KR20140046554A - Leaf-, stem- or both- specific promoter, expression vector comprising the same, transformed plants 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

{Leaf-, stem- or both-specific promoter, expression vector comprising the same, transformed plants thus and method for preparation kind}

The present invention relates to a promoter specifically expressed on a leaf, stem or both, an expression vector comprising the promoter, a transformant 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 pathway and microinjection method.

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 transformation basic carrier. In the research aspect, 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 is the root specific expression promoter. Although there is no commercialized case, Arabidopsis peroxidase (peroxidase, prxEa) was isolated and confirmed root-specific expression. Recently, the sweet potato-derived Maz gene (ibMADS) and sugar-induced ADP-glucose pyrophosphatase (ADP-glucose pyrophosphatase) , AGPase) gene is isolated to confirm that the promoter induces specific expression in the roots and induces root-specific transient expression in carrots and radishes (patent registration No. 10-0604186, 10-0604191) There is a bar.

Fourth, other tissue specific promoters, such as a leaf, 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 regulating 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 provides a promoter capable of expressing a foreign gene in a leaf, stem, or both of the plant, but not in the whole body of the plant, an expression vector including the promoter, a transformant transformed with the expression vector, and a method of preparing the same. I would like to.

In order to achieve the above object, the inventors of the present invention have discovered that promoters capable of regulating plant tissue or organ-specific gene expression can be identified, thereby specifically expressing or inhibiting a gene of interest, And to establish a system with In this process, it was confirmed that the promoter of LOC_Os02g03670 of rice is not expressed in petals, lobules, roots, surgery, pistils, seeds, etc., but specifically induces expression in leaves or stems, thereby securing the promoter and expressing the same. The present invention was completed by confirming that reporter genes were specifically expressed in leaves, stems, or both in transformants transformed by preparing vectors and then transforming them into 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 plant. Since the rice transformation of the vector for the verification of the promoter activity is difficult to take a long time, the present inventors have utilized the fusion ohmic analysis technique that has been actively tried recently by a method different from the existing method.

Since the GUS gene without a promoter is adjacent to the right end of the T-DNA of pGA2707, pGA2715, pGA2717, and pGA2772, the promoter search vector, when T-DNA is inserted into the promoter 3 'in the forward direction, the promoter is activated through GUS expression. Can be measured. In addition, since these promoter trap strains can be identified by genomic DNA PCR and sequencing of large-scale T-DNA insertion sites, it is possible to search for related strains by indexing gene locus ids in a database.

The isolated 547 leaf and stem specific genes were selected from over 280 lineages for 250 genes through the relevant promoter search line. Seeds of these strains were sterilized and whole rice husks were used for GUS analysis for 7 days in nutrient medium.

As a result, experimental group 1B-21622 was inserted with T-DNA into the second intron of LOC_Os02g03670, which had no homology with the existing gene, and fusion of GUS protein was expected after 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.

With reference to nucleotide sequence information, promoters up to 2 kb in front of ATG of LOC_Os02g03670 were isolated by genomic DNA PCR, and cloned into pGEM T-easy vectors for nucleotide sequence translation.

By using such a separated promoter, it was found that gene expression patterns specific to the leaves, stems, or both of the plants can be controlled.

The present invention is a promoter of the LOC_Os02g03670 of rice, a promoter specific to the leaves, stems or both to induce the expression of the foreign genes specifically to the leaves, stems or both (hereinafter referred to as the 'promoter of the invention') To provide.

The genomic DNA size of LOC_Os02g03670 is 4123 bp and the representative coding sequence (LOC_Os02g03670.1) is 969 bp, which is divided into 9 exons and 8 introns and translated into 323 amino acids to make proteins, but its function is unknown. Encrypt the DUF1995 domain (PF09353.3). In addition, the genetic information from the University of Michigan's rice gene function assignment task was estimated to be plastid from the estimated intracellular organelles. Thus, the function associated with photosynthesis can be predicted. However, no other information related to the function of this gene is known.

That is, the present invention provides a promoter for specific expression of leaves, stems, or both of the foreign genes, including the promoter of LOC_Os02g03670 of rice, which is the nucleotide sequence represented by SEQ ID NO: 1.

Promoters are essential elements for regulating the environmental, temporal and histological expression of genes. The promoters specific for leaves, stems, or both, according to the present invention can be used to control the expression of foreign useful genes in plants, such as leaves, stems, or both. Can be specifically induced.

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 that, upon introduction into an appropriate host cell, results in the expression of the cloned DNA. 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.

The conventional vector may be any one capable of introducing the promoter of the present invention, but preferably, a vector of the pUC family, such as pBR322, pBI121, pBGWFS7, pCAMBIA, Gateway vector, Ti-plasmid and a vector derived therefrom. It may be any one selected from the group consisting of.

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. Such nucleic acid fragments have more than 99% sequence homology compared to the nucleotide sequence set forth in SEQ ID NO: 1, and such nucleic acid fragments can be easily produced by molecular biological methods well known in the art.

The expression vector of the present invention allows the gene for the target protein to be expressed 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 leaf and stem characteristics such as color and development of photosynthesis, photosynthesis, and the like. Toc75, CIA2, GLK, 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 Genes related to photosynthesis such as PEP carboxylase, Magnesium Chelatase H subunit, Magnesium Chelatase I subunit, Magnesium Chelatase D subunit, Chlide a monooxygenase, glutamyl-tRNA reductase, NADPH: Pchlide oxidoreductase, glutamic acid 1-semialdehyde aminotransferase, Uro-III decarboxylase One or more selected from the group consisting of Mg-Proto-IX reductase, Pchlide a reductase, Chlide areductase, Chl a reductase, Protogen-IX oxidases, PASA, PSAB and the like 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 hosts include, but are not limited to, bacterial cells such as Escherichia coli, Streptomyces and Salmonella typhimurium cells and plant cells.

Any plant cell can be used as "plant cell" used for transformation of a plant. 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 plant transformed with said vector or said transformed cell.

Such transgenic plants exhibit the characteristic that the introduced foreign genes are specifically expressed in leaves and stems.

"Transformed plant of the present invention" and "functionally equivalent transgenic plant" is a trait produced using a nucleotide sequence having a sequence homology of 99% or more compared to the variant of the nucleic acid consisting of the nucleotide sequence represented by SEQ ID NO: 1 As conversion plants, they are transgenic plants whose specific promoter effects on leaves and stems have substantially the same characteristics.

Transformation of a plant of the present invention can be carried out by any method known in the art for 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. 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) Infections caused by (non-integrative) viruses in Agrobacterium tumefaciens mediated gene transfer (EP 0 301 316), and the like.

Plants to be transformed in the present invention can be used without limitation dicotyledonous plants as well as monocots including rice (Oryza sativa L.) used in this embodiment.

Preferably, monocotyledonous plants are used, more preferably rice (Oryza sativa L.).

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

In another aspect, the present invention provides a method for transforming a plant comprising the step of transforming the plant with the vector to specifically express the foreign gene in the leaves, stems, or both of the plant.

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 it provides a method for transforming a plant comprising the step of introducing a rice transformant is introduced to the expression vector is introduced specifically to the leaves, stems or both of the expression vector.

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.
Figure 2 is a diagram showing the T-DNA insertion position for confirming the promoter activity of the present invention.
Figure 3 is a diagram showing the GUS staining results and PCR results showing that the 1B-21622 line co-segregation to LOC_Os02g03670 gene.
Figure 4 shows leaf preferential expression of the 1B-21622 strain.
FIG. 5 is a diagram showing a cross section of a mature branch showing expression throughout mesophyll. FIG.
FIG. 6 shows microarray results showing preferential expression in leaves and stems of LOC_Os02g03670 gene.
Fig. 7 shows the ATG front 2Kb promoter of the LOC_Os02g03670 gene.
8 is a diagram showing a vector used for the production of LOC_Os02g03670 promoter GUS plants.

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 Figure 1, the closer to yellow corresponds to the gene showing preferential expression, the closer to blue, the lower the 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  Confirmation of the system

Since the GUS gene without a promoter is adjacent to the right end of the T-DNA of the promoter searching vectors pGA2707, pGA2715, pGA2717, and pGA2772, when the T-DNA is inserted into the rear of the promoter in the direction in which the gene is expressed and in the forward direction, GUS expression is expressed. Promoter activity can be known from this.

Through this method, T-DNA insertion sites were determined on a rice chromosome on a large scale by inverse-PCR and sequencing of rice plants into which T-DNA was inserted. This was analyzed through T-DNA index mutation information corresponding to 1/2 of the genome.

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. Each plant was treated with 15 ml of GUS staining solution for 24 hours, followed by decolorization 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. Thus, 100 mg of the leaves of the 7 days of cultivation were taken, quenched in liquid nitrogen, crushed, and some of the leaves were used for GUS staining. DNA was extracted from the ground leaves using CTAB buffer (CTAB 10g, NaCl 40.91g, 0.5M EDTA (pH8.0) 20ml, PVP 10g and 0.44g of ascobic acid and 500ml water).

DNA was extracted from 25 strains of selected rice seeds. Among them, experimental group 1B-21622 was a strain in which the GUS reporter gene was bound to the second intron of the LOC_Os02g03670 gene (SEQ ID NO: 2 and sequence). Genotyping was carried out by PCR using No. 3) and NGUS1 primer (SEQ ID NO: 4). The insertion position and corresponding sequence of LOC_Os02g03670 gene are shown in FIG. 2 and SEQ ID NO: 5, and the primers used are shown in Table 2 below.

Table 2. Primers of 1B-21622

2 is nucleotides 88 to 156 of SEQ ID NO: 5, T-DNA is nucleotides 167 to 710 of SEQ ID NO: 5, 2 is nucleotides 256 to 437 of SEQ ID NO: 5, and 3 is sequence Nucleotides 1048 to 1095 of SEQ ID NO: 5, nucleotides 1177 to 1218 of SEQ ID NO: 5, 5 is nucleotides 1318 to 1416 of SEQ ID NO: 5, 6 is nucleotides 1625 to 1817 of SEQ ID NO: 5 , 7 is nucleotides 1897 to 1995 of SEQ ID NO: 5, 8 is nucleotides 2301 to 2420 of SEQ ID NO: 5, and 9 is nucleotides 3011 to 3129 of SEQ ID NO: 5. Each of 1 to 9 in FIG. 2 is a sequence corresponding to exon.

PCR reactions were performed after 5 minutes at 95 ° C; Repeated 37 times for 30 seconds at 95 ° C, 30 seconds at 57 ° C, 1 minute 30 seconds at 72 ° C, and finally 5 minutes at 72 ° C. PCR reaction products were stored at 4 ℃ and some of them were confirmed by electrophoresis.

The results are shown in Fig.

As shown in Figure 3, it was confirmed that the PCR results of the experimental group 1B-21622 and the GUS staining results are the same.

FIG. 3A shows GUS staining results for five week old individuals of Experimental Group 1B-21622, and FIG. 3B shows the results of analyzing genotypes of individuals used in the experiment using PCR.

In FIG. 3A, the wild type has no T-DNA insertion, so that GUS expression does not appear and staining is not performed, and hybridization (he) and obedience (ho) were confirmed as blue staining results of GUS expression on T-DNA.

The upper band of FIG. 3B corresponds to the PCR product of the gene specific primers (SEQ ID NO: 2 and SEQ ID NO: 3) of LOC_Os02g03670, and the lower band shows NGUS1 (SEQ ID NO: 4) on RB (right border) of SEQ ID NO: 3 and T-DNA. Corresponds to the PCR product. The wild type was amplified only by gene specific primers, and the hybrid was amplified by both gene specific primers (SEQ ID NO: 2 and SEQ ID NO: 3) and NGUS1 (SEQ ID NO: 4) on the right border (RB) of SEQ ID NO: 3 and T-DNA. I could confirm this. Finally, obedience (ho) was confirmed that only amplified by NGUS1 (SEQ ID NO: 4) on the right border (RB) of SEQ ID NO: 3 and T-DNA.

That is, the above results confirmed that the 1B-21622 line was co-segregated to the LOC_Os02g03670 gene.

In addition, as shown in Figure 4, 7 days of individual staining results of the 1B-21622 strain GUS expression is not seen in the root, it was confirmed that it is specifically expressed in the leaves.

In order to confirm the expression pattern in the leaf as described above, the cross section of the mature leaf (flag leaf) was examined through the hand section.

1B-21622 strains matured in the rice paddies were cut into several pieces of 1-3cm in size, soaked in GUS staining, and stained for 24 hours. 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 Figure 5, it was possible to confirm the expression in the whole leaf tissue (mesophyll) among the leaves.

Finally, tissue expression patterns of LOC_Os02g03670 genes were analyzed by microarray database analysis, and the results are shown in FIG. 6.

As shown in Figure 6, it can be seen that the leaves and stems are yellow, which means that the expression in the leaves and stems is preferred.

Based on the results of FIGS. 3 to 6, experimental group 1B-21622, a strain to which the GUS reporter gene is bound to the second intron of the LOC_Os02g03670 gene, showed preferential expression in leaves and stems.

< Example  5> Promote promoter for leaf and stem preferential expression Cloning

PCR was performed using primers prepared for cloning the promoter of LOC_Os02g03670 and genomic DNA of Oryza sativa L. japonica cultivar-group. Template DNA was used at 100 ng and each primer was used at 5 pmol. PCR conditions were 5 min at 95 ° C; Repeat 5 times 30 seconds at 95 ° C, 30 seconds at 55 ° C, 5 minutes at 68 ° C; Repeat 35 seconds at 95 ° C., 30 seconds at 62 ° C., 5 minutes at 68 ° C .; Finally, it proceeded for 10 minutes at 68 ℃. The primer for promoter cloning is shown in Table 3 below.

Table 3. Promoters Cloning Primers for LOC_Os02g03670

The PCR product was electrophoresed to confirm its size and cloned into a pGEM T-EG vector and sequenced. After digesting the cloned DNA with Xba1 and Xho1, ligation with the pGA3383 vector (promoter-GUS cloning vector) digested with the same restriction enzyme was carried out at 14 ° C for 12 hours. The ligation product was mixed with 50 μl of Top10 E. coli competent cells, transferred to a 1.5 ml tube, and left on ice for 15 minutes. Subsequently, 1 minute of standing in a 37 degree oven again, 1 ml of LB liquid medium was further added to the tube, and then left in a 37 degree 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 obtained by miniprep was digested with restriction enzymes Xba1 and Xho1 and then agarose gel was separated by electrophoresis to identify the bands. The cloned DNA thus obtained was subjected to sequencing again to select a DNA in which no error occurred. The diagram of the vector used for the preparation of LOC_Os02g03670 promoter GUS plant thus made is shown in FIG. 8. Sequencing was performed using a 3730XL DNA analyzer (AB, USA) and BigDye v3.1 (AB, USA), and primers using NGUS1, RB (right border), and sequences in the promoter were used. Indicated.

Table 4. Primers for Sequencing

The results of the analysis are shown in FIG. 7 and SEQ ID NO: 1, and the promoter corresponding to the 2Kb promoter sequence before the ATG of the LOC_Os02g03670 gene corresponds to a promoter that preferentially expresses the leaves and stems.

Table 5 shows RNA-seq expression analysis showing preferential expression in the leaves of LOC_Os02g03670 gene.

Table 5. RNA-seq expression analysis showing preferential expression in leaves of LOC_Os02g03670

The data was derived from the Gene Expression Analysis Service (http://rice.plantbiology.msu.edu/expression.shtml) provided by the Rice Genome Annotation Project team at Michigan State University. RNA-seq analysis based on technology. Indicate the expression level of the genes examined by the frequency of RNA-seq identified for each library listed in Table 5. RNA-seq analysis showed the highest levels of expression in the 20-day-old leaves and seedlings of the four leaf stage. In comparison with other organs, the expression in leaf tissues was high. This confirms the expression pattern confirmed in the microarray.

< Example  6> Construction of Transgenic Cells

The plant expression vector produced in Example 5 was extracted.

50 아 of Agrobacterium tumefaciens LB4404 and 2 의 of plant expression vector were mixed and left on ice for 15 minutes. Subsequently, after 75 seconds in liquid nitrogen, it was left for 5 minutes in an oven at 37 degrees, and then 1 ml of LB liquid medium was placed in a 28 degree shaking incubator for 3 hours. Subsequently, the platelets were plated in tetracycline-resistant LB solid medium and waited for 36 hours to prepare mini-prep after cell culture in 1 mL of LB liquid medium, and the size was determined after enzyme cleavage using Xba1 and Xho1.

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

< Example  7> Construction of Transgenic Plants

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 cells grown with Agrobacterium transformed with the plant expression vector obtained in Example 6 for 72 hours and left in a dark treatment room at 22 ° C. 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 cleaved callus was transferred to MSR (hygromycin 40 mg / L) solid medium, which is a regeneration medium, to induce re-differentiation in the growth room at 28 ° C. for 4 weeks, and then the plants were transferred to MS solid medium and grown in 28 ° C. Transferred to grow replanted plants.

< Example  8> Verification of Leaf and Stem Priority Expression Using Transgenic Plants

In order to confirm the organ specific GUS expression of regenerated plants, 10 seeds harvested were cultured at 28 ° C for 7 days and all individuals were stained with GUS.

<110> University-Industry Cooperation Group of Kyung Hee University <120> Leaf-, stem- or both specific promoter, expression vector          comprising the same, transformed plants enhance and method for          preparation <130> P-12-076-KHU <160> 10 <170> Kopatentin 2.0 <210> 1 <211> 2320 <212> DNA <213> Oryza sativa <220> <221> promoter (222) (1) .. (2320) <223> Promoter of LOC Os02g03670 <400> 1 gatggagaag tcgatgttcg atagggtggt tattcagaca atcggcagag agaacatccg 60 gttgcacaac cacctgctca tgtcggtcct ccgcaatgca tctctcccag ctccccatcc 120 aggtgcaccg ccaggagcca tgtgattttt tgcgacaagt ttgtttcagg gtttttagaa 180 atctcttgga aggagcggat tttgcaggat ttttctgata aattagtcat ggggaaaatt 240 ctatttgttc ggtaatagag ttcttggtga tgtttgttag gaagaaccac catttagttt 300 gattgttggt gttacttcgt cacctattcg aggatatgta tcctttagcg ttaaactata 360 attttgattg ttaaattcat ttttttccct agatttaatt catctcctgg ttttggatgc 420 aagtagcagg tgatttattt gcaatgagga tgttgaagtt tttatagttg aatcattatg 480 attaagcttg ctattgcaaa acacttattg tttttctttt ccatttctac acctacattt 540 gcataaaaga tctagtaccc attgatgcat gtgaaatgta ctatacttac ttcagagcat 600 gtacaatagc aggctataag ccagctataa acacatttcg aagagataaa agagaagaga 660 gaagggcaac ggcttacaga tttgtagcca gctgtagcac ggacttaagg ttcctccaag 720 gtgcatgtgt atgacatgtg ggaccagata ttaattatgc agtatatgtt tataagtaac 780 tattgtatga attagctatt aagttgacta tagatgattt agagccagca gttggctata 840 ctattaaact tcctctcata taatactccc tccgtttttt aatagatgac gctgttgact 900 ttttctcaca tatttgacca ttcgtcttat tcaaaaaatt tatgtaatta taatttattt 960 tgttatgagt tgttttatca ttcatagtac tttaagtgtg atttatatct tatacatttg 1020 tataaaattt ttaaataaga cgaatgatca aatatgtgag aaaaagtcaa cggcgtcatc 1080 tattaaaaaa cggaggtagt atgtttggat gcaagaattc tgtggtatct ttccatagta 1140 agtttaagaa actctaggtc gttggatgcg cctggaaaca ttccgagatt cgtacacgtg 1200 gcgaaacacg ctacccgaag cagacgccac cgccgctcct ctcctccgcc gccgtcggag 1260 cacgccgccg gcgatatcct ccggtagacc tcaggcatcg cggcatcacc accctctgcc 1320 cggctagcgg agcggagtag tccccgccgc gcgttcgcgt ggagctcgca agcctgcaag 1380 tctgcaacca gcatggctct gggcttcatc tggaaccgga tgctcgcccg ccgacgcccg 1440 cgtggtgttc gacgtaatgc cgacgaggga ttccgctcgt ggcccgagcc cccgcgagga 1500 gcaggagggg cgccgaggcc agggagctct ttagtttgac atgatgccat cggggaatgg 1560 ctaatcgtgg ctgacaatga tcttctagct aaaccaagag gaagcatatc tgcagagcgg 1620 ggggagcttt ctgacttcgg tttcggtgtg cgacgctgta ctgtattgct gtcaaattct 1680 gtcgagcttt agcagatact tggattgaga cttgagcaga taagccgaag tagaagaggc 1740 agagtgcttt ggtgctatgg tcgtcagaat ggattcggaa agtttctgtg tgatctttct 1800 actatttttg acgatcttca tggcttcaga gttcagacac cgatttttgg tcgaggccag 1860 gatatgccca gatggaaatt gcttactgaa agtattcatt atgctggacc tcactccgtt 1920 ctattctgag gattccttac aaggctaaca tcgagaagct caaaacaaca ttttgccaat 1980 tggtaggtat cctctgtgtc catggctgac agaagcaaag gcaaggtgaa gtactggagc 2040 attaaaccgc ttgtttttac tcagcttctg ttcaggcaac tcatttctca accaagattt 2100 taagctgcta tatgcaaaat actagatttc ctctgtatag ttgtccacca tcttaaaatt 2160 tttaaaacaa aactagcaga gaaaacgtct ccatcttatc acccaatggc agcatatgca 2220 cagaactgtc ataacagaca agatattttc ccccatctct ctccaattgg agaccacaca 2280 gtaaggcccc acaaacagat tgctgctcaa acatttctac 2320 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of 1B-21622 <400> 2 tttctcagca gggacaaagc 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of 1B-21622 <400> 3 aagggcaact acactggtgg 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer of NGUS1 <400> 4 aacgctgatc aattccacag 20 <210> 5 <211> 4123 <212> DNA <213> Artificial Sequence <220> <223> Recombinant DNA of LOC Os 02g03670 <400> 5 aaacatttct acattaccac atagccttct ttgtttctcc ctccctccct ctccaacaca 60 ccaatcccat ccccagtgtt cagtgaccat ggcaaccagt tattgctcca tctccaatcc 120 tccattatcc aaaacttcct ttcccaataa acaggtatgc tatcacaaac aagctacaac 180 atcttttgtt cttaatgttg tacttattat cagcatttct gcttatgcat tgtcttggat 240 tttttttttc aaggtcccag gttgggtact tcgagcaatc agcaaaggca agggcaacta 300 cactggtggt atttatacta caacaaagag gaatttaaga actgggttcc atgtttgtgc 360 cgtcaatgga ggtcagggca ctcgcaatgt gtctggggca gagttcccaa gcgattacac 420 tgaacttcta gcgcaggtac ttaatgtcag agtgtgaaaa cttgttcact gatagttgat 480 aatcataaag catgcttatc ttagtctctt tacttgagta tatataacta taagcatgta 540 gctcgctatt gcaactgctc aattaagata atccagttaa cccacttaaa aaattcagac 600 agtggcagca ttactgaatt aacaatgcca gctgtttgtt ttaagtgatg atattactga 660 aatatgggtg gttcatgtac attttggtca aaattttgtg agatctcacc tcataaaaag 720 aatcttgatg ggttgcttat cagcaagact gaacttttcc gtgcatgtta cctgttctga 780 tggagcacca taggtgtata atcaaaggca ccgcatttag atgtctcatt ttttgcactc 840 tactgaacta gttgcgaagt catcgttgtg gatagatgtc caaaaaataa catatgaagg 900 gtttatgtgt aattttgtac tgggatatcc aatatactga agctggaaac tgtatttctg 960 taataaccag aaactatgca cttgtttagg gatggaggga gtagaataat cgaatgacta 1020 attcactata catgtagtga tttcaggcaa aagaagctgc tgagtcagct tttaaggatg 1080 ggaagcagct attggtaaag ccttaatctg tccactgtct gacttatttt tttattcctt 1140 tttttgcaca agaaattgtg atgcaatgta tttcaggaaa ttgagttccc tacagcagga 1200 ctacaatctg taccaggttc tacatagcta acctttatgt atattacctt gttataagat 1260 attggtcagg actcaggtgg aaaggatacc atataagatt ctattttact tttaggtgac 1320 agtgaaggtg gaattgagat gactggaagc atgcttctta taagagaatt ctgtgatcgc 1380 tttgtccctg ctgagaaagc tacaagaacc agaatcgtat gttctgacca ttttctagta 1440 actagtacta caggctaatc tgtaaatgta ttcctgactc tcatttataa acagcaaaaa 1500 gacaaagttg tacatggata atcactggca gttttctacc agtgattgaa gcacaaacca 1560 gtgtgctgaa attaaacttg aaatgtcctg cttgattcca tttttattat ctgacatgtt 1620 tcagttcttc cctgaggcaa atgaagtttc atttgcaaga caatctgcct ttgaaggatg 1680 ttccctgaag ctagattatc taacaaaacc atccctattt gaagattttg gttttacaac 1740 aaaggtcaaa atgtcagacc gtgtgcgtcc agaagacgag atattcctcg tggcttatcc 1800 ctatttcaat gtcaatggta tgtgtttctg tttattatta ttgctctatc gaagttgagt 1860 ttaccacttc actaatatga tgatgatgtt ttccagaaat gcttgtggtt gaagagcttt 1920 acaaggaagc aattgttagc acagaccgaa aactgataat attcaacggg gaactagatc 1980 gaataagaag tgggtgtatc cttgtttgta atttagagtt tgtatttcat atgatgacaa 2040 aactgctaat tattctgtgc taaactgcta attctagtgc atatatgatg ccaaacagtt 2100 gatcttatgc caaacaagtt attaaatttg tttttaaatc aaacctgttt tcctgcatca 2160 caaatgatgt gtaaattttg cagtgctagt gactttcctc aacaaaagag aagctgcgct 2220 gatgatgttt gaaaatagta tgcttgtgaa attctagtta gctatcattt gtgttggttc 2280 atttccttga ccaatcaaag actaccctcc tttcttctac ccaaagctag cggaactttc 2340 caaaacattt cttccaaagc tggagacagt ttattatatt cacaatttta agggactcaa 2400 aggaggaaca cttttcaggt ttgcatattc cagccttctt ctgttgctct ccaaaattag 2460 caccactgac tcatgtctat gcttttgcat gagcaagttt tggtctagct ataattaatt 2520 aattcattga ctgattatac agttttatgt tggccaatca ttcttgaatc aatgtaccaa 2580 tgatatggca cccaaattag atgccttcag ctgccatact ttttggagga tccgattaac 2640 tgtgtttggc aagataatta acttacttac actccaataa ccctagttga ttttgtctgt 2700 tacccagttt gaatgatgct ctagttaaca tgacaacttg aatgttgcgt tctatccttt 2760 attgttatta ttgtcaccat cgccagcatg gctattgttt cttgatatgt aatgctttga 2820 aacaaatata ttcagcttgt tgttaccatc aactcaccat accaagatgt agttgcagaa 2880 atttagtttg gtataagttg cagtatgttt atctcatcag gagaaaaaca tgattgcatt 2940 cagaaccttt atggagcaaa ctgtggagat taaacgtgtg attttgccga aactttgttg 3000 tctggcaggt gttatcctgg gccttggaag gttctgagaa acataggtgg cagtttcttc 3060 tgcttgcatg aacaagaaga gatgccatca ctgaaggaag tggccctcga catccttcct 3120 tctgcctaga acttttcatg agtcattcca agcataagcc aagaaaatgg agaagggaaa 3180 aagcctaaaa gactacaggt tatttctccc tatatgttga tggactttcg ttgtacataa 3240 ggagagctag gaacggatgt attgctctga tgtgtacatg catctgtgca tcaaagtgaa 3300 tgcatggttt ggtgccttga tggcgatcaa tatttgtcct gccaaaaatt aagacttttt 3360 tttttgtcgg gatgaccgaa ggaggtcatc caatgttatt aagatagagg aggaaaaaaa 3420 tgttacaaga aggtttttca tcgacaacaa gttgtcgagg aaggatgtgc accaaagcac 3480 actccccgac accacatgcc aaccgcacaa acacaagcta caaagaagag agccgaacac 3540 cgaaccggcc gccgctatgc aaggcaggtc gtcgccgggc ttacaacaac accacataac 3600 ggatattcta aaaaagacaa cgccttcacg aaggtagtga cgtcaaaaga cgccgtcgtt 3660 gtccgtccaa agagactaga catggttttc acctagagat cctcgtcgaa ggaggagggt 3720 acctcgacaa tgcccccaag agggttaccg cgcctgaagg cgtagccgct gccgaccaag 3780 tgttttgtta ccgtgcttga aggcgtagcc gctgccggtc cagtgaaaca ctgggctagg 3840 ctttcgcctc ctctaccccc gtcgtcgtgg tccgtcgccc accaactctg ataccaccag 3900 cccagtgtat gagaagtaag gatccgttgg gtaaactgaa atgctagtgt gtaagagaga 3960 tcggataatg gacagctcag gatttatgca aatttgagta ttttggccag gataaaaaca 4020 tcaagaggaa gaaccggcct tcctttctgt taattgagat cgaagtgttg caaattgctt 4080 agaagaacga taccactaag aacaagttaa caattgatct aaa 4123 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer of 02g03670-pro_xba1 <400> 6 gtctagagat ggagaagtcg atgttcg 27 <210> 7 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Primer of 02g03670-pro_xho1 <400> 7 ctcgaggtag aaatgtttga gcagca 26 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer of RB_backbone-F2 <400> 8 tcgcacggaa tgccaagca 19 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer of 02g03670-seq_1 <400> 9 tgtatgacat gtgggaccag 20 <210> 10 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer of 02g03670-seq_2 <400> 10 ggctctgggc ttcatctg 18

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. A vector comprising the promoter of claim 1 and a foreign gene operably linked thereto. The transformed cell transformed by the vector of claim 2. A transformed plant transformed with the vector according to claim 2 or the transformed cell according to claim 3. The transformed plant of claim 4, wherein the transformed plant is rice (Oryza sativa). A method for transforming a plant, comprising the step of transforming the plant with the vector of claim 2 to express the foreign gene in the plant. 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 a nucleotide sequence represented by 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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