KR101497832B1 - Leaf-, stem- or both- specific promoter, expression vector comprising the same, transformed Oryza sativa thereby and method for preparation thereof - Google Patents

Abstract

The present invention relates to a promoter which specifically expresses a leaf, a stem or both of them, an expression vector containing the promoter, a transformant transformed with the expression vector, and a method for producing the same.
The promoter of the present invention can be used for researches for enhancing disease resistance or environmental stress tolerance to leaves or stems of major cereals including rice, by specifically controlling gene expression necessary for improving the traits of leaves, stalks or both of them It can be used for research on biomass through promotion of photosynthesis efficiency.

Description

The present invention relates to an expression vector containing the promoter, the expression vector comprising the promoter, the transformed rice, and a method for producing the same, for preparation thereof}

The present invention relates to a promoter that specifically expresses a leaf, a stem, or both of them, an expression vector containing the promoter, rice plants transformed with the expression vector, and a method for producing the same.

Rice (Oryza sativa) is one of the most important food crops in the world. It has been steadily increasing its yields over the past two decades, but by 2020 it will have to produce more than 70% have. However, the agricultural land where rice is cultivated is getting smaller and smaller as the industrialization phenomenon of each country in the world, and the new genetic resource which is the subject of breeding is depleted and the introduction of the foreign useful gene is required. Fortunately, the development of molecular biology has enabled the isolation and manipulation of exogenous useful genes, transforming useful genes into many plant cells, including rice, to obtain transformants with new genes, Research has become a good source of breeding as well. The production of new varieties using this transformation will not only lead to a high value-added industry in the coming 21st century, but will also solve some of the food problems that are the biggest problem of humanity.

In the 1980s, transgenic plants were obtained by using polyethylene glycol (PEG) and electroporation in protoplasts. In the late 1990s, gene gun utilization became popular, and in recent years, The use of Agrobacterium, which has been widely used in plants, is also widely used. Other methods include pollen pathways and microinjection methods.

The promoter can achieve the transformational purpose by locating the expression of the foreign gene only at the whole body of the plant or a specific tissue, and can be classified as follows according to its function.

First, systemic expression-inducible promoters can be mentioned. As a plant systemic expression-inducing promoter, a promoter of 35S RNA gene of cauliflower mosaic virus (CaMV) is used as a typical promoter for dicotyledonous plants. Actin and maize ubiquitin gene promoters have been mainly used as promoters for the expression of the whole plants in rice plants and rice plants. Recently, a promoter of the rice cytochrome C gene (OsOc1) has been developed by domestic researchers, (Cf. Reg. No. 10-0429335). 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. Because these promoters lack tissue-selectivity or organ-selectivity, transgenic selectable markers and the like are expressed in whole plants, resulting in delayed plant growth. In addition, due to the nature of the promoter, the expression of the introduced gene is limited to the target organ, and can not be expressed sufficiently, resulting in an inferior economic efficiency of the transformant.

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 patent application (Patent Application No. 10-2006-0000783) for seed-specific expression induction of carrot-derived DC-3 promoter and perilla derived oleosin promoter used in the study promoting the production of vitamin E by inducing gene expression . 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, it is a root specific expression promoter. Although there are no commercial examples yet, root-specific expression of pyruvate peroxidase (prxEa) has been isolated, and recently, it has been confirmed that the expression of maz genes (ibMADS) derived from sweet potatoes and ADP-glucose pyrophosphatase , AGPase) gene was isolated to induce a specific expression of the promoter in the root, leading to root-specific transient expression in carrot and radish, and was registered as a patent (Korean Registration No. 10-0604186, No. 10-0604191) There is a bar.

Fourth, other tissue-specific promoters such as leaves can be mentioned. (RbcS: ribulose bisphosphate carboxylase / oxygenase small subunit) promoter which induces expression of a strong gene only in photosynthetic tissues such as leaves, RolD promoter inducing expression of plant roots derived from Agrobacterium, potato-derived tuber A specific expression-inducible patatin promoter, and a tomato-derived fruit maturation-specific expression-inducing PDS (phytoene synthase) promoter.

In addition, although the development is currently underway, new promoters capable of controlling the location of gene expression more precisely according to the intention of the developer, for example, genes that should be expressed only in specific organs (petals, roots, leaves, stems, etc.) , Infertility, burning, specific metabolism-related substances, defensive substances, etc.), there is a need to continue to develop promoters that operate only at specific institutions or periods.

KR 10-2009-0029296 KR 10-2008-0007421

The present invention is intended to provide a promoter capable of expressing the expression of a foreign gene only in leaves and stems, not in the whole body of rice, an expression vector containing the promoter, a rice transformed with the expression vector, and a method for producing the same.

In order to achieve the above object, the present inventors have discovered that promoters capable of regulating tissue or organ-specific gene expression of rice can be specifically identified and restricted to specific tissues or organs of rice to express or inhibit a target gene And to establish a system with In this process, it was confirmed that the promoter of LOC_Os02g38020 of rice was not expressed in the petal, navel, and root but induces expression specifically in the leaf or stem. Especially, it was confirmed that the expression was strong in the entire mesophyll tissue, The present inventors have completed the present invention by confirming that reporter genes are specifically expressed in leaves, stems, or both of them in the transgenic rice plants prepared by introducing them into rice plants.

In more detail, we collected 983 species of published rice affymetrix data and reclassified them into 17 organelles. The affymetrix array allows the analysis of gene expression represented by 57,000 probes in a single experiment and the organ-specific expression pattern produced by reconstruction of the large-scale collection of affymetrix arrays is quite high Reproducibility. Through analysis of the expression pattern of this data, various organ and tissue specific genes were isolated. Among them, the present inventors have been interested in finding promoters necessary for expressing important traits in leaves or stems.

Through this method, 547 genes showed high expression patterns in leaves and stems compared to other genes.

The gene expression pattern regulated by these genes was precisely analyzed by expressing the GUS reporter gene in the 3 'direction of these promoters and measuring its activity in the rice. Because of the time-consuming difficulty of transgenic vectors for the verification of promoter activity, the present inventors have utilized a fusion omics analysis technique which has been recently actively pursued by a method different from the conventional method.

Since the GUS gene without the promoter is adjacent to the right end of the T-DNA of the promoter search vectors pGA2707, pGA2715, pGA2717 and pGA2772, when the T-DNA is inserted in the direction of the promoter 3 'in the forward direction, the promoter activity Can be measured. In addition, since the promoter trap system can confirm large-scale T-DNA insertion sites through genomic DNA PCR and base sequence analysis, it is possible to search the related system by indexing the gene locus id in the database.

The isolated 547 leaf and stem - specific genes were selected by over 280 lines of 250 genes through the related promoter search line. The seeds of these lines were sterilized and the entire rice nipple was used for GUS analysis in the nutrient medium for 7 days.

As a result, in Experiment 1C-01149, T-DNA was inserted into the second exon of LOC_Os02g38020, which is not homologous with the existing gene, and fusion of GUS protein was expected in the second exon of this gene. Through GUS expression analysis and genomic DNA PCR, it was confirmed that GUS expression in this strain shows the promoter activity of this gene.

The promoter up to 2kb in front of ATG of LOC_Os02g38020 was isolated by genomic DNA PCR and cloned into pGEM T-Ease vector and base sequence was decoded by referring to base sequence decode information.

It was found that the effect of the isolated promoter on the regulation of leaf and stem specific gene expression pattern of rice was greatly influenced.

The present invention relates to a promoter of LOC_Os02g38020 of rice, which comprises a promoter (hereinafter referred to as 'promoter of the present invention') that is a leaf, a stem or a promoter specific to all of them, which induces expression of a foreign gene to be specific to leaves, Lt; / RTI >

That is, the present invention provides a promoter for specific expression on the leaves, stems, or all of the foreign genes, including the promoter of LOC_Os02g38020 of rice, which is the nucleotide sequence shown in SEQ ID NO: 1.

The promoter of the present invention expresses specifically in mesophyll, especially in tissues contained in leaves, stems or both.

The promoter is an essential element for regulating the environmental, temporal, and systemic expression of the gene. The expression of the exogenous gene of interest in the rice can be expressed in leaves, stems, or both , Particularly in leaf, stem or tissue contained in both of them. C3 photosynthetic metabolism of rice leaves is composed mainly of chloroplasts, which are key cell organelles that perform photosynthesis reaction. In the chloroplasts of these leaf tissues, there is an emerging reaction to produce photosynthetic fuel materials. And it can contribute to the enhancement of photosynthesis efficiency and productivity by the related tissue specific expression.

The present invention also provides expression vectors specific for leaves, stems or both, including the promoter and operatively linked foreign genes.

The term "vector" refers to a DNA fragment (s), nucleic acid molecule, which transfers into a cell, which can be cloned and independently reprogrammed in host cells. "Expression 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. The expression vector may generally be derived from a plasmid or viral DNA, or may contain both elements. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, phage, recombinant virus or other vector resulting in the expression of the cloned DNA upon introduction into an appropriate host cell. Suitable expression vectors are well known to those skilled in the art and include those that replicate in eukaryotic and / or prokaryotic cells and those that remain as episomes or that are integrated into the host cell genome.

Such a vector may be any vector as long as it can introduce the promoter of the present invention. Preferably, the vector may be a vector such as pCAMBIA family, pGA family, pGWB family such as pGA3383, pCAMBIA3301, pCAMBIA3300, pGA3426, pGA3780, pGWB12, pGWB14 Ti-plasmid, and vectors derived therefrom.

The expression vector of the present invention includes a nucleic acid consisting of the nucleotide sequence shown in SEQ ID NO: 1 and a functionally equivalent fragment thereof.

Means a fragment or a fragment of a nucleic acid consisting of the nucleotide sequence shown in SEQ ID NO: 1, which exhibits substantially equivalent effect to the promoter of the present invention. These nucleic acid fragments are 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99% or more of sequence homology. Such a nucleic acid fragment can be easily prepared by molecular biological methods well known in the art. In addition, the promoter represented by SEQ ID NO: 1 of the present invention is a sequence located in front of ATG of LOC_Os02g38020 on the published rice genome sequence, and even if some of the nucleotide sequences shown in SEQ ID NO: 1 are changed, added or deleted, It can be included in the scope of the present invention if homology with the sequence located in the promoter of LOC_Os02g38020 on the genomic sequence is maintained and exhibits substantially equivalent effect.

The expression vector of the present invention allows expression of a foreign gene for the target protein in the 3 'direction.

Preferably, the expression vector may comprise useful foreign genes which are intended to specifically express on the leaves and stems.

The foreign gene may be anything related to the characteristics of leaves and stems such as the color and development of leaves and stems, photosynthesis and the like, preferably genes related to development of leaves such as Arabidopsis, CONSTANS-LIKE, KNOX, Tic21, Toc33 , Tic40, Toc75, CIA2, GOLDEN2-LIKE, Stromal 70-kD Heat Shock Protein, Glutamine Phosphoribosyl Pyrophosphate Amidotransferase, Chloroplastic Adenylate Kinase, Differential Development of Vascular Associated Cells 1, Phosphoenolpyruvate-Phosphate Translocator, Carbamoyl Phosphate Synthase subunit, The genes involved in the photosynthesis of photosynthesis include Pyruvate orthophosphate dikinase, Phosphoenolpyruvate carboxylase, NAD-malic enzyme, NAD-malate decarboxylase, Alanine aminotransferase, Magnesium Chelatease H subunit, Magnesium Chelatease I subunit, Magnesium Chelate D subunit, Chlide a monooxygenase, glutamyl-tRNA reductase , NADPH: Pchlide oxidoreductase, glutamic acid 1-semialdehyde aminotransferase, Uro-III decarboxylase, Mg-Proto-IX reductase, Pchlide a reductase, Chlide areductase, Chl a reductase, Protogen-IX oxidase, PASA and PSAB Any one or more can be used.

The above expression vector may be one in which the promoter of the present invention is introduced into a conventional vector, and the expression vector may be prepared by introducing the promoter of the present invention into such a manner as will be readily apparent to those skilled in the art. It is possible.

The present invention also provides a transformed cell transformed with said vector.

Such recombinant vectors may be introduced into host cells cultured using well known techniques such as infection, transfection, transfection, electroporation and transformation. Representative examples of a host include, but are not limited to, bacterial cells such as E. coli, Streptomyces and Salmonella typhimurium cells and plant cells.

Any plant cell can be used as "plant cell" used for transformation of rice. The plant cell may be any type of cultured cell, cultured tissue, cultured organ or whole plant, preferably cultured cell, cultured tissue or culture organ, and more preferably cultured cell. "Plant tissue" refers to a tissue of a differentiated or undifferentiated plant, such as, but not limited to, stem cells, leaves, cancer tissues, and various types of cells used in culture, such as single cells, protoplasts, Callus tissue.

The present invention also provides a transgenic rice transformed with said vector or said transformed cell. These transgenic rice plants exhibit characteristics in which introduced foreign genes are specifically expressed in leaves and stems.

"Transgenic rice functionally equivalent to the transformed rice of the present invention" is 60%, 65%, 70%, 75%, 80%, 85% or more of the nucleic acid sequence variants comprising the nucleotide sequence shown in SEQ ID NO: Transgenic rice produced using a nucleotide sequence having a nucleotide sequence homology of 90%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% , The transgenic rice having the characteristics that the promoter effect is substantially equivalent in leaves and stems.

Transformation of the present invention rice can be carried out by any method known in the art for transferring DNA to rice. 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. For example, 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. (Klein et al., 1987, Nature 327, 70), infiltration of plants or mature pollen of various plant elements (DNA or RNA-coated) And infection by (non-integrative) viruses in Agrobacterium tumefaciens mediated gene transfer (EP 0301316).

Such mutants can be easily produced by molecular biological methods well known in the art.

The present invention also provides a method for transforming rice, comprising transforming rice with the vector and specifically expressing the foreign gene in leaves, stems or both of the rice.

Preferably, the method comprises the steps of: preparing a vector comprising a promoter comprising the nucleotide sequence shown in SEQ ID NO: 1 and a foreign gene operatively linked thereto; Introducing the expression vector into rice; And selecting a rice transformant to which the expression vector is introduced to express a foreign gene specifically in leaves, stems, or both of the rice, and a method of transforming rice.

The promoter of the present invention is a promoter that induces specific expression on leaves, stems or both of them, and specifically expresses genes necessary for improvement of traits of leaves and stems from leaves, stems or both of them, It can be used for research on the resistance to diseases such as leaves and stems, resistance to environmental stress, and research on biomass through promotion of photosynthesis efficiency.

FIG. 1 is a heat map showing a separated gene showing preferential expression in leaves and stems and its expression pattern. FIG.
FIG. 2 is a diagram showing a T-DNA insertion site for confirming the promoter activity of the present invention. FIG.
FIG. 3 shows GUS staining results and PCR results showing that 1C-01149 strain co-segregates with LOC_Os02g38020 gene.
4 is a diagram showing leaf preferential expression of the line 1C-01149.
FIG. 5 is a diagram showing the predominant expression of the LOC_Os02g38020 gene in the mesophyll tissue.
6 is a diagram showing microarray results showing preferential expression in leaves and stems of LOC_Os02g38020 gene.
7 shows a 2Kb promoter in front of ATG of LOC_Os02g38020 gene.
8 is a diagram showing a diagram of a vector used for producing LOC_Os02g38020 promoter GUS rice.
9 is a diagram showing predominant expression in leaves of leaves, flowers and roots of transgenic rice plants in which a promoter of LOC_Os02g38020 was introduced

Hereinafter, the present invention will be described in more detail with reference to examples. However, the following embodiments are only examples for helping understanding of the invention, and thus the scope of the present invention is not limited thereto.

< Example  1> Large selection of rice genes specifically expressed on leaves or stems

To select genes that specifically express on leaves or stems, 983 species of rice epimetrics collected from NCBI GEO (http://www.ncbi.nlm.nih.gov/geo/), a published microarray database The database for gene expression analysis was constructed by reconstructing the affymetrix microarray data by 17 organizations / organizations.

This is shown in Table 1.

Table 1. Database for gene expression analysis reconstructed by institution / organization

Using the K-means clustering analysis method, gene groups having high expression in leaves and stems of the database for gene expression analysis shown in Table 1 were isolated.

This was performed using MeV's K-means clustering analysis software for microarray data analysis, and 547 genes with preferential expression in leaves and stems were isolated from the database for expression / organization-specific expression analysis.

The results are shown in Fig.

As shown in FIG. 1, the closer to yellow, the higher the preferential expression is, and the closer to blue the gene has the lower priority. That is, among the 17 reconstructed gene databases, 547 genes with high preferential expression in leaves, end leaves, seeds and stems were identified.

< Example  2> Promoters for genes preferential to leaves and stems trap  Identification of the system

Since the GUS gene without the promoter is adjacent to the right end of the T-DNA of pGA2707, pGA2707, pGA2715, pGA2717, and pGA2772, which are vectors for the promoter search, GUS expression is observed when the T- DNA is inserted in the forward direction and the forward direction of the promoter The promoter activity can be known.

Through inverse PCR and sequence analysis of T-DNA-inserted rice lines, T-DNA insertion sites were determined on the rice chromosome on a large scale. The mutation information of the T-DNA index corresponding to 1/2 of the genome was analyzed.

As a result, it was possible to search for a gene having a locus id name from the above 547 kinds of leaf and stem-preferential genes and potentially trapping the promoter. Through this, 280 promoter trap lines were selected for 250 genes.

< Example  3> GUS  Analysis of organ-specific expression of leaf and stem preferentially expressed genes by staining

In order to confirm the organ specificity of the selected gene, 10 seeds of 280 promoter trap lines for 250 genes expressed preferentially in leaves and stems were cultured at 28 ° C for 7 days, and all individuals were stained with GUS .

The GUS solution was prepared by mixing 200 ml of 0.5 M sodium phosphate buffer, 100 ml of 12.5 mM potassium ferricyanide, 100 ml of 12.5 mM potassium ferrocyanide, 20 ml of 0.5 M EDTA, 50 ml of 10% Triton X-100, 1 g of X-glucin dissolved in 10 ml of DMSO, 200 ml of distilled water was added to make 1 L volume. The rice of each line was treated with 15 ml of GUS staining solution for 24 hours and decolorized at room temperature using 70% ethanol and 100% ethanol sequentially.

As a result, GUS expression was confirmed in 36 strains among 280 strains subjected to GUS staining. Among them, 25 strains with T-DNA insertion in the forward direction were selected.

< Example  4> Leaves and stem lines showing preferential expression GUS  Expression and T- DNA  Cover co - segregation  Verification

The selected 25 lines of rice seeds were cultured in MSO medium at 28 占 폚 for 7 days. 100 mg of leaves of the 7 day cultivated leaves were sampled and pulverized in liquid nitrogen, and a part of the leaves were used for GUS staining. DNA was extracted from the pulverized leaves using CTAB buffer (CTAB 10 g, NaCl 40.91 g, 0.5 M EDTA (pH 8.0) 20 ml, PVP 10 g and ascorbic acid 0.44 g, and 500 ml of water).

DNAs were extracted from selected 25 species of rice seedlings. Among them, 1C-01149 in the experimental group was a system in which a GUS reporter gene was ligated to a second exon of LOC_Os02g38020 gene, and primers (SEQ ID NOS: 2 and 3 ) And the NGUS1 primer (SEQ ID NO: 4). The inserted positions were shown in FIG. 2 and the primers used were shown in Table 2 below.

Table 2. Primers of 1C-01149

2 is nucleotides 2103 to 2299 of SEQ ID NO: 5, T-DNA is nucleotides 2080 to 2275 of SEQ ID NO: 5, 3 is nucleotide 219 to 2299 of SEQ ID NO: 5 to nucleotides 2465 to 3285. Each of those corresponding to 1 to 3 in Fig. 2 is a sequence corresponding to exon.

After 5 minutes at 95 ° C, the PCR reaction was repeated 37 times at 95 ° C for 30 seconds, at 57 ° C for 30 seconds, at 72 ° C for 1 minute 30 seconds, and finally at 72 ° C for 5 minutes. The PCR reaction product was stored at 4 ° C and a part thereof was confirmed by electrophoresis.

The results are shown in Fig.

As shown in FIG. 3, it was confirmed that the PCR result of the test group 1C-01149 and the GUS staining result were in agreement.

FIG. 3A shows the result of GUS staining for one week of 1C-01149, and FIG. 3B shows the genotype analysis result of the individual used in experiment A of FIG.

The upper band in FIG. 3B is the PCR product of the gene specific primer (SEQ ID NOS: 2 and 3) of LOC_Os02g38020, and the lower band is the PCR product of SEQ ID NO: 3 and NGUS1 (SEQ ID NO: 4) on the RB PCR products. By comparing the results of the two products, genotype analysis of the rice is possible.

Only the products of SEQ ID NOS: 2 and 3 are wild type (wt), and the products of SEQ ID NOS: 2 and 3 and the products of SEQ ID NOS: 3 and 4 are hybrid, respectively.

In the wild type, there is no T-DNA insertion, so GUS expression does not appear. In hybrids, GUS expression on T-DNA appears, so that it is stained blue.

That is, it was confirmed from the above results that the 1C-01149 strain was co-segregated with the LOC_Os02g38020 gene.

In addition, as shown in Fig. 4, GUS expression was not observed in the roots of the 1C-01149 strain for 7 days, but it was confirmed that the expression was specifically in the leaves.

In order to confirm the expression pattern in the leaves, a cross section of a mature flag leaf was examined through a hand section.

The mature ripening leaf of 1C-01149 line developed in the rice paddy was cut into several 1-3cm size and stained with GUS staining solution for 24 hours to decolorize. The stained area of the discolored leaves was cut with a double blade razor to within 0.5 mm by hand, placed on a slide glass, fixed with a cover glass, and observed with an optical microscope at 20-40 magnification.

The results are shown in Fig. As shown in Fig. 5, it was confirmed that the expression was high throughout the whole mesophyll of leaves.

< Example  5> Expression analysis of genes by microarray data analysis

The expression pattern of LOC_Os02g38020 gene was analyzed by microarray database analysis and the results are shown in FIG.

As shown in FIG. 6, it can be seen that the leaves and the stem are yellowish, indicating that the expression of the gene by the LOC_Os02g38020 promoter occurs specifically in leaves and stems.

Based on the results of FIG. 3 to FIG. 6, it was confirmed that the test strain 1C-01149, in which the GUS reporter gene was linked to the second exon of the LOC_Os02g38020 gene, showed preferential expression in leaves and stems.

< Example  6> Promoter for preferential expression of leaf and stem Cloning

PCR was performed using the primers prepared to clone the promoter of LOC_Os02g38020 and genomic DNA of Oryza sativa L. japonica cultivar-group. 100 ng of template DNA was used and 5 pmol of each primer was used. The PCR conditions were 95 ° C for 5 minutes-> (95 ° C, 30 seconds -> 55 ° C, 30 seconds -> 68 ° C, 5 minutes and 00 seconds) 30 seconds to 68 占 폚, 5 minutes and 00 seconds), 35 times and 68 占 폚 for 10 minutes.

Primer for promoter cloning is shown in Table 3 below.

Table 3. Primers for cloning promoter of LOC_Os02g38020

The PCR product was electrophoresed to confirm its size and cloned into a pGEM T-EG vector and sequenced. The cloned DNA was digested with BamH1 and Xho1, followed by ligation with a pGA3383 vector (vector for promoter-GUS cloning) digested with the same restriction markers at 14 DEG C for 12 hours. The ligation product was mixed with 50 [mu] l of Top10 E. coli competent cells, transferred to a 1.5 ml tube, and left on ice for 15 minutes. Subsequently, the plate was allowed to stand in a 37 ° C oven for 1 minute, and then 1 ml of LB liquid medium was further added to the tube and left in a 37 ° C shaking incubator for 3 hours. Then, the cells were plated on tetracycline-resistant LB solid medium, and the resulting colonies were awaited for 12 hours and then mini-prepared after cell culture in 1 ml of LB liquid medium. DNA from mini - preparations was digested with restriction enzymes BanmH1 and Xho1, and then separated into agarose gels by electrophoresis, and the bands were confirmed. The cloning DNA thus obtained was subjected to base sequence analysis again to select DNA that did not cause an error. A diagram of the vector used for the production of the LOC_Os02g38020 promoter GUS rice thus produced is shown in FIG. Sequence analysis was performed using a 3730XL DNA analyzer (AB, USA) and BigDye v3.1 (AB, USA). Primers using NGUS1, RB (right border) .

Table 4. Primers for sequencing

The results of the analysis are shown in FIG. 7 and SEQ ID NO. 1, and a promoter corresponding to the sequence of 2,182 bp in front of the ATG of the LOC_Os02g38020 gene corresponds to a promoter preferentially expressed on leaves and stems.

< Example  7> Production of transformed cells

The rice expression vector prepared in Example 6 was extracted.

50 아 of Agrobacterium tumefaciens LB4404 and 2 의 of rice expression vector were mixed and left on ice for 15 minutes. Subsequently, the substrate was placed in liquid nitrogen for 75 seconds. Then, the substrate was allowed to stand in an oven at 37 ° C for 5 minutes. Then, 1 ml of LB liquid medium was added thereto, followed by incubation in a 28 ° C shaking incubator for 3 hours. Next, the cells were plated on tetracycline-resistant LB solid medium and incubated for 36 hours. The resulting colonies were cultured in 1 mL of LB liquid medium, mini-preparations were performed, and BamH1 and Xho1 were used for enzyme cleavage.

The resulting transformed Agrobacterium was used for transgenic rice transformation experiments.

< Example  8> Production of transgenic rice

In the N6D solid medium, Dongjinbyeon seeds were grown in a 28 ℃ growth chamber for 7 days to produce calli of rice. The resulting callus was mixed with the cells cultured for 72 hours with Agrobacterium transformed with the rice expression vector obtained in Example 6 and left in a 22 ° C cancer-treated growth chamber in a medium containing N6D-Acetosyringone.

The callus contaminated with Agrobacterium was rinsed 5 times with tertiary distilled water. Subsequently hygromycin selection was performed in N6D solid medium (hygromycin 30 mg / L) and subculture (hygromycin 40 mg / L) . Selection was made in the 28 ℃ growth room for 2 weeks each for 4 weeks. The divided callus was transferred to the MSR (hygromycin 40 mg / L) solid medium for regeneration. After induction of regeneration in the light condition growth chamber for 4 weeks at 28 ° C., the rice was transferred to MS solid medium, grown in a light condition growth room for 7 days, And raised the regrowth rice.

< Example  9> Validation of leaf and stem preferential expression using transgenic rice

In order to confirm organ specific GUS expression of regenerated rice, leaf, flower, and mature root of transformant were collected and stained with GUS staining solution for 24 hours. In the case of leaves that had undergone decolorization, the stained sections were cut within 0.5 mm by hand using a double-edged razor blade, fixed on a slide glass, fixed with a cover glass, and observed at 20-40 magnification in an optical microscope. The results are shown in Fig. Each of FIGS. 9 (1), (2), (3) and (4) represents a mature branch of a mature leaf, a cross section of a leaf of the mature leaf, and a flower. As shown in FIG. 9, GUS expression of 1C-01149, a promoter-introduced transformant and a promoter trap line, in LOC_Os02g38020 was dominant in the leaf tissue of the leaves.

< Example  10> LOC _ Os02g38020   Gene RNA - seq  Expression analysis

The results of RNA-seq expression analysis showing preferential expression in the leaves of LOC_Os02g38020 gene are shown in Table 5.

Table 5. RNA-seq expression analysis showing preferential expression in leaves of LOC_Os02g38020

The above data is based on the gene expression analysis service (http://rice.plantbiology.msu.edu/expression.shtml) provided by the Rice Genome Annotation Project team of Michigan State University. The data used in the expression analysis is the next generation sequence analysis Technology-based RNA-seq analysis. The frequency of RNA-seq identified for each library listed in Table 5 indicates the degree of expression of the examined gene. The RNA-seq analysis showed that the gene was expressed at the highest level in 20-day-old leaf and four-leaf stage seedling. Expression in other tissues was also higher in leaf tissues. This confirms the expression patterns observed in the microarray.

<110> University-Industry Cooperation Group of Kyung Hee University <120> Leaf-, stem- or both- specific promoter, expression vector          comprising the same, transformed Oryza sativa thereby and method          for preparation thereof <130> P12-073-KHU-REA <150> KR KR 12 / 113,571 <151> 2012-10-12 <160> 10 <170> Kopatentin 2.0 <210> 1 <211> 2182 <212> DNA <213> Oryza sativa <220> <221> promoter &Lt; 222 > (1) .. (2182) <223> LOC_Os02g38020 Promoter <400> 1 gagtccgagt gtaggacttg gaatggtcgg ctacacaagg cccaggtgat agagttgcga 60 gcacaggctg atcccaaggg cctcaggccc atgaaatgac caccacagtc cacatacagc 120 gaatactgat ctgttttcaa aacgctttca ttggctattt gcgttacctt ccaaaacaag 180 gagtacctat gcatctttcg aaagatttct atgatcatat catataaatt tttaatcttt 240 ggattaaaat ccaatagaca tagtaaaagg caaaaagcaa ctaagaaaca ccataataga 300 tactaaatta ccatatcttt aagaaaaaga ccaaatccaa tataaatttc aaaacttaca 360 tgcgaagatt caaaaattac actgtaagtt tcataaatta cactataatt tacaagaaaa 420 tacactgtaa atttgagtga gaaaatcgag tgagagataa agaaaaaatc tttcaagtga 480 gatatagcaa aatcgcagaa caattcgagt ccaagattat tattcattaa atggacttca 540 ctgagtcatt tcttgtgaag ggatttcact atgttatttt gatcataaat ctagagttac 600 cacttttact ccccataatt tgattagact ttcaaggaaa tattgtacgt caaaatttga 660 tatctcaaaa tcaaaccgat aaataaacat ggtttgctta aaatataaca ccacattttt 720 cacgtaattt caatcatttt tttcctatgt tattgcgctc tctcccctat atatataagt 780 atgcttctat ctctcctgtt tgcggccgtc gaacttgcaa atggctcagt ggtgccctgc 840 aatttggtcg agttacaaat ctccatttgg agctacaagt atactactac tcagtatctt 900 tgtttttaag ttatcgtaca aattcagaag caacaccaaa ctgaaatcac aggaagattc 960 ttatacctaa ctacagattc gaaagtgaag tcggcaaaag gagaatcagc cataagaaat 1020 cttgaaactg tgcacgcttt aaagacacaa tacttcatcc acaacacacg acttgcaggg 1080 aaaaaaaaaa tgtcgacctg agccctggcc aactcgatga gcagaaaatg tgtctagtgc 1140 ggtgtctgct aaatgataaa ttgtggccaa tctctagtga tcgtagaaga caagatcaag 1200 tattccatac ctgataaatt gtggccaatc tctagtgatc gtagaagaca agatcaagta 1260 ttccatacct gatacaggga caaagacgct gattattctt ctcttcagca tccggcctgg 1320 agcacacaag ttgattttct gttctgtggt ccttggccag cagcgctcca atcgctactg 1380 ccaaacggga accaccggtc catctcacca acaaaaagaa acacgaagca aacgcataag 1440 catgcttaca atcttattct tggaccacag ctgcgttcac ttgttaaaca tttattagtc 1500 gaacaagagg aaatatgcat accctgatac ccatgagaag ccagaccagt gttgacttcc 1560 tctgcaatta aatagatttc tgagcatgga aaatcttgat tactaacacc gactgtatat 1620 ctgaagagtc accgtttgtg tgtcataaga acagtacgtt tttctgtcat aagaacagta 1680 catataacat gagaacttca catgtaactg tattagtttc atttatctgc acagtagatg 1740 gcatcagctt tagaaccaca gacgcaagta tcttcagaag ccttccatat taaactgatg 1800 caagatgtta atgtcatgct aaatattgca ctggagaatc aaaacaacag aaacagacaa 1860 cagttagatc tttaaaaaag acgatggagg gaagggtggc cctgatggta acgcagactg 1920 aaaacagtca agtggtcagc tgcaacccca ggttatggat taggctggaa tcttgctcgt 1980 atccccttcc accactcatt ccaagccacc actcgttcat ctcagcccga gctgggccac 2040 cacagaaagc aagccctttc ctccatccac aaaacaatgc tcacttcatt ccacaaacac 2100 ctatggccac cgcttctcat tcaccttcct cctctcctcc cccttaatct catctttttc 2160 ttctcggtgc ttctctgctc aa 2182 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 1C-01149 Forward Primer <400> 2 aacaattcca aaacctcccc 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 1C-01149 Reverse Primer <400> 3 ttgcgggaaa atttaaatgc 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NGUS1 <400> 4 aacgctgatc aattccacag 20 <210> 5 <211> 3734 <212> DNA <213> Oryza sativa <220> <221> gene &Lt; 222 > (1) .. (3734) <223> LOC_Os02g38020 <400> 5 atcttcagaa gccttccata ttaaactgat gcaagatgtt aatgtcatgc taaatattgc 60 actggagaat caaaacaaca gaaacagaca acagttagat ctttaaaaaa gacgatggag 120 ggaagggtgg ccctgatggt aacgcagact gaaaacagtc aagtggtcag ctgcaacccc 180 aggttatgga ttaggctgga atcttgctcg tatccccttc caccactcat tccaagccac 240 cactcgttca tctcagcccg agctgggcca ccacagaaag caagcccttt cctccatcca 300 caaaacaatg ctcacttcat tccacaaaca cctatggcca ccgcttctca ttcaccttcc 360 tcctctcctc ccccttaatc tcatcttttt cttctcggtg cttctctgct caaacctgta 420 ctttgcttct gccactccat gtctcaatct tcccctttct tctccgttgc ccgagcacat 480 gctggagcag gagggcgagc tgcggctgcc gctctcctgc tccgtcaccc tgttgctcag 540 ctgccacctc ggattcatgg cctgaggtac tacccttcag ccattgtgtc acctgccaag 600 acactgaact cacacctgag ccttccccat gccacaatat cctcctttgc caatgctgat 660 aatggctcca gcgggcaagc agatgccaca gagtctgagg aggagcagaa cggggaatct 720 gaactgtcgg agatggccaa ggcattccat atatcgcccc ggatggcaat gtcaatctca 780 gtggtgattg catttgcagc gctcactgtg ccactggcga tgcagtcagt ggtcttccat 840 gggacgaaca aaatgaaggc actggcatat ctgacactgc tttcagggtt ctacatggca 900 tggaacattg gggcaaatga tgtggccaat gccatgggga cgtcagtagg gtctggtgct 960 ttgacactcc ggcaagcagt gcttactgcg gcggtgctag agttctctgg tgcattcctt 1020 atgggcaccc atgtcaccag caccatgcag aagggcatcc tagtcgcatc tgtcttccaa 1080 ggaaaggact ctctgctctt cgctggattg ttgtcatccc tcgctgctgc tggaacatgg 1140 ttgcaggtaa tattccaatt agatcacctt atcagttacc gcccttttga gctttgctgg 1200 aatggtgacg atcagaggat gcaatcatgt agtgccatta gcagtacata attactgaga 1260 cttctgcaag tttcagtata tgaaacaaaa tatttaagac attccatcag cgacacattc 1320 atgatgcaag aatagcatga tctacctagc aaggcaaaat caatagacct ttgcacttga 1380 agatgctagg ttaagcccca gaaataaatg atagcagtaa gttgtagcag ctttattata 1440 ttcagtgaaa cctactgcat gagaaccttc tatgagttga ttgtactata ttacacaatt 1500 tcctttgagg tataaactcc acaatacggt aaacaacttc catggtagga agtgaaaact 1560 gaaaagggaa aggaacttaa cagcatagct ttaaacttgt tgaagtcttt taacgtaagg 1620 attttaatcc aattatatgc agtttcagga tgttgtgtaa catccatccg tgaatgttgt 1680 gcagctcact tgcaattacc agagtttgat gtctatctta tcaacaatcc caactagcag 1740 aagtacagaa gcaacaaaat gagacaatta aagacaatgc tcactagaaa tatagcaact 1800 tttacaaggg atagactaat ggaatcttga atcattagac aactaggcat gcacaatttt 1860 gatgaagaca tttttggtgg gactctaatt gcgggagaat gtttcatctg cttaatcatt 1920 ggaattaaga tattttcatg attgcgggaa aatttaaatg catacatcat gcctcaaaga 1980 aatatttaac ataaaattaa atgcctctgc caaagatcca attgtgtgct cccttttacg 2040 gttatctgag catagcatat taacatttct cagagtcaat atgaattaac gcttgctatt 2100 tctaggttgc ttcctcctac ggctggcctg tttcaactac acattgtatt gtcggggcca 2160 tggttggttt tgggatagta tttggaggcg taaatgcagt gttctggagc tccttggcaa 2220 gagtatcttc atcatgggtc atttcaccat tgatgggtgc tgcagtctca tttattgttt 2280 acaagggtat acgaagggta agcactagac taactttact tgcagcatgc tagacagcat 2340 ttttaattta agagttactt agttaggcag tcaccttatt gaacacacat cataaataga 2400 ggacatagga ctgttcaaga caagacaata agttaattct ctgcttaccc tatttcattt 2460 cagttcgtat atagtgcacc aaatccaggt caggctgcag ctgctgctgc accaattgca 2520 gtttttactg gtgttactgc aatatcattt gccgcttttc ctctcagcaa gactttttca 2580 atcgctatcc tgcaagcatt agcatgtggc gcaataggag ccgtaatcgt taacagagtg 2640 atccaaaaac agcttggtga cctactgtcc tcagaagcag aaaagatagc atctgctgat 2700 aaggcaaatg ctcagcaagt tggatttctt tcggacatag ctggaccaac aggagctcaa 2760 ttgcagattg tatatggtgt atttggatac atgcaggtct tgtcagcatg cttcatgtca 2820 tttgctcatg gaggcaatga tgtctccaat gccataggcc ccctggcggc agctttgtcc 2880 attcttcaag gtgtagcaag cagtgctgag atagtcatac ctactgaagt ccttgcttgg 2940 ggaggttttg gaattgttgc agggcttaca atgtggggtt acagggtcat agcaacaatt 3000 ggcaagaaaa tcaccgaact aacaccaact agaggttttg cagcagagtt tgcagcagct 3060 tctgtagtat tgtttgcatc aaaacttgga ttgccaatat ctgctacaca tacacttgtt 3120 ggtgcagtga tgggagtggg atttgcaaga gggctcaata gggtcagagc agagacagtg 3180 cgtgaaattg tggcctcctg gttagtcaca attccagttg gtgctgtgtt atctatcttc 3240 tacacattgc tcttcacaaa gattctggca tactttatgt gagtgattgt gaaatgtaat 3300 tagtgttttg agaggtgtac agcccattgc acttcataaa aatttccgca atggcaaaga 3360 gataggctga ctccaaaaag tttcgaccat aaaatttttc aggattggcc agagtaaata 3420 atcatttctt tggcaacaaa acacatgatt aggtaaaaag cttttacagc cagttacaat 3480 gttacatact ccagcttgtt cgtccgttta tctctaatgt actgccttgt atcttttgtt 3540 ttatatcaca aggtggacca taataatttc ttgtagagat acaattatac ataatagaca 3600 acaatggttc ttctatcctt gcagttatgt tacatcaagg acatggttct ttaatagcca 3660 ctgaaaatta ataatgtttt tgtggtatgg tgcaagataa tagcaagttc actgctagca 3720 agaattcaga aaaa 3734 <210> 6 <211> 26 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > 02g38020-pro_BamH1 <400> 6 ggatccgagt ccgagtgtag gacttg 26 <210> 7 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> 02g38020-pro_Xho1 <400> 7 ctcgagttga gcagagaagc accgag 26 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > RB_backbone-F2 <400> 8 tcgcacggaa tgccaagca 19 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 02g38020-seq_1 <400> 9 ttattgcgct ctctccccta 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 02g38020-seq_2 <400> 10 cctgataccc atgagaagcc 20

Claims (7)

A promoter that is specifically expressed on a leaf, stem, or all of them including the nucleotide sequence shown in SEQ ID NO: 1. 2. The promoter according to claim 1, wherein the promoter is expressed specifically in mesophyll tissue in leaves, stems, or both tissues. A vector comprising the promoter of claim 1 or 2 and an operably linked foreign gene. A transformed cell transformed by the vector of claim 3. Transgenic rice (Oryza sativa) transformed with the transformed cell according to claim 4. A method for transforming rice (Oryza sativa) comprising transforming rice (Oryza sativa) with the vector of claim 3 and expressing the foreign gene in rice (Oryza sativa). A method for producing a rice plant transformant that is expressed specifically on leaves, stems, or both of rice plants comprising the steps of:
Preparing a vector comprising a promoter comprising the nucleotide sequence shown in SEQ ID NO: 1 and a foreign gene operatively linked thereto;
Introducing the vector into rice; And
The step of selecting the rice transformants into which the vector is introduced and in which the gene is expressed specifically in leaves, stalks or both of them.

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