KR20150048947A - Pollen specific promoter, Expression vector comprising the same, transformed plants thereby and method for preparation thereof - Google Patents

Abstract

The present invention relates to a pollen specific promoter, an expression vector comprising the promoter, transformants transformed with the expression vector, and a manufacturing method thereof. The promoter of the present invention can be helpfully used for growing crops with the increased productivity by specifically controlling gene expression required for improving a pollen trait, and improving pollen development or the like of main grain including rice.

Description

A pollen-specific promoter, an expression vector containing the pollen-specific promoter, and a method for producing the pollen-specific promoter,

The present invention relates to a promoter specifically expressed in a pollen, an expression vector containing 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 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. (IbMADS) and sugar-induced adipose-glucose pyrophosphatase (AGPase) genes were isolated from the sweet potato-derived rice paddy rice, and the peroxidase (prxEa) (Korean Registration Nos. 10-0604186 and 10-0604191), confirming that the promoter induces specific expression in roots and induces root-specific transient expression in carrot and radish.

Fourth, other tissue-specific promoters such as leaves may 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.

On the other hand, the development process from the microspores to the mature pollen is an important biological process that is based on the existence of a relatively short cycle or seed plant belonging to the germ generation of a plant which makes a male spouse. It is very useful. In particular, a transformant vector containing a promoter that specifically expresses only in the microspores and pollen tissues can be prepared, and thus the transformant can be used for cultivating an increased pollen activity. Individuals cultivated in this way have a great agricultural use, for example, by increasing the activity of pollen from major crops such as rice to increase productivity.

However, there is a lack of research on promoters that can act specifically on pollen in contrast to other tissue - specific promoters. The development of the pollen is closely related to the temperature, especially at high temperatures, the pollen development is inhibited. Hyundai is at a high risk of exposure to high temperature crops due to global climate change, which can lead to reduced productivity due to pollen developmental disabilities. To solve this problem, it is important to develop a promoter capable of regulating gene expression in pollen. Therefore, it is necessary to study a new promoter that can control the expression in the pollen more precisely according to the intention of the developer.

Korean Patent No. 10-1185845 Korean Patent No. 10-1015972

The present invention is intended to provide a promoter capable of expressing expression of a foreign gene locally to a flower pot, not a whole body of the plant, an expression vector containing the promoter, a transformant transformed with the expression vector, and a method for producing the same.

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_Os11g20384 of rice was expressed in a pollen-specific manner from other tissues of plants. Thus, an expression vector containing the promoter was prepared and introduced into a plant to transform Confirming that the reporter gene is expressed in a pollen-specific manner in the transformant, thereby completing the present invention.

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 expression of important traits in pollen.

Through this method, 672 genes showed higher expression patterns in pollen than 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.

With reference to the base sequence decode information, it is estimated that about 2,500 in front of ATG of LOC_Os11g20384 The promoter of bp was isolated by genomic DNA PCR and cloned into pGEM T-Ease vector to decode the base sequence.

The thus isolated promoter was inserted into pGA3383 vector to prepare a transformed plant. By verifying the GUS reaction in the transgenic plants, it was found that the pollen-specific gene expression of the plants can be induced using the corresponding promoter.

The present invention provides a promoter specifically expressed in a pollen containing a promoter of LOC_Os11g20384 of rice, which is the nucleotide sequence shown in SEQ ID NO: 1.

A promoter is an essential element for controlling the environmental, temporal, and systemic expression of a gene. Using the pollen-specific promoter according to the present invention, a pollen-specific expression site of the foreign useful gene can be induced in the plant.

The present invention provides a pollen-specific promoter (hereinafter referred to as "the promoter of the present invention") which induces the expression of a foreign gene to be specific to pollen, as a promoter of LOC_Os11g20384 of rice.

LOC_Os11g20384 encodes the SacI homology domain containing protein and consists of 17 exons. The promoter of this gene consisting of 597 amino acids has the nucleotide sequence of SEQ ID NO: 1.

The invention also provides a vector comprising said 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 of SEQ ID NO: 1 of the present invention is a sequence located in front of LOC_Os11g20384 on the published rice genome sequence, and even if some of the nucleotide sequences shown in SEQ ID NO: 1 are added or deleted, It can be included in the scope of the present invention if homology with the sequence located before LOC_Os11g20384 is maintained and exhibits substantially equivalent effect.

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 be expressed specifically in pollen.

The foreign gene may be any gene that desires expression in a flowerpot.

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 the host include 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 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.

The "transgenic plants of the present invention" and "functionally equivalent transgenic plants" are 60%, 65%, 70%, 75%, 80%, 85% or more of the variants of the nucleic acid comprising the nucleotide sequence of SEQ ID NO: As a transgenic plant using a nucleotide sequence having a nucleotide sequence homology of at least 90%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% , And the specific promoter effect in the pollen is substantially equivalent.

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 plants to be transformed in the present invention can be used not only in monocotyledonous plants including rice (Oryza sativa L.) used in this embodiment but also in dicotyledonous plants.

Preferably, a monocot plant, more preferably rice (Oryza sativa L.) is used.

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

The present invention also provides a method for transforming a plant, comprising transforming the plant with the vector and expressing the foreign gene in 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 selecting a rice transformant to which the expression vector has been introduced and which is specifically expressed in the pollen of rice, thereby providing a method of transforming the plant.

The pollen-specific promoter of the present invention is useful for cultivating crops with increased productivity by controlling pollen development and pollen development of major cereals including rice by specifically controlling the gene expression necessary for improvement of pollen traits .

FIG. 1 is a diagram showing a separated gene showing preferential expression in the pollen region of rice and its expression pattern as a heatmap.
FIG. 2 is a diagram showing a T-DNA insertion site for confirming the promoter activity of the present invention. FIG.
FIG. 3 shows PCR results showing that the strain 1A-13819 was co-segregated with the LOC_Os11g20384 gene.
FIG. 4 shows RT-PCR results showing the expression pattern of GUS and the expression pattern of RNA to show that LOC_Os11g20384 gene is expressed in mature pollen. FIG.
FIG. 5 shows pollen-specific expression of LOC_Os11g20384 gene through GUS staining.
6 shows microarray results showing pollen-specific expression of the LOC_Os11g20384 gene.
7 is a diagram showing a diagram of a vector used for the production of a GUS plant containing the LOC_Os11g20384 promoter.
8 is a diagram showing a promoter of about 2.5 Kb in front of ATG of LOC_Os11g20384 gene.

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 in pollen

To select the genes specifically expressing in the pollen, 983 species of rice epimetric microarrays were collected from NCBI GEO (http://www.ncbi.nlm.nih.gov/geo/), an open microarray database (affymetrix microarray) data were reconstructed by 17 organizations / organizations to construct a database for gene expression analysis.

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 the pollen of the database for gene expression analysis shown in Table 1 were isolated.

This was performed using the K-means clustering method of MeV, a microarray data analysis software, and 672 genes with preferential expression in pollen were isolated from the database for expression / organization-specific expression analysis.

The results are shown in Fig.

As shown in Fig. 1, a gene which shows stronger expression toward yellow is closer to blue, and the expression becomes lower toward blue. That is, among the 17 reconstructed gene databases, 672 genes showing preferential expression in pollen were identified.

< Example  2> Promoter for the pollen-preferential gene 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 system having a locus id name and potentially trapping the promoter from the above 672 pollen-specific genes.

< Example  3> lines showing preferential expression GUS  Expression and T- DNA  Co-segregation verification of the label

GUS expression and co-segregation were verified by randomly selecting 26 promoter trap lines for 672 pollen-specific genes. 26 selected lines were developed in rice paddy fields, mature leaves were picked, quenched into liquid nitrogen and pulverized. 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). In addition, the flowers from the transplantation to the flowering stage were collected to verify the expression of GUS in pollen.

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.

Of the 26 promoter trap lines selected, the 1A-13819 strain was inserted into the 17th intron of the LOC_Os11g20384 gene and inserted into the GUS reporter gene. Genomic analysis was performed by PCR using F / R primers (SEQ ID NOs: 2 and 3) and F / NGUS primers (SEQ ID NOs: 2 and 4) made before and after the T-DNA insertion site.

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. 3, and the primers used are shown in Table 2 below.

Table 2. Primers for genotype analysis of 1A-13819

The T-DNA insertion position in FIG. 2 is a sequence corresponding to nucleotides 14,231 to 14,379 of SEQ ID NO: 5.

The results of the co-segregation verification of GUS expression and T-DNA labeling in lines exhibiting a flower-primed expression are shown in FIG. In FIG. 3, "+" means that GUS staining is confirmed, and "-" means that GUS staining is not confirmed. 3, the band of F / R is a PCR product of a gene specific primer (SEQ ID NOs: 2 and 3) of LOC_Os11g20384, the band of F / NGUS is a PCR product of SEQ ID NO: 3 and NGUS1 SEQ ID NO: 4). By comparing the results of the two products, genetic analysis of the plant 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 the T-DNA appears, resulting in blue staining.

That is, when the results of FIG. 3 were examined, it was confirmed that the PCR result and the GUS staining result coincided, and it was confirmed that the 1A-13819 strain was co-segregated with the LOC_Os11g20384 gene.

< Example  4> Expression patterns of the lines showing flower-preferential expression by period

In order to confirm the pollen-specific expression patterns of 1A-13819 strain, flowers were collected from the pollen development period (meiosis, 4 minute magnetism, vesicle magnetization, before and after heading) to perform GUS staining and verify RNA expression RT-PCR was performed to confirm the results.

GUS staining and discoloration were performed as in Example 3 and the results are shown in Figure 4 (A).

In order to verify the RNA expression of LOC_Os11g20384 gene at the molecular level, transgenic shoots, 4 minute magnetism, vesicle magnet, flowers before and after the outflow were sampled and pulverized after being cooled to liquid nitrogen. Total RNA was extracted from the pulverized sample using RNAiso (Takara). Extracted total RNA was quantified using nanodrop and 1 ug of RNA from each sample was used for cDNA synthesis. cDNA synthesis was performed using the reverse transcriptase M-MLV RTase (Promega) and divided into two steps. In order to synthesize cDNA in two steps, M-MLV RTase, dNTP, RNase inhibitor and reaction buffer were added to the poly (A) tail of the mRNA and the oligo (dT) Lt; 0 &gt; C for 60 min. For inactivation of RTase, the cells were further treated at 95 ° C for 10 minutes and the synthesized cDNA was stored in the refrigerator. After 5 minutes at 95 ° C, the synthesized cDNA was repeated 25 times at 95 ° C for 30 seconds, at 57 ° C for 30 seconds, at 72 ° C for 25 minutes, and finally at 72 ° C for 5 minutes to perform PCR . The PCR reaction product was stored at 4 ° C and a part thereof was confirmed by electrophoresis. In order to compare the relative expression of LOC_Os11g20384, the Ubi5 (LOC_Os01g22490) gene was used as a control and the results are shown in FIG. 4 (B).

1 to 6 in Fig. 4 (A) are each 1; meiosis, 2; tetrad (4 min self), 3; microspore, 4; vacuolated pollen, 5; late pollen mitosis (late somatic cell division), 6; The results of GUS staining of mature pollen (mature pollen) are shown. As shown in FIG. 4 (A), GUS expression was strongly observed only at the stage where the pollen was matured, which means that the LOC_Os11g20384 gene was specifically expressed in the pollen instead of the operation.

FIG. 4 (B) shows RT-PCR results showing the expression of the LOC_Os11g20384 gene by pollen development time. As shown in FIG. 4, the before heading of the cone is corresponding to 5 in FIG. 4 (A), and the after heading corresponds to 6 in FIG. 4 (A) , Indicating that the pollen is strongly expressed in the post-emergence period, which is the maturation period of the pollen as in the GUS expression pattern.

FIG. 5 shows the expression of GUS in the microparticle unit of the pollen. As a result of the experiment, it was confirmed that the expression of GUS was increased in the pollen depending on the passage of time. In Fig. 5, UC (Uninucleate microre) represents a 1-nucleus microsphere, BC (Bicellular microspore) represents a 2-nucleotide microparticle, and MP (Mature pollen) represents a mature flower pollen. As shown in FIG. 5, it was confirmed that GUS expression increased in pollen as the pollen matured.

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

The expression pattern of LOC_Os11g20384 gene was analyzed by microarray database analysis and the results are shown in FIG.

As shown in FIG. 6, it can be confirmed that the pollen tissue is yellowish, indicating that the expression of the gene by the LOC_Os11g20384 promoter occurs in the pollen.

On the basis of the results of FIGS. 4 to 5, in the system 1A-13819 It can be confirmed that the LOC_Os11g20384 gene is expressed specifically in the pollen, which is also consistent with the result of FIG. 6 through microarray database analysis.

< Example  6> Promoter for pollen-specific expression Cloning

PCR was performed using the primers prepared for cloning the promoter of LOC_Os11g20384 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. The primers used for the promoter cloning at this time are shown in Table 3 below. PCR conditions were 5 min at 95 ° C; Repeating 4 times at 95 캜 for 30 seconds, at 55 캜 for 30 seconds and at 68 캜 for 300 seconds; 30 seconds at 95 ° C, 30 seconds at 62 ° C, and 300 seconds at 68 ° C for 35 times and finally 68 ° C for 10 minutes. 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 BamHI and XhoI, followed by ligation with pGA3383 vector (vector for promoter-GUS cloning) digested with the same restriction enzyme at 14 DEG 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, the plate was allowed to stand in a 37 ° C oven for 1 minute, and then 1 ml of LB liquid medium was further placed in 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. The DNA of the mini preparation was digested with restriction enzymes BamHI and XhoI, and then subjected to electrophoresis to separate the agarose gel and identify bands. The cloning DNA thus obtained was subjected to base sequence analysis again to select DNA that did not cause an error. Nucleotide sequencing was performed by capillary sequencing (Macrogen). Primers using sequences NGUS1, RB (right border), and promoter sequences were used for the sequencing.

Table 3. Primers for promoter cloning of LOC_Os11g20384

Table 4. Primers for sequencing

The above analysis result is shown in Sequence Listing 1, and the approximately 2,500 bp sequence preceding the ATG of the LOC_Os11g20384 gene corresponds to a promoter sequence preferentially expressed in a flowerpot.

< Example  7> Production of transformed cells

The plant expression vector prepared in Example 6 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, 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. Then, 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 BamHI and XhoI were used for enzyme cleavage.

The resultant Agrobacterium was used for transformation of rice.

< Example  8> Production 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 the cells cultured for 72 hours with Agrobacterium transformed with the plant expression vector obtained in the above Example 7, and left in a 22 占 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 and induced to regenerate in a light condition growth chamber for 4 weeks at 28 ° C. The plants were transferred to MS solid medium, grown in a light condition growth room for 7 days, To grow regenerated plants.

< Example  9> Verification of expression preference using a transgenic plant

In order to confirm the organ specific GUS expression of the regenerated plant, samples were taken at various times and tissues and GUS staining was performed to confirm GUS expression in the flowers.

<110> University-Industry Cooperation Group of Kyung Hee University. <120> Pollen specific promoter, Expression vector comprising the same,          transformed plants by and method for preparation thereof <130> P13-093-KHU <160> 10 <170> Kopatentin 2.0 <210> 1 <211> 2666 <212> DNA <213> Oryza sativa <220> <221> promoter <222> (1) .. (2666) <223> Loc_Os11g20384 promoter <400> 1 ccgtacattg caactgactc atgataaaaa taaatacttt ttatggtttc aaattttagt 60 attcactata aaaatccccg tgcattgcaa tggatgagag ttattttaat cttattattg 120 ttatactgtt taattaatgt gaaattcact atttaaattt gcttagatat atatattttt 180 agaaagtcat aagctgcaat taggagtccg atcatctcaa gctagcatgc gagttttttt 240 aaagagattt cttatacgac tccttctatg tttccaaaaa cgaacaaact taaaaactaa 300 ctcaaatacg aaaatgtatt tccaaaagta aacgaagtta aaaaccgact catacacgga 360 tgacgtacta aagtaccggc aaaaaaattt atttttttat attatagaga ttttatagtg 420 gtattaattg atttcttata ataatccatg taagtggatt tagagcattc ttgcaccggg 480 aatattcgat ttgactatga ggagctcaat taaccttggt ggctttacac tcctcccaca 540 cacccattac tactttttct tttttttgct tcacccaatg caacgcatgg gcattttgct 600 tgtgtgtgtg tgtataatgc agattcgcac tcgggttcat atgtacctac aatctacact 660 gttgatgtgt tccaagattc ctgctgcaaa atgtagctaa ccttaacgaa atgaatgctt 720 actataggga gaaaagataa ttaggattag gttggttcac tatatgtata acatgtatgt 780 tgcatttttt accaaataaa tttttcaata ataatcatat ataacattta tatatctagt 840 gcattgtatt tttctaaccc ttattttgtt tataacatga tatttagtca aaatgagaaa 900 acaatatatc tactatatca ccaaattgtc atcttgcaaa aatgatattt ccaaaatgat 960 atatatccta ccatcaaaaa ctacataaac aagataattt atataaacaa ggtaaactac 1020 ataaacaagt tttgcataag aaatacaaaa attatataaa caaggtattt tacacttgta 1080 aaaaatatag tataacatgt atgttacatt ttgttagcac ctaatatact agccttgtgg 1140 gtacatgtga acttgcgtgc aaaaaatcca actcttatat atatatatat gataaaagta 1200 acttagtata taattaaagt gatttacaag cataaatatt tttttcatca taatataatc 1260 atgtaagatc ttgatgtaca gatataattg caacgaaacg atgtaatcgg gtcgtagatc 1320 agataagtaa tttaagagaa aaaattattt gaaatttaga tgggtaatga ggatgcaagt 1380 ggcaggtgct actactttta gtaagaaggg taacaagttg attacacact gcactatttt 1440 tggtaaaaag catggtcgct ggttagcaca accgagtgca tgcgctccct gtagtcgcat 1500 ccataagaaa aataatcaaa aaaaaaaagg aaaagtacta gaacgtgagg attttatcag 1560 ccaggcctcc gacattgttg ctacccacca aaaatggcca ccatcgcccc gcttctagtg 1620 tggcccttcc ctctcccgcg ttcatctcac taccgctagt tccatcccct tctccacttc 1680 tccgaggcga ttccccttct ccaatccatt cattccaccc tcttccgatc cccaaccgcc 1740 gctggtccgt atccattcca ctctcctccg acccctaccg ctgccggagt gctaggtgcg 1800 tcaggatcta atccctcttt gtgtgcttcg tttcctacgg caattttgaa ttgtttatac 1860 ggcgcatgat acgtactgct tgccaaaatt tctcggtttt gcgtcgtggt gcgggtgtgt gaggagcgtt 1980 ttgcatttta attttagggt atgaatattg acttgtcagt gcataattat atgaaatcat 2040 ggaatagcat tttgtagaaa aaaagaaaat gtgagtccta ggcactgttg gcatgattga 2100 tagaaaactg tgtacatttt ttatgatgca aggtcaggat catgtgatga gtattaaaat 2160 caggttatgg attttctttt cacattatat tatctgtgca ttcttatgta atagctttct 2220 tgtttatttg acatttatag ttatccggtt tgatttccct atctatctat tgtgataaaa 2280 aggatacatg gcagggtacc ttccctggcc cttttcgtca tacttgtttg gcttcttgtc 2340 tcatatgctc tttttgtctt gctatatgcg atatgatatg atcatcggac catggctatc 2400 taacagttct agatgctgaa ggatgttggt tccatggccc acaaatccca attagttttt 2460 taaggatgtt agtttaatgt gcatgtgttt gtattctatg attacaatag gcttatcctt 2520 actaggaaaa tcctgacact tgtactgtta ttggttattt taagagcacg ataacattat 2580 tcaggtattc atctgttact attgttccag tatattatgg gcatcacatg tcttgactgg 2640 ttatatctgt ggcccatatt tatcat 2666 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 1A-13819-Foward primer <400> 2 catggatctt cttcaaggac 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 1A-13819-Reverse primer <400> 3 cattgccatt tcggtaggat 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NGUS1 <400> 4 aacgctgatc aattccacag 20 <210> 5 <211> 15647 <212> DNA <213> Oryza sativa <400> 5 actaccgcta gttccatccc cttctccact tctccgaggc gattcccctt ctccaatcca 60 ttcattccac cctcttccga tccccaaccg ccgctggtcc gtatccattc cactctcctc 120 cgacccctac cgctgccgga gtgctaggtg cgtcaggatc taatccctct ttgtgtgctt 180 cgtttcctac ggcaattttg aattgtttat acaaaatttt ggggcgcatg atttgacagt 240 gagaccgtga tggatcgatc ttgaggcgct gaacgtactg cttgccaaaa tttctcggtt 300 ttgcgtcgtg gtgcgggtgt gtgaggagcg ttttgcattt taattttagg gtatgaatat 360 tgacttgtca gtgcataatt atatgaaatc atggaatagc attttgtaga aaaaaagaaa 420 atgtgagtcc taggcactgt tggcatgatt gatagaaaac tgtgtacatt ttttatgatg 480 caaggtcagg atcatgtgat gagtattaaa atcaggttat ggattttctt ttcacattat 540 attatctgtg cattcttatg taatagcttt cttgtttatt tgacatttat agttatccgg 600 tttgatttcc ctatctatct attgtgataa aaaggataca tggcagggta ccttccctgg 660 cccttttcgt catacttgtt tggcttcttg tctcatatgc tctttttgtc ttgctatatg 720 cgatatgata tgatcatcgg accatggcta tctaacagtt ctagatgctg aaggatgttg 780 gttccatggc ccacaaatcc caattagttt tttaaggatg ttagtttaat gtgcatgtgt 840 ttgtattcta tgattacaat aggcttatcc ttactaggaa aatcctgaca cttgtactgt 900 tattggttat tttaagagca cgataacatt attcaggtat tcatctgtta ctattgttcc 960 agtatattat gggcatcaca tgtcttgact ggttatatct gtggcccata tttatcattc 1020 tgatgattta acatttactc atgtattcct ctatcagggc atcacacgat gagtgttgca 1080 actgaagcat gcccttcacc aaagctacac acaagactaa ggctgtggga gtttgcagac 1140 cgttacgtat ttgaaccagt tgacggtctt gctgatttgt tcctgtcagt tgatcggaca 1200 aatggatcaa tgagtctagt ggaagagttg ccacctcgtg gtccctctac aaatcctaaa 1260 gttagaatag tttttggtgt gattggagtg ctcaaacttg cagttcggtc gtactttctt 1320 gtgatcactg gccgtgattg tgttggatcc tacttggggc atgcaatttt caaattgacg 1380 ggactaaaag ttctgccatg caataactcg ttaaatactt cttctgctga gcaggtttgg 1440 aagtgattat ttgggcctta tttacaattt ctttccatcc atattccatt agtcattcca 1500 taaagctctt gcagaagaaa atggaatcag aattttcgga acttctggat gcagcagaga 1560 ggactatagg cctgtacttc tcctatgatg tcaacttaac actaacgtgg gtacctcaat 1620 gaatattatg cctttttttt tcttgcaaac atttgcctga agtgtataga gggggttcca 1680 tctttctttt ttttttgtta tcttcctctt ctttcgtttt aagttggtta agcatttatc 1740 tctgcccctt atatgttgta gttcacagag gctgcatgat ctaggagatc agtttaagtc 1800 acttccactt tggagacagg tttgttgcct agctccttac ataattccgt ggtttgcttg 1860 tgcatgaaat tcctgatctt ttttatcctt tcttatataa tctgcaagct tctatagctt 1920 gtcctcaaca tcttcacatg atttataccg ctttccttat tatggaacct aattgtatat 1980 ttggttatta cttattagat cagtggatta cacttgtgat gtagaaagat ggaattgaag 2040 ttaaaatttt ctaaatgcaa tgtgtttcat ttggccgata tttgtttttt agataatgga 2100 agctttatta agactcagtt aaatacacca agatgataca ttccaactga gtccaccccc 2160 ggcctctgca tagaatgcac acaaccaaaa aactggacct aagaacactt ccccatcaca 2220 aaacatagaa actagtggct atcaatccgt agactagatc gccacccatg ctcctgggta 2280 aaaaactcct gtgccaccta atccaaacgt tgcgatacca ccgcaatagt atcccgaagt 2340 ccctgtttgt ggaggatggc ccaggagcgt agccaccggg tgactaaagt agtaacctgc 2400 aaaggagaag acaccacctt gttatcaaaa attacctcgt ttctgcttag ccatatggac 2460 caacaaagag atgctgcacc catcaaaagc agatttctca tgtctttagg aacacaagta 2520 agccaacgcc caaacaagtg agtaatattt tgaggaggtt gtatgttcga agccatatga 2580 acaacagacc ataccaagcg agaaacacga cattgaaaaa acaaatgttg aatggtctct 2640 tccttgtgac aaaagccatg ttgaatggcc gatatttgta gttgcagcta aaaatttttg 2700 atgctcaatt tgtggttaat attttttcat gagaagtcac gtgtgacaaa attgtaacga 2760 tgtgattttc catttaagaa actcaattta taataccaca cactcctagt tagcatattc 2820 cttcctgaga aaatagctat catacaaatc caagcgaaat gaaccaaatg catgaaagct 2880 atatcggaac atgtcacaca atagattctt acttaatttg taaattatag tttctagttt 2940 taaatagact gtcaaatgga agattctcag ttcaatgaac aggtccaaga agcaagcaga 3000 agtccatagc gtacctagaa taaaatattc atgtgcttca gtttaaaaat attttctaga 3060 tgtacaatat agtatgtttg cttttatttc cttgtcctga tttcttaaga aggatgtggt 3120 gggagaaatt agcaaaagct gatgttacat tttgattttt ccgtgatgac atttgcaccg 3180 tattttagaa tattgctaac actgtgccca ttggtaatga ttctactagc atcctgcaaa 3240 atcttcaagc ttatcctttg gattctttta aggttcattg accttttttt tcttgaatat 3300 gcaagagaaa tacgtatcat ttcattaaga caggaaaaaa aaacgaggag ggaagaaaaa 3360 ccctccctca ccctacaaac aaacaggacc tgaggttaca tatgaaaagg aacaaaaaca 3420 accttaccca aacggggtta gcgacctact agggaaatat gtgagcttag aagctccggc 3480 catacaccat aggtcatact catttgccac cgtttggatg acagacatta cattaggatt 3540 gacgccgtta aacaaacagt cgttcctatg cttccagatt tcccaagcca ccaaaatgat 3600 aactgtattg agcccttttt ttttggagtt ttgatcaatg cctcttacac tgccttaggc 3660 caccaacagc ataagcaagt agaattttga cttggagaaa tggaaatcag ccccaacctt 3720 tggagaatta gtgaccacac ttgttgggaa aagacacaag taactaggat gtgattaatg 3780 gattcctcag cctggtcaga aagcatacaa gtagcaggat gtggcagtcc acgcttggca 3840 aggcgattag ccgtccagca atggttatta attggaagac atagaaaaac ttgcaccaca 3900 agggcgccta gctctcccaa agtcttctgc atagtgagaa tctgattgtg ccaacaaagg 3960 actcatatgc tggtttgcac gtgtacgatt ccgactttgt gaacttccaa cgttaccatc 4020 ctgcaagacc gcattggcca ctcagtacca aacttgcaag tattctataa tgacctggac 4080 agtgaaatgt tcattgactt attttacgct ttcttattaa atgttataac tctgtgaggg 4140 acagtgagaa aggtggtgca aaaaaaggaa agaacacaca gatggagccc atgtcatgat 4200 taagacatct tgtttttcat aaagacaatc ggaaatagaa atgaacgcta gctacgacat 4260 acaaatgaaa gacaaacatg tcatagcatg gttagcaatc acagtgaata tcttttaaaa 4320 ctttgtttga tggtattcac tgaaaaaact gttccgtaaa caaaatgaaa aattgagttg 4380 taacaacata tgcaaataat ttttcaatcc accgcttatt tttattacct ttttacaatt 4440 aagtcattga ccacactttt agtaatacta aggactgttt tggtttgata ccaagacccc 4500 ttccaatatt ttggccactt gaatagtagt tgtgatgcat ttgggttgga gccatatttt 4560 tggcaatgcc catgtccaaa ttggcagcag tcgaaagctg tcttaaccta caaagaattg 4620 gctcataatt ggcatcaaac caaaccaaat caatacaatc ctatggaccc taccaaataa 4680 ttggtagtac tgaaacttgc ctaggtttca gcactaccaa aacagtgtag ggtagtaatc 4740 caaaacaact ttaatcaact aatagagttt gatggtagtg gtaaaagtga taggagcatg 4800 attttttttc tactactttg aagacacagt tgcctcctcc accctatgca tgtagtgcga 4860 gaaaatccca taaacccctc aacaccttat ttgccactat atacttccca tttcttgaac 4920 gtcatttgag tgcaaatttt atttctcttg tgccattttc atacataacc agttaagtga 4980 gtgcaaaagc aggagcataa taaggaattg aaggaacgct cagtggcata ggaggaattt 5040 tctgcagaaa atgaaatggt ttaataaact acatgagtac actttggaca tgttaataag 5100 tcttttgatg ctttactggt tacttttgca ccagattaat taattatacc ttttgcaggc 5160 agaaccaaga tttctttgga acggttactt gttggagcct ctaattgaga acaaagtaag 5220 attagtgttg ttattaactg tggatcgtgt gaccaatatt tacaattaat tttgatttcc 5280 cttttgtccc tcaacagctg caccaatact tactaccagt cattcaagga agtatccttc 5340 acctcatcgt aacatctcaa cttattacct gagatgttag atgacttatg atagtttata 5400 tttaggttac ctttctgtta aggtcatatg tcaatttgaa cttctgcaac ttaactaaat 5460 atcaggtttt cagagtatcc atgcagaagt tgggtcagag aaggtaaatg tgaccctgat 5520 tgcacgtcgg tgtacacgta ggataggtaa gagttacgaa ccatcaaaag cttattcgcc 5580 attttctttt cattaaatgt taccgtagtt agaacctaag gtaaatttgt attcatttgc 5640 tattttacta tttttggaaa ctgaatgtca atgatcgttt aggaatttac gagctgttct 5700 ttgttgaagg tacccggatg tggaggcgag gagctgatcc agagggttat gctgcaaatt 5760 ttgttgaatc agagcagata atgcaatcga aagaatttac agcatcatat gtgcaagtaa 5820 gtttcttcac tcaattgaat gacacatagg ggagctagtg tccccgtttg aggaattgac 5880 atgatgcaat gtcccatttc tttttttgaa aaaataatga tttgcataca cataaagtta 5940 tgttaacctt catattttgt ttccaattta tctttggaat caacaatcga aaaggaaaaa 6000 agagtcaatc atgcacagct catgtacgct aaaagatttc ttgtcttcct atttgtatct 6060 tgatactagc ggtgaatatg acactaaaag tttgcaggtt ttcccaactt ttttctagca 6120 cctttcatat ttattttggc ttacagttta tttttatatt ctgtgataac tttgttacat 6180 tgatgtgtat tgcaggttcg aggttctatg ccttttttgt gggaacaaat tgttgatttg 6240 acatataaac ctagttttga tgttgtcaga gtagaggagg cggtgagtgc tctcatcata 6300 cgattttttt tcctttttat cgttgttgct ctactaattt ctcgttgttg ctccactaat 6360 ttctacccct tcagttcatg tacatattat gaagatctaa cttaagtatt tgcaatgtag 6420 ccattgctta tacttttctt gtcataaatt ttcttggatt tgatttctgc agcctcgtgt 6480 acttgaacgg cattttcatg atttacaaaa gaaatatgga gctgtcgtgg ctgttgatct 6540 tgtcaataca gtaagtattg aattcttgga cccaaaccag tctcttcatt ctgtttgtca 6600 tgtataacag tcatgttggg attccctacc attggtgtgc aagttgactg gcctcaaggc 6660 agcttgtcag actttacaca gtcttcagaa gtctaattgt tggcatattt agataaaact 6720 ggtcttaatt atgcatatct ctggataagt cattttgttg tttcattaga tgttagtggt 6780 tttagataac ttcatctatg aaagtcctgc cgcatgattg aagaaagaaa taccagtttc 6840 acctatccct tgccatctgg aaccacatca tagatcctta gccattccac agggtccagg 6900 gcctcatctt gatacttcac acagttgcaa catcaaaccc acccccctac aaatcatcta 6960 ggctgtgtca tatggttgtc ttgattgcag gaccagaatg agagttcttc atttttttcc 7020 tcgaaaaagc aagagaaatg tgtttcattt cattaagggg aagaaaaata aagaacaaaa 7080 atacaaagta ggagaaccag agtactctca ccgaaaaaac acacccacac accccagcca 7140 atactggcaa gcgctagctg cctaggtttg ctgctccatt aagaaacttt tggagggcaa 7200 atgcccctgc taaacaccaa aggttaccct cattctctac cacctgcaag accgccgcaa 7260 ttctaggatt ggcgccattg aaaacacagt catttgtagc ttggctccac tacctcaaaa 7320 aacaaatagt tgaaggttga ggcactgcat cagatatccc ttgcttcaag aatagtgccg 7380 tccacacttg gcaagaaaag acacaagaaa tcaaaatgtg tttgacggat tcttcacttt 7440 aatcacaaag gggacatgcc gcggggtact gcaatccgcg cttagcaagt tggttggctg 7500 tctaacatct attattgacg gccaaccaga tgaagaactt acagcgaaga ggagcccaac 7560 tcttccaaat gtgattccaa ggagcaaact tgatcgatcc caggaaataa gcttcatagg 7620 tagatttgca agaatacatc ccatcagccg ttagcttcca aacatgttga tcttgtacac 7680 cttgtagcag aaccacctct tataccaaaa cccaaatggt catgtactcg taaagaacct 7740 gaatagatag ctccccatgt atgtcagcca ccgatctgtt gttctccaaa gcctgcgccg 7800 cagtacgttg cttccttgcc tcttagagat caaggcaaag agattagggg ccagttttgc 7860 tattgttttc ccttgcagct acctatcgct ccaaaatttg gtattttctt cattcgcaat 7920 tatagttttg acagccacat tgaaaagaga atggacgttc tgaggcacat gaaaagataa 7980 actagcccaa ggtctagcaa aatcagtttt ttgaagccat aaccacctca tgcataatgc 8040 tcatcccatc ttttctaggt taaggatacc aaggcctcca taggacaaag gccgacacac 8100 acgatccaaa gagatcaaac aattttctcc attagtgttt tctcttccct tccataagaa 8160 gccgcgatgc cttttatcta tagccttgac cacccactta gagatccaac gccatcattg 8220 cataaaagcc gagaggacca ccattactgt tataagccag cacgccctat tcattaaaga 8280 ggctttccaa catggtaaat tatctgccac tttgtccaca aggggcaaca attctgtctt 8340 ggttggttta catattctaa gaggaagtcc gagataagag caagggaagc tcatcacttt 8400 acaagacaac tcattagaaa tcacttccag atcatcttcc aagcagtgaa taggaatgca 8460 agagctcttc atcatattgg ttcttagtcc agaaatctga ccaaaaactc caaaatctgc 8520 ttgataagag ataggtcccc acttgtagga cacaagaaca tgaccacatc agccgcatac 8580 agagaaaccc ggtgtgttgt ttgcgtgaca gcaagaggct ccaggagata tccatgatcc 8640 gcagactgcc acaactgagt tcagtatgtc cataaccaag ataaaaaaaa gaaaaagaaa 8700 aaagagttct tcactttgac gaatagtgtc ccatgttcat tccataaatt gcatttttat 8760 ctccgatttt gtatattctg ttccttgaaa tagaattaac tagttcattt cagcttatat 8820 tacataacga taattgtttg ctctagaatt agttaaatgt ttaggagttt tgcctcttct 8880 ttttttaac atgctagtaa tatgcggtgg tggtactagt ttgttctact cctgtgtgat 8940 atttttcacc tatcatatac cttgttaata tattcagcat ggtggcgaag gtcgcctgta 9000 tgaaagatat gcaaaatcaa ttgagcctat cttgagtgaa gatataaggt aagttttgct 9060 ccactctaga gttattctat tgtggtttac tgtttataat tgcattaaac ctaattattg 9120 cagatttgtg cattttgact tccatcaaat ctgtggtcat attcactttg aacgcctttc 9180 tcaactgtat gatcaaattg aggattatct taagaagcat aggtatgttg ctagaagata 9240 caaagtacta catgttttat catttataat ttttgtataa tacctcttca catgacattg 9300 gtacattgac agtctctaaa gcacatgttt gatcatcgcc ttctcaaatt atatatttat 9360 caaaataatt agaaatataa ttgcatgaaa atgaaatata ttaataacat tttacatatc 9420 aaatcttaat atctttgcat ttattataag attgtggggt tgattttaca cattcaaaaa 9480 atgccatttg atttggtaga gtatatgtta gcatgagttt tcttctacag aatttttctc 9540 tgtttaacat ttctgcaagt gtgagtatct gcattttcta tttgaaaaat cagtgcattt 9600 catgtagaga ataatgtagc tgatttacta ttcagttcct tcttttgcca aactactata 9660 gcatatatgc agtaatgtgc tttcgtttaa taacccttgt ttcgtttaat atttctttca 9720 ttataactta taattattgg tagtagggac tagcatacat gcattgaaaa gcgtacacat 9780 attttgctat atgcttccta gttttggccc tgttcctgtc aaagggaaag caaatttatc 9840 gtacattagt caaactattt tgcttctttt tctatgttct tatgttgcca tctctacatg 9900 gcgagaaaaa aaaaacattt cacatctacc tttcccacac aacaagaaaa caaatggcaa 9960 aatggtagct gcatctgaca cttttggtca catatgttat ataaaacctc ctagctgaaa 10020 tttggtgagg aatttggatt aaaacccaag gaatgtccca aaagaatccc tttcatccaa 10080 actcatatta tttctttgtt ctcatagtct ttttaattgt atggcatagg tacttccttt 10140 taaatagtaa aggtgagaaa atggaagagc agaccggaac cgttagaaca aattgtgtcg 10200 attgtctgga tcgtactaat gtaactcagg tacatacttc caaggacagc tttttcatga 10260 tattttgaaa tgtaattcta gatactaaca aaataattgc tgatgtggta actatcgctt 10320 taattttttt ttgaaagaat gggatgtact ttagtacagt attgtaatct aagtgcagtt 10380 ttcaaaagat acagaacatc acataatcgc tcctaggttc ttctagatag tgtttagttt 10440 ggacgaggcc ctcaccctga atttggttgg taggacgaag tggaatgagc cgggacgagt 10500 tcatccaaca tgatagaata ttcgaccaag atgcaaaacg gtgttgtacc agaaattttg 10560 ctagatgagc tcttcccacc tcgcacgtga tagcttttgc tatgtctggt cgctgcgtgg 10620 atatcagccg gtgagagaaa agcgatggtg catgcatgac tctggtctat gaggagacgt 10680 tgaagctagt gaagaggatg ccgcacgctg cgccagatgg ggagaggccg tagtgccctg 10740 tcctagagtt ggacaaggag gccgacgtca ttgcgtgtgc cagaggtgga ggcagctaga 10800 acaatggagc agtctgggga ggcgaaatca tccaactgcc ttaaaggtga tatgatagac 10860 ttctgaccgc gtgggggaag ggcgcaaggc atcattcatc caaccaacac agaaaataga 10920 gaggaggcga tcctagaaga agcactctgc catggatgag atttttcgat gtggctgcct 10980 tgaatgagat tgatgtgttt gccatgtaga gaagtagagg aaaaatgcgg ggagaatgga 11040 aagttgaaaa tagaacataa ttctcacctc ctaaagagaa ggttttctta accaatcctg 11100 ttccaattta atttcttcaa tcaaacaatg ggattgatgg ggttatatgc attggctcat 11160 ggaggtcata aaacttggtt atgtacctag gggtggttct agacattagt tatgcactgc 11220 catggtgtca aaccttcacg gggtgaattt aatactaggc ccaataatag tggctcttgt 11280 agcccggact gtggttttgg tgcgattagt cccacatata gagtttaggt aggtacaaac 11340 tgagggattt ctgaccatag tatcttcgtt taacacttct acacaacgtg aggagtttag 11400 tcgatatgga ctttggttgg tagtagagca taaaccttcg ttacacaaga atgtgcgagc 11460 agatcaccac attctagatt gatcttgtca ttggacattc aaaaaataaa tgatatgttg 11520 tttcatcctt gtcattggat aacttcagtt tggatccatt tgagagaagg gttgttggca 11580 gtactaggtc acccacattc cttgtaaatg tatagccatg caccatgtcc agacaatagg 11640 ttaaatagtc cacattgaag tctaaacata gagtaactag acatgcataa tataaacata 11700 aagtgtgaat agacgaggta ccaacagata acaaatagta aactagaaag aaataataag 11760 atcacatcag aattttaccg gattgaaggg ataggagttt tattccatcg gggagaaaaa 11820 atcatcgaag tggtacaaat ttggagtgtt agcattccgg cctatagtat ttttctggtc 11880 tctgagattt ctttccttgt caactatctt ttcctagctg tttccaccct ctcgagaaga 11940 ggggcgagtg aagtgagagt ggagccccct atttatatta gaagtgcctt gggcatatgg 12000 gtttcatctc caccaggcat ggaggaatga agtggcatga ggcatttctc ccacactgca 12060 tggagcatac aaggaggtgg agtagcagct aaggcatctt gtggggctag ccaacttatg 12120 tcggtgtggg atcgactggg ctacggagtg gaccggctgg gctgtaagct tgggctggcc 12180 aggctgcatg cttggtttgg gaccggccgg actgcatccc ttggttggct aggctggttg 12240 ggccgttgca tacgttgcct ggcattgttc ttgcattttt tttgaacttc ctgattttgc 12300 ttgaatatgt tgatgcactt attaccggct taccgccaaa aacttgttga cacaccaaaa 12360 ctcgtggata cattaaggtt agtgccaaaa taaatacagc atttatcaat acctcattta 12420 ttcataagta tttaaggagt acaaaatatc acttttagca ttttacgggc gtcaacaatg 12480 ctccatgtgg aactaatctc accaaaccac accatgggtt acatcactag caagtagcaa 12540 ctactgggct agaatattac attcaaagta tttgtagttg tgtggggatc cttatgtctt 12600 tctcgagcaa ttactttgtt tctttggcct tctccatttt tttaatgtgt atgtaactac 12660 aatagtctcc ttaatacaca aaacagagca tgatcgggag aaaaatattg gaaagccaac 12720 tgcaaaaaat cagtgttctt ggtgacaaca atacaatcag cgattatccg gcatttgatg 12780 cagattacaa agtttgtaag tgttgcatat gatattcact aaacatgttt gttagtatgt 12840 cccccacatt tgtctcacaa tgttgctggt cctaaacaat tgctgacttc tttttcagtg 12900 tgggccaacc atggcgatgc aataagcact cagtactctg gaactcctgc cttgaaggga 12960 gactttgtgc ggtaatttgt gttttcaagt ttttgtgaat tctcttattt agcccctttt 13020 cctttgcaca atttggacct actaatttca gaaaaagaag taatgatttg attgcaacca 13080 taagcacgtt taagctgagt tgctaatgtt tccttatgaa gcaaaggttt tgtaacttgt 13140 aagatccttt aacacctagc ctgctaaaca ttttctctag tttcatgttg tgaatctcct 13200 caatagttaa acctttataa tggcaacttt taaactgatt tatttcttgc tcgctgtgaa 13260 aagttatctt ctcgtcacca tcagaaaaat tacttgttgg attatgttta gttgcttact 13320 tcccagtatc aggactgagc tgacacctaa gatgttgaca cagttctaat gcatgagagt 13380 gcagtattat aattaaatta ctgacaaact acatgatcac ataaggtgca aaataatact 13440 atcgttctgc caatggcatt gccacatgtt ttcacttttt cctctattaa tccgctgtct 13500 atgatctatt tcttcttttg gttgttggat ctttagaaac aaataatgat taattagtta 13560 tttgttgttc ttaacctaac tatatcttat ttatttatta ggtacggaaa gcgaactaca 13620 cagggaattt taaatgatct gtggaatgca atggctagat attacctgaa caactttgca 13680 gatggtacta aacaggtatg ttgcttactt ggtctcacca ttgatgggct ggagtgagtt 13740 ggattttgct attttaggtt tgctatttta tccaagacat catggacgag tgaagaaaag 13800 atcccttacg ttggtaatga tggatcaagg catagtgata actcattatt tgtttattgt 13860 cttgtatagg atgccatgga tcttcttcaa ggacatcaca tatcctcagt tagtcgggat 13920 atgccaactc caaccaaagg acttatagag aatcacgcgg ttggttattt tctttctatt 13980 acttgagatt tttttttaca gatgaataat ccttaagttg cataccgtga attgcattta 14040 ttgttcttac tcttggttct gaattctcat gcttaaaagg attgcaattt acaatgaatt 14100 tctatgcata attttgatgt ttccatctac ttactaggat aagttgacag cggtgatgct 14160 atgatccata ccaaggtttt gaataccagt ctttctggaa aaccactata tccatttttt 14220 ttaattcaca aaccatttac cagccgtaac cgaccatgaa ccagtttttg atggaaaatt 14280 ttgtattaaa acttatggca tggataatgc gtgctttttt ttaatgactt agtatttatc 14340 acacttaatg ctgcaaaaaa tatacagctc ataatgttct gaccggaaat accagtaact 14400 ggtttggagc tcctggccgg taaccagcag tgtaattacc tgcgaagatt tccacccctg 14460 atgtattccc tcaatttagt tctttcgtgt tgatagagtt acatgggcat ttgaatgtcg 14520 ttatgcatat cttagtggag atatttgttg aatagtccca cattggttgt ggaaggacct 14580 aagatataag tgggggagcc cctcacctca atggctagct tttggggtgg gaaaggccct 14640 ttacggtctt acaattggta tcagagcctg gctaggttaa catctctggc ccgatggaca 14700 cagtatgtga aggtttgatg gaccgtgggt gcctgcgggg cccgtatacc taatggaccg 14760 tgggcctgtt ctgtggggtc tatacacggt gaaaggctga gggacttaag atataagtgg 14820 gggagcccct cacctcaatg gctagctttt ggggtgggaa aggcccttta cgatcctaca 14880 atatttttcc atatttaaat taggatattt tagtggagaa cttacttgag ttttcatttg 14940 cagtccttcc gccttgcttt tgctctgctt ttggctgccg tgatattctt gataatgtca 15000 ctgagaagag gtactatttc atcctaccga aatggcaatg atttaagtta atttatgcag 15060 attagaggca ctcaactgtt gtaaacttga cgagttgttt gcagccaaca tattactatg 15120 tggaaacaaa tacataacca agtcactagg ccaaattcac tccaacaagt ttaatttgat 15180 ccttcttcat gctttgtcat actaacttga ttcatttcct tgcagcacgt aatgatgttt 15240 tccatttggt gctgtcacta ctatggtctg gtttttgctt tggcatcacg cgttatgtca 15300 aagctaacgg acggaagttc acgaaccgtc ctcgctttca cctatcgcgc cattgatgcg 15360 aacaaaaggg attatataca cactcatggt cgactcacct ttctgggcat gcatgtgtgc 15420 tctcagtttt gtatcaagtt taactttccc tctttgtaag attttgcaca tgttctgacc 15480 atcacggctt tatattttct tgttgaatgg gaacatcagg aattgatttg ttctattgca 15540 tttatttatt tatttattta tttatataaa cgttttgcag aaaatgttgc aaagatgtat 15600 atgatgatca aaattgcaaa tggtttaggg tttcatatat tttgggc 15647 <210> 6 <211> 26 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > Os11g20384-BamHI-Foward primer <400> 6 ggatccccgt acattgcaac tgactc 26 <210> 7 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Os11g20384-BamHI-Reverse primer <400> 7 ctcgagatga taaatatggg ccacag 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> Os11g20384-seq_1 <400> 9 ttaggattag gttggttcac 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Os11g20384-seq_2 <400> 10 ctcactgtca aatcatgcgc 20

Claims (7)

A promoter which is specifically expressed in a pollen containing a nucleotide sequence represented by SEQ ID NO: 1. A vector comprising the promoter of claim 1 and an operably linked foreign gene. A 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. 5. The transgenic plant according to claim 4, wherein the transgenic plant is rice (Oryza sativa). A method for transforming a plant, comprising transforming a plant with the vector of claim 2 and expressing the foreign gene in a plant. A method for producing a rice plant transformant specifically expressed in rice pots 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
Selecting the rice plant transformants into which the vector is introduced and the gene is expressed specifically in the flowerpot.

Images