KR101825960B1 - Root―specific promoter derived from Oryza sativa and use thereof - Google Patents

Abstract

The present invention relates to a root-specific promoter derived from Oryza sativa and to uses thereof, and more specifically, to an OsGASA1 promoter, which specifically induces the expression of a target gene in an Oryza sativa seedling meristem, particularly, in root tissues, to a recombinant expression vector containing the promoter, to a method for producing a transgenic plant using the recombinant expression vector, and to a plant transformed by the method. By using the OsGASA1 promoter specifically expressed in the Oryza sativa seedling meristem particularly, in root tissues, it is possible to specifically express the target gene and to usefully use the same for growth regulation.

Description

Root-Specific Root-Specific Promoters and Their Uses {Root-specific promoter derived from Oryza sativa and use thereof}

The present invention relates to a root-specific promoter derived from rice ( Oryza sativa ) and a use thereof, and more particularly, to a method for promoting the expression of a OsGASA1 promoter which specifically induces expression in a cleavage structure of rice seedlings, A recombinant expression vector containing the recombinant expression vector, a method for producing a transformed 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 such background, the present inventors have developed a novel promoter capable of expressing a target gene in a tissue-specific manner in a plant, specifically, a cleavage structure of a rice seedling, particularly, a useful promoter capable of specifically expressing a target gene in root tissue As a result of intensive efforts, the present invention has been accomplished by isolating the OsGASA1 promoter capable of specifically expressing a target gene in root tissue in rice.

Korean Patent No. 0429335

It is an object of the present invention to provide a method for producing a transformant which comprises a promoter which specifically expresses a target protein in a fissured tissue of a plant, particularly a root tissue, an expression vector containing the promoter, a transformant transformed with the expression vector, And a method for inducing the expression of a target protein using the promoter and the vector.

In one aspect for accomplishing the above object, the present invention provides a promoter comprising a nucleotide sequence of SEQ ID NO: 1, which specifically induces expression in the root tissue of a plant.

The term "root" in the present invention refers to a root of a plant, usually an organ that is buried in the ground or put in another object to suck up water and nutrients and to support the stem, and belongs to a meristem. "Fragmented tissue" refers to tissue that divides into cells to produce new cells. Split tissue is the part that causes cell division and growth of the plant, and it belongs to stem or growing point at the end of root.

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 which specifically induces expression only in a specific part of a plant, particularly in a root tissue of a plant, and has a sequence having the nucleotide sequence of SEQ ID NO: 1 but having a homology of 80% or more with the above sequence, Or more homology, more preferably at least 95% homology, even more preferably at least 98% homology, and most preferably at least 99% homology to a sequence having substantially the same or corresponding biological activity Base. ≪ / RTI > 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 base sequence, and may include a sequence having the same sequence as the above-mentioned base sequence. This comparison of homology can be performed using a visual program or a comparison program that is easy to purchase. Commercially available computer programs can calculate homology between two or more sequences as a percentage, and homology (%) can be calculated for adjacent sequences.

The promoter of the present invention can express the target protein or the target gene RNA in the root tissue of the plant, and more preferably, can express the target protein specifically in the root tissue of the plant.

Specifically, in one embodiment of the present invention, rice transformed with the recombinant expression vector containing the OsGASA1 promoter of the present invention showed that the target protein operably linked to the promoter was the root tissue of the rice seedling, specifically the root of the root And the locus forming region (Figs. 6 and 7).

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 OsGASA1 promoter consisting of 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 OsGASA1 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-pOsGASA1, which was linked as much as possible, was prepared (Example 3-1 and Fig. 4).

In another aspect, the present invention provides a transformant transformed with a recombinant expression vector containing the OsGASA1 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 expressed specifically in tissues, particularly in root tissues of plants.

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 transformants, and microorganisms are preferred, but are not limited to, Escherichia coli (Escherichia coli), Bacillus subtilis (Bacillus subtilis), Streptomyces (Streptomyces), Pseudomonas (Pseudomonas), Proteus Mira Billy's (Proteus mirabilis ), Staphylococcus and Agrobacterium tumefaciens , and more preferably Agrobacterium tumefaciens . The term " Agrobacterium tumefaciens "

Specifically, in one embodiment of the present invention, Agrobacterium tumefaciens LBA4404 was used (Example 3-2).

In another embodiment, the present invention provides a transgenic plant by introducing into a plant a recombinant expression vector comprising a nucleotide sequence encoding an OsGASA1 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 the conventional method for inducing callus, rooting, and soil purification, through a silent propagation method. 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, one embodiment of the present invention, to recombinant expression transformed by the vector microorganism transformed chain, Agrobacterium tumefaciens (Agrobacterium tumefaciens) LBA4404 containing OsGASA1 promoter inoculated to rice (Oryza sativa) transgenic rice (Example 4-1).

In another aspect, the present invention provides a method for producing a transgenic plant in which a target protein is specifically expressed in the root tissue of a plant, 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 monocot plant. Preferably, the plant may be rice, wheat, barley or corn, and more preferably rice ( Oryza sativa ).

In another aspect, the present invention provides a method for inducing expression of a target protein in root tissues of rice seedlings.

The OsGASA1 promoter for target protein-specific expression in the root tissue of the rice seedlings containing the nucleotide sequence of SEQ ID NO: 1 according to the present invention, and the structural gene encoding the target protein are operatively linked A method of inducing the expression of a target protein in the root tissue of rice seedlings can be provided by transforming rice with a 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, a root or a stem, a useful protein can be expressed in a specific tissue of the plant, Can be developed.

Using the OsGASA1 promoter specifically expressed in the cleavage tissue of the rice seedlings of the present invention, particularly the root tissue, the target gene can be specifically expressed in the root tissue and can be used for growth control by using the gene.

1 shows microarray analysis results showing expression patterns of abscisic acid (ABA) -treated rice seedlings (A) and expression patterns of OsGASA1 gene (LOC_Os09g24840) in roots of rice seedlings after ABA treatment B).
FIG. 2 is a diagram showing the results of analysis of the expression pattern of OsGASA1 gene (LOC_Os09g24840) in rice by public DB analysis.
3 is a diagram showing the base sequence of the OsGASA1 promoter of the present invention.
FIG. 4 is a schematic diagram showing an expression vector of the OsGASA1 promoter for rice transformation produced through a gateway system.
5 is a diagram showing the result of genomic PCR analysis of the transgenic rice of the present invention.
FIG. 6 is a graph showing the results of GUS staining analysis of transgenic rice plants into which the OsGASA1 promoter of the present invention was inserted.
7 is a graph showing the results of assaying the expression of OsGASA1 promoter by the hormone response and GUS staining of transgenic rice plants in which the OsGASA1 promoter of the present invention was inserted. Here, DJ is a wild-type Dong-Jin rice, and T228 and T299 are transgenic rice plants in which the OsGASA1 promoter is inserted. DW (distilled water) was used as a control group in the experimental group treated with auxin.

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: Identification of Rice Genes Expressed in Roots and Expression Patterns by Tissue

Abscisic acid (ABA) is one of the plant hormones that is involved in the overall growth of plants. High concentrations of ABA inhibit plant growth and root development.

In order to select the genes specifically expressed in the roots, microarray analysis of roots treated with ABA was performed to select genes whose expression was suppressed by ABA in the root of rice, The expression level of the selected gene was analyzed by tissue.

Example  1-1: OsGASA1  Selection of genes

ABA is a major hormone that inhibits the growth of root and stem length in rice. ABA of 3 μM was treated with wild type Dongjin rice seedlings and then the phenotype of Dongjin rice seedlings was observed. As a result, the growth of roots and stem lengths of rice seedlings was compared with the control without ABA treatment (Fig. 1 (A)).

In addition, gene expression profiling was analyzed by microarray method in the roots of Dongjin rice which were treated with ABA for 6 hours (6h) and 48 hours (48h). As a result, among the genes affected by ABA treatment, GASA family transcription factors involved in signal transduction of gibberellic acid (GA), a hormone that is known to regulate length growth, are remarkably noted and remarkably expressed by ABA (LOC_Os09g24840), which is a GASA (Gibberellic acid-stimulated Arabidopsis ) gene, is suppressed (Fig. 1B).

Example  1-2: OsGASA1  Analysis of gene expression patterns in tissues

The expression pattern of the OsGASA1 gene selected in Example 1-1 was analyzed through public database (RiceXPro2, http://ricexpro.dna.affrc.go.jp/) analysis. As a result, As shown in FIG. 2, OsGASA1 gene is a relatively highly expressed gene in root than other tissues (FIG. 2).

Example  2: Genomic DNA In PCR  by OsGASA1  Of the promoter region Cloning  And base sequence analysis

In order to select a root-specific expression promoter regulated by the hormone in rice, the OsGASA1 gene (LOC_Os09g24840), which is inhibited by ABA in the root of rice, was selected in Example 1, and root-specific Expression promoter, and OsGASA1 promoter.

Example  2-1: OsGASA1  Of the promoter region PCR  Amplification

In order to isolate the promoter region of the OsGASA1 gene (LOC_Os09g24840) expressed in the root of rice, the nucleotide sequence information of 2,019 bp at the start of translation was extracted from the rice genome database, and primers for amplifying the fragment (SEQ ID NO: 2) and a reverse primer Os09g24840-PR: 5'-TTCTCTGAAGCTCCACTAACTGAAGA-3 '(SEQ ID NO: 3) were synthesized by designing a forward primer Os09g24840-PF: 5'-CACCGTCAGATGCAACCTCGGGAGTG- Respectively. The genomic DNA isolated from the leaves of Dong Jin-jin was used as a template and primer polymerase (Takara) was added to the PCR reaction solution using the synthesized primer set, and the gene was amplified using an Applied Biosystem 9700 machine. 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 assumed to be the OsGASA1 promoter was cut out and purified using a QIAquick gel extraction kit and then used for cloning.

Example  2-2: OsGASA1  Of the promoter region Cloning cloning and sequencing

The PCR product of the OsGASA1 promoter region amplified in Example 2-1 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 OsGASA1 promoter gene (Fig. 3).

Example  2-3: OsGASA1  Search for motifs in the promoter region

In order to confirm the reaction regulatory factors present in the OsGASA1 promoter gene obtained in Example 2-2, PLACE (Plant Cis-acting Elements database: http: // www. Dna.affrc.go.jp/PLACE/) and Gene expression motifs were analyzed using PlantCARE (http://bioinformatics.psb.ugent.be/webtoo ls / plantcare / html /) program.

As a result, it was confirmed that ABA reaction motif (ABRECE1HVA22), GA reaction motif (GARE2OSREP1), auxin reaction motif (ARFAT, CATATGGMSAUR) existed as shown in Table 1 below.

These results indicate that the OsGASA1 promoter can be involved in the regulation of root and length by confirming the presence of a hormone responsive motif involved in root growth of the rootstock.

Example  3: Construction of transformant vector and transformant

Example  3-1: OsGASA1  Production of Promoter Carrier

A recombinant expression vector was constructed to express the reporter gene GUS using the OsGASA1 gene promoter isolated in Example 2-1.

Specifically, a recombinant vector in which the promoter was inserted was constructed in pBGWFS7 vector (PSB company, Plant System Biology) for expression of a gateway promoter (Fig. 4).

Example  3-2: Agrobacterium ( Agrobacterium tumefaciens ) Production of Transformants

The OsGASA1 promoter expression vector prepared in Example 3-1 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  4: Production and analysis of rice plant transformants using Agrobacterium

Example  4-1: Construction of transgenic rice plants using Agrobacterium

Agrobacterium LBA4404 into which OsGASA1 promoter expression vector prepared in Example 3-2 was introduced was inoculated into a callus of Dongjinbyeo and infected. Then, 6 쨉 g / ml of PPT (phosphinothricin, phosphinotricin) L, and finally the selected transformants were grown in a greenhouse to produce transformed rice.

Specifically, in order to induce the callus of Dongjinbyeon in 2N6 medium, Agrobacterium-mediated transformation was carried out by washing the seeds in the lactose and drying them appropriately. The 2,4-D hormone was added to 2N6 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, in a 2N6 medium, a culture medium containing cefotaxime and PPT (phosphinothricin) at 6 / / L was added 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 cells were transferred to MSR medium (containing Maltose and Sorbitol in Duchefa MS medium) supplemented with cefotaxime and PPT for induction of shoots in the calli, and then cultured for 4 weeks. After rooting, rooting was performed prior to transfer to the greenhouse Treatment.

Example  4-2: OsGASA1  Analysis of promoter-inserted rice plant transformants

In order to confirm whether the OsGASA1 promoter expression vector was properly inserted into the transgenic rice produced using the Agrobacterium transformant into which the OsGASA1 promoter expression vector was introduced in Example 4-1, Genomic DNA of an OsGASA1 promoter-introduced rice transformant was isolated and subjected to PCR analysis.

Specifically, the genomic DNA was extracted from the leaves of transgenic rice plants into which the OsGASA1 promoter was inserted using an Inclone Genomic DNA prep kit (Inclone Cat # IN1003-0200), and an antibiotic gene Were designed and synthesized. PCR was performed using the extracted genomic DNA as a template using PPT5: 5'-GGTCTGCACCATCGTCAACC-3 '(SEQ ID NO: 4) and PPT3: 5'-TCAGATCTCGGTGACGGGCA-3' (SEQ ID NO: 5) Respectively. At this time, the PCR was performed under the conditions of PCR amplification for 5 minutes at 94 ° C., followed by heating at 94 ° C. for 30 seconds, at 57 ° C. for 30 seconds, and at 72 ° C. for 30 seconds, After that, it was further heated at 72 캜 for 5 minutes. 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. 5, the PCR amplification product was not observed in the wild type DJ used as the control group, whereas the transgenic rice (15YS227, 15YS228, 15YS229) inserted with the OsGASA1 promoter expression vector ), It was confirmed that the product band of PCR amplification was distinct.

These results indicate that the OsGASA1 promoter expression vector was correctly inserted into the transgenic rice produced using the Agrobacterium transformant into which the OsGASA1 promoter expression vector was introduced.

Example  5: OsGASA1  Expression analysis by GUS staining of promoter

In order to test the expression of the OsGASA1 promoter, seeds of transgenic rice in which the OsGASA1 promoter insertion was confirmed in Example 4-2 were germinated for 7 days, and the roots of the seedlings were analyzed by GUS protein staining. At this time, the tissue was clarified to observe the stained area, and the site where the OsGASA1 promoter was activated was stained blue by GUS staining.

As a result of the analysis of the expression of the promoter, it was confirmed that GUS staining was strongly stained at the root and anterior root formation positions of the root of the rice seedlings germinated for 7 days as shown in Fig.

Example  6: OsGASA1  Hormonal Response of Transgenic Transgenic Rice Plants Embedded with Promoters and GUS Staining OsGASA1  Tissue expression assay of promoter

Auxin is a hormone that regulates cell division, kidney, and differentiation. For the expression of the OsGASA1 promoter and the expression of the OsGASA1 promoter, the transgenic rice seeds in which OsGASA1 promoter insertion was confirmed in Example 4-2 were transformed with PPT (phosphinothricin) In the included 1/2 MSO-Agar medium Germinated and grown for 7 days. The seedlings grown for 7 days were cultured in a solution containing oxalate (IAA, Indole acetic acid) for 16 hours, and the roots of the seedlings were analyzed by GUS protein staining. At this time, the tissue was clarified to observe the stained area, and the site where the OsGASA1 promoter was activated was stained blue by GUS staining. On the other hand, the control group treated distilled water (DW) instead of auxin in the seeds of the transgenic rice plants.

As a result of analysis of the expression of the promoter, auxin, which is a hormone that promotes growth and cell division, was treated. As shown in FIG. 7, GUS expression by auxin treatment in the cell division zone of roots Which is a significant increase.

These results indicate that the OsGASA1 promoter can express the target gene in the rice plant, which is a typical monocotyledonous plant, in the cleavage structure of rice seedlings, specifically in the apical and anterior roots of roots, and the regulation of the concentration of oxine, It is possible to induce the expression more strongly at a specific position.

SmSpR: spectinomycin and streptomycin resistant
LB: left T-DNA border
Bar: bialaphos resistance gene
attR1: gateway attR1
OsGASAP: OsGASA1 promoter
attR2: gateway attR2
Egfp: enhanced green fluorescence protein
gus: beta-glucuronidase A
T35S: CaMV 35S terminator
RB: right T-DNA border

<110> Republic of Korea <120> Root-specific promoter derived from Oryza sativa and use thereof <130> P16R12D1629 <160> 5 <170> KoPatentin 3.0 <210> 1 <211> 2019 <212> DNA <213> Artificial Sequence <220> <223> OsGASA1 promoter <400> 1 gtcagatgca acctcgggag tgaaggcttg ctggatctgg ggcatccatg cctagatccg 60 gtagctccct ggctcggtta gtgatgatag cagctccatt ttattagaga atgccggtgg 120 caatggtgca cgaaagtagg tcacctcgac gtgttgcatt gggctagggc ggttgcaaac 180 acacatgcga gcgagcacaa ctgacagtgg gactgcttta acattggtaa ctcggtggag 240 gccaaggtga aagcctagat ggcgacacct gtgggcgtcg ttcccttctt ggaggcatct 300 tttgtgtctc cccatcttgc cgtgttttga ttcgtggtag tccctagctt taccttgcat 360 tgacacataa gaatggcatt tgggaagttg gagctgcaac atcgaggcct ttgacaagct 420 tggcaacaat taatgactcg aagctttgct tgatttcatt tggggtttgg ccgttgtcct 480 ttgggaaatc ggagctgctg cgcgattgcg agcttggcag cgatgaccca agtcttaccc 540 gcgtcagtcg tgttttgctc cagtgtgagg tggtgagttt tttagtttgc aaggcttgta 600 ggtctagctc gcccatgcac cagtaagact agtgatcgtg aagtcggagc tgcggcatcg 660 acggggcgtg acatgcggca acgatgacat gagccctttt gaattgtgct tcagcgtggg 720 tcggatcttt gtccttactt ggtgtttcta ggcgaagtcg gagccgcccg atcacgtagg 780 tgatggtgtg atgacgataa tgtcaagtat atttgccagt ctgggagtct tgttttcttc 840 taatttgctg cttggccttg tcgtttttgt gttgagcagc gtttgttgag tgttgtacat 900 ggttgtcctt tttcgttgct gtggttgttt atgctagtta gaaagtttgt tgtggttttc 960 gctcaatttt tcctaattaa cgtatattgt aaggtctggt ctcattttct ctttaaaatg 1020 tgatattttc ttctttatat aaaacgtcag taacaacgtg accgttcaaa gaaaagaaca 1080 tgttttggtt gtccgtaaaa acaattttca tagtaaagct ttagcataaa ttaaaaaata 1140 agttatatat tgttccggcc agtgtataat actacgatct attcatcctc ctacaagctg 1200 agaacagatc ctttcagata gcatattagc atgcagtgct gatcgtagat ctcacactct 1260 tactgattca ctgaagtgtc tatgttaagc tcacttgcaa gtgttcccgg attgggcgtg 1320 cgtccacgtg aagcggcaca tcacatgtgc tgtgcacgcg cgcaaactgg caacctgcag 1380 gcagcagcta gccgcgcaaa tgcatcagtt caccaccacc gcccatatgc attcatggtc 1440 ctaagctcaa ttagctacta agatctgtgc cgatctgagc ggtggagtgt cccaaaaata 1500 aaaattctgg gatctctgct tgagcacaac ggcgaccgta aatgatgcga tcaatgcaga 1560 cttaattaaa ttaggataaa gttagtaaat ttacctgtca agacatggcc aagtgattgg 1620 taaatggaga tcgagacaac cttaaagaac ataagatcga atctttccag cacgaatgct 1680 atgatgaaaa ggttagatcg atgggggtga gggtgaactg aacatggtta catgcatgcg 1740 tatccatgag tactgaatta gccagctcct tgtcactgtg ttattccttt tgacgcatgt 1800 tgggagatga tgcatcgatg cgccctgtac ctgcttcgct gttttcccca acttgccagc 1860 ttctccatgg cgagaaagaa cagtggcaat gcaaactcgt cttggaaccc actgctttgc 1920 ctataaatat gtagctatag cttctctgct ttccgtcgct taacacagtt aacaccaacg 1980 cagtggtgct ctctcttcag ttagtggagc ttcagagaa 2019 <210> 2 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Os09g24840-PF <400> 2 caccgtcaga tgcaacctcg ggagtg 26 <210> 3 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Os09g24840-PR <400> 3 ttctctgaag ctccactaac tgaaga 26 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PPT5 <400> 4 ggtctgcacc atcgtcaacc 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PPT3 <400> 5 tcagatctcg gtgacgggca 20

Claims (9)

A promoter comprising a nucleotide sequence of SEQ ID NO: 1, which specifically induces expression in the root tissue of rice seedlings.
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 .
3. A rice ( Oryza sativa ) plant transformed with the vector according to claim 2 or the transformant according to claim 3.
delete delete 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) transforming Agrobacterium tumefaciens with the recombinant expression vector to produce transformed Agrobacterium; And
c) infecting the rice plant with the transformed Agrobacterium;
Wherein the target protein is specifically expressed in the root tissue of the rice seedling seedlings.
A method for inducing expression of a target protein in a root tissue of a rice seedling by transforming rice with the promoter of claim 1 or the vector of claim 2.

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