3 or the composition according to claim 8 or 9 into the plant body. The first polypeptide, the second polypeptide and the third polypeptide are expressed, and the second polypeptide or the third polypeptide is expressed as a fusion protein with the target protein,
The first polypeptide is
(I) a protein comprising the amino acid sequence shown in SEQ ID NO: 1, 3, 5 or 7, or (ii) one or several amino acids of the amino acid sequence shown in SEQ ID NO: 1, 3, 5 or 7 are substituted, A protein consisting of an added or deleted amino acid sequence and having the ability to form an ER body,
The second polypeptide is
(Iii) a protein comprising the amino acid sequence shown in SEQ ID NO: 11, 13 or 15, or (iv) one or several amino acids of the amino acid sequence shown in SEQ ID NO: 11, 13 or 15 are substituted, added or deleted A protein having an ER body accumulation ability,
A third polypeptide is
(V) a protein consisting of the amino acid sequence shown in SEQ ID NO: 9, or (vi) an amino acid sequence in which one or several amino acids of the amino acid sequence shown in SEQ ID NO: 9 are substituted, added or deleted, A plant that is a protein having an ER body accumulation ability.   A method for storing a target protein in a plant comprising the step of introducing the DNA construct according to claim 3 or the composition according to claim 8 or 9 into the plant.