KR20140049128A - 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

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 is to provide a promoter capable of expressing the expression of foreign genes only in the leaves and stems, not the whole body of the plant, an expression vector comprising the promoter, a transformant transformed with the expression vector and a method of producing the same. do.

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_Os06g49160 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, T-DNA was inserted into the 3rd intron of LOC_Os06g49160, which was not homologous to the existing gene, and GUS expression in this line was confirmed by GUS expression analysis and genomic DNA PCR. It confirmed that it showed.

With reference to nucleotide sequence information, promoters up to 2 kb in front of ATG of LOC_Os06g49160 were isolated by genomic DNA PCR, and cloned into pGEM T-easy vectors for nucleotide sequence translation.

The isolated promoters were found to have a significant effect on the regulation of leaf and stem-specific gene expression patterns of plants.

The present invention is a promoter of LOC_Os06g49160 of rice, a promoter specific to leaves, stems, or both, which induces the expression of a foreign gene to be specific to leaves, stems, or both (hereinafter referred to as the 'promoter of the present invention'). To provide.

The genomic DNA size of LOC_Os06g49160 is 1580 bp and the coding sequence is 720 bp, which translates into 239 amino acids and is expected to produce thylakoid lumenal 16.5 kDa protein. In addition, the location information in the intracellular organelles estimated by the Gene Ontology analysis provided by the Rice State Function Assignment Project, administered by the University of Michigan, is thylakoid, and the specific function of the thylakoid lumenal 16.5 kDa protein is not known, but photosynthesis and It is assumed to have an associated function.

Compared to the protein sequence of LOC_Os06g49160, 71% of the ortholog, AT4G02530, and 82% of the ortholog, Sb10g029300, were 82%.

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_Os06g49160 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 provides a promoter specifically expressed on leaves and stems including the promoter of LOC_Os06g49160 of rice, which is the nucleotide sequence represented by SEQ ID NO: 1.

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. An expression vector thus refers to a recombinant DNA or RNA construct such as a plasmid, phage, recombinant virus or other vector that results 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.

The above-mentioned normal vector may be any vector capable of introducing the promoter of the present invention, preferably a vector derived from a Ti-plasmid such as a pCAMBIA family or a pGA family vector such as pGA3383, pCAMBIA1381, pCAMBIA1391, pGWB3 And the like. More preferably pGA3383 can be used.

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 transformed transgenic plant transfected with said vector or said transformed cell.

Such transformed monocotyledonous plants exhibit the property of the foreign genes to be specifically expressed in leaves and stems.

"Transformed monocotyledonous plant of the present invention" and "functionally equivalent transgenic plant" is prepared 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 Transformed monocotyledonous plants, which are transformed monocotyledonous plants with substantially equivalent characteristics of specific promoter effects on leaves and stems.

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.

The monocotyledonous plants transformed in the present invention can be used without limitation, monocotyledonous plants including rice (Oryza sativa L.) used in the present 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.

The present invention also provides a method for transforming monocotyledonous plants comprising the step of transforming monocotyledonous plants with the vector to specifically express the foreign genes on the leaves, stems, or both of the plants.

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.
3 is a diagram showing GUS staining results and PCR results indicating that the 2D-10589 strain co-segregates to the LOC_Os06g49160 gene.
4 is a diagram showing leaf preferential expression of the 2D-10589 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_Os06g49160 gene.
Fig. 7 shows the ATG front 2Kb promoter of the LOC_Os06g49160 gene.
8 is a diagram showing a vector used for the production of LOC_Os06g49160 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 2D-10589 is a strain in which the GUS reporter gene is bound to the third intron of the LOC_Os06g49160 gene (SEQ ID NOs: 2 and 3). ) And NGUS1 primer (SEQ ID NO: 4) was used for genotyping by PCR. The insertion position is shown in FIG. 2 and the primers used are shown in Table 2 below.

Table 2. Primers of 2D-10589

2 is nucleotides 148 to 541 of SEQ ID NO: 5, 2 is nucleotides 653 to 794 of SEQ ID NO: 5, T-DNA is 463 to 990 of SEQ ID NO: 5, and 3 is SEQ ID NO: Nucleotides 906 of 979 to 979, and 4 is nucleotides 1122 to 1232 of SEQ ID NO: 5. Each of 1 to 4 of FIG. 2 is a sequence corresponding to exon.

The PCR reaction was repeated 5 minutes at 95 ° C., 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 FIG. 3, it was confirmed that the PCR result of the experimental group 2D-10589 and the GUS staining result were identical.

FIG. 3A shows the GUS staining results for 5 weeks old individuals of 2D-10589, and FIG. 3B is a result of genotyping analysis of the subjects used in the experiment A of FIG.

In Figure 3B, the upper band is the PCR product of the gene specific primers (SEQ ID NOs: 2 and 3) of LOC_Os06g49160 and the lower band is the NGUS1 (SEQ ID NO: 4) on RB (right border) of SEQ ID NO: 3 and T-DNA. PCR product. Comparing the results of the two products allows genotyping of the plant.

If only the products of SEQ ID NO: 2 and 3 are amplified wild type (wt), and if both the products of SEQ ID NO: 2 and 3 and the products of SEQ ID NO: 3 and 4 are amplified hybrid (he).

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.

In other words, the 2D-10589 strain was co-segregated to the LOC_Os06g49160 gene.

In addition, as shown in Figure 4, 7 days of individual staining results of the 2D-10589 strain GUS expression is not found in the root, but it was confirmed that it is specifically expressed 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.

Several mature ripening lobes of 2D-10589 strains developed in rice fields were cut into 1-3cm 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_Os06g49160 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, it was confirmed that the experimental group 2D-10589, a line in which the GUS reporter gene was coupled to the third intron of the LOC_Os06g49160 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_Os06g49160 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.

Promoter cloning primers are shown in Table 3 below.

Table 3. Promoters Cloning Primers for LOC_Os06g49160

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 and then ligated with pGA3383 vector (promoter-GUS cloning vector) digested with the same restriction mark at 14 ° 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 obtained by miniprep was digested with restriction enzymes BanmH1 and Xho1 and then separated by agarose gel by electrophoresis. 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 LOC_Os06g49160 promoter GUS plant preparation thus made is shown in FIG. 8. 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 the promoter corresponding to the 2Kb promoter sequence before the ATG of the LOC_Os06g49160 gene corresponds to a promoter that is preferentially expressed in leaves and stems.

Table 5 shows RNA-seq expression analysis showing preferential expression in the leaves of LOC_Os06g49160 gene.

Table 5. RNA-seq Expression Analysis with Preferred Expression in the Leaves of LOC_Os06g49160

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. Expression in other tissues was also higher in leaf tissues. 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 generate minipreps after cell culture in 1 mL LB liquid medium, and then the size was determined after enzyme cleavage using Xba1 and Xho1.

The resulting transformed Agrobacterium was used in the transformation experiment of rice.

< 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> p12-075-khu <160> 10 <170> Kopatentin 2.0 <210> 1 <211> 2005 <212> DNA <213> Oryza sativa <220> <221> promoter <222> (1) .. (2005) <223> Promoter of LOC Os06g49160 <400> 1 cgcgccctct gccatggtgg gcttcgtgcc ccggatgcac tggcttgctg acccggtatg 60 cacaaatgca ctgcacactg acgaacattt ctgaatgttt tggtgcatca tgagtaatat 120 ctcgctgaat ttttggtgca tcataagtaa tctgcacatt aatttatttt tgaaagacca 180 catgatacgt atactaattg aggatgacta cacatgggtg gctacttctt ggtgtaattg 240 ctgaatttta ttgaggaagt tagttatgcc aattggagtg tctgcttctg cagggaagta 300 atgcaaagga atacagatat ggaagctggt ggtcagtttg gtggacaggg acttatagca 360 tggttctctc caaagcaagt tttttccaca ggcagtactt ggatctgtat accattcgca 420 tgcttccgtc tattcgtgat tatgtgaatg aaaacaggtg agattcatat cttactagtt 480 actaccgcaa catttcttca gaaagtttct tcagctgcgg gtattattat gctatctgaa 540 taatctgatg gtcatttgtg tagtatacta gtaagtctta ccattttaac tgattattga 600 taggaattgc gaggatattg caatgtcctt tcttgtggca aatgtaactg gatctccacc 660 aatatgggtt caaggtcagt aatcatcctt gtgcagcaat gaaatcattt tcagatcgag 720 aaacttgcag acccctattt agttcaatta gtcacacatc atgcaacgac actttgcatt 780 tcttcccagt attggagaat ttcctcttgg agtatgagca gcagcaaatc aatgtagttg 840 tctgaaactt tgctatcctt ttatatgctt gtcagttttt gtgatacctt ctgtattacc 900 ttgaatcagc aagaaccatg tggaagcact gtctaaattc atgtgacaaa ccaacaacga 960 agatcaataa aataagagta taagatgatt tgctatttag cctgctgcta ttgttgtcag 1020 gaggccatac tgaagttccc tttcgtgatg ttactgcagg aaggatattc gagattggat 1080 caagcggcat tagcagtttg aaaggccatg atttacagag atcaaaatgt ctcaatacat 1140 tttctgccat gtatgggcat atgcctcttg tagccaccac agtcaaagct gttgatagcc 1200 gcacaagctg gttctggtga tggttctgtt ttgccttccc ttaggtgctg cctgcctatt 1260 tgcctagcca atattataag gctagtgtaa tcatgtgcat gattcatcaa cagctaatga 1320 caaggattat attgtacaaa ctgattcttt ggaggagata ttgtacacct gaaactaaag 1380 gctttgattg attttctaat caattgataa atgagattta tgtggctaag tacttgtcct 1440 aactgtttgg aaacgtgcac atctgggggt tcataacgga aggcaaatgt acctaccaaa 1500 tcactaaagt gagttcatca tatggcgtgt tacgtgtaca tgtgtctaga gtttgaaatt 1560 tagttttttt tttctcaatt tctttcttct tgaaatacat cacattatgt gttctcctat 1620 ggataattgc ttcatccatg tataaatggc aagaaataaa agagtaagtt taaatagggc 1680 caacaaaaaa caaactaaat tgagtttaaa tttcacccaa atttatttct acgaaattta 1740 aatttatcag gaattaagtt caattctaac ttcaaaattt acaaacttcg tttcccgtaa 1800 tttttatttt tgttcttttc aaatgtcgaa cgctgatggt gtccatggct aactcgtgtg 1860 tggctgcgac ggttgcacca agattgtgag gataaggaga gctttgctcc agtctaacca 1920 ggctgaactg ccacgctcac acccttctcc tcctcctctg ctatctgctg ctgctgctga 1980 aaaattctct cgaatcgatc gcccg 2005 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of 2D-10589 <400> 2 tccattgagt tcttttgggg 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of 2D-10589 <400> 3 catgccagga aaaagtagcc 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer of NGUS1 <400> 4 aacgctgatc aattccacag 20 <210> 5 <211> 1580 <212> DNA <213> Oryza sativa <220> <221> gene (222) (1) .. (1580) <223> LOC_Os06g49160 <400> 5 gtgtggctgc gacggttgca ccaagattgt gaggataagg agagctttgc tccagtctaa 60 ccaggctgaa ctgccacgct cacacccttc tcctcctcct ctgctatctg ctgctgctgc 120 tgaaaaattc tctcgaatcg atcgcccgat ggtggtggcc atcgctaccg aagcgtgggc 180 gctcgccgga tgcggcgcgg cggccaagtc ggcggccgcg gcggcgcagg aggcgccggt 240 gcagctgcag cagcatagtc tttctgcagc cagggccaag aaaccgattt cgttcagagc 300 ggtggcggcg gcggcggtca gtagccaatg ccaccaagaa cggcgcgccg tcgtcgtcgg 360 gagacgcagc ggcatggcgt cctgcctgct cgccgccgtc gctgcctccc tctccggcgc 420 cggtgaagcg cgggccgcgg tcctggaggc cgacgacgac atcgagctcc tggagcgcgt 480 caaggaggac aggaagaagc ggctccagaa gcagggcgtc atcagctcct ctggcaccga 540 gacaggtcgg ctcatcatac atcagagcaa tttcatctag ctaatttctt caatgattat 600 cgattaatct ctcttgtgct tgttaattgc actctagttt taattacaag ccacagggta 660 cttgcaggat ctcatctaca agctgagcaa ggtagggcaa gccattgaca agaatgacct 720 ccctgctgca agcagtgtcc taggcccaaa ctctgacgct cagtgggttc agaacatcaa 780 tgtagctttc accaaggttc catttctcca tcctggactc agtttcttct gaatattatt 840 tgatatttcc ttgtcaagtt gtgtagttta tttcatctga ttcagaacca ctgtttgcat 900 gcaacagttt agctctagcc cagaggagaa gaacatggtc gatagcttca attcctcctt 960 ggcctccttg attacatctg gtacactggc tcttgttctt cttctgttct ctagaatctg 1020 aacttaccct tttctgtcta aaaaaggaag attctctcag tgatttttct gttatgccaa 1080 tctgaatctg aaacactgca tgtatgtatc atcttgttgc agtgaacaag agtgatgttg 1140 attcatccaa gtcggcgttt gtgtcgtcag ccacaacgct ggagaaatgg atagcttcag 1200 ccggtttgag tggtcagctc aaaggattct aaatttctga cgccaattga atctgaatcg 1260 cctgacaagt acatgatcgg agtagcattt ccttgaagca atggtctata tataatgcgt 1320 caataccagt aagaatgttt tgtccaagaa aaagaatttg aagatatgga ttcggctgct 1380 gtgaatcttc gaagagggta ttgtaactga atcttcagtt cagacagaat atgtgataat 1440 agccatggct ctgttttttc tctctctccg tgggaagtaa attgcatcat ttcgtatatc 1500 acgagattct ttgcaatgca gaagaaaatt tagcaaagtt tctttgtttt catgtctata 1560 tagctttcgt ttgcaagcaa 1580 <210> 6 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of LOC_Os06g49160 Bamh1 <400> 6 ggatccatgc actgcacact gacgaa 26 <210> 7 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of LOC_Os06g49160 Xho1 <400> 7 ctcgagagtt cagcctggtt agactg 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> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer of Os06g49160_seq1 <400> 9 gtctagagat ggagaagtcg atgttcg 27 <210> 10 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Primer of Os06g49160_seq2 <400> 10 ctcgaggtag aaatgtttga gcagca 26

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 monocotyledonous plant transformed with the vector according to claim 2 or the transformed cell according to claim 3. 5. The transgenic plant of claim 4, wherein the transgenic plant is rice (Oryza sativa). A method of transforming a monocotyledonous plant comprising transforming the monocotyledonous 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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