KR101522603B1 - Promoter to dorminantly express the gene in root stellar cells and use thereof - Google Patents

Abstract

The present invention relates to a vector comprising a OsRS promoter specifically expressed in root center of rice and specifically expressing the plant specifically in the root center, And can be usefully used for the development of transgenic plants and their seeds which are intended to produce useful substances specifically from roots.

Description

The present invention relates to an OsRS promoter for expressing a target protein mainly in the central structure of plant roots and a use thereof.

TECHNICAL FIELD The present invention relates to a technique for producing a plant expressing an exogenous protein specifically at the root center of a plant using an OsRS (Oryza sativa Root Specific) promoter that is specifically expressed in plant root central tissues isolated from rice will be.

Expression of a gene refers to a series of processes for synthesizing a protein according to a code entered into the gene through transcription and translation occurring within the cell. In particular, the transcription process is the initial stage of gene expression, in which the RNA polymerase is initiated by binding to a promoter sequence located on top of the gene with the aid of several cofactors, and the transcription factor (TF) One such cofactor is known to bind directly to the promoter sequence.

Crop molecular breeding can use all kinds of genes as a material and it is possible to control the effect of breeding infinitely by dragging the breeding technique which was possible only by the existing genome unit as a gene unit, Technology. In order to maximize the effect of these crop molecular breeding techniques, it is necessary to 1) accumulate a large amount of gene data that can represent various plant genes, 2) construct a transformation system for various crops, 3) The development of various promoters capable of regulating the expression of the inserted gene should be preceded.

Known promoters used to achieve the transfection purpose by limiting the expression of foreign genes to specific tissues of plants can be classified as follows according to their functions.

First, it is a root tissue specific expression promoter. Although there are no commercially available examples, it has been confirmed that the peroxidase (prxEa) is isolated and specific expression of root center main tissues has been confirmed. Recently, the expression of maz genes (ibMADS) derived from sweet potatoes and the sugar-induced ADP-glucan pyrophosphatase ADP-glucose pyrophosphatase, AGPase) gene has been isolated and it has been confirmed that the promoter induces specific expression in roots and induces root-specific transient expression in carrot and radish (Korean Patent Publication No. 10-18771 and 10-604191). These promoters can be expected to be mainly used for the purpose of improving agricultural traits, accumulating useful proteins, and producing useful substances in major root crops used for food, food, or food.

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 has been widely used to induce seed-specific expression of monocotyledonous plants so far. 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. A DC-3 promoter derived from carrot used in the study of promoting the production of vitamin E by inducing gene expression, and the case of confirming the induction of seed-specific expression of an oleosin promoter derived from perilla. The seed-specific promoters are mainly used for the purpose of accumulating useful proteins and producing beneficial substances in major crops in which seed itself is used as a food, a food, or a raw material for food.

Third, a systemic expression-inducible promoter can be mentioned. As a promoter for plant systemic expression induction, 35S RNA gene promoter of cauliflower mosaic virus (CaMV) has been used as a typical promoter for dicotyledonous plants. As a promoter inducing systemic expression for rice plants such as rice, rice actin ) And maize ubiquitin gene promoters have been mainly used. Recently, a promoter of rice cytochrome C gene (OsOc1) has been developed and used by domestic researchers (Korean Patent Publication No. 10-429335). They are already inherent in inducing the expression of antibiotics, herbicide resistance genes and reporter genes used as selection markers in the plant transgenic carrier. From a research point of view, Promoters considered.

Fourth, other tissue-specific promoters such as leaves may be mentioned. RbcS (ribulose bisphosphate carboxylase / oxygenase small subunit) promoter derived from rice and maize, which induces expression of a strong gene only in photosynthetic tissues such as leaves, RolD promoter inducing expression of plant roots derived from Agrobacterium, induction of specific expression of potato-derived tuber A phattoin promoter, a PDS (phytoene synthase) promoter derived from tomato-derived fruit-specific expression, and a cabbage-derived oleosin promoter whose function is confirmed to be a pollen-specific plant of a Chinese cabbage. A male sterile plant has been developed by inducing the expression of a Bt protein gene having toxicity to cells in a carpet-specific tissue-specific BcA9 promoter (Korean Patent Registration No. 10-435143).

In addition, a large number of promoters have been reported for various plant-derived plant tissues for various purposes and are still under development. However, the types of promoters commercialized or commonly used among plant researchers are not limited to those mentioned above And new promoters that can regulate gene expression sites more precisely according to the intention of the developer need to be developed in the future.

Recently, many studies have been conducted to produce useful substances using foreign gene introduction in plants using genetic engineering technology. In this process, when a foreign useful gene is expressed in a transgenic plant, a promoter involved in gene expression is required. For this purpose, a promoter derived from cauliflower mosaic virus (CaMV35S), in which expression is induced in whole tissues of plants, is widely used. However, in case of producing a useful substance only at the root, or when the foreign gene is expressed in a tissue other than the root to harm the plant, a promoter which is specifically expressed in the root only is required. Korean Patent Registration No. 0604186 discloses a method for transiently expressing in a storage root of a plant using a high-efficiency expression promoter base sequence for sweet potato-derived plant storage root, a high-efficiency transient expression vector containing the plant, and the above expression vector, Patent Publication No. 0574563 discloses a plant high-efficiency expression promoter derived from Arabidopsis thaliana and a plant high-efficiency expression vector containing the same, and Korean Patent Registration No. 12110308 discloses a root-specific expression promoter derived from the rice EXPB5 gene. Lt; / RTI > promoter.

The central cylider stele is the central part of the main axis of the plant that surrounds the cortex, and contains a tubular body part, a body part, and a cambium layer. The central region can be divided into the primordial region, the radiative central region, and the coronal region. The central part of the primordial main body is surrounded by the body part and can be seen from the root of the root part of the plant. The central part of the radial part is developed in the central part of the protoplasm and is surrounded by the body part and the body part. It can be seen in the root of the dicotyledonous plant. And can be seen in the roots of monocotyledonous plants. Thus, the root-centered plant plays an important role in the growth and development of the plant because it contains the organ that is essential for the nutrient transfer of the plant.

Thus, the present inventors have searched for a promoter that specifically expresses the root center of rice from the rice, prepared a vector containing the same and a transformant, and confirmed specific expression and activity of root center in the transgenic plants. Therefore, Can be utilized in the production of plant specifically expressing the root center of the root.

It is an object of the present invention to provide an OsRS (Oryza sativa root specific) promoter having a nucleotide sequence of SEQ ID NO: 1 and specifically expressed in root center main tissues of plants

Still another object of the present invention is to provide a primer comprising the sequence of SEQ ID NO: 2 and SEQ ID NO: 3 for amplifying the OsRS promoter.

It is still another object of the present invention to provide a plant-specific root expression-specific expression vector into which the OsRS promoter is introduced.

It is another object of the present invention to provide a transgenic plant transformed with the plant root center specific expression vector.

It is a further object of the present invention to provide a method of specifically expressing a foreign gene in root-center in a transgenic plant.

It is another object of the present invention to provide a transgenic plant or seed thereof in which a foreign gene is specifically expressed at the root center.

In order to achieve the above object, the present invention provides an OsRS (Oryza sativa Root Specific) promoter having a nucleotide sequence of SEQ ID NO: 1 and being specifically expressed in root center main tissues of plants.

In addition, the present invention provides a primer comprising the sequence of SEQ ID NO: 2 and SEQ ID NO: 3 for amplifying the OsRS promoter.

In addition, the present invention provides a plant-specific root expression vector having the OsRS promoter introduced therein.

The present invention also provides a transgenic plant transformed with the plant root center specific expression vector.

The present invention also provides a method for expressing a foreign gene locally in a transgenic plant.

In addition, the present invention provides a transgenic plant or seed thereof in which a foreign gene is expressed specifically in root center.

When a plant is transformed with a plant-specific root-specific expression vector into which a root-specific, OsRS (Oryza sativa Root Specific) promoter isolated from rice of the present invention is introduced, the gene introduced into the vector is specific to the root center Therefore, it can be usefully used to enhance the growth and development of plants by controlling the expression of foreign genes specifically in root centers of plants, and to enhance various environmental stress resistance.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic diagram of the plant root center specific expression vector pBGWFS7-pOsRS promoter.
2 is a photograph showing the activity of the OsRS promoter by GUS staining;
(A): Donggin Rd.
(B): 1st seedling roots;
(C): root cross section; And
(D): Root longitudinal section.

Hereinafter, the present invention will be described in detail.

The present invention provides an OsRS (Oryza sativa Root Specific) promoter having a nucleotide sequence of SEQ ID NO: 1 and specifically expressed in root center main tissues of plants.

The gene is preferably isolated from rice, but is not limited thereto.

Variants of the above base sequences are also included within the scope of the present invention. Specifically, the gene has a nucleotide sequence having a sequence homology of 70% or more, more preferably 80% or more, still more preferably 90% or more, and most preferably 95% or more, with the nucleotide sequence of SEQ ID NO: 1 . "% Of sequence homology to polynucleotides" is ascertained by comparing the comparison region with two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region is the reference sequence for the optimal alignment of the two sequences (I. E., A gap) relative to the < / RTI >

In addition, the present invention provides a primer comprising the sequence of SEQ ID NO: 2 and SEQ ID NO: 3 for amplifying the OsRS promoter.

In addition, the present invention provides a plant-root-center-specific expression vector into which the OsRS promoter of the present invention has been introduced.

The expression vector preferably includes a bar gene, which is a herbicide resistance gene, but is not limited thereto.

The root-center-specific expression vector of the present invention may be used as a transient expression vector capable of transient expression in a plant into which a foreign gene is introduced and a plant expression vector capable of permanently expressing the foreign gene in the introduced plant .

The binary vector that can be used in the present invention may be any binary vector containing the RB (right border) and LB (left border) of T-DNA which, when present together with the Ti plasmid of A. tumefaciens, Preferably, pBI101 (Cat #: 6018-1, Clontech, USA), pBIN19 (Genbank Accession No. U09365), pBI121, pCAMBIA vector and the like which are frequently used in the art are preferably used.

The root-center-specific expression vector according to an embodiment of the present invention may be, but is not limited to, pBGWFS7. In the present invention, a vector (FIG. 1) in which the GUS reporter gene and eGFP are inserted into the pBGWFS7 vector and the promoter of the present invention is positioned at the front of the gene is used as a foreign gene for plant transfection and transient expression . It is also apparent to those skilled in the art that the GUS reporter gene and the eGFP gene can be replaced with other foreign gene.

The term "vector" is used to refer to a DNA fragment (s), nucleic acid molecule, which is transferred into a cell. The vector replicates the DNA and can be independently regenerated in the host cell. The term "expression vector" is often used interchangeably with a "recombinant vector ". The term "recombinant vector" means a recombinant DNA molecule comprising a desired coding sequence and a suitable nucleic acid sequence necessary for expressing a coding sequence operably linked in a particular host organism. Promoters, enhancers, termination signals and polyadenylation signals available in eukaryotic cells are known.

The vectors that can be used in the present invention include plasmids such as pSC101, ColE1, pBR322, pUC8 / 9, pHC79, pGEX series, pET series and pUC19 which are frequently used in the art, phages such as λgt4 · λB ,? -charon,?? z1, and M13), or a virus (e.g., SV40, etc.).

A preferred example of a plant expression vector is a Ti-plasmid vector that is capable of transferring a so-called T-region into a plant cell when it is present in a suitable host such as Agrobacterium tumefaciens. Other types of Ti-plasmid vectors (see EP 0 116 718 B1) are currently used to transfer hybrid DNA sequences to plant cells or protoplasts in which new plants capable of properly inserting hybrid DNA into the plant's genome can be produced have. A particularly preferred form of the Ti-plasmid vector is a so-called binary vector as claimed in EP 0 120 516 B1 and U.S. Patent No. 4,940,838. Other suitable vectors that can be used to introduce a gene according to the invention into a plant host include viral vectors such as those that can be derived from double-stranded plant viruses (e. G., CaMV) and single- For example, from non -complete plant virus vectors. The use of such vectors may be particularly advantageous when it is difficult to transform the plant host properly.

The expression vector preferably comprises one or more selectable markers. The marker is typically a nucleic acid sequence having a property that can be selected by a chemical method, and includes all genes capable of distinguishing a transformed cell from a non-transformed cell. Examples include antibiotic resistance genes such as herbicide resistance genes such as glyphosate or phosphinotricin, kanamycin, G418, Bleomycin, hygromycin, chloramphenicol, It is not.

In a plant expression vector according to an embodiment of the present invention, the terminator can be a conventional terminator, such as nopaline synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, Agrobacterium And the terminator of the Octopine gene of agrobacterium tumefaciens, but is not limited thereto.

The present invention also provides a transgenic plant transformed with the plant-root-center-specific expression vector and seeds thereof.

The transgenic plants include root crops including radish, turnip, cabbage, cabbage, carrot, burdock, sweet potato, maize, lotus root, potato and ginger; Medicinal plants including ginseng, 칡, and udon; Forage crops including corn and soybeans; Dominant crops including rice; And oilseed crops including rapeseed, but is not limited thereto.

The root-specific plant expression vector of the present invention can transform any plant regardless of dicotyledonous or monocotyledonous plants, but in the present invention, transformation was carried out in rice (Oryza sativas). The plant according to an embodiment of the present invention may be a terminal plant such as rice, barley, corn, wheat, rye, oats, canola, soybean, forage, millet, sorghum, rice paper, and orchardgrass .

When the vector of the present invention is transformed into eukaryotic cells, yeast ( Saccharomyce cerevisiae) and the like insect cells, human cells (e.g., CHO cells (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cell lines), plant cells and plants can be used. The host cell is preferably a plant.

Transformation of a plant means any method of transferring DNA to a plant. Such transformation methods do not necessarily have a regeneration and / or tissue culture period. Transformation of plant species is now common for plant species, including both terminal plants as well as dicotyledonous plants. In principle, any transformation method can be used to introduce the hybrid DNA according to the present invention into suitable progenitor cells. The method is based on the calcium / polyethylene glycol method for protoplasts (Krens, FA et al., 1982, Nature 296, 72-74; Negrutiu I. et al., June 1987, Plant Mol. Biol. 8, 363-373) (Shillito RD et al., 1985 Bio / Technol. 3, 1099-1102), microinjection into plant elements (Crossway A. et al., 1986, Mol. Gen. Genet. 202,179-185 (Klein et al., 1987, Nature 327, 70), the infiltration of plants or the transformation of mature pollen or vesicles into Agrobacterium tumefaciens Infection by viruses (non-integrative) in virus-mediated gene transfer (EP 0 301 316), and the like. A preferred method according to the present invention comprises Agrobacterium mediated DNA delivery. Particularly preferred is the use of so-called binary vector techniques as described in EP A 120 516 and U.S. Pat. No. 4,940,838.

"Plant cell" used for transformation of a plant may be any plant cell. The plant cell may be any of a cultured cell, a cultured tissue, a culture or whole plant, preferably a cultured cell, a cultured tissue or culture medium, and more preferably a cultured cell.

"Plant tissue" refers to a tissue of differentiated or undifferentiated plant, including but not limited to roots, stems, leaves, pollen, seeds, cancer tissues, and various types of cells used for culture, protoplasts, shoots and callus tissue. The plant tissue may be in planta or may be in an organ culture, tissue culture or cell culture.

In addition,

1) recombining a foreign gene into a plant-specific expression vector according to the present invention; And

2) A method for specifically expressing a foreign gene in a transgenic plant in root center main tissues, comprising the step of transforming the recombinant vector in step 1) into a plant using Agrobacterium.

The plant of step 2) is selected from the group consisting of rice, Arabidopsis, cabbage, cabbage, mustard, rapeseed, radish, Brassica napobrassica ) and turnip ( Brassica rapa or Brassica campestris ), and Dong Jin rice is the most preferable, but is not limited thereto.

In addition, the present invention provides a transgenic plant or seed thereof in which a foreign gene is expressed specifically in root center.

In a specific example of the present invention, the present inventors isolated the OsRS promoter specifically expressed in the root center of rice from the rice and confirmed its base sequence (SEQ ID NO: 1), the vector containing the OsRS promoter (see FIG. 1) And transformed into Dong Jin - in. As a result of confirming the root-specific expression of the transgenic rice at the protein level and the gene level, it was confirmed that the protein and the gene were expressed specifically in the root center main tissue (see FIG. 2).

Therefore, since the vector comprising the OsRS promoter of the present invention is specifically and strongly expressed at the root center of the transgenic plant, the transgenic plant in which the foreign gene contained in the vector expresses specifically in the root and the seed thereof And can be usefully used for manufacturing.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Example  1 > OsRS (&lt; RTI ID = 0.0 &gt; Oryza sativa  Root Specific ) Isolation of Promoter

<1-1> motif search and promoter region PCR  Amplification

The motif of the promoter region of the gene was searched using PLACE (a database of Plant Cis-acting Regulatory DNA Elements) program and a PCR product of about 2 Kb was amplified to amplify a sufficient region including main motifs such as TATA box The primer was designed so that it could be obtained, and the promoter region of the gene was isolated by PCR.

Genomic DNA was extracted from rice leaves (Qiagen, DNeasy Plant Mini kit). A primer for amplifying a fragment of about 1378 bp predicted to be a promoter region of the gene was designed to synthesize a sense primer OsSKOR promoter-forward (pOsRSF: SEQ ID NO: 2) and an antisense primer OsSKOR promoter-reverse (pOsRSR: SEQ ID NO: 3) Are shown in Table 1 below. Then, the gene was amplified using an Applied Biosystem 9700 machine in the PCR reaction solution together with Invitrogen's Pfx polymerase. The PCR conditions used were denaturation at 95 ° C for 10 min, followed by 30 cycles of 95 ° C for 1 min, 55 ° C for 1 min, and 68 ° C for 2 min, followed by treatment at 68 ° C for 5 min. After the PCR reaction, the amplified gene was electrophoresed on 1% agarose, and the band, which was supposed to be the promoter, was cut out from the agarose gel, purified using a QIAquick gel extraction kit, and then used for topo cloning.

SEQ ID NO: Name of sequence Sequences (5 '-> 3') 2 pOsRS forward primer CACCCAAGAGAATTATGTGGTACAATGGGACA 3 pOsRS reverse primer  CTAACTGTGTCTCTCTGCCTGCTT

<1-2> In the gate introduction vector Cloning

The gene amplified in Example <1-1> was cloned into pENTR D-topo vector (Invitrogen, direct entry TOPO Cloning vector kit), which is a gateway entry vector.

The amplified gene was subjected to topoisomerase reaction at room temperature for 30 minutes, transformed into DH5 alpha strain by heat shock method, plated on LB medium, and colonies were selected and subjected to colony PCR. As a result, the plasmid of the identified colony was extracted and used for sequencing.

<1-3> OsRS  Sequence analysis of promoter

The 2099 bp OsRS promoter was isolated and named OsRS (Oryza sativa Root Specific) promoter. The entire nucleotide sequence thereof was determined by using an ABI 3700 sequencer to determine the nucleotide sequence thereof.

< Example  2> OsRS  Production of promoter expression vector and transformant

 <2-1> OsRS  Production of promoter expression vector

The inventors constructed a vector to confirm the function of the OsRS promoter isolated in Example 1 above.

Specifically, a recombinant expression vector was constructed to express the reporter gene GUS using the OsSKOR gene promoter isolated in Example 1 above. For this purpose, a recombinant vector in which the promoter was inserted into pBGWFS7 vector for expression of a gateway promoter purchased from Gent University of Belgium was prepared (Fig. 1).

First, LR (Invitrogen) was reacted to transform the vector pBGWFS7 (PSB, Plant System Biology) as a target vector. The genes were identified by colony PCR and sequencing, and then transformed into Agrobacterium for transformation into rice.

In order to transform the completed binary vector into Agrobacterium (LBA4404), the cells were subjected to freezing and thawing 2-3 times, transformed by heat shock method at 37 ° C, and transformed into YEP medium (Yeast 10 g, NaCl 5 g, peptone 10 g, Agar 15 g / 1 L) for two days to confirm colonies. Colony transformed by colony PCR was transformed into AB medium (AB buffer (K2HPO4 60 g, NaH2PO4 20 g / l), AB Salts (60 g NH4Cl, 6 g MgSO4 7H2O, 3 g KCl, CaCl2.2H2O 0.265 g, FeSO4.7H2O 50 mg / l, Glucose 5 g / l).

 <2-2> OsRS  Production of a promoter-expressing transformant

The present inventors produced transgenic rice to confirm the function of the OsRS promoter isolated in Example 1 above.

Concretely, rice seeds used for transformation of rice (Dongjin rice) are peeled brown rice. After the rice seeds are immersed in 70% ethanol for 1 minute, they are sterilized in water containing 50% of lactose for 40 minutes. At this time, 10 uL Tween 20 per 30 mL of lactose was used by dropping.

After disinfecting all the disinfectants, the rice seeds were washed 5 times with sterilized water. When the disinfectant solution was well washed, the water was removed from the rice seed using a filter paper, and rice seed was applied to the 2N6 medium. At this time, rice seeds were put into the medium to make contact with the medium, and the seeds were turned upward. The 2N6 medium is made from the medium described in the literature with some modified medium (Hiel et al., Plant J., 1994). 4 g / L of a mixture containing a large amount of elements, trace elements and vitamins of N6 medium (Chu et al., Scin Sin, 1975, 18: 659-668), 30 g / L sucrose, 0.5 g / L proline l glutamine, 0.3 g / L casamino acids, 0.01 g / L myo-inositol, 2 mg / L 2,4-D, 4 g / L phytagel (pH 5.8). The incubation temperature was 28 ° C and cancer treatment was performed.

After culturing in 2N6 medium for 5 days, the callus is removed and the callus is removed. Carefully remove the endosperm using tweezers so that the callus is not damaged, and keep the cotyledon and root parts intact to prevent the callus from being damaged.

Agrobacterium was prepared by inserting the desired gene into AB solid medium for 3 days, and then collected in an AAM liquid medium to prepare a suspension. The concentration of the suspension was measured using an absorbance meter. First, OD595 was adjusted to 0.1, and then diluted 10 times with AAM to obtain OD595 = 0.01.

The callus, from which the ending oil was removed, was immersed in the suspension for 2 minutes to inoculate Agrobacterium, and the solution was gently shaken for 2 minutes. After the inoculation, only the suspension was discarded from the callus, and the filtrate was removed using a filter paper. Do this quickly and be careful that the callus is not overly dry on the filter paper. Callus was transferred to 2N6-AS medium and co-cultured. 2N6-AS medium was adjusted to pH 5.2 with medium prepared by adding 10 g / L glucose and 100 uM acetosyringone to 2N6 medium. The filter paper was placed on the medium in advance, so that the media components were sufficiently impregnated, and then the inoculated callus was placed on the filter paper. The filter paper is intended to prevent the agglutination of callus by excessive growth of Agrobacterium during co-cultivation. Co-culture was performed at 25 ° C for 7 days.

After co-cultivation, Agrobacterium, which was responsible for inserting useful genes into the callus, was no longer needed and was removed by washing. Washing was performed with 1 L of sterilized distilled water and 500 mg of carbenicillin as an antibiotic. The callus was washed three times and then all the filtrate was removed using a filter paper. The callus is transferred to 2N6-CH medium supplemented with 200 mg / L of cefotaxime and 40 mg / L of hygromycin, which are antibiotics, in 2N6 medium. At this time, cotyledons and roots were removed using tweezers, and the callus was divided into 2 ~ 3 pieces and cultured by moving to the maximum area of contact with the medium. The culture temperature was 28 ° C, and the transformed callus was induced by culturing in a cancer incubator for 14 days.

Then, 14 days later, in the 2N6-CH medium supplemented with the hygromycin, which is an antibiotic, the transformed callus grew while dividing, and subcultured with MSR medium, which is a regeneration medium capable of inducing shoots and roots. The distinction of the transformed callus is judged by the presence of a newly differentiated callus mass and discarding the callus dead. The seedlings grown in the MSR regeneration medium were transferred to the soil and grown in the greenhouse.

< Example  3> OsRS  Analysis of tissue-specific activity of promoter

The present inventors stained GUS for analysis of the tissue-specific activity of transgenic rice plants prepared in Example <2-2>.

Specifically, the rice transformed with the pBGWFS7-pOsRS promoter vector prepared in Example 2 was analyzed by histochemistry of the promoter region by GUS staining for 2 weeks.

As a result, the region where the OsRS promoter is activated is stained in blue by GUS staining. When the leaves of the second parked seedling are stained in blue, the OsRS promoter expresses the gene of interest in the leaves Could know. However, it was found that the OsRS promoter mainly expresses the target gene on the leaf, since it is not stained at the root portion. The expression of the promoter was analyzed in various tissues using adults grown for about 2 months, and the root was strongly stained. However, it was found that weak staining was found in seeds and flower embryo, and that the other tissues were not stained. As a result, the OsRS promoter was able to express the target gene mainly in the root portion in the representative monocotyledonous rice plant Respectively. In order to observe the root structure more closely, the plant tissues embedded with paraffin after GUS staining were micro - cut into cross sections and longitudinal sections. Microscopic observation of the cut tissues confirmed that GUS staining was mainly performed only in the center of the root (Fig. 2).

<110> 2-1998-005031-4 <120> Promoter to dorminantly express the gene in root stellar cells          and use thereof <130> P130419 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 2000 <212> DNA <213> OsRS (Oryza sativa ROOT specific) promoter <400> 1 caagagaatt atgtggtaca atgggacaat agtacaatta tgcagccgga tacatgtact 60 aatcaaaata gaggttcaga acccttcatt ttggagccta agcatcaagc actagtttat 120 tagtgcaagc aaaacattag catatcatgg tcatacatga tagaaaaatt aaaggaacaa 180 agcagctcga gtgagtgtca gatgcactag ctgctactgc accttcgtcc taatccgaac 240 acttaacctg ctcaaaacca caacttagta aagctaaacc tccatgaaac ctctcattaa 300 ctatgaatca tgtattattt gtctaatcca attggcactc attagtggaa caatcggcca 360 tggccatcca tttctctcca ctaggacaac ataatgttgc tagtttaagc catcatgttt 420 aaaagagaca ccaaatggta ctagattagt ttggatttca gactttcagt cggtgcatgc 480 aatgccttga tcaaaaggtt tagagatcac cagctgaatt cagcactgtt cttctaatgt 540 aaagctgcct cgagttatga caatgcatta cagcacaggc aatctactca tatattaaca 600 cccagtaagc accaatcagc tgttcattag ctaaagctta gtttgaacca actccttgaa 660 agtgaaagtt tcagtcagct acaatttttt ttagcctcgt caactcaaaa gctaatccaa 720 gtgaaaggcc aggctaattg gctcatctag cagaccccct tgttgatctt ctgtgctctg 780 aactctgaag atgacacgca cctgcgcact agaaagatac ctgacacatg acaagcctga 840 aatggactgt cctgagccaa gctttgatct gcttttgagt tttgaccagt ttttacttgc 900 aattagtatc tggttcatga acctgttggg taagttttgc tggattttgg agagctttaa 960 aatggttggc cgagctccat gatcttgctg cactcagcgt taaggctaaa agatttaggt 1020 agccaaacta tcaaccgtag attcctctgc acccaaatta tagtcatgga aatattgtca 1080 gtataaacca aacgaactta attaagagta taacttttac ctttcaccta catccaactt 1140 gatgtactgt aacagtgccg aatgcttctg ggaagcattg atgcagtaac catccagatc 1200 tgatgaggca ggattcagtc gatccccttg aggatctttg accagcacaa gtgagtataa 1260 gtgacctgtc cttgcagcta tgatgcctag actacagctt gatgtgtaga accagtacca 1320 cttggctgca gtgcataatt gctttgtagc atgtgagaat tctgttgcaa acttgcaatg 1380 ctgcggtaag tttttccagc tctgggccgg gaggcgtgca gcagctaaca ataacataga 1440 tagctaacca gtgacgatgc atgccaaggt cactgaattt cacaaccgaa tttgatgtct 1500 tcttacattg caagttgcct acttgcaaag atgcaaaccc ccattcagca agcaaaaggt 1560 gtcaatttta aacggccatc catgtcacgt agccgtaggc ggacgccgaa ctagtccaaa 1620 actgcatgga ggagaagaaa agaaaaatgg cagctgctga tttgctccag tgaacagaaa 1680 aaaacgaaaa aaagaaagcc agtgcaccat gctgcaacca tcaccatgtc aatacgagta 1740 gtttggaagc atttaaaatg gggaggagag gagatgaggt gatgacagca tttgcaacta 1800 tttgaactga actcacctca cccccagttg agttgagcct gaatgctagc ttggtagtgc 1860 catttgtgca gtgcccaatt gctcttctgc aacagaaagc ggcaggtcca gtgctttgct 1920 ttgcattgag aatctgcaat gcatatatag aggaatttga cacttgcaaa ggagagaagc 1980 aggcagagag acacagttag 2000 <210> 2 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> OsRS forward primer <400> 2 cacccaagag aattatgtgg tacaatggga ca 32 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> OsRS reverse primer <400> 3 ctaactgtgt ctctctgcct gctt 24

Claims (12)

An OsRS (Oryza sativa Root Specific) promoter that is specifically expressed in the root-center main tissue of a plant, comprising the nucleotide sequence of SEQ ID NO: 1.
The method of claim 1, wherein the promoter is selected from the group consisting of rice (Oryza sativa L.). &Lt; / RTI &gt;
A primer set as set forth in SEQ ID NO: 2 and SEQ ID NO: 3 for amplifying the OsRS promoter of claim 1.
A plant-specific root expression vector-specific expression vector into which the OsRS promoter of claim 1 is introduced.
[Claim 5] The plant-root-center-specific expression vector according to claim 4, wherein the expression vector comprises a bar gene which is a herbicide resistance gene.
A transgenic plant transformed with the plant-root-center-specific expression vector of claim 4.
[Claim 7] The transgenic plant according to claim 6, wherein the transgenic plant is a root crop plant including radish, turnip, cabbage, cabbage, carrot, burdock, sweet potato, maize, lotus root, potato and ginger; Medicinal plants including ginseng, 칡, and udon; Forage crops including corn and soybeans; Dominant crops including rice; And oilseed rape. The transgenic plant according to claim 1, wherein the plant is a plant.
1) recombining a foreign gene into a plant-specific expression vector according to claim 4; And
2) A method for specifically expressing a foreign gene in a transgenic plant in root center main tissues, comprising the step of transforming the recombinant vector in step 1) into a plant using Agrobacterium.
The method of claim 8, wherein the plant of step 2) is rice, Arabidopsis, Chinese cabbage, cabbage, mustard, rapeseed (rapeseed), radish, homu (Brassica napobrassica ) and turnip ( Brassica rapa or Brassica campestris ), wherein the foreign gene is specifically expressed in the root center main tissue in the transgenic plant.
[Claim 9] The method according to claim 8, wherein the plant of step 2) is Dongjinbyeo, wherein the foreign gene is specifically expressed in the root center main tissue in the transgenic plant.
9. A transgenic plant produced by the method of claim 8, wherein the foreign gene is specifically expressed in root center main tissues.
11. Seeds of transgenic plants according to claim 7 or 11.

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