JP2004097005A - VACUOLAR TRANSPORT SIGNAL PEPTIDE OF SOYBEAN beta-CONGLYCININ AND UTILIZATION THEREOF - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for mass-synthesizing the objective protein in a plant, especially a soybean seed and to provide a peptide having a specific amino acid sequence for promoting stable accumulation thereof in the plant (especially the soybean) seed. <P>SOLUTION: A specific amino acid sequence or a sequence composed of at least ≥4 residues on the N-terminal side in the specific amino acid sequence as a vacuolar transport signal peptide for a protein storage vacuole is added to either of the C-terminus and the N-terminus. The resultant protein is expressed in the plant seed in a genetic engineering manner. <P>COPYRIGHT: (C)2004,JPO

Description

【００４０】

【図面の簡単な説明】
【図１】図１は、使用したα’サブユニットの野生型および末端切断型変異体タンパク質と、同じく使用したＧＦＰ変異体とを模式的に示す図である。
【図２】図２は、Ｔ_１植物から得られたＴ_２種子中に野生型および末端切断型（（が発現、蓄積されているかどうかを確認するために、抗α’抗体を用いた完熟種子抽出液のウエスタンブロッティングを行った結果である。レーン１は、ダイズより精製した天然型α’サブユニット、レーン２は大腸菌で発現させた組換え型α’サブユニット、レーン３は大腸菌で発現させた組換え型α’サブユニットＣｏｒｅ、レーン４は非形質転換シロイヌナズナ種子抽出液、レーン５はα’サブユニットを発現されたシロイヌナズナ種子抽出液、レーン６はΔＰｒｏを発現されたシロイヌナズナ種子抽出液、レーン７はΔＧｌｙを発現されたシロイヌナズナ種子抽出液、レーン８はΔＣＴ６を発現されたシロイヌナズナ種子抽出液、レーン９はΔＣＴ１０を発現されたシロイヌナズナ種子抽出液、レーン１０は、ΔＥｘｔを発現されたシロイヌナズナ種子抽出液、レーン１１は、Ｃｏｒｅを発現されたシロイヌナズナ種子抽出液、レーン１２は、Ｃｏｒｅ　ΔＧｌｙを発現されたシロイヌナズナ種子抽出液である。
【図３】図３は、野生型及び各種変異型α’のＴ_２完熟種子抽出液を用いてショ糖密度勾配遠心分離を行い、ウエスタンブロッティングを行った結果である。
【図４】図４は、野生型及び各種変異型α’が種子細胞内でＰＳＶへ輸送されたかどうかを完熟種子細胞の免疫電子顕微鏡観察により解析した結果を示す。Ａは、野生型α’サブユニットを発現させたシロイヌナズナ種子の切片を抗α’サブユニット抗体で処理したもの、Ｂは、野生型α’サブユニットを発現させたシロイヌナズナ種子の切片を抗グリシニン（ダイズ１１Ｓ）抗体で処理したものであり、Ｃは、非形質転換シロイヌナズナ種子の切片、ＤはΔＰｒｏＥはΔＥｘｔ、ＦはΔＧｌｙ、ＧはＣｏｒｅ、Ｈは、Ｃｏｒｅ　ΔＧｌｙ、ＩはΔＣＴ６、ＪはΔＣＴ１０を、それぞれに発現させたシロイヌナズナ種子の切片を抗α’サブユニット抗体で処理したものである。Ｋは、Ｊの細胞間隙の部分の拡大図である。Ｌは、ΔＣＴ１０を発現させたシロイヌナズナ種子切片を抗グリシニン（ダイズ１１Ｓ）抗体で処理した図である。
【図５】図５は、形質転換シロイヌナズナ種子中でのダイズ由来のシグナルペプチドとＧＦＰとのキメラタンパク質の蓄積を、種子抽出液の抗ＧＦＰ抗体を用いるウエスタンブロッティングによって解析した結果である。レーン１はｓｐＧＦＰ、レーン２はｐｒｏＧＦＰおよびレーン３はＧＦＰ−ＣＴ２４である。レーン４は非形質転換シロイヌナズナ種子抽出液である。
【図６】図６は、シロイヌナズナ種子中でのＧＦＰキメラタンパク質である、ｓｐＧＦＰ　（Ａ）、ｐｒｏＧＦＰ　（Ｂ）　ＧＦＰ−ＣＴ２４（Ｃ）の局在部位を乾燥種子の共焦点レーザー顕微鏡観察により解析した結果を示す。
【図７】図７は、ＧＦＰ−ＣＴ２４をコードする遺伝子（ＣおよびＤ）またはシグナルペプチドのみを連結したｓｐＧＦＰをコードする遺伝子（コントロール；ＡおよびＢ）をパーティクルガンでダイズ登熟種子に導入し、２４時間後に、発現したＧＦＰ−ＣＴ２４の細胞内での存在部位を共焦点レーザー顕微鏡観察により確認した結果である。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for synthesizing a target protein in large amounts in plants, particularly soybean seeds, and a peptide having a specific amino acid sequence for promoting stable accumulation in plant (particularly soybean) seeds.
[0002]
[Prior art]
Soybean seeds are the most abundant protein-rich seeds, are also very useful as mass-produced and inexpensive raw materials for vegetable oils, and are used in a wide variety of countries and regions. BACKGROUND ART The development of effective uses of defatted soybeans in which oil and fat components are separated from soybean seeds has attracted attention in the food industry.
[0003]
Production of a desired protein in a plant, and isolation and purification of the protein have been made possible by recent advances in genetic engineering techniques and the like. Therefore, the mechanism by which soybean major storage proteins (11S globulin, 7S globulin, etc.), which are abundantly stored in the protein storage vacuole (PSV) of soybean seeds, are transported to PSV is elucidated and applied. Thus, it is considered that the desired protein can be accumulated in the PSV of soybean seed, and the desired protein can be inexpensively and efficiently purified from the defatted soybean from which the fat component has been separated.
[0004]
In plants, the function of vacuoles is diverse. Previous studies have shown that there are at least two types of vacuoles, one of which is degraded (LV) and the other is protein storage (PSV). (Vitale and Raikhel, 1999). It has also been reported that these vacuoles coexist in barley and pea root tip cells and in pea-ripening cotyledon cells (Hoh et al., 1995; Paris et al., 1996). In cotyledon cells, as the seeds ripen, LV disappears and PSV develops (Hoh et al., 1995). PSV stores 11S and 7S globulin, which are main seed storage proteins, lectins, various enzymes and protease inhibitors (Herman).
and @Larkins, $ 1999).
[0005]
It is believed that the transport of soluble proteins to LV and PSV is performed by different mechanisms. Transport to LV is performed by clathrin-coated vesicles (CCV) that germinate from the trans-Golgi network, and the uptake of soluble proteins into CCV depends on the signal (vacuolar transport signal) of the transported protein is sequence-specific (Vitale and Raikhel, 1999; Ahmed et al., 2000). The transport of PSV proteins such as seed storage proteins is carried out by dense vesicles (DV) that germinate from the Golgi apparatus (Hohl et al., 1996; Neuhaus and Rogers, 1998; Hillmers et al., 2001). Also, in pumpkin seed cells, it has been suggested that a PAC vesicle formed in the endoplasmic reticulum has a transport pathway that does not pass through the Golgi apparatus (Hara-Nishimura et al., 1998). However, little is known about the transport mechanism of PSV proteins by DV or PAC vesicles.
[0006]
Vacuolar transport signals of plant vacuolar proteins examined in previous studies are classified into three types: sequence-specific vacuolar transport signal (ssVSD), C-terminal vacuolar transport signal (ctVSD), and structure-dependent signal (psVSD). (Neuhaus and Rogers, 1998). The sequence of ssVSD is considered to be recognized by the receptor, and one containing an NPIR motif or a sequence similar thereto has been found. ctVSD is a strongly hydrophobic sequence present in the C-terminal propeptide, but varies in length, and no common sequence has been found. Little is known about these transport mechanisms. For psVSD, its structural features have not yet been found.
[0007]
Vacuolar transport signals have also been reported for a number of soluble proteins present in seed PSV (tobacco chitinase, tobacco glucanase, barley lectin, kidney bean phaseolin, Brazil nut 2S albumin, castor seed lysin, barley phytepsin). (Matsuoka and Neuhaus, 1999; Frigerio et al., 1998, 2001, Tormakangas et al., 2001). In lysine and phytepsin, a signal is present in the internal propeptide. Among them, in lysine, it has been suggested that isoleucine conserved among lysine-like proteins of various plant species is involved in transport and is ssVSD (Frigerio et al, 2001a). In chitinase, glucanase, lectin, phaseolin, and Brazil nut 2S albumin, the C-terminal propeptide serves as a signal, has no common sequence, and has high hydrophobicity, and is considered to be ctVSD.
[0008]
However, all of these signals were analyzed using cells other than seeds. Whether cells similar to DV or PAC vesicle-specific transport to PSV occur in cells of tissues other than seeds at present stage has not yet been elucidated about molecules involved in the formation of DV and PSV. Can not judge. For this reason, it is considered necessary to analyze the PSV protein signal of the seed using the seed.
[0009]
In the present invention, β-conglycinin, one of the major seed storage proteins of soybean, used for the study of vacuolar transport signals, is also called 7S globulin, and its main constituent subunits are α, α ′, There are three types of β, and various molecular species in which these subunits are randomly combined exist in soybean seeds (Thanh and Shibasaki, 1976, 1978). Although it is not known whether any of these three types of subunits are propeptides or not, it is known that a sequence similar to ctVSD (AFVY) of phaseolin, which is a kidney bean 7S globulin, exists. (Doyle et al., 1986; Sebastiani et al., 1990; Maruyama et al., 1998).
[0010]
[Problems to be solved by the invention]
The present invention provides a means for mass-producing a target protein by accumulating the target protein in a plant, particularly soybean seed in a large amount. That is, the present invention provides a vacuolar transport signal peptide capable of accumulating a target protein in a large amount in seeds, a protein containing the peptide, a nucleic acid encoding the peptide, and use thereof.
[0011]
[Means for Solving the Problems]
In order to solve the above problems, the present inventors attempted to identify a sequence involved in transport to PSV using the α ′ subunit among the three subunits of soybean β-conglycinin, The present inventors have found that the amino acids present at positions 7 to 10 counted from the terminal are involved in the transport of PS to the PSV as a vacuolar transport signal peptide of the α 'subunit, thereby completing the present invention. The amino acid sequence at positions 1 to 10 (SEQ ID NO: 1) counted from the C-terminus has 100% homology between the α subunit and the α ′ subunit, and further has a similar sequence at the C-terminus of the β subunit. Since (SEQ ID NO: 2) is present, it is considered that the peptide having the sequence shown in SEQ ID NO: 1 and the peptide having the sequence shown in SEQ ID NO: 2 have similar functions.
[0012]
Therefore, the present invention provides the following aspects 1 to 25:
1. A protein in which the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 2 is added to the amino acid sequence of the target protein as a vacuolar transport signal peptide to a protein storage vacuole at either the C-terminus or the N-terminus ,
2. In the amino acid sequence of the target protein, a sequence consisting of at least four N-terminal residues or more of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, or Pro \ Xaa \ Xaa \ Ser (Xaa is any A protein having a sequence represented by amino acid) added to either the C-terminus or the N-terminus as a vacuolar transport signal peptide to a protein storage vacuole
3. The protein according to the above 1 or 2, wherein a specific protease recognition sequence exists between the amino acid sequence of the protein of interest and the vacuolar transport signal peptide sequence.
4. The protein according to the above 3, wherein a plurality of amino acid sequences of the target protein are linked in tandem, and a specific protease recognition sequence is present between the amino acid sequences.
5. The protein according to any one of the above 1 to 4, wherein the protein is transported to a protein storage vacuole when expressed in a plant seed.
6. The protein according to the above 5, wherein the plant is a dicotyledonous plant.
7. The plant according to the above 6, wherein the plant is soybean or Arabidopsis thaliana,
8. A nucleic acid encoding the protein according to any one of the above 1 to 7,
9. A plant transformation vector containing the nucleic acid according to the above item 8,
10. The vector according to the above 9, wherein the nucleic acid is contained in a form that can be expressed in a plant cell.
11. The vector according to the above 10, wherein the plant cell is a cell derived from a dicotyledon,
12. The vector according to the above 11, wherein the plant cell is a cell derived from soybean or Arabidopsis thaliana,
13. A plant cell containing the nucleic acid according to the above 8 as a foreign gene,
14． A plant cell, comprising the vector according to any of the above 9 to 12,
15. The plant cell according to the above 13 or 14, wherein the plant cell is a dicot plant cell.
16. The plant cell according to the above 15, which is a cell of soybean or Arabidopsis thaliana,
17. A plant containing the plant cell according to any of 13 to 16,
18. The plant according to the above 17, wherein the plant is a dicotyledon,
19. The plant according to the above 18, wherein the plant is soybean or Arabidopsis thaliana,
20. A method for producing a target protein,
i) {a step of obtaining the nucleic acid according to the above item 8;
ii) introducing the nucleic acid into an expressible form into a plant cell;
iii) a step of regenerating the plant cells under conditions capable of expressing the nucleic acid to obtain seeds thereof;
iv) {separating or isolating the target protein from the obtained seed;
The above method, comprising:
21. The method according to the above 20, wherein the plant is a dicotyledonous plant,
22. The method according to the above 21, wherein the plant is soybean or Arabidopsis thaliana,
23. The protein encoded by the nucleic acid in the step (i) is the protein according to the above 2 or 3, and
v) {a step of cleaving the specific protease recognition site in the protein obtained in the above step (iv) with a cleavable protease to obtain a protein having a desired amino acid sequence;
The method according to any one of the above 20 to 22, further comprising:
24. It is represented by the amino acid sequence represented by SEQ ID NO: 1 or the amino acid sequence represented by SEQ ID NO: 2, a sequence consisting of at least four N-terminal residues of the amino acid sequence, or Pro @ Xaa @ Xaa @ Ser (Xaa is any amino acid) And a nucleic acid encoding one or more desired proteins of interest can be inserted in-frame under the control of a plant promoter. Expression of a protein to which an amino acid sequence represented by SEQ ID NO: 1 or a sequence consisting of at least four residues on the N-terminal side of the amino acid sequence, or a sequence represented by Pro \ Xaa \ Xaa \ Ser (Xaa is an arbitrary amino acid) is expressed To enable, plant transformation expression vector, and,
25. SEQ ID NO: 1 or SEQ ID NO: 2, or at least the N-terminal side of the amino acid sequence, or a sequence consisting of a sequence represented by Pro @ Xaa @ XaaSer (Xaa is any amino acid) The vector according to the above 24, further comprising a sequence encoding a specific protease recognition site in-frame between the nucleic acid sequence encoding the protein and the insertion part of the nucleic acid encoding the protein of interest.
About.
[0013]
BEST MODE FOR CARRYING OUT THE INVENTION
The sequence consisting of the 10 amino acid residues Pro Leu 末端 Ser Ser Ile Leu Arg Ala Phe Tyr (SEQ ID NO: 1) at the C-terminus of the α 'subunit of β-conglycinin, a major storage protein of soybean, is sufficient for transporting the protein to PSV Since the protein having the sequence as a transport signal at the C-terminus is expressed in the seed of a plant (especially, soybean or Arabidopsis thaliana), the protein is transported to and accumulated in PSV in the protein. Can be produced in large quantities in the body. Furthermore, our studies show that even without the C-terminal 6 residues of the sequence of SEQ ID NO: 1, the protein is transported to PSV, and at least the N-terminal of the sequence of SEQ ID NO: 1 It is thought that a sequence consisting of four residues Pro {Leu} Ser {Ser} is responsible for transporting the protein to PSV. Therefore, a protein having at least the sequence containing at least 4 residues or the sequence containing the 10 residues at the C-terminus of the α ′ subunit at the N-terminus or C-terminus as a vacuolar transport signal peptide is also produced in a large scale as described above. can do. It also contains the N-terminal 4 residues Pro \ Phe \ Pro \ Ser of the sequence consisting of 10 amino acid residues Pro \ Phe \ Pro \ Ser \ Ile \ Leu \ Gly \ Ala \ LeuTyr at the C-terminal of the β-subunit of β-conglycinin (SEQ ID NO: 2). Since it is considered that the sequence can also function as a vacuolar transport signal peptide, it can be used in the same manner as the above sequence of the α ′ subunit. Furthermore, only the four residues from the N-terminal side of SEQ ID NO: 1 and SEQ ID NO: 2, ie, the common first residue Pro and the fourth residue Ser of Pro \ Leu \ Ser \ Ser \ and Pro \ Phe \ Pro \ Ser The sequence of Pro \ Xaa \ Xaa \ Ser (Xaa is any amino acid) in which is conserved could be used as well.
[0014]
Plants used for the production of such proteins are not limited, but are preferably dicotyledonous plants, more preferably legumes, and particularly preferably soybeans.
[0015]
Such a protein can have an arbitrary amino acid sequence by using a conventional genetic engineering technique. For example, the amino acid sequences of various proteins are published in various databases available in the art, and the amino acid sequences of the target proteins can be obtained. Obtaining a nucleic acid encoding it based on the amino acid sequence can be easily achieved by those skilled in the art. Further, those skilled in the art can easily obtain a nucleic acid encoding the amino acid sequence of the target protein by cloning in various ways.
[0016]
The obtained nucleic acid is linked in frame with the nucleic acid encoding the vacuolar transport signal peptide, and the nucleic acid encodes a protein in which a sequence consisting of the amino acid residue is added to the end of the amino acid sequence of the target protein. Can be obtained. Furthermore, a protein in which an amino acid sequence that can be recognized and cleaved by a specific protease (also called endopeptidase, for example, Factor Xa, thrombin, etc.) is inserted between the amino acid sequence of the target protein and the vacuolar transport signal peptide. Can be prepared. Various types of such proteases are known in the art, and those having no recognition site in the amino acid sequence of the target protein and in the vacuolar transport signal peptide may be selected. By inserting such a specific protease recognition site and allowing the protease to act on a protein separated from the seed, the added vacuolar transport signal peptide can be cleaved from the target protein.
[0017]
Further, in order to obtain a target protein in a larger amount, a nucleic acid encoding the protein is tandemly linked in-frame, and a specific protease recognition site is inserted into the link site so as to be also in-frame. It is also advantageous. In this case, it is necessary to design a nucleic acid sequence encoding from the amino acid sequence of the target protein to the terminal of the added vacuolar transport signal peptide so that a stop codon is not included. That is, the amino acid sequence of the protein of interest is tandemly linked across a specific protease recognition site, and the amino acid sequence consisting of 10 residues of SEQ ID NO: 1 or SEQ ID NO: 2 or the N-terminal It becomes possible to express as a protein to which a sequence consisting of at least 4 residues or a sequence represented by Pro @ Xaa @ Xaa @ Ser (Xaa is an arbitrary amino acid) is added as a vacuolar transport signal peptide. When this protein is expressed in plant seeds, it is transported and accumulated in PSV by the vacuolar transport signal peptide. Therefore, after separating or isolating the protein from the seed, the protein is cleaved with a protease that cleaves the protease recognition site. By the reaction, the vacuolar transport signal peptide portion is removed by cleavage, and a plurality of linked target proteins are also cut one by one to obtain a target protein. .
[0018]
The protein of interest may be any desired protein. For example, all kinds of proteins such as bioactive peptides (including various growth factors and peptidic hormones), antibodies (including fragments containing variable regions), and enzymes can be used. Things.
[0019]
As described above, downstream of the nucleic acid used to produce the protein, ie, the nucleic acid encoding the amino acid sequence of the protein of interest, in-frame (ie, in the same reading frame as the amino acid sequence) SEQ ID NO: 1 or Nucleic acid sequence encoding a sequence consisting of 10 residues shown in SEQ ID NO: 2 or at least 4 residues on the N-terminal side thereof, or a sequence represented by Pro @ Xaa @ Xaa @ Ser (Xaa is any amino acid) (vacuum transport signal peptide A nucleic acid to which a nucleic acid comprising a coding sequence) is linked is also included in the present invention. A sequence encoding one or more amino acids may be inserted in frame between the signal peptide encoding sequence and the nucleic acid sequence encoding the amino acid sequence of the target protein, and the specific peptidase recognition site may be It may be advantageous to include a coding sequence (see above). Further, two or more nucleic acid sequences encoding the amino acid sequence of the protein of interest (which may be the same or different) were linked in tandem, and a sequence encoding a specific peptidase recognition site was inserted between each of them. Nucleic acids may also be advantageous (see above). The nucleic acid is preferably ligated under the control of a promoter that functions in plant cells, preferably a plant-specific promoter. Such a nucleic acid can be used for various plant transformation methods (for example, a protoplast method, a particle gun method, etc.). A method for obtaining a transformed plant by regenerating a plant from transformed cells is as follows. It is known in the art and is disclosed in many protocol collections (for example, "Experimental protocol of model plants-from genetic techniques to genome analysis-" (Hidejunsha, published in 2001, Isao Shimamoto, Kiyotaka Okada) Supervision) etc.).
[0020]
The nucleic acid may be incorporated into a plant cell transformation vector. Examples include various Ti plasmids for transformation with Agrobacterium, binary plasmids such as pBI101, and Ri plasmids. Such a vector is preferably linked to the nucleic acid in a form that allows expression of the nucleic acid in plant seeds, that is, a form in which a suitable expression promoter can function in plant cells. Various expression promoters suitable for plant cells are known to those skilled in the art. Among them, plant seed-specific promoters are preferable. For example, promoters of each subunit of the soybean major storage protein (α ′ subunit of β-congoricinin) are preferable. Unit promoter).
[0021]
Further, such a vector may contain a selection marker (such as a nutrition selectivity or drug resistance marker).
Techniques for transformation using Agrobacterium or the like are also known to those skilled in the art.
[0022]
Therefore, cells transformed with the above-described genes via these vectors and having the genes as foreign genes are also included in the scope of the present invention. Plant cells containing the gene in an expressible form (ie, under the control of an appropriate promoter) are preferred. As used herein, the term "foreign gene" refers to a gene artificially introduced into a cell (including the genome) other than a gene naturally present in the genome of the plant. Furthermore, plants regenerated from such cells are also included in the present invention.
[0023]
From such a plant, a seed of a plant expressing a protein having a signal peptide can be harvested and the protein can be separated or isolated therefrom. Such plants are preferably dicotyledonous plants such as Arabidopsis and soybean. Also, legumes are preferred. Particularly, soybean is advantageous in that the protein can be obtained in large quantities and at low cost because the protein can be separated or isolated from the residue obtained by extracting the seed from soybean oil. Separation or isolation of the protein from the seed can be accomplished by techniques well known in the art.
[0024]
Accordingly, the present invention includes a method for producing a protein of interest using the above-described technique. In addition, the present invention also provides an expression vector designed so that a protein linked to the signal peptide can be expressed only by inserting a gene encoding the protein in order to produce a desired protein by the method. include.
[0025]
That is, the vector encodes a desired protein under the control of a promoter (see above) that enables expression in plant cells, particularly seed cells, and upstream of the signal peptide-encoding sequence. This is a vector for inserting a sequence in frame with the signal peptide. Therefore, it may be a circular vector or a linear vector having a restriction enzyme recognition site for insertion into the insertion site. Furthermore, if a sequence encoding a sequence-specific protease recognition site in frame is included between the insertion site and the signal peptide coding sequence, the signal sequence linked to the expressed protein can be cleaved off. Is advantageous.
[0026]
【Example】
Hereinafter, although an example is described in order to make an understanding of the present invention easy, the present invention is not limited to the following example.
Wild type, truncated and mutant α ' Gene construction and production of transformed Arabidopsis
To identify the C-terminal region involved in the transport of the α ′ subunit to PSV, wild-type (Wild), deletion of 6 residues at the C-terminal (ΔCT6) and deletion of 10 residues at the C-terminal (ΔCT10) The subunit gene was constructed (Fig. # 1). Each gene was operably linked downstream of the α 'promoter, which is known to regulate seed-specific expression. The α ′ promoter, signal peptide, and propeptide coding regions were prepared by PCR from the genome of soybean (variety: Wessuzunari). On the other hand, genes encoding the extension region and the core region were prepared from the cDNA of the α 'subunit by PCR. These genes were simultaneously inserted into the plant transformation vector pBI101. The space between the left and right border sequences of these plasmids was introduced into Arabidopsis nuclear DNA by the Agrobacterium method. Transformed plants were selected using kanamycin, and 3 to 7 transformants (T₁Plant). Similarly, other constructs as shown in FIG. 1 were also constructed to obtain transformants thereof, which were used in the following experiments.
[0027]
Wild-type and mutant α in transgenic Arabidopsis seeds ' Expression and accumulation
T₁T obtained from plants₂Whether or not wild-type and mutant α ′ were expressed and accumulated in the seeds was confirmed by Western blotting of anti-α ′ (a matured seed extract using an antibody. Wild-type α ′ was purified from soybean. A band was detected at the same size position as that of, but several bands were also detected on the lower molecule side, and a main band was detected at the position of the core size (Fig. 2, lane 5). , ΔCT6 and ΔCT10, bands were detected at positions having the same molecular weight as that of soybean α ′ and at the position of the core size (FIG. 2—lanes 6, 8, and 9). A band was detected at the same size position as the mature α 'expressed in Escherichia coli, and a band was also detected at the same size position as the α' core expressed in Escherichia coli (FIG. 2, lane 7). From this, it was confirmed that no sugar chain was added in ΔGly, and that such a stepwise degradation to the size of the core was also observed when α ′ was expressed in petunia seeds. It has already been reported that this is a unique phenomenon observed when α 'is expressed in heterologous plants, and the extension region protrudes from the surface of the molecule, so it may be susceptible to degradation by proteases. In both ΔExt and Core, there was no extension region, so that a band was detected in the core size (lanes 10 and 11 in FIG. 2), and since the size of ΔExt was the same as the core, the pro region of ΔExt was It can be said that only the core region has been accumulated by the processing, and CoreΔGly {({lane 12)} in FIG. A band was detected at the same size as that of α'core to which no sugar chain was added, and no band was detected on the lower molecular weight side of the core size in both wild type and mutant α '. Therefore, these proteins are T₂It was confirmed that it was stably accumulated in the seed.
[0028]
Wild type and mutant α ' Structure forming ability
T of wild-type and mutant α '₂Sucrose density gradient centrifugation was performed using the ripe seed extract. As a result, the wild-type and ΔPro, ΔGly, ΔCT6 and ΔCT10 mainly precipitated at the position of 7S, which is the size of the trimer (FIG. 3), and the wild-type and these three mutant α ′ soybeans It was confirmed that a trimer was formed similarly to α '. ΔExt, Core, and CoreΔGly mainly settled at the 9S position (FIG. 3). The present inventors have previously confirmed that α 'cores expressed in E. coli sediment at the 9S position and that they are two trimers associated. Therefore, it was considered that the present ΔExt, Core and Core ΔGly also formed a trimer, and then they were associated with each other to show a size of 9S. From the above, it was confirmed that the wild type and all the mutant α's were correctly molecularly assembled in Arabidopsis seeds. In other words, it can be said that the mutation performed this time did not cause any inconvenience to the structure formation.
[0029]
Wild type and mutant α ' of PSV Transport to the cell and extracellular secretion (confirmation of the region where the vacuolar transport signal exists)
Whether the wild type and the truncated α ′ were transported to PSV in the seed cells was analyzed by immunoelectron microscopic observation of the mature seed cells. An ultra-thin section of an Arabidopsis thaliana ripe seed for electron microscopy was reacted with an anti-α ′ antibody, and then reacted with a secondary antibody with gold particles. As a result, many gold particles were present in the PSV of Arabidopsis seed cells expressing the wild type (FIG. 4A). In addition, the gold particles were particularly abundant in a section that is apparently different from other parts in the PSV (this section is referred to as “crystalloid-like structure” in this specification) (FIG. 4A). The sections were similarly treated with an antibody against soybean glycinin (11S globulin) (anti-glycinin antibody) and observed. (FIG. 4B). On the other hand, gold particles showing the presence of a crystalloid-like structure or α 'were not detected in non-transformed Arabidopsis seeds. This confirmed that wild-type α ′ was transported to PSV in Arabidopsis seeds and accumulated in a crystalloid-like structure.
[0030]
Also for ΔPro, ΔExt, ΔGly and Core, many gold particles are present in the crystalloid-like structure of PSV of the mature seed cells (FIG. 4D, D, E, F, G), and these mutant α ′ also It was confirmed that it was transported to PSV and accumulated. From this, it was found that none of the pro region, extension region, and sugar chain were necessary for transporting α ′ to PSV. This was also confirmed by CoreΔGly accumulating in the crystalloid-like structure of PSV (FIG. 4H). That is, the transport of α ′ to PSV involves only the core region excluding the sugar chain.
[0031]
On the other hand, regarding the C-terminal part, it was confirmed that ΔCT6 in which six residues were deleted was transported to PSV (FIG. 4I). However, in ΔCT10 in which 10 residues were deleted, gold particles were detected in the intercellular space (FIG. 4 {J, {K)}), confirming that ΔCT10 was secreted into the intercellular space without being selectively transported to PSV. Was done. Similar results were obtained in analysis using three independent lines. Furthermore, as a result of performing the same treatment using an anti-glycinin antibody, Arabidopsis endogenous 11S globulin was detected only in PSV but not in the intercellular space (FIG. 4L). This result indicates that the extracellular secretion of ΔCT10 is not due to cell abnormalities, but to the lack of a vacuolar transport signal for ΔCT10 to be transported to PSV. . From this, it was found that a region necessary for transport to PSV was present in 10 residues at the C-terminal portion of α '.
[0032]
PSV In transport C Confirmation of independent involvement at the end
Next, in order to further confirm the involvement of the C-terminal part of the α ′ subunit in the selective transport to PSV, transformed Arabidopsis expressing three chimeric proteins (FIG. 1A, ΔspGFP, ΔproGFP, ΔGFP-CT24) was used. Created. Green fluorescent protein GFP was used as a reporter. Since GFP is a cytoplasmic protein, an α 'signal peptide was added to the N-terminal side to transfer it into the endoplasmic reticulum. In FIG. 4, the vacuolar transport signal for α ′ may be a sequence longer than the sequence in the C-terminal 10 residues required for α ′ transport. In addition, 24 residues were added, including the 14 residues just before the sequence. A DNA was prepared by adding the DNA sequence encoding the 24 residues (SEQ ID NO: 3) to a sequence encoding GFP by PCR, and this was expressed to produce a GFP-CT24 protein. In addition, it is suggested that the C 'terminal 24 residue of α' fluctuates on the molecular surface. As a control, proGFP in which a pro-region known to be unnecessary for vacuolar transport was added to the N-terminal of GFP was used.
[0033]
The accumulation of the chimeric protein in the transformed Arabidopsis seeds was confirmed by performing Western blotting of the seed extract and using an anti-GFP antibody. As a result, in each of the chimeric proteins, a main band was detected around 27 kDa, which is the molecular weight of GFP (FIG. # 5). In addition, in GFP-CT24, a band was detected on the side of about 3 kDa macromolecule (Fig. # 5, #lane # 3). This band is almost the same as the molecular weight of GFP with the addition of 24 C-terminal residues. From these results, it was confirmed that all the chimeric proteins were accumulated in the transformed Arabidopsis seeds.
[0034]
The localization site of the chimeric protein in Arabidopsis seeds was confirmed by confocal laser microscopic observation of the dried seeds. In spGFP で (FIG. 6A) and proGFP (FIG. 6B), particularly strong fluorescence was detected in the intercellular space, but not in PSV. On the other hand, with GFP-CT24, fluorescence was detected in intracellular PSV (FIG. 6C). This confirmed that the 24 residues at the C-terminal portion of α 'were sufficient to transport GFP to PSV.
[0035]
In soybean seed C At the end PSV Involvement in transport and confirmation of the region where the vacuolar transport signal exists
In order to confirm whether the C-terminal 24 residues of α 'transport GFP to PSV even in soybean seed cells, a gene encoding GFP-CT24 (see FIG. 1) was introduced into soybean ripening seeds using a particle gun. Then, 24 hours later, the location of the expressed GFP-CT24 in the cells was confirmed by confocal laser microscopy. As a result, GFP fluorescence was detected by PSV (data not shown), confirming that GFP-CT24 was transported to PSV and accumulated as in Arabidopsis seeds. As a control, spGFP (see FIG. 1) to which only the signal peptide was linked was observed in the same manner. As a result, in spGFP, GFP fluorescence was mainly detected in the endoplasmic reticulum and the intercellular space (FIG. 7A, B). From this, it was confirmed that the C-terminal 24 residues of α ′ added to GFP-CT24 can transport GFP to PSV even in ripened soybean seeds. In addition, the fact that both spGPF and GFP-CT24 expressed in soybean matured cotyledons showed the same results as those observed using the transformed Arabidopsis thaliana (Fig. # 6) means that the analysis using the transformed Arabidopsis thaliana (Fig. # 4) Strongly suggests that the phenomena occurring in soybean seed cells can be reproduced.
[0036]
Analysis using transgenic Arabidopsis thaliana seeds (Fig. 4) confirmed that 10 residues at the C-terminal part of α 'were required to transport α' to the vacuole (Fig. 4 J, K). Whether or not GFP can be transported to PSV with only the 10 residues at the C-terminal part was observed by introducing a gene encoding GFP-CT10 (FIG. 1) into soybean ripening seeds in the same manner. Was. As a result, GFP fluorescence was mainly detected by PSV and was not detected in the intercellular space (FIGS. # 7, #C and #D), confirming that GFP-CT10 was transported to PSV and accumulated. This confirmed that the C-terminal 10 residues can transport GFP to PSV.
[0037]
From the above results, it was found that six residues at the C-terminal were insufficient for transporting protein to PSV in plant seeds, and that ten residues were necessary and sufficient for transport to PSV. Was. It was suggested that a vacuolar transport signal to PSV was present in this region.
[0038]
【The invention's effect】
The present invention enables the production of a desired protein, including the accumulation of a large amount of the desired protein, in a plant, particularly in the seeds of soybean or Arabidopsis thaliana. Soybean is a crop plant that is cultivated in large quantities worldwide, and also enables large amounts of desired proteins to be separated from the residue obtained after extracting soybean oil from soybean seeds.
[0039]
[Sequence list]

[0040]

[Brief description of the drawings]
FIG. 1 is a diagram schematically showing the wild type and truncated mutant proteins of the α ′ subunit used and the GFP mutant used.
FIG. 2 shows T₁T obtained from plants₂Lane 1 shows the results of Western blotting of a mature seed extract using an anti-α ′ antibody in order to confirm whether wild-type and truncated forms ((are expressed and accumulated in the seeds. Lane 2 is a recombinant α ′ subunit expressed in E. coli, lane 3 is a recombinant α ′ subunit expressed in E. coli, and lane 4 is non-transformed. Arabidopsis seed extract, Lane 5: Arabidopsis seed extract in which α ′ subunit was expressed, Lane 6: Arabidopsis seed extract in which ΔPro was expressed, Lane 7: Arabidopsis seed extract in which ΔGly was expressed, Lane 8: Arabidopsis seed extract expressing ΔCT6, lane 9 shows Arabidopsis seed extract expressing ΔCT10, lane 0, Arabidopsis seed extract is expressed DerutaExt, lane 11, Arabidopsis seed extract is expressed Core, lane 12 is the Arabidopsis thaliana seed extract is expressed Core ΔGly.
FIG. 3 shows T of wild-type and various mutant α ′.₂It is the result of performing sucrose density gradient centrifugation using the ripe seed extract and performing Western blotting.
FIG. 4 shows the results of analysis of whether or not wild-type and various types of mutant α ′ were transported to PSV in seed cells by immunoelectron microscopic observation of mature seed cells. A shows a section of an Arabidopsis thaliana seed section expressing a wild-type α ′ subunit treated with an anti-α ′ subunit antibody, and FIG. Soybean 11S) antibody, C is a section of a non-transformed Arabidopsis seed, D is ΔProE is ΔExt, F is ΔGly, G is Core, H is Core ΔGly, I is ΔCT6, and J is ΔCT10. And Arabidopsis seeds expressed in each of the sections were treated with an anti-α ′ subunit antibody. K is an enlarged view of the portion of the cell gap of J. L is a diagram obtained by treating an Arabidopsis thaliana seed section expressing ΔCT10 with an anti-glycinin (soybean 11S) antibody.
FIG. 5 shows the results of analyzing the accumulation of a chimeric protein between soybean-derived signal peptide and GFP in transformed Arabidopsis seeds by Western blotting of a seed extract using an anti-GFP antibody. Lane 1 is spGFP, lane 2 is proGFP and lane 3 is GFP-CT24. Lane 4 is a non-transformed Arabidopsis seed extract.
FIG. 6 shows the localization sites of spGFP 部位 (A) and proGFP (B) GFP-CT24 (C), which are GFP chimeric proteins in Arabidopsis seeds, were analyzed by confocal laser microscopic observation of dried seeds. The results are shown.
FIG. 7 shows that genes encoding GFP-CT24 (C and D) or genes encoding spGFP linked only with a signal peptide (controls; A and B) were introduced into soybean ripening seeds using a particle gun. 24 shows the results of confirming the location of the expressed GFP-CT24 in cells 24 hours later by confocal laser microscopy.

Claims (25)

A protein in which the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 2 is added to the amino acid sequence of the target protein as either a C-terminal or an N-terminal as a vacuolar transport signal peptide to a protein storage vacuole . In the amino acid sequence of the target protein, a sequence consisting of at least four or more N-terminal residues of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, or Pro \ Xaa \ Xaa \ Ser (Xaa is any (Amino acid) as a vacuolar transport signal peptide to a protein storage vacuole at either the C-terminus or the N-terminus. The protein according to claim 1 or 2, wherein a specific protease recognition sequence exists between the amino acid sequence of the protein of interest and the vacuolar transport signal peptide sequence. 4. The protein according to claim 3, further comprising a plurality of amino acid sequences of the protein of interest linked in tandem, and a specific protease recognition sequence existing between the amino acid sequences. The protein according to any one of claims 1 to 4, wherein the protein is transported to a protein storage vacuole when expressed in a plant seed. The protein according to claim 5, wherein the plant is a dicotyledon. The protein according to claim 6, wherein the plant is soybean or Arabidopsis thaliana. A nucleic acid encoding the protein according to any one of claims 1 to 7. A plant transformation vector containing the nucleic acid according to claim 8. The vector according to claim 9, wherein the nucleic acid is contained in a form that can be expressed in a plant cell. The vector according to claim 10, wherein the plant cell is a cell derived from a dicotyledon. The vector according to claim 11, wherein the plant cell is a cell derived from soybean or Arabidopsis thaliana. A plant cell containing the nucleic acid according to claim 8 as a foreign gene. A plant cell containing the vector according to any one of claims 9 to 12. 15. The plant cell according to claim 13, which is a dicotyledon cell. The plant cell according to claim 15, which is a cell of soybean or Arabidopsis thaliana. A plant comprising the plant cell according to claim 13. 18. The plant according to claim 17, which is a dicotyledonous plant. The plant according to claim 18, wherein the plant is soybean or Arabidopsis. A method for producing a target protein,
i) obtaining the nucleic acid according to claim 8;
ii) introducing the nucleic acid into an expressible form into a plant cell;
iii) a step of regenerating the plant cell under conditions capable of expressing the nucleic acid to obtain a seed thereof;
iv) separating or isolating the target protein from the obtained seed;
The above method, comprising: 21. The method according to claim 20, wherein the plant is a dicotyledonous plant. The method according to claim 21, wherein the plant is soybean or Arabidopsis thaliana. The protein encoded by the nucleic acid in the step (i) is the protein according to claim 2 or 3, and
v) cleaving a specific protease recognition site in the protein obtained in the above step (iv) with a cleavable protease to obtain a protein having a desired amino acid sequence;
23. The method according to any one of claims 20 to 22, further comprising: It is represented by the amino acid sequence represented by SEQ ID NO: 1 or the amino acid sequence represented by SEQ ID NO: 2, or a sequence consisting of at least the N-terminal four residues of the amino acid sequence, or Pro @ Xaa @ Xaa @ Ser (Xaa is any amino acid) And a nucleic acid encoding one or more desired proteins of interest can be inserted in-frame under the control of a plant promoter. An amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, a sequence consisting of at least four residues on the N-terminal side of the amino acid sequence, or a sequence represented by Pro \ Xaa \ Xaa \ Ser (Xaa is any amino acid) An expression vector for plant transformation, which is capable of expressing the expressed protein. The amino acid sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 2, or a sequence consisting of at least four N-terminal residues of the amino acid sequence, or Pro @ Xaa @ Xaa @ Ser (Xaa is any amino acid) 25. The method according to claim 24, further comprising a sequence encoding the specific protease recognition site in-frame between the nucleic acid sequence encoding the represented sequence and the insertion site of the nucleic acid encoding the protein of interest. The described vector.

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