KR20160057657A - Seed-specific promoter derived from oryza sativa and use thereof - Google Patents

Abstract

The present invention relates to a seed-specific promoter derived from Oryza sativa and uses thereof and, more specifically, to an Oryza sativa pre harvest sprouting 1 (OsPHS1) promoter which induces the specific expression of a target gene in embryos and scutellum tissues of mature seeds, to a recombinant expression vector including the promoter, to a method for manufacturing a transformed plant using the recombinant expression vector, and to a plant transformed by the method. According to the present invention, the OsPHS1 promoter for seed-specific expression in Oryza sativa is used: for controlling a target gene to be mainly expressed in embryos and scutellum tissues of mature seeds, thereby being usefully used for the trait improvement of crops such as the production of a seed-specific substance, dormancy, germination, growth control, etc.; and for gene function analysis of the target gene in mature seeds.

Description

Rice-derived seed-specific promoters and their uses {Seed-specific promoter derived from Oryza sativa and use thereof}

The present invention is rice (Oryza The present invention relates to a seed-specific promoter derived from Saliva sp. and its use, and more particularly relates to a seed-specific promoter derived from OsPHS1 ( Oryza), which induces expression specifically in embryo and scuttellum tissues of mature seeds sativa Pre Harvest Sprouting 1) promoter, a recombinant expression vector containing the promoter, a method for producing a transgenic plant using the recombinant expression vector, and a plant transformed by the method.

Recently, as the genetic engineering technology has developed, many researches have been carried out to improve the characteristics of plants. In particular, there is much interest in techniques for obtaining a useful gene from a transgenic plant. A promoter involved in expression of the gene is required when the desired gene is expressed in a transgenic plant.

In general, a promoter is a kind of regulatory region having a function of regulating the expression of a nucleotide sequence, that is, a gene, which determines when and where a gene is to be expressed. Numerous types of promoters have been identified through endless research. Conventionally reported promoters can be classified according to their features and functions such as a promoter that induces expression at all times and a promoter that induces expression in time or position specific manner.

More specifically, the first is a strong promoter capable of maximizing the expression level. This was initially developed as a promoter that is used to maximize the expression level of the target gene, and is representative of the 35S RNA gene promoter (P35S) of flower cabbage mosaic virus and the actin gene promoter of rice. They are mainly used for the purpose of production of antibiotic resistance genes used as selection markers in the transformation of plants, genes for inhibiting insect pests of plants, or plants.

Second, promoters that limit the time of expression can be mentioned. This has been reported as a promoter that restricts the expression of a gene of interest only to a specific stage of the developmental stage of the plant. For example, promoters of genes involved in flowering can express genes only during the flowering season, and promoters of genes expressed by various biotic or abiotic stimuli can be included in the timing-regulated promoters.

Third, tissue-specific promoters that limit expressed tissue are examples. This is the promoters that are used to achieve the transfection purpose by limiting the expression of the desired gene only to specific tissues of the plant. A number of examples of promoters inducing such tissue-specific expression have been reported for each tissue of a plant according to various purposes to be used.

In particular, the promoters inducing tissue-specific expression among the promoters classified according to functions and characteristics are classified as follows according to the tissues expressed in the plant.

First, a systemic expression-inducible promoter can be mentioned. As a promoter that induces expression in plant whole body, a promoter of 35S RNA gene of cauliflower mosaic virus (CaMV), which is also a promoter used to maximize expression amount, is used as a representative promoter for a dicotyledonous plant. Promoters of rice actin and maize ubiquitin genes have been mainly used as promoters for systemic expression of camphor plants. In recent years, promoters of rice cytochrome C gene (OsOc1) have been developed 0429335). These genes are used for the purpose of inducing the expression of antibiotics, herbicide resistance genes and reporter genes used as selection markers in the transformation as in the case of the promoters maximizing the expression level. From the viewpoint of research, These are the promoters that are considered to be the priority when attempting to reveal.

Second, seed-specific promoters can be mentioned. As a representative example, the rice glutelin promoter used for the development of golden rice as promoters of rice major storage protein gene is widely used to induce seed-specific expression of monocotyledonous plants. Promoters that are mainly used to induce seed-specific expression include soybean-derived lectin promoter, cabbage-derived napin promoter and γ-tocopherol methyl transferase (γ-TMT) in Arabidopsis seeds. There are carrot-derived DC-3 promoter and perilla derived oleosin promoter used in studies to promote vitamin E production by inducing gene expression.

Third, root-specific expression promoter has not yet been commercialized. However, root-specific expression of pyruvate peroxidase (prxEa) has been isolated and confirmed recently. In recent years, the expression of maz genes (ibMADS) It has been reported that the ADP-glucose pyrophosphatase (AGPase) gene is isolated and the corresponding promoter induces a specific expression in the root and induces root-specific transient expression in carrot and radish .

Fourthly, there are other tissue specific promoters such as leaves. The related promoters include ribosyl bisphosphate carboxylase / oxygenase small subunit (rbcS), which induces expression of strong genes only in photosynthetic tissues of leaves and the like. ) Promoter, tomato-derived fruit maturation-specific expression-inducing phytoene synthase (PDS) promoter, and pollen-specific plant-derived cabbage-derived oleosin promoter.

Such seed, root or leaf-specific expression promoters are expected to be used for the purpose of improving agricultural traits, accumulating useful proteins, and producing useful substances in major crops used for food, food, or food.

However, conventional promoters known to induce tissue-specific expression in the known promoters, particularly plants, have the advantage of being capable of expressing a target gene in a tissue-specific manner, but have a disadvantage in that the expression amount thereof is insignificant. Therefore, it is urgent to find useful new promoters capable of expressing a gene of interest in a plant in a tissue-specific and large-scale expression.

Under these circumstances, the inventors of the present invention analyzed the mutants showing the herbal phenotype of the rice seeds in order to develop a new promoter capable of expressing the target gene in a tissue-specific manner in the plant, and as a result, The OsPHS1 promoter which can be expressed in the embryo of the seeds and in the blastocysts was separated, thereby completing the present invention.

It is an object of the present invention to provide a promoter that specifically expresses a target protein in the embryo and blastocyst of mature seed of rice, an expression vector containing the promoter, and a transformant transformed with the expression vector.

Another object of the present invention is to provide a method for producing the transformant.

It is still another object of the present invention to provide a method for inducing expression of a target protein using the promoter and a vector.

As one embodiment for achieving the above object, the present invention provides a promoter that specifically induces expression in embryo or scutellum tissue of rice seed mature seed having the nucleotide sequence shown in SEQ ID NO: 1 do.

The term " embryo "in the present invention means a part where the seed germinates and becomes a young plant, which matures at a later stage to become a leaf, a stem, and a root. The term" scutellum " It is the part that has the enzyme that ages the plant.

The term "promoter" in the present invention is a genome region linked to the upper side of a structural gene, and regulates the transcription of the structural gene linked thereto to mRNA. Promoters are activated by the binding of several common transcription factors, such as the TATA box that controls gene expression, the CAAT box site, the site that affects gene expression in response to external stimuli, And an enhancer that promotes the expression of almost all of the genes regardless of the gene expression. Since proteins necessary for basic metabolism of living organisms must maintain a constant concentration in cells, the promoter linked to these genes is always activated by the action of common transcription factors. On the other hand, proteins that do not have a role in normal times and require functions only under specific circumstances are connected to a tissue specific promoter or an inducible promoter that induces the expression of the structural gene. In other words, tissue specific promoters are activated by the binding of specific transcription factors activated by external stimuli from environmental factors from the environment in the developmental process of the organism.

The promoter of the present invention is a promoter that specifically induces expression in a specific region of a plant, particularly in embryo and scutellum tissues of mature seed, and has a nucleotide sequence of SEQ ID NO: 1, but more than 80% Preferably 90% or more homology, more preferably 95% or more homology, even more preferably 98% or more homology, and most preferably, 99% or more homology. As well as bases having the same or corresponding biological activity. In addition, it is obvious that the promoter having a base sequence in which some sequences are deleted, modified, substituted or added is also included in the scope of the present invention if it is a base sequence having substantially the same or corresponding biological activity as a sequence having such homology.

In the present invention, the term " homology "refers to a degree of similarity to a wild-type nucleotide sequence, and may include a nucleotide sequence of the present invention having the same sequence as the above-mentioned nucleotide sequence. A commercially available computer program can calculate the homology between two or more sequences as a percentage (%), and the homology (%) is the number of sequences in an adjacent sequence / RTI >

The promoter of the present invention can express the target protein or the target gene RNA in the splenic tissue derived from the embryo of the rice seed, the blastocyst or the blastocyst, more preferably the target protein or the target gene RNA in the dividing tissue derived from the embryo, Protein can be expressed.

Specifically, in one embodiment of the present invention, rice transformed with a recombinant expression vector containing the OsPHS1 promoter of the present invention showed that the target protein operably linked to the promoter was a ribosomes, , And was specifically expressed in the mitotic cells derived from the seed embryo (Fig. 5).

The tissue specific promoter of the present invention may serve as a promoter of a conventional recombinant expression vector.

In another embodiment, the present invention provides a recombinant expression vector comprising a foreign gene encoding a target protein operably linked to the OsPHS1 promoter having the nucleotide sequence shown in SEQ ID NO: 1.

As used herein, the term "recombinant expression vector" refers to a gene construct that contains an essential regulatory element operably linked to express a desired protein or RNA of interest in a host cell, such that the gene insert is expressed.

The recombinant expression vector of the present invention may contain a promoter of the present invention and a plasmid, virus, and the like known in the art into which a nucleotide sequence encoding a target protein operably linked to the promoter can be inserted or introduced Or other media. The nucleotide sequence encoding the plant-specific expression promoter of the plant according to the invention and the target protein operably linked to the promoter may be operably linked to an expression control sequence, wherein the operably linked gene sequence and the expression control sequence May be included in an expression vector that also contains a selection marker and a replication origin.

Said "operably linked" may be a gene and expression control sequence linked in such a way as to enable gene expression when a suitable molecule is coupled to an expression control sequence. "Expression control sequence" means a DNA sequence that regulates the expression of a polynucleotide sequence operably linked to a particular host cell. Such regulatory sequences may include a promoter for carrying out transcription, any operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence controlling the termination of transcription and translation.

The expression vector according to the present invention may be prepared by inserting a promoter of the present invention using a conventional vector used for protein expression as a basic framework and inserting a nucleotide sequence encoding a target protein downstream of the promoter have. Therefore, plasmids derived from Escherichia coli (pBR322, pBR325, pUC118 and pUC119), Bacillus subtilis -derived plasmids (pUB110 and pTP5), yeast-derived plasmids (YEp13, YEp24 and YCp50 ) And a Ti plasmid, and may be a plant virus vector, and binary vectors such as pCHF3, pPZP, pGA and pCAMBIA sequences may be used. However, any person skilled in the art can use any vector as long as it can introduce the promoter of the present invention and the base sequence encoding the target protein operably linked to the promoter into the host cell.

The target protein that can be used in the present invention may be any target protein derived from the outside, that is, an exogenous protein or an endogenous protein and a reporter protein, which is desired to be expressed specifically by a plant tissue by the promoter of the present invention. The exogenous protein refers to a protein that does not naturally exist in a specific tissue or cell, and the endogenous protein refers to a protein that is expressed by a gene naturally present in a specific tissue or cell. In addition, the reporter protein is a protein expressed by a reporter gene, and refers to a marker protein that shows its activity in a cell by its presence.

In addition, the expression vector of the present invention may preferably comprise one or more selectable markers. The marker may be any gene capable of distinguishing a transfected cell from a non-transfected cell, typically a nucleic acid sequence having a selectable characteristic by a chemical method. Examples include herbicide resistance genes such as glyphosate or phosphinotricin, antibiotics such as G418, kanamycin, Bleomycin, hygromycin, cholramphenicol, Resistant genes, but are not limited thereto.

Specifically, in one embodiment of the present invention, the OsPHS1 promoter of the present invention having the nucleotide sequence of SEQ ID NO: 1 is inserted into a known vector having a reporter system in which a blue coloring gene, Gus, The recombinant expression vector, pBGWFS7-pOsPHS1, possibly linked, was prepared (Example 2-1 and Fig. 3).

In another embodiment, the present invention provides a transformant transformed with a recombinant expression vector comprising the OsPHS1 promoter of the present invention.

The term "transformation" in the present invention means genetically changing the trait of an individual or a cell by DNA, which is a genetic material given from the outside. In the present invention, by the introduction of the promoter shown in SEQ ID NO: 1 Means that the gene of interest is specifically expressed in a tissue-specific manner, particularly in a mature seed embryo or a blastocyst.

Transformation with the recombinant expression vector of the present invention in the present invention can be carried out by a transformation technique known to a person skilled in the art. For example, microprojectile bombardment, particle gun bombardment, silicon carbide whiskers, sonication, electroporation, PEG-mediated fusion PEG-mediated fusion, microinjection, liposome-mediated method, In planta transformation, Vacuum infiltration method, floral meristem dipping method, Agrobacterium sp. mediated method, and the like, but the present invention is not limited thereto.

The term "transformant" in the present invention means a host cell transformed with a recombinant expression vector comprising a promoter of the present invention and a base sequence encoding a target protein operably linked to the promoter.

The transformant is preferably a microorganism, including, but not limited to Escherichia coli), Bacillus subtilis (Bacillus subtilis), may be a Streptomyces (Streptomyces), Pseudomonas (Pseudomonas), Proteus Mira Billy's (Proteus mirabilis), Staphylococcus (Staphylococcus) and Agrobacterium Tome Pacific Enschede (Agrobacterium tumefaciens), and more preferably Agrobacterium tumefaciens . ≪ / RTI >

Specifically, in one embodiment of the present invention Agrobacterium tumefaciens (Agrobacterium tumefaciens LBA4404 was used (Example 2-2).

In another embodiment, the present invention provides a transgenic plant by introducing into a plant a recombinant expression vector comprising a nucleotide sequence encoding a OsPHS1 promoter and a promoter operably linked to the promoter of the present invention.

The transformed plant according to the present invention can be obtained by a conventional method in the art, such as an ovine reproduction method or an aseptic reproduction method. More specifically, the plant of the present invention can be obtained through reproductive process, which is a process of producing seeds through the moisture process of flowers and propagating from the seeds. Also, the plant can be transformed with the recombinant expression vector according to the present invention, and then obtained by a conventional method, such as induction, rooting and soil purification of the callus. That is, a fragment of a plant transformed with the recombinant expression vector of the present invention is pelleted in a suitable medium known in the art, and cultured under appropriate conditions to induce callus formation. When shoots are formed, the plant is transferred to a hormone-free medium Lt; / RTI > After about two weeks, the shoots are transferred to rooting medium to induce roots. The transformed plants according to the invention can be obtained by roots being induced and then transplanted into the soil for purification. Transgenic plants in the present invention may include whole plants as well as tissues, cells or seeds obtainable therefrom.

Specifically, in one embodiment of the present invention, Agrobacterium ( Agrobacterium tumefaciens), which is a microorganism transformation transformed with a recombinant expression vector containing OsPHS1 promoter tumefaciens ) LBA4404 in rice ( Oryza sativa ) to produce transgenic plants (Example 3-1).

In another aspect, the present invention provides a method for producing a transgenic plant in which a target protein is specifically expressed in rice embryo or blastocyst tissue, comprising the following steps.

a) preparing a recombinant vector comprising a promoter consisting of the nucleotide sequence of SEQ ID NO: 1 and a foreign gene encoding a target protein operably linked thereto;

b) As the recombinant expression vector, Agrobacterium tumefaciens ) to produce transformed Agrobacterium; And

c) infecting the plant with the transformed Agrobacterium.

In the present invention, the plant may be a dicotyledonous plant or a monocotyledonous plant. It may be, but not limited to, a monocotyledonous plant, more preferably rice, wheat, barley or corn, Rice ( Oryza sativa .

In another aspect, the present invention provides a method for inducing the expression of a target protein in the embryo or blastocyst of a rice seedling.

The OsPHS1 promoter for expression of the target protein in the splenic tissue derived from the embryo of the rice seed, the blastocyst derived from the embryo or the blastocyst including the nucleotide sequence of SEQ ID NO: 1 according to the present invention, and the structural gene encoding the target protein are operably linked the present invention can provide a method for inducing expression of a target protein in a splenic tissue derived from a seed of a rice seed, a blastocyst or a blastocyst, by transforming rice with an operatively linked recombinant expression vector.

The target protein may be any kind of protein. For example, the target protein may be a protein capable of accumulating a large amount of nutrients that can be used for medical purposes such as enzymes, hormones, antibodies, cytokines, etc., , But is not limited thereto. Examples of the target protein include interleukin, interferon, platelet-derived growth factor, hemoglobin, elastin, collagen, insulin, fibroblast growth factor, human serum albumin and erythropoietin.

When the promoter of the present invention is used, in the case of a crop consuming a specific part of the plant, for example, a seed, a leaf or a stem, a useful protein can be expressed in a seed of a plant, Can be developed.

The OsPHS1 promoter specifically expressed in the rice seeds of the present invention can be used to regulate expression of the target gene in the embryo and blastocyst of the mature seed, thereby improving the production of seed specific substances, dormancy, germination, Not only can it be usefully used for trait improvement, but it can also be useful for analysis of target gene function in mature seeds.

1 is a diagram showing the base sequence of the OsPHS1 promoter of the present invention.
2 is a diagram showing a motif associated with seed germination found in the OsPHS1 promoter of the present invention.
3 is a schematic diagram showing an expression vector of the OsPHS1 promoter for rice transformation produced through a gateway system.
FIG. 4 is a diagram showing the result of genome PCR analysis of the transgenic rice of the present invention. FIG.
FIG. 5 is a graph showing the results of GUS staining analysis of transgenic rice plants into which the OsPHS1 promoter of the present invention was inserted.

Hereinafter, the constitution and effects of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example  1: genome DNA of PCR On by OsPHS1  Of the promoter region Cloning  And base sequence analysis

Example  1-1: OsPHS1  Of the promoter region PCR  Amplification

Total RNA was isolated from rice seedlings mutant seeds distributed by the RDA and analyzed for gene expression pattern by QRT-PCT method to select OsPHS1 ( Oryza sativa Pre Harvest Sprouting 1) gene. In order to isolate the promoter region of the selected OsPHS1 gene, nucleotide sequence information of the nucleotide sequence of 2022kb at the start of translation was extracted from a rice genome database, and a primer for amplifying the fragment was designed, and forward primers (forward primer OsPHS1P-F: 5'-CACCCAGACTACAGTCGTCATCCAGAATATGC-3 '(SEQ ID NO: 2) and reverse primer OsPHS1P-R: 5'-GGCCGGCGCGCGCGCTCTCCGGAGTTCAATTGG-3' (SEQ ID NO: 3). Using the primer set, the genomic DNA of Dongjin rice was used as a template, and the gene was amplified using a Applied Biosystem 9700 machine in a PCR reaction solution together with a Primestar polymerase (Takara). The reaction conditions for PCR were denaturation at 94 ° C for 5 minutes, followed by 40 cycles of 94 ° C for 30 seconds, 57 ° C for 40 seconds, and 72 ° C for 2 minutes, Min. After completion of the PCR reaction, the amplified gene was electrophoresed on 1% agarose, and the band predicted to be the OsPHS1 promoter was cut out, purified using a QIAquick gel extraction kit, and then used for cloning.

Example  1-2: OsPHS1  Of the promoter region Cloning ( cloning ) And base sequence analysis

The PCR product of the OsPHS1 promoter region amplified in Example 1-1 was cloned into a gateway donor vector (pENTER / D-TOPO, Invitrogen) and the whole nucleotide sequence was determined. Thus, the nucleotide sequence of SEQ ID NO: 1 OsPHS1 promoter gene (Fig. 1).

Example  1-3: OsPHS1  Motif of promoter region ( motif ) Search

To confirm the regulatory factors present in the OsPHS1 promoter gene obtained in Example 1-2, a PLACE (Plant Cis-acting Elements database: http://www.dna.affrc.go.jp/PLACE/) program Were used to analyze gene expression motifs. As a result, as shown in Fig. 2, it was confirmed that seed or papillary reaction motif (GLMHVCHORD or DPBFCOREDCDC3) was present and seed germination such as amylase box, GARE (GA responsive element) and ABRE (ABA responsive element) There is also a motif that responds to hormones involved in regulation. Through this, it was found that the OsPHS1 promoter could induce expression specifically in seeds and could participate in seed germination, dormancy and growth regulation.

Example  2: Transformation vector ( vector ) And transformant production

Example  2-1: OsPHS1  Production of Promoter Carrier

A recombinant expression vector was constructed to express the reporter gene GUS using the OsPHS1 gene promoter isolated in Example 1-1 .

Specifically, a recombinant vector in which the promoter was inserted was constructed in the pBGWFS7 vector (PSB, Plant System Biology) for expression of the gateway promoter (Fig. 3).

Example  2-2: Agrobacterium ( Agrobacterium tumefaciens ) Production of Transformants

The OsPHS1 promoter expression vector prepared in Example 2-1 was transformed into Agrobacterium tumefaciens (LBA4404) using the freeze-thaw method.

Specifically, YEP medium (10 g of Yeast, 5 g of NaCl, 10 g of peptone, 15 g of Agar, and 10 g of Agar) was added to the medium after transfection by freeze and thaw two to three times, followed by heat shock at 37 캜. 1 L) overnight to identify colonies. Colony transformed by colony PCR was transformed into AB medium (AB buffer (K ₂ HPO ₄ 60 g, NaH ₂ PO ₄ 20 g / 1 L), AB Salts (NH ₄ Cl 60 g, MgSO ₄ 7H ₂ O 6g, KCl 3g, CaCl ₂ 2H ₂ O 0.265g, FeSO ₄ .7H ₂ O 50mg / 1L), Glucose 5g / 1L).

Example  3: Construction and analysis of rice plant transformants using Agrobacterium

Example  3-1: Construction of transgenic rice plants using Agrobacterium

Agrobacterium LBA4404 into which OsPHS1 promoter expression vector prepared in Example 2-2 was introduced was inoculated into a callus of Dongjinbyeon and infected. Then, 6 ㎍ / ml of PPT (phosphinothricin, phosphinotricin) L, and finally the selected transformants were grown in a greenhouse to produce transformed rice.

Specifically, transformation of rice with Agrobacterium was carried out by washing the seeds in a lactose to induce the callus of Dongjinbyeong in 2N6 medium, drying it appropriately, and allowing the 2,4-D hormone 2 to be added to N6 medium (Duchefa vitamin-containing medium) mg / L. < / RTI > The embryogenesis callus was subcultured in 2N6 fresh medium after inducing callus by incubation at 27 DEG C for 3 to 4 weeks. Agrobacterium-transformed genes containing the transgene were cultured in AB liquid medium, co-cultured with embryogenic calli for 20 minutes, and then cultured for 3 days. Cefotaxime was added to the sterilized water to wash the Agrobacterium until the Agrobacterium was completely removed. Subsequently, the cells were washed with 2N6 medium supplemented with 6 ㎍ / L of cefotaxime and PPT (phosphinothricin) For 3 weeks. The callus surviving without browning in 2N6 medium supplemented with cefotaxime and PPT was transferred to 2N6 medium supplemented with cefotaxime and PPT and cultured for 2 weeks. The shoots were transferred to MSR medium (containing maltose and sorbitol in Duchefa MS medium) supplemented with cefotaxime and PPT for induction of shoots in the callus and cultured for 4 weeks. , Purification treatment was carried out prior to transfer to the greenhouse.

Example  3-2: OsPHS1  Analysis of promoter-inserted rice plant transformants

In order to confirm whether the OsPHS1 promoter expression vector was correctly inserted into the transgenic rice produced using the Agrobacterium transformant into which the OsPHS1 promoter expression vector was introduced in Example 3-1, Genomic DNA of a rice plant transformant into which an OsPHS1 promoter was introduced was isolated and subjected to PCR analysis.

Specifically, the genomic DNA was extracted from the leaves of transgenic rice plants into which the OsPHS1 promoter was inserted using an Inclone Genomic DNA prep kit (Inclone Cat # IN1003-0200), and a portion GUSA-anti) and the promoter portion (OsPHS1P-5) were designed and synthesized. The genomic DNA extracted using the synthesized primer set GUSA-Anti: 5'-CGCGATCCAGACTGAATGCCC-3 '(SEQ ID NO: 4) and OsPHS1P-5: 5'-GCCATGGCTTGAAGATAGGTTGCC-3' (SEQ ID NO: 5) And PCR was performed. At this time, the PCR was performed by heating the PCR amplification conditions at 94 ° C for 5 minutes, and then heating the reaction mixture at 94 ° C for 40 seconds, 60 ° C for 40 seconds, and 72 ° C for 40 seconds. Then, it was incubated at 72 캜 for 7 minutes Heating was carried out. After the PCR reaction, the amplified PCR products were developed on a 1% agarose gel using an electrophoresis apparatus and the band of the reaction product developed under UV was confirmed. As a result, as shown in FIG. 4, the PCR amplification product was not observed in Dongjinbang, which is the wild type (WT) used as a control, whereas the PCR amplification product band was clearly observed in the transgenic rice with the OsPHS1 promoter expression vector inserted therein I could confirm that it appeared. As a result, it was found that the OsPHS1 promoter expression vector was correctly inserted into the transgenic rice produced using the Agrobacterium transformant into which the OsPHS1 promoter expression vector was introduced.

Example  4: OsPHS1  Promoter GUS  Analysis of Expression by Dyeing

In order to test the expression of the OsPHS1 promoter, the transgenic rice in which OsPHS1 promoter insertion was confirmed in Example 3-2 was analyzed by GUS protein staining. At this time, the site where the OsPHS1 promoter was activated was stained blue by GUS staining .

As a result of the analysis of the expression of the promoter, as shown in FIG. 5, GUS staining was not observed in rice leaves grown in the packaging for 2 months, but it was confirmed that the staining was strong in mature seeds. Especially, it was confirmed that endosperm was hardly stained even in seeds, and stained specially and strongly only in embryo and scutellum.

In addition, when mature seeds of transgenic rice plants were exposed to 2N6 medium, callus was induced in the blastocysts. On the other hand, almost no GUS staining was observed in most of the reproductive organs, such as unmatured rice seeds, stamens, and pistils, and GUS staining was also observed in seeds of early development (2 days after flowering, 2 DAF) Respectively. In addition, GUS staining was not observed in most vegetative organs such as stem, leaf, and root of seedlings grown for 2 weeks in transgenic rice plants transfected with the OsPHS1 promoter. From these results, it was found that the OsPHS1 promoter is able to express the target gene mainly in the callus tissue derived from the embryo of the mature seed, the blastocyst, and the blastocyst from the rice plant, which is a typical monocotyledonous plant.

LB: left T-DNA border
Bar: bialaphos resistance gene
35S: cauliflower mosaic virus (CaMV) 35S promotor
attB1: gateway attB1
pOsPHS1: OsPHS1 ( Oryza sativa Pre Harvest Sprouting 1) promoter
attB2: gateway attB2
eGFP: enhanced green fluorescence protein
GUSA: beta-glucuronidase A
T35S: CaMV 35S terminator
RB: right T-DNA border

<110> Republic of Korea <120> Seed-specific promoter derived from Oryza sativa and use thereof <130> P14R12D1320 <160> 5 <170> Kopatentin 2.0 <210> 1 <211> 2022 <212> DNA <213> Artificial Sequence <220> <223> OsPHS1 promoter <400> 1 cagactacag tcgtcatcca gaatatgctg aacggcaagt cttcgaattg aatacgtata 60 gttgaagttg ttcttttctc caatagagca tggggtcaat tccctgcgtg ttctctgata 120 gagtagaaaa gaagtggtta cgttactgct taatcagggt tttaacaaag ctgatggaaa 180 ccgctcggtt ttgcacaaat cggttaagta aaaaaaaaac tcaattttga caaaatttag 240 tcgaatttca caagtattaa ttcagctatt tcgtgaaaac taagaaaact gctctggagt 300 ttttgaaggc gtttttttta gagtcaatcc attttatgac atcgacaaaa cgcgggttct 360 acaatatgcc actgacaaaa ctcagttcca cactatacca tcgaatgact cattttcttc 420 cttatatgcc atcgacgtta gtcaaccgcc gttaaaagat gaaaaatgac cattttaccc 480 ctagcataaa attgagattt tgcataatgc catatcgcgg taaaaaaatc ttggatgata 540 aaaagacaag caatttcaat ttttcatcaa tgagaataaa tagtgaatca tgatcagtga 600 aatcgatgag gatgcatcgt accctagggg taaaatagac atttttttgc tcttaacaga 660 atttgactaa ctgttgactg gcggcgatgg catgtaagga agaaaagtag ttgttcgatg 720 gtatattata gaactgagct ttatcaatgg tatattgttg aatccatgct ttgtcaatgg 780 catagaatgg attatctctt tttttttaac cctgctgctt actccctcca tcctataaaa 840 aaccaaccta atactggatg tgatacatcc tagtactact aatctggaca tacctctgtc 900 cagattcatt gtactagaat gcgtcacatc caattctaga ttcgtttttt tggacggagg 960 gagtacatct ttgttaacat aatctgatgt gagactggtc accaaatata tatatttgca 1020 cttcctgaat tttgtgtgct ctctttgtgg gtagtgttac gtgctattta ttgagcggat 1080 atttgtaata agtggttgta acttctttga agtaaaggtc gagctttatt ctatctaaaa 1140 atatatttgg tcactaaata attgtacgaa catatacagg agaaagtcaa cactagcaac 1200 tttataattg ccacgagtgt caactgtacg tacacaatga cagtaatgat tgtggcagtc 1260 tattgtttat aacgcactac tgtgctagct gtatagaata cgtatctata tacctcgagt 1320 ttacgtatta tacacctcaa atacttgatt tggtgacata tactgtacca gtttgatcca 1380 ctctctttct gaagggagac gggcaatttt tcgttagacg atgacggcga cggagaggtg 1440 gtctctatac gcacgcacat acataagcgt gtgtatattc acggatttag tcgagttttt 1500 tctatgtctt cttttgattc accaaacgaa gcattggtcc ttttgaaatt agcaaggggg 1560 taatcattta ggtgagccat tagggcgctt gaagggtcac ttatgaatat agtacaatca 1620 tgctcctaat acacacgcta ctcttgattt gcaggaacac tgtcggaaac tgcacagtat 1680 ttcttgattt gcgaatggta gagtttacta cacatgtggc aacaccaggg attaatctac 1740 ctctatcact gtcctgactg aatactcgag acacctcctc ttgaaacatt cagaactcga 1800 ggtaattaag ctgaatgagt actattatat cgataaaaca atggattgcc atggcttgaa 1860 gatgagttgc cggcaattga gggacctgat cctgcgcgac gcgacgcccc agaagggatt 1920 tctctttgtt atccactgcc ccctctcgag tcgagataaa aaaccacatg ccgcttgccg 1980 tcttcctgcc caattgaact ccggagagcg cgcgcgccgg cc 2022 <210> 2 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> OsPHS1P-F <400> 2 cacccagact acagtcgtca tccagaatat gc 32 <210> 3 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> OsPHS1P-R <400> 3 ggccggcgcg cgcgctctcc ggagttcaat tgg 33 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GUSA-Anti <400> 4 cgcgatccag actgaatgcc c 21 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> OsPHS1P-5 <400> 5 gccatggctt gaagataggt tgcc 24

Claims (8)

A promoter that specifically induces expression in embryo or scutellum tissues of rice seed mature seeds having the nucleotide sequence shown in SEQ ID NO: 1.
A recombinant expression vector comprising a foreign gene operably linked to the promoter of claim 1 and encoding a protein of interest.
A transformant transformed by the vector of claim 2.
4. The transformant according to claim 3, wherein the transformant is Agrobacterium tumefaciens .
A transformed plant transformed with the vector according to claim 2 or the transformant according to claim 3.
6. The method of claim 5, wherein the transgenic plant is selected from the group consisting of rice sativa ).
a) preparing a recombinant vector comprising a promoter consisting of the nucleotide sequence of SEQ ID NO: 1 and a foreign gene encoding a target protein operably linked thereto;
b) As the recombinant expression vector, Agrobacterium tumefaciens ) to produce transformed Agrobacterium; And
c) infecting the plant with the transformed Agrobacterium;
Wherein the target protein is expressed specifically in the embryo or blastocyst of rice seed mature seeds.
A method for inducing the expression of a target protein in the embryo or blastocyst of a rice seedling by transforming rice with the promoter of claim 1 and the vector of claim 2.

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