JP2010104363A - Promoter of seed storage protein arcelin 2 of phaseolus, and utilization of the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a means for the production and accumulation of a foreign useful protein in a plant seed which is an excellent storage part of the protein. <P>SOLUTION: This promotor sequence of gene of a seed storage protein arcelin 2, of phaseolus is isolated and the gene encoding the foreign useful protein is seed specifically expressed under the expression control of the promotor. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a novel DNA comprising a promoter sequence of a gene encoding the kidney bean seed storage protein Arserin 2. The present invention also relates to a vector containing the DNA. Furthermore, the present invention relates to the use of the DNA and vector.

  A promoter is DNA having a function of controlling gene expression, and various promoters are known as promoters that can function in plant cells. A recombinant gene can be prepared by linking a structural gene intended to be expressed downstream of various promoters and inserting it into a plasmid vector. Then, by introducing the recombinant vector into a plant cell, the intended structural gene can be expressed in the plant cell in a manner according to the characteristics of the promoter. In recent years, plant cultivar improvement by genetic recombination technology has not only improved plant traits but also research and development for producing and accumulating useful proteins such as pharmaceutical raw materials, health food materials and enzymes in plants. For the production of recombinant useful proteins in plants, seeds and leaves are used as the production and accumulation sites. In particular, seeds are excellent as protein storage organs, and are stable in large quantities of target foreign proteins. Can be accumulated. Moreover, since it is a seed, it has high storage stability and is easy to process.

  On the other hand, in order to produce and accumulate the target useful protein in a seed-specific manner, it is necessary to express a foreign gene under the control of a promoter that induces gene expression in the seed. As known promoters for inducing seed-specific gene expression, rice glutelin promoter (see Patent Document 1), flax seed-specific promoter (see Patent Document 2), kidney bean arserin 5 promoter (Patent Document 3) See).

  However, when a foreign protein is expressed using the rice glutelin promoter, the accumulation site is the endosperm which is a starch layer, which is not suitable for leguminous plants which are an endosperm seed. Flax seed specific promoters have not been demonstrated to be usable in legumes. The kidney bean arserin 5 promoter has no report on promoter activity in recombinants, and it is unclear how much foreign protein can actually be accumulated in seeds.

JP 2005-130833 A Special table 2003-525030 gazette International Publication WO2002 / 50295 Pamphlet

  An object of the present invention is to provide a novel DNA having a legume seed-specific promoter activity. Another object of the present invention is to provide a vector containing the DNA and a transformant into which the vector has been introduced. Another object of the present invention is to provide an inexpensive method for producing a foreign protein by expressing a foreign protein at a high rate in plant seeds, which are excellent protein storage sites. A further object of the present invention is to provide a foreign protein by expressing a target foreign gene in seeds even in plants other than legumes.

The inventors of the present invention have intensively studied to solve the above problems.
As a result of the research, the inventors succeeded in isolating a novel DNA having a promoter sequence of the kidney bean seed storage protein Arselin 2 by a method described later. Furthermore, as a result of determining the base sequence of the DNA, it was found that the DNA was a 3860 bp DNA fragment having the base sequence of SEQ ID NO: 1 in the sequence listing.

  Further, as a result of producing a vector in which the base sequence contained in the DNA is bound upstream of the reporter gene and introducing it into soybean cultured cells and seeds, the base sequence induces seed-specific gene expression, The present inventors have found that its promoter activity is higher than that of a known seed-specific expression-inducing promoter.

That is, the present invention
[1]
DNA containing the base sequence shown in the following (A), (B) or (C):
(A) the base sequence of SEQ ID NO: 1,
(B) a base sequence encoding a DNA having a seed-specific promoter activity, wherein one or several bases are deleted, substituted, inserted or added in the base sequence of SEQ ID NO: 1;
(C) a partial sequence of the base sequence described in (A) or (B) above, which encodes a DNA having seed-specific promoter activity,
[2]
DNA comprising the base sequence set forth in base numbers 1399 to 3860 of SEQ ID NO: 1,
[3]
A vector comprising the DNA according to [1] or [2], a foreign gene encoding a foreign protein linked downstream of the DNA, and a DNA having terminator activity linked downstream of the foreign gene;
[4]
The DNA having the terminator activity is one in which one or several bases are deleted, substituted, inserted or added in the base sequence of SEQ ID NO: 2 or the base sequence of SEQ ID NO: 2, and has a terminator activity. The vector according to [3], comprising a base sequence encoding DNA;
[5]
A transformed plant into which the vector described in [3] or [4] is introduced;
[6]
Transformed seeds into which the vector according to [3] or [4] is introduced,
[7]
A method for producing a foreign protein, characterized in that a foreign gene encoding the foreign protein is expressed in plant seeds under the control of the DNA according to [1] or [2],
I will provide a.

  Since the promoter of the present invention has a seed-specific promoter activity, a vaccine, an antibody, an antibiotic, a seed, which is an original storage organ, is transformed by using a vector in which a foreign gene is linked to the promoter. It becomes possible to efficiently produce and accumulate useful proteins derived from foreign genes, represented by functional proteins and functional peptides.

It is the photograph of the CBB dyeing | staining after SDS-PAGE which shows the accumulation amount of the arserin 2 protein in the seed of the transformation soybean which introduce | transduced pUHGArc2PCT. The arrow in the figure indicates the arserin 2 protein. It is the photograph of the western blotting after SDS-PAGE which shows the accumulation amount of the arserin 2 protein in the seed of the transformation soybean which introduce | transduced pUHGArc2PCT. The arrow in the figure indicates the arserin 2 protein.

The present invention is described in detail below.
(1) The promoter according to the present invention and the terminator incorporated in the vector according to the present invention The DNA having the seed-specific promoter activity according to the present invention (hereinafter referred to as the DNA according to the present invention) is a promoter of kidney bean arserin 2 gene DNA containing a sequence, specifically, DNA containing the base sequence of SEQ ID NO: 1 or a partial sequence thereof.

  Moreover, the DNA having the terminator activity incorporated into the vector according to the present invention is a DNA containing the terminator sequence of kidney bean arserin 2 gene, specifically, a DNA containing the base sequence of SEQ ID NO: 2.

  It is known that there are a plurality of homologous genes from Arserin 1 to 7 (Non-Patent Document Alian et al .; Plant Physiology 120, 1095-1104 (1999)). The base sequence homology is very high.

  In general, DNA containing a promoter sequence of a specific structural gene can be isolated by a screening operation from a genomic DNA library. When the base sequence of the structural gene is known, a probe based on the base sequence is used. Can be isolated by screening from a genomic DNA library and determining the base sequence of the selected clone. However, when there are a plurality of genes having high homology to the structural gene, such as the promoter of the arserin 2 gene of the present invention, the probability that a clone containing the target promoter region is selected is low.

  Isolation of DNA containing the promoter sequence and terminator sequence of the arserin 2 gene of the present invention is performed by binding an adapter to genomic DNA treated with a restriction enzyme and designing a primer based on the known sequence and the adapter sequence. It can be performed using a method of cloning an unknown region, that is, a genome walking method using PCR technology. Specifically, by performing PCR using an adapter sequence-specific primer and an arserin 2 gene-specific primer designed based on a known arserin 2 gene cDNA base sequence (GenBank accession No. M28470), By amplifying the upstream region and downstream region of the kidney bean arserin 2 gene, cloning this into a vector and determining the base sequence, isolation of the promoter and terminator region of the kidney bean arserin 2 gene and determination of the base sequence It can be carried out.

  In general, even when one or several bases are deleted, substituted, inserted or added in the base sequence of DNA having promoter activity and DNA having terminator activity, the promoter activity and terminator activity are not affected. It will be widely recognized by those skilled in the art that it can be maintained. Therefore, the DNA according to the present invention includes DNA having a base sequence formed by deleting, substituting, inserting or adding one or several bases in the base sequence of SEQ ID NO: 1 and The DNA having specific promoter activity is included, and in the DNA having terminator activity incorporated in the vector according to the present invention, one or several bases are deleted or substituted in the base sequence of SEQ ID NO: 2. DNA containing a base sequence formed by insertion or addition and having terminator activity is included. Such DNA can be produced using methods well known to those skilled in the art, such as site-directed mutagenesis. The “one or several” here is preferably an integer of 1 to 9, more preferably an integer of 1 to 4.

  In the present invention, a partial sequence of the base sequence of SEQ ID NO: 1 or a base sequence of SEQ ID NO: 1 in which one or several bases are deleted, substituted, inserted or added, and is seed-specific The partial sequence of a base sequence encoding DNA having promoter activity (hereinafter referred to as a homolog sequence of SEQ ID NO: 1) refers to a continuous base sequence contained in SEQ ID NO: 1 or the homolog sequence of SEQ ID NO: 1. Among the DNAs containing the partial sequence, those having seed-specific promoter activity are included in the DNA according to the present invention. Examples of such DNA include DNA containing the base sequence described in base numbers 1399 to 3860 of SEQ ID NO: 1.

  Once the base sequences of the promoter and terminator of the present invention have been determined, the primers are synthesized by chemical synthesis or using DNA consisting of a part of the base sequence as a primer, and the whole kidney bean DNA is used as a template. It can be easily obtained by PCR using

(2) Assay for promoter activity Confirmation that the isolated DNA has a seed-specific promoter activity is carried out by introducing a transformed plant into which a vector incorporating a reporter gene capable of quantifying the expression level is introduced downstream of the DNA. This can be done by examining the expression site and expression level of the reporter gene in the body.

  Examples of the reporter gene include β-glucuronidase (GUS) gene, chloramphenicol acetyltransferase (CAT) gene, luciferase (LUC) gene, and GFP gene.

  Production of a transformed plant by introducing a vector can be performed by various methods already reported and established. Preferred examples thereof include the Agrobacterium method, PEG method, electroporation method, particle gun method and the like.

  Plants that can be transformed to measure seed-specific promoter activity include rice, barley, wheat, corn, soybean, kidney bean, rapeseed, Arabidopsis and the like. Examples of plant materials to be transformed include plant tissues such as roots, stems, leaves, seeds, embryos, ovules, ovary, shoot tips, buds, pollens, etc., undifferentiated callus, protoplasts, etc. A plant culture cell is mentioned.

  When a GUS gene is used as a reporter gene, by histochemical staining using 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid (X-Gluc) as a substrate by the catalytic action of the gene expression product, Promoter activity can be measured. In addition, in the case of confirmation of transient expression, whether or not seed-specificity is confirmed is obtained by transforming seeds or each of the above other plant tissues with a vector, for example, 3-5 days after histochemistry This can be confirmed by performing a manual staining. Furthermore, after transformation with a vector, a transformed plant body is obtained by a step of regenerating the plant body reported for each plant species, and other tissues including seeds formed by the transformed plant body are examined. Can also be confirmed.

(3) Vector according to the present invention The vector according to the present invention has the DNA according to the present invention, a foreign gene encoding a foreign protein located downstream of the DNA, and a terminator activity located downstream of the foreign gene. DNA. The foreign gene includes genes derived from kidney beans, as well as genes derived from kidney beans and having a base sequence different from that of the arserin 2 gene. The foreign gene is preferably a gene encoding a useful protein such as a vaccine, enzyme, antibody, functional protein, functional peptide and the like. In these genes, a base sequence encoding a signal peptide for arranging an expression product of the gene at a specific position in a cell, and a tag peptide (GST, his for facilitating isolation and purification of the expression product) -Tag etc.) can be combined and expressed as a fusion protein. The vector according to the present invention preferably further contains a selection marker. Examples of the selection marker include a hygromycin resistance gene and a kanamycin resistance gene. By introducing the vector according to the present invention into a plant, a transformed plant described below can be produced.

  The vector according to the present invention can be prepared using means known to those skilled in the art. For example, a vector comprising a DNA comprising the nucleotide sequence of SEQ ID NO: 1, a foreign gene located downstream thereof, and a DNA comprising the nucleotide sequence of SEQ ID NO. 2 located further downstream thereof can be obtained as follows. it can.

First, from a first primer having a base sequence continuous from the 5 ′ end to the 3 ′ side of SEQ ID NO: 1 and a complementary strand of the base sequence continuous from the 3 ′ end to the 5 ′ side of SEQ ID NO: 2. Using the second primer, PCR is carried out using the kidney bean genomic DNA as a template, and consists of the nucleotide sequence of SEQ ID NO: 1, the nucleotide sequence of SEQ ID NO: 2, and the arserin 2 gene sandwiched between these two sequences. DNA was obtained, and this was designated as pBluescript II.
It is incorporated into a commercially available cloning vector such as SK (-). This was introduced into Escherichia coli as a host, appropriately replicated and then extracted again. Using this as a template, a third primer consisting of a base sequence continuous from the 5 ′ end to the 3 ′ side of SEQ ID NO: 2, PCR is carried out using a fourth primer consisting of a complementary strand of a base sequence continuous from the 3 ′ end to the 5 ′ end of SEQ ID NO: 1. A vector according to the present invention can be prepared by ligating this amplification product with a separately isolated foreign gene to obtain a circular plasmid vector. Here, a restriction enzyme recognition sequence can be added to the first to fourth primers as necessary.

  If the 5 ′ end of the first primer is set downstream from the 5 ′ end of SEQ ID NO: 1, DNA consisting of a partial sequence of the base sequence of SEQ ID NO: 1, a foreign gene located downstream thereof, and further A vector containing DNA consisting of the base sequence of SEQ ID NO: 2 located downstream can be prepared.

The base sequence in the vector prepared as described above has one or several bases deleted, substituted, inserted or added in the base sequence by techniques well known to those skilled in the art such as site-directed mutagenesis. It can be mutated to the base sequence.
In addition, a gene encoding a selection marker can be a gene having a known nucleotide sequence, and since vectors containing various selection marker genes are also commercially available in the commercially available vectors, The selection marker gene can be easily inserted by molecular biological techniques well known to those skilled in the art.

(4) Transformed plant according to the present invention Using the recombinant vector prepared as described above, a target plant can be transformed to prepare a transformed plant. Various methods already reported and established can be used to prepare transformed plants. Preferred examples thereof include the Agrobacterium method, PEG method, electroporation method, particle gun method and the like.

  Plants used for transformation in the present invention include plants such as rice, barley, wheat, corn, soybean, kidney beans, rapeseed, and Arabidopsis. Examples of plant materials to be transformed include plant tissues such as roots, stems, leaves, seeds, embryos, ovules, ovary, shoot tips, buds, pollens, etc., undifferentiated callus, protoplasts, etc. A plant culture cell is mentioned. After transformation into the tissues and cells, transformed plant cells are selected using the resistance effect caused by the selection marker gene as an index. A transformed plant body can be obtained from the selected cells through a step of regenerating the plant body reported for each plant species.

  The transformed plant thus obtained is cultivated and the seeds are ripened to obtain the transformed seed according to the present invention, and the desired foreign protein is obtained in the transformed seed.

  Whether or not a gene has been incorporated into a plant can be confirmed by PCR, Southern hybridization, Northern hybridization, Western blotting, or the like. For example, protein is extracted from the seeds of the transformed plant and subjected to Western blotting. After Western blotting, foreign staining using the vector according to the present invention is performed by immunostaining using a primary antibody specific to the produced foreign protein and a secondary antibody labeled with horseradish peroxidase (HRP), etc. It is possible to confirm that the gene has been appropriately introduced, and that useful proteins derived from the foreign gene have been accumulated in the seed, and the amount of accumulation thereof.

Examples of the present invention will be specifically described below, but the scope of the present invention is not limited thereto.
In addition, the isolation operation of DNA containing the promoter region and terminator region of the arserin 2 gene performed in the following examples is performed by binding an adapter to a genomic DNA fragment subjected to restriction enzyme treatment, based on the known sequence and the adapter sequence. Primers were designed, PCR was performed using these primers, and a commercially available kit for cloning an unknown region was used, using Right Walk Kit ^™ manufactured by Bex Corporation.

[Example 1]
(1) Preparation of Genomic DNA of Kidney Bean Wild Species Genomic DNA 50 μg was extracted from 1 g of fresh leaves of kidney bean wild species (line number: G12866) using DNeasy Plant Maxi kit (Qiagen).

(2) Isolation of DNA containing the promoter sequence of Arselin 2 gene from kidney bean species (i) First extension reaction After digesting 280 ng of the genomic DNA with the restriction enzyme SauIIIAI, dGTP was added, and klenow enzyme (Promega) 1 base extension reaction was carried out. Thereafter, a ligation reaction was performed using the RWA-1 adapter attached to the RightWalk Kit ^™ and Ligation high (manufactured by Toyobo Co., Ltd.) to obtain a PCR template for isolating the DNA in the upstream region of the arserin 2 gene.

  Next, oligonucleotides (named as primer SP1 and primer SP2 respectively) consisting of the nucleotide sequences of SEQ ID NO: 4 and SEQ ID NO: 5 based on the known nucleotide sequence of the kidney bean arserin 2 gene (GenBank accession No. M28470) It was made using the custom synthesis contract service of Fasmac Co., Ltd. Since then, unless otherwise stated, primer synthesis was performed using the same company.

Next, PCR was performed using 2.8 ng of genomic DNA linked with the adapter constructed as described above as a template, and using primer WP-1 and primer SP1 attached to Right Walk Kit ^™ . In PCR, 50 μL of reaction solution was used per reaction, denaturation reaction was performed at 94 ° C. for 2 minutes for 1 cycle, denaturation reaction at 94 ° C. for 30 seconds, annealing at 65 ° C. for 30 seconds, and extension reaction at 68 ° C. Then, 35 cycles were performed with 5 minutes as one cycle. The reaction solution was KOD-Plus-Ver. Containing 200 μM dNTP mixed solution, 1.5 mM MgSO ₄ solution, 1 μM each of the above primers, and 1 unit of KOD-Plus- (manufactured by Toyobo Co., Ltd.). Contains 2 buffers.

After completion of the PCR, the reaction solution was diluted 100-fold, and 1 μL thereof was used as a template for the second PCR, and PCR was performed using the primers WP-2 and SP2 attached to the Right Walk Kit ^™ . The solution composition and temperature conditions for the second PCR were the same as those for the first PCR except for the template and primer.

  The amplified DNA fragment was subjected to phosphorylation with T4 Polynucleotide Kinase (manufactured by Takara Bio Inc.) in the presence of ATP at a final concentration of 1 mM, and then pBluescript II SK (−) (manufactured by Stratagene) previously treated with SmaI. A ligation reaction was performed.

  The reaction product was Arc2P (i), and the nucleotide sequence was determined using DNA sequencing service of Fasmac Co., Ltd. As a result, it was confirmed that a novel region of 844 bp upstream of the start codon of the arserin 2 gene was included. Thereafter, unless otherwise specified, the nucleotide sequence was determined using the company's sequencing service.

  Next, in order to further isolate the upstream region, a new primer was prepared and a second extension reaction was performed.

(Ii) Second extension reaction After 280 ng of the genomic DNA was digested with the restriction enzyme BglII, dGTP was added, and a one-base extension reaction was performed using klenow enzyme (manufactured by Promega). After the reaction, a ligation reaction was carried out with the RWA-1 adapter attached to the Right Walk Kit ^™ , and used as a PCR template for promoter isolation.

  Next, based on the known nucleotide sequence of the kidney bean arserin 2 gene (GenBank accession No. M28470), an oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 6 (designated as primer secondSP1) was also subjected to the first extension reaction. Based on the obtained base sequence of 844 bp upstream of the start codon, an oligonucleotide having the base sequence of SEQ ID NO: 7 (designated as primer secondSP2) was prepared.

Next, PCR was performed using 2.8 ng of genomic DNA linked with the adapter constructed as described above as a template, and using primer WP-1 and primer secondSP1 attached to Right Walk Kit ^™ . The PCR solution composition and temperature conditions were the same as those in the first extension reaction (i) except for the template and primer.

After completion of the reaction, the PCR solution was diluted 100 times, and 1 μL thereof was used as a template for the second PCR, and PCR was performed using the primer WP-2 and the primer secondSP2 attached to the Right Walk Kit ^™ . The solution composition and temperature conditions for the second PCR are the same as those for the first PCR except for the template and primers.

  The amplified DNA fragment was subjected to phosphorylation with T4 Polynucleotide Kinase (manufactured by Takara Bio Inc.) in the presence of ATP at a final concentration of 1 mM, and then pBluescript II SK (−) (manufactured by Stratagene) previously treated with SmaI. A ligation reaction was performed.

  The reaction product was Arc2P (ii) and the nucleotide sequence was determined. As a result, it was confirmed that a new region of 197 bp upstream (total 1041 bp) of Arc2P (i) was included. Next, in order to further isolate the upstream region, a new primer was prepared and the third extension reaction was performed.

(Iii) Third extension reaction After digesting 280 ng of the genomic DNA with the restriction enzyme XbaI, dCTP was added, and a one-base extension reaction was performed with klenow enzyme (manufactured by Promega). After the reaction, a ligation reaction was carried out with the RWA-2 adapter attached to the Right Walk Kit ^™ , and used as a PCR template for promoter isolation.

  Next, based on the 197 bp base sequence obtained by the second extension reaction, oligonucleotides consisting of the base sequences of SEQ ID NO: 8 and SEQ ID NO: 9 (named as primer thirdSP1 and primer thirdSP2 respectively) were prepared.

Next, PCR was performed using 2.8 ng of genomic DNA linked with the adapter constructed as described above as a template, and using primer WP-1 and primer thirdSP1 attached to the Right Walk Kit ^™ . The PCR solution composition and temperature conditions were the same as those in the first extension reaction (i) except for the template and the primer.

After completion of the reaction, the reaction solution was diluted 100-fold, 1 μL thereof was used as a template for the second PCR, and PCR was performed using the primer WP-2 and the primer thirdSP2 attached to the Right Walk Kit ^™ . The solution composition and temperature conditions for the second PCR are the same as those for the first PCR except for the template and primers.

  The amplified DNA fragment was subjected to phosphorylation with T4 Polynucleotide Kinase (Takara Bio) in the presence of ATP at a final concentration of 1 mM, and then pBluescript II SK (-) (Stratagene) previously treated with SmaI. A ligation reaction was performed.

The reaction product was Arc2P (iii), and the base sequence was determined. As a result, it was confirmed that a novel region of 2819 bp upstream (3860 bp in total) of Arc2P (ii) was included. As described above, DNA containing 3860 bp of a novel promoter sequence including the 5′-untranslated region upstream of the initiation codon of the arserin 2 gene could be obtained by three extension reactions (SEQ ID NO: 1). Arselin 1 (GenBank accession No. M68913), Arselin 3 (GenBank accession No. AX463292), Arselin 4 (GenBank accession No. AX463293) and Arserin 5-1 (GenBank accession) which are known genes having homology with the Arserin 2 gene
As a result of examining the homology with the promoter sequence of No. Z50202), the homology is 55.4% or less in comparison with any of the promoter sequences, and the DNA described in SEQ ID NO: 1 is a DNA containing a novel arserin promoter sequence It became clear that.

[Example 2]
(1) Isolation of DNA containing the terminator sequence of Arserin 2 gene from kidney bean species After digesting 280 ng of genomic DNA extracted in Example 1 (1) above with restriction enzyme NheI, dCTP was added, and klenow enzyme (Promega) 1 base extension reaction was carried out. After the reaction, a ligation reaction was performed using an RWA-2 adapter attached to the Right Walk Kit ^™ and Ligation high (manufactured by Toyobo Co., Ltd.) to obtain a PCR template for isolation of the terminator gene.

  Next, oligonucleotides (named as primer SP3 and primer SP4, respectively) comprising the nucleotide sequences of SEQ ID NO: 10 and SEQ ID NO: 11 based on the known nucleotide sequence of the kidney bean arserin 2 gene (GenBank accession No. M28470). Produced.

Next, PCR was performed using 2.8 ng of genomic DNA linked with the adapter constructed as described above as a template, and using primer WP-1 and primer SP3 attached to Right Walk Kit ^™ . In PCR, 50 μL of reaction solution was used per reaction, denaturation reaction was performed at 94 ° C. for 2 minutes and 1 cycle, denaturation reaction was performed at 94 ° C. for 30 seconds, annealing was performed at 65 ° C. for 30 seconds, and extension reaction was performed at 68 ° C. Then, 35 cycles were performed with 5 minutes as one cycle. The reaction solution was 200 μM dNTP mixed solution, 1.5 mM MgSO ₄ solution, 1 μM of each primer, 1 unit of KOD-Plus- (manufactured by Toyobo Co., Ltd.), KOD-Plus-Ver. Contains 2 buffers.

After completion of PCR, the reaction solution was diluted 100-fold, and 1 μL thereof was used as a template for the second PCR, and PCR was performed using Primer WP-2 and Primer SP4 attached to Right Walk Kit ^™ . The solution composition and temperature conditions for the second PCR were the same as those for the first PCR except for the template and primer.

  The amplified DNA fragment was subjected to phosphorylation with T4 Polynucleotide Kinase (manufactured by Takara Bio Inc.) in the presence of ATP at a final concentration of 1 mM, and then pBluescript II SK (−) (manufactured by Stratagene) previously treated with SmaI. A ligation reaction was performed.

  The reaction product was Arc2T, and the base sequence was determined. As a result, it was confirmed that a novel region of 795 bp downstream of the stop codon including the 3′-side untranslated region of the arserin 2 gene was included (SEQ ID NO: 2). As a result of searching for the homology with the terminator region of arserin 1 (GenBank accession No. M68913) and arserin 5-1 (GenBank accession No. Z50202) which are homologous to the known arserin 2 gene, it is homologous in any comparison The sex was 49.1% or less, and it was revealed that the DNA described in SEQ ID NO: 2 was a DNA containing a novel terminator sequence.

[Example 3]
(1) Synthesis and Isolation of DNA Corresponding to Arselin 2 Genomic Gene PCR was performed using 80 ng of genomic DNA extracted in Example 1 (1) as a template. Based on the base sequences described in SEQ ID NO: 1 and SEQ ID NO: 2, oligonucleotides consisting of the base sequences of SEQ ID NO: 12 and SEQ ID NO: 13 (named as primer Arc2P and primer Arc2T, respectively) were prepared.

In PCR, 50 μL of reaction solution was used per reaction, denaturation reaction was performed at 94 ° C. for 2 minutes and 1 cycle, denaturation reaction was performed at 94 ° C. for 30 seconds, annealing was performed at 57 ° C. for 30 seconds, and extension reaction was performed at 68 ° C. Then, 25 cycles were performed with 4 minutes as one cycle. The reaction solution was KOD-Plus-Ver. Containing 200 μM dNTP mixed solution, 1.5 mM MgSO ₄ solution, 1 μM each of the above primers, and 1 unit of KOD-Plus- (manufactured by Toyobo Co., Ltd.). Contains 2 buffers.

  The amplified DNA fragment was subjected to phosphorylation with T4 Polynucleotide Kinase (manufactured by Takara Bio Inc.) in the presence of ATP at a final concentration of 1 mM, and then pBluescript II SK (−) (manufactured by Stratagene) previously treated with SmaI. A ligation reaction was performed.

  The reaction product was Arc2PCT, and the base sequence was determined. As a result, it contains a promoter region of 2461 bp upstream of the initiation codon including the Arserin 2 gene and its 5 ′ untranslated region, and a novel region of 795 bp downstream of the stop codon including the 3 ′ untranslated region. Confirmed (SEQ ID NO: 3). Escherichia coli DH5α (manufactured by Takara Bio Inc.) into which the recombinant vector Arc2PCT thus obtained was introduced was named Escherichia coli DH5α / Arc2PCT, and was issued to the National Institute of Advanced Industrial Science and Technology Patent Biological Depositary Center on July 30, 2008. From the date of deposit, it has been deposited as deposit number P-21624.

(2) Preparation of promoter activity assay vector in plant (i) Preparation of arselin 2 promoter activity assay vector PCR using 10 ng of Arc2PCT obtained in Example 3 (1) as a template to amplify the promoter, terminator and vector part went. Based on the base sequence described in SEQ ID NO: 3, oligonucleotides of SEQ ID NO: 14 and SEQ ID NO: 15 (named as primer Arc2P3 and primer Arc2T5, respectively) were prepared.

In PCR, 50 μL of reaction solution was used per reaction, denaturation reaction was performed at 94 ° C. for 2 minutes and 1 cycle, denaturation reaction was performed at 94 ° C. for 30 seconds, annealing was performed at 57 ° C. for 30 seconds, and extension reaction was performed at 68 ° C. 25 cycles were performed with 6 minutes 30 seconds as one cycle. The reaction solution was 200 μM dNTP mixed solution, 1.5 mM MgSO ₄ solution, 1 μM each of the above primers, 1 unit of KOD-Plus- (manufactured by Toyobo Co., Ltd.), KOD-Plus-Ver. Contains 2 buffers. Thus, a DNA fragment Arc2PT containing the promoter 2461 bp and the terminator 795 bp of the arserin 2 gene and the vector sequence therebetween was obtained.

  Next, PCR amplification of the β-glucuronidase (GUS) gene of the plant cell transformation vector pBI221 (Clontech) was performed.

  Based on the known base sequence of pBI221 (GenBank accession No. AF502128), oligonucleotides of SEQ ID NO: 16 and SEQ ID NO: 17 (named as primer GUS5 and primer GUS3, respectively) were prepared.

In PCR, 50 μL of reaction solution was used per reaction, denaturation reaction was performed at 94 ° C. for 2 minutes and 1 cycle, denaturation reaction was performed at 94 ° C. for 30 seconds, annealing was performed at 57 ° C. for 30 seconds, and extension reaction was performed at 68 ° C. Then, 25 cycles were performed with 2 minutes as one cycle. The reaction solution was KOD-Plus-Ver. Containing 200 μM dNTP mixed solution, 1.5 mM MgSO ₄ solution, 1 μM each of the above primers, and 1 unit of KOD-Plus- (manufactured by Toyobo Co., Ltd.). Contains 2 buffers. In this way, a gene fragment GUS1 of β-glucuronidase (GUS) gene 1812 bp was obtained.

  The GUS1 gene fragment was phosphorylated with T4 Polynucleotide Kinase (manufactured by Takara Bio Inc.) in the presence of ATP at a final concentration of 1 mM, and then ligated with the Arc2PT. In this way, a promoter activity assay vector Arc2PGUS containing a promoter 2461 bp of the arserin 2 gene upstream of the β-glucuronidase gene and a terminator of 795 bp downstream was prepared.

(Ii) Preparation of an arserin 5 promoter activity assay vector In confirming the promoter activity of a DNA containing an arserin 2 promoter sequence in the present invention, comparison with other promoters having seed-specific promoter activity is performed. A known arserin 5 promoter and terminator (international publication WO02 / 50295 pamphlet), whose expression was recognized, was isolated.

  50 μg of genomic DNA was extracted from 1 g of fresh leaves of kidney bean wild species (line number: G02771) using DNeasy Plant Maxi kit (Qiagen). PCR was performed using 80 ng of the genomic DNA as a template. Based on the nucleotide sequence of the known kidney bean arserin 5-1 gene (GenBank accession No. Z50202), two types of oligonucleotides (named as primer Arc5P and primer Arc5T, respectively) consisting of the nucleotide sequences of SEQ ID NO: 18 and SEQ ID NO: 19 Produced.

In PCR, 50 μL of reaction solution was used per reaction, denaturation reaction was performed at 94 ° C. for 2 minutes and 1 cycle, denaturation reaction was performed at 94 ° C. for 30 seconds, annealing was performed at 57 ° C. for 30 seconds, and extension reaction was performed at 68 ° C. for 4 minutes. As a cycle, 25 cycles were performed. The reaction solution was KOD-Plus-Ver. Containing 200 μM dNTP mixed solution, 1.5 mM MgSO ₄ solution, 1 μM each of the above primers, and 1 unit of KOD-Plus- (manufactured by Toyobo Co., Ltd.). Contains 2 buffers.

  The amplified DNA fragment was subjected to phosphorylation with T4 Polynucleotide Kinase (manufactured by Takara Bio Inc.) in the presence of ATP at a final concentration of 1 mM, and then pBluescript II SK (−) (manufactured by Stratagene) previously treated with SmaI. A ligation reaction was performed. The reaction product was Arc5PCT, and the base sequence was determined. As a result, it was confirmed that the gene contains the 1818 bp promoter region upstream of the start codon including the 5 ′ untranslated region and the 1192 bp region downstream of the stop codon including the 3 ′ untranslated region. .

  PCR was performed using 10 ng of Arc5PCT obtained above as a template. Two types of oligonucleotides (named as primer Arc5P3 and primer Arc5T5, respectively) consisting of the nucleotide sequences of SEQ ID NO: 20 and SEQ ID NO: 21 were prepared from the known nucleotide sequences of the arserin 5-1 gene.

In PCR, 50 μL of reaction solution was used per reaction, denaturation reaction was performed at 94 ° C. for 2 minutes and 1 cycle, denaturation reaction was performed at 94 ° C. for 30 seconds, annealing was performed at 57 ° C. for 30 seconds, and extension reaction was performed at 68 ° C. 25 cycles were performed with 6 minutes 30 seconds as one cycle. The reaction solution was 200 μM dNTP mixed solution, 1.5 mM MgSO ₄ solution, 1 μM each of the above primers, 1 unit of KOD-Plus- (manufactured by Toyobo Co., Ltd.), KOD-Plus-Ver. Contains 2 buffers. Thus, a gene fragment Arc5PT containing the promoter 1818 bp and terminator 1192 bp of the arserin 5 gene was obtained.

  The phosphorylated GUS1 gene fragment was ligated to this to prepare a promoter activity assay vector Arc5PGUS containing the 1818 bp promoter of the arserin 5 gene upstream of the β-glucuronidase gene and 1192 bp of the terminator downstream.

[Example 4]
(1) Assay of promoter activity in soybean seeds The recombinant vector Arc2PGUS obtained in Example 3 above and Arc5PGUS as a comparative group were introduced into soybean seeds by the whisker method (Japanese Patent No. 3321867).

  25 days after the flowering of soybean (variety: Jack), the straw was immersed in a 70% ethanol solution for 1 minute and then in a sodium hypochlorite 0.5% (effective chlorine concentration) solution for 30 minutes to sterilize the straw. Aseptically matured seeds were removed and then the seed coats were removed. Seeds were cut into 5 to 7 mm squares to obtain gene transfer samples.

  Potassium titanate whisker LS20 (manufactured by Titanium Industry Co., Ltd.) was placed in a 1.5 mL tube and allowed to stand for 1 hour, after which ethanol was removed and completely evaporated to obtain a sterilized whisker. 1 ml of sterilized water was put into the tube containing the whisker and stirred well. Whisker and sterilized water were centrifuged and the supernatant water was discarded. In this way, the whisker was washed. This whisker washing operation was performed three times. Thereafter, 0.5 ml of a known MS liquid medium was added to the tube to obtain a whisker suspension.

  After putting the seed slices of the above two seeds into the tube containing the whisker suspension obtained above and stirring, the mixture is centrifuged at 1000 rpm for 10 seconds to precipitate the seed slices and whiskers. The seeds were discarded and a mixture of seed slices and whiskers was obtained.

  To the tube containing the mixture, 20 μL (20 μg) of the above-described recombinant vector (that is, the above-described recombinant vector Arc2PGUS or Arc5PGUS) was added and sufficiently shaken to obtain a uniform mixture. The tube with the homogeneous mixture was then centrifuged at 18000 × g for 5 minutes. The centrifuged mixture was shaken again and this operation was repeated three times.

The tube containing the seed slice, whisker, and vector obtained as described above was placed so that the tube was sufficiently immersed in the bathtub of the ultrasonic generator. Ultrasonic waves having a frequency of 40 kHz were irradiated at an intensity of 0.25 W / cm ² for 1 minute. The mixture was kept at 4 ° C. for 10 minutes after irradiation. The mixture thus sonicated was washed with the MS liquid medium to obtain a transformed seed slice.

  The seed slice obtained by introducing the recombinant vector as described above was placed on an MS agar medium obtained by adding 30 g / L sucrose and 8 g / L agar to MS medium. Thereafter, the cells were cultured for 3 days under conditions of 28 ° C., 2000 lux, and 16 hours of day length.

  GUS staining in soybean seeds transformed with the recombinant vector obtained above was performed according to a known method (Plant Genetic Transformation and Gene Expression A laboratory manual, pages 324 to 327, Blackwell Scientific Publications LTD).

A soybean seed slice was placed in a petri dish having a diameter of 3.5 cm, and a staining buffer (2 mM X-Gluc (5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid), 50 mM NaPO ₄ , pH 7.0) was used. 3 mL each was added and immersed. After incubating overnight at 37 ° C., the staining buffer was removed, and 3 mL of 70% ethanol was added and washed. After this washing operation was repeated three times, the number of blue spots in the soybean seed slice was examined. The results are shown in Table 1.

  As described above, the number of blue spots in the seed section into which Arc2PGUS of the present invention is introduced is higher than that of Arc5PGUS linked to the seed-specific expression promoter Arserin 5-1 promoter, and shows high expression in seeds. Was recognized.

(2) Assay of promoter activity in soybean somatic embryos Next, in order to confirm the expression time of the promoter of the present invention, genes were introduced into soybean spherical somatic embryos, which are in the early stage of embryo development. In addition, gene transfer was also carried out for pBI221 (Clontech) in which a cauliflower mosaic virus 35S promoter was linked upstream of the β-glucuronidase (GUS) gene as a comparative plot.

  Known methods (K. Nishizawa, Y. Kita, M. Kitayama, M. Ishimoto. (2006) A red fluorescent protein, DsRed2, as a visual reporter for transient expression and stable transformation in soybean. Plant Cell Reports 25: 1355- 1361), 30 somatic embryo masses (diameter 3 mm or less) derived from soybean (variety: Jack) immature seeds were added to a 1.5 mL tube, and the gene was transformed in the same manner as in Example 4 (1) above. The introduction operation was performed.

  The somatic embryo mass after gene introduction was placed in a petri dish having a diameter of 3.5 cm, 3 mL of a known somatic embryo growth medium was added, and the cells were cultured for 3 days under conditions of 25 ° C., 500 lux, and 23 hours of day length. GUS staining of soybean somatic embryos transformed with the recombinant vector obtained above was performed by the method of (1) above. The results are shown in Table 2.

  As described above, no blue spot was observed in Arc2PGUS of the present invention group, and expression was not observed in soybean spherical somatic embryos in the early stage of embryogenesis. From the above, it was proved that the arserin 2 promoter of the present invention is a high expression promoter showing specific expression in the late stage of seed ripening.

[Example 5]
(1) Construction of expression plasmid of kidney bean arserin 2 protein An expression plasmid for expressing a kidney bean arserin 2 storage protein in soybean seeds was constructed under the control of the promoter of the arserin 2 gene.

  As described above, Arc2PCT obtained in Example 3 (1) is a known pUHG vector (Y. Kita, K. Nishizawa, M Takahashi, M. Kitayama, M. Ishimoto, (2007) Genetic impulse. soybean and transformed of the improved breeding lines. Plant Cell Reports 26: 439-447), and a plant transformation vector for expressing the arserin 2 protein by the promoter of the arserin 2 gene was constructed.

  Arc2PCT was cleaved with restriction enzymes SpeI and HindIII, and ligated to the SpeI and HindIII sites of the pUHG vector to prepare a plant transformation vector pUHGArc2PCT. That is, 2 μg of Arc2PCT was cleaved with 20 units of restriction enzymes SpeI and HindIII at 37 ° C. for 2 hours, and then subjected to agarose electrophoresis. Only about 4.0 kb fragment was excised from Arc2PCT, and Gene Clean (Bio101) was used. Purified. On the other hand, 2 μg of pUHG plasmid was digested with 20 units of SpeI and HindIII at 37 ° C. for 2 hours and purified by Gene Clean. Each DNA fragment was reacted at 16 ° C. for 3 hours using Takara Ligation kit.

  In this way, a plant transformation vector pUHGArc2PCT in which an approximately 4.0 kb SpeI and HindIII cleavage fragment derived from Arc2PCT was incorporated into the SpeI and HindIII cleavage DNA fragment of the plasmid pUHG was prepared.

(2) Transformation of soybean by expression plasmid of kidney bean arserin 2 protein A known method (K. Nishizawa, Y. Kita, M. Kitayama, M. Ishimoto. (2006) A red fluorescent protein, u. fortified expression and stable transformation in soybean. Plant Cell Reports 25: 1355-1361), 30 somatic embryo blocks (3 mm or less in diameter) derived from immature seeds of soybean varieties Jack were added to a tube for 1.5 ml, Gene introduction was performed by the whisker ultrasonic method (Japanese Patent No. 3312867).

  Potassium titanate whisker LS20 (manufactured by Titanium Industry Co., Ltd.) was placed in a 1.5 ml tube and allowed to stand for 1 hour, after which ethanol was removed and completely evaporated to obtain a sterilized whisker. 1 ml of sterilized water was put into the tube containing the whisker and stirred well. The whisker and sterilized water were centrifuged and the supernatant water was discarded. The whisker was washed in this way. This whisker washing operation was performed three times. Thereafter, 0.5 ml of a known MS liquid medium was added to the tube to obtain a whisker suspension.

  In a tube containing the whisker suspension obtained above, 30 somatic embryo masses (diameter 3 mm or less) were placed and stirred, and then the mixture was centrifuged at 1000 rpm for 10 seconds to obtain somatic embryo masses and whiskers. Precipitation was carried out, and the supernatant was discarded to obtain a mixture of somatic embryo mass and whisker.

  To the tube containing the mixture, 20 μl (20 μg) of the pUHGArc2PCT vector prepared in Example 5 (1) was added and sufficiently shaken to obtain a uniform mixture.

  The tube with this homogeneous mixture was then centrifuged at 18000 × g for 5 minutes. The centrifuged mixture was shaken again and this operation was repeated three times.

The somatic embryo mass, whisker, and the tube containing the vector obtained as described above were placed so that the tube was sufficiently immersed in the bathtub of the ultrasonic generator. Ultrasonic waves having a frequency of 40 kHz were irradiated at an intensity of 0.25 W / cm ² for 1 minute. The mixture was kept at 4 ° C. for 10 minutes after irradiation. The sonicated mixture was washed with the MS liquid medium.

  After treatment, the somatic embryo mass is cultured with known somatic embryo growth liquid medium for 1 week by shaking (100 rpm), and then hygromycin B (15 mg / l) (Roche Diagnostics, Mannheim, Germany) is contained. And cultured for 1 week in fresh somatic embryo growth liquid medium. Furthermore, after culturing for 4 weeks in a somatic embryo growth liquid medium containing 30 mg / l hygromycin B (changing the medium every week), it was selected for 1 week in a somatic embryo growth liquid medium containing 45 mg / l hygromycin B. Culture was performed. In addition, the gene transfer was performed on 12 microtubes for each vector.

  The somatic embryo mass resistant to hygromycin was transferred to a known somatic embryo maturation liquid medium, and the somatic embryo was matured by continuing shaking culture (100 rpm) for 4 weeks. Mature somatic embryos were placed in a sterile petri dish for 3 to 5 days, dried, and then transferred to a known germinating solid medium. After germination culture for 7 to 10 days, it was transferred to a known rooting medium to grow germinating seedlings. After the roots and shoots grew, the plants were transferred to pots containing soil and maintained at high humidity until acclimated.

In this manner, three transgenic soybean plants were produced in which pUHGArc2PCT was introduced into the Jack variety. These transformed soybean plants were cultivated under an environment of 10000 lx and 16 hours of day length after adaptation to environmental humidity, and seeds were harvested from all individuals. Thus, to obtain a T ₁ generation of seeds of the transformed soybean plants.

(3) Evaluation of accumulated amount of arserin 2 protein in transformed soybean seeds Total protein was extracted from the transformed soybean seeds obtained above, and the accumulated amount of arserin 2 protein was determined by CBB (Coomassie Brilliant Blue R- after SDS-PAGE). 250) Evaluated by staining and Western blotting using specific antibodies.

  20 μg of total protein extracted from the transformed soybean seeds was separated by SDS-PAGE and stained with Rapid CBB KANTO (manufactured by Kanto Chemical Co., Inc.) (FIG. 1). In addition, after reacting with a specific antibody against Arserine 2 (obtained from Hokkaido National Agricultural Research Center, National Agriculture and Food Research Organization), ECL Advance Western Blotting Detection Kit (manufactured by GE Healthcare Bioscience Co., Ltd.) was used. Detection was performed. The chemiluminescence image was taken in with LAS4000miniPR (Fuji Film Co., Ltd.) and then analyzed using the analysis software MultiGage attached to the same machine (FIG. 2). As a control, the whole protein extracted from the seed of wild beans (line number: G12866) from which the Arselin 2 gene was isolated and the soybean cultivar Jack which was not transformed was used.

  As a result, a signal band corresponding to the kidney bean arserin 2 storage protein was confirmed in each line of the transformed soybean seeds obtained above, and a high concentration accumulation of the arserin 2 storage protein in the soybean seeds was confirmed. When comparing lanes, the expression level of Arserin 2 storage protein varies, but it is a common phenomenon that in transgenic plants, the expression level of the transgene varies from individual to individual. This is the common technical knowledge of contractors. In FIG. 1, the presence of the arserin 2 protein in the transformed soybean is clearly recognized by CBB staining after SDS-PAGE. Usually, in plants, proteins that are expression products of introduced foreign genes. Is difficult to detect as a major band by protein staining after SDS-PAGE, it can be understood that the accumulated amount of the arserin 2 protein in this example is extremely large.

  From the above results, it was shown that the promoter of the arserin 2 gene highly expresses the target gene linked downstream thereof in soybean seeds and causes accumulation of the expressed protein in the seeds.

  The present invention makes it possible to produce and accumulate useful proteins such as vaccines, antibodies and antibiotics in the seeds of plants, which are the original storage organs, and to enable efficient and stable supply of pharmaceutical raw materials and the like. Become.

Claims (7)

DNA containing the base sequence shown in the following (A), (B) or (C):
(A) the base sequence of SEQ ID NO: 1,
(B) a base sequence encoding a DNA having a seed-specific promoter activity, wherein one or several bases are deleted, substituted, inserted or added in the base sequence of SEQ ID NO: 1;
(C) A partial sequence of the base sequence described in (A) or (B) above, which encodes a DNA having seed-specific promoter activity.   A DNA comprising the base sequence set forth in base numbers 1399 to 3860 of SEQ ID NO: 1.   A vector comprising the DNA according to claim 1 or 2, a foreign gene encoding a foreign protein linked downstream of the DNA, and a DNA having terminator activity linked downstream of the foreign gene.   The DNA having the terminator activity is one in which one or several bases are deleted, substituted, inserted or added in the base sequence of SEQ ID NO: 2 or the base sequence of SEQ ID NO: 2, and has a terminator activity. The vector according to claim 3, comprising a base sequence encoding DNA.   A transformed plant into which the vector according to claim 3 or 4 is introduced.   A transformed seed into which the vector according to claim 3 or 4 has been introduced.   A method for producing a foreign protein, comprising expressing a foreign gene encoding a foreign protein in plant seeds under the control of the DNA according to claim 1 or 2.

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