KR101486825B1 - Promoter of gene specifically expressed in early flower stage in rice and uses thereof - Google Patents

Abstract

A promoter specifically expressed in a flower of an early stage of rice contributed to quantity decision is provided to increase yields by controlling differentiation of cells and organs. Provided in the present invention are a promoter of a gene specifically expressed in an early stage of a rice flower developmental stage, a recombinant plant expression vector including the promoter, a plant transformed by the recombinant plant expression vector, a method for manufacturing the transformed plant using the recombinant plant expression vector, a transformed plant manufactured by the method, and a seed of the same.

Description

[0001] The present invention relates to a promoter of a gene specifically expressed at an early stage of a rice flower development stage and a use thereof,

The present invention relates to a promoter of a gene specifically expressed in an early stage of rice flower development and a use thereof. More particularly, the present invention relates to a promoter of a gene specifically expressed in an early stage of rice flower development, A plant transformed with the recombinant plant expression vector, a method for producing a transgenic plant using the recombinant plant expression vector, a transgenic plant produced by the method, and seeds thereof.

In the early stages of rice flower development, many genes involved in differentiation are expressed, and the regulation of these genes is a key factor in determining yield. Particularly, it is possible to use the panicle branch initiation, organ primordia formation, meristem organization, cell specialization in anther, and determination of the number and type of fire (R. Sharma et al., 2012, Funct. Integr. Gemonics, 12: 229-248). By understanding the regulation of these genes, you can identify potential factors that can improve yields.

On the other hand, retinoblastoma-related protein has similar structure to human tumor suppressor protein and is known to functionally similar to animal's role. It is mainly involved in cell cycle regulation and cell differentiation. In plants, it plays an important role in determining the progress of growth and development in cell division. In addition, it plays a role in switching between heterotrophic growth and independent nutrient growth after germination (R. Gutzat et al., 2012, Trends Plant Sci, 17: 139-147). Plant-tumor suppressor-associated proteins were first found in wheat and were reported to modulate gene expression, chromosomal breakdown, number, size and death of embryonic cells. In connection with this, understanding how the cell cycle regulates during embryonic development can lead to increased yield (Savelli PA et al., 2013, PNAS, 110: 1827-1836).

A large number of promoters have been reported for various tissues from various plant-derived plants for various purposes. Although a number of promoters have been reported and are still under development, the present inventors have developed promoters specifically expressed in rice early stage flowers, It is possible to improve the yield by controlling the organ differentiation.

Korean Patent Registration No. 0892904 discloses a gene regulating flowering time, a transgenic plant using the same, and a method for regulating flowering time. In Korean Patent No. 0996667, Specific promoter and transcription factor 'have been disclosed. However, the promoter of the gene specifically expressed at the early stage of the rice flower development stage of the present invention and the use thereof have not been disclosed.

SUMMARY OF THE INVENTION The present invention has been made in view of the above-described needs, and it is an object of the present invention to select a gene specifically expressed in an early stage of rice flower development through a rice 3'-tiling 300k DNA microarray, And the target gene linked to the promoter was specifically expressed at the early stage of flower development, thereby completing the present invention.

In order to solve the above problems, the present invention provides an early stage specific promoter during rice flower development stage.

The present invention also provides a recombinant plant expression vector comprising the promoter.

The present invention also provides a plant transformed with the recombinant plant expression vector.

In addition, the present invention provides a method for producing a transgenic plant using the recombinant plant expression vector.

The present invention also provides a transgenic plant produced by the above method and seeds thereof.

According to the present invention, the tissue-specific promoter sequence can serve as an important marker gene that can be used by researchers as an important factor for determining each developmental stage, as well as controlling the developmental stage or gene expression at a particular stage It can also be used as useful information and tools for controlling artificial genetic manipulation. In addition, the method of isolating the tissue-specific promoter using the rice 3'-tilting 300k microarray of the present invention may be utilized as a useful tool for further searching for the early stage-specific promoter during the flower development stage.

Fig. 1 shows a method of producing a probe of a rice 3 ' tilting 300k microarray.
Figure 2 shows 11,840 hierarchical clustering of genes doubled or doubled compared to rice leaves.
Figure 3 shows RT-PCR results for gene expression at the early stages of leaf, root and pre-fertilization stages. P1cm; Flower size 0-3cm, P8cm; Flower size 8-15cm, P20cm; Flower size 20-22cm, P22cm; Flower size 22cm or more, Leaf; Leaves, Root; Root, RBR1; Retinoblastoma-related protein 1, rbcS; Rubisco small subunit.
4 shows a schematic diagram of a recombinant vector in which the GUS gene is linked to a promoter of a plant tumor suppressor associated protein gene.
FIG. 5 shows the results of a histochemical analysis for the expression of GUS protein under RBR promoter control in transgenic rice plants. pRBR: Developmental stage of plant transformed with a recombinant vector, in which the GUS gene is linked to a plant tumor suppressor associated protein gene promoter. Early stage flower, NT: Initial stage flower of the control plant.

In order to accomplish the object of the present invention, the present invention provides an early stage specific promoter of the rice flower development stage comprising the nucleotide sequence of SEQ ID NO: 1.

The CaMV35S promoter derived from the previously widely used CaMV virus expresses a gene introduced in whole tissues. In contrast, the promoter derived from the early stage of the rice flower development stage of the present invention is a promoter derived from the foreign gene Lt; / RTI > can be expressed specifically at the early stage of the process.

The "early stage" of rice flower development stage was classified according to the size of rice flower (0-3cm, 8-15cm, 20-22cm and 22cm or more) It means the previous stage. Rice generally begins to bloom after about 60-70 days after planting seeds. Rice begins to bloom 3 days for blooming, 7 days for blooming, 10 to 14 days for blooming in one package It is known to be consumed.

The term "specific" means that the expression activity of a promoter is higher in a particular tissue than in at least one other tissue in the same plant. The level of expression activity of the promoter is assessed by comparing the expression level of the promoter in tissues previously measured using methods commonly used with those in other tissues. Generally, the expression level of the promoter can be measured by the amount of the gene product expressed under the control of the promoter, such as protein and RNA.

Also, homologues of the promoter sequences are included within the scope of the present invention. The homologue is a base sequence having a functional characteristic similar to that of SEQ ID NO: 1, although the base sequence is changed. Specifically, the promoter sequence has a nucleotide sequence that has at least 70% homology, more preferably at least 80% homology, more preferably at least 90% homology, and most preferably at least 95% homology with the nucleotide sequence of SEQ ID NO: 1 .

"% Of sequence homology to polynucleotides" is ascertained by comparing the comparison region with two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region is the reference sequence for the optimal alignment of the two sequences (I. E., A gap) relative to the < / RTI >

The present invention also provides a recombinant plant expression vector comprising an early stage specific expression promoter during the flower development stage of the present invention.

The initial stage specific expression vector of the present invention is a transient expression vector capable of transient expression in a plant into which a foreign gene is introduced and a plant expression capable of being permanently expressed in a plant into which a foreign gene is introduced It can be used as a vector.

The term "recombinant" refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, heterologous peptide or heterologous nucleic acid. The recombinant cell can express a gene or a gene fragment that is not found in the natural form of the cell in one of the sense or antisense form. In addition, the recombinant cell can express a gene found in a cell in its natural state, but the gene has been re-introduced into the cell by an artificial means as modified.

The term "vector" is used to refer to a DNA fragment (s), nucleic acid molecule, which is transferred into a cell. The vector replicates the DNA and can be independently regenerated in the host cell. The term "carrier" is often used interchangeably with "vector ". The term "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. Promoters, enhancers, termination signals and polyadenylation signals available in eukaryotic cells are known.

A preferred example of a plant expression vector is a Ti-plasmid vector that is capable of transferring a so-called T-region into a plant cell when it is present in a suitable host such as Agrobacterium tumefaciens. Other types of Ti-plasmid vectors (see EP 0 116 718 B1) are currently used to transfer hybrid DNA sequences to plant cells or protoplasts in which new plants capable of properly inserting hybrid DNA into the plant's genome can be produced have. A particularly preferred form of the Ti-plasmid vector is a so-called binary vector as claimed in EP 0 120 516 B1 and U.S. Patent No. 4,940,838. Binary vectors which can be used in the present invention Agrobacterium tyumeo Pacific Enschede (Agrobacterium tumefaciens may be any binary vector containing RB (right border) and LB (left border) of T-DNA that can transform a plant when present in combination with the Ti plasmid of Tumefaciens , (Cat #: 6018-1, Clontech, USA), pBIN19 (Genbank Accession No. U09365), pBI121, pCAMBIA vector and the like. Other suitable vectors that can be used to introduce a gene according to the invention into a plant host include viral vectors such as those that can be derived from double-stranded plant viruses (e. G., CaMV) and single- For example, from non -complete plant virus vectors. The use of such vectors may be particularly advantageous when it is difficult to transform the plant host properly.

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

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

In a recombinant plant expression vector according to an embodiment of the present invention, the plant expression vector may be prepared by operably linking a foreign gene encoding a target protein downstream of the promoter of the present invention. In the present invention, "operably linked" refers to a component of an expression cassette that functions as a unit for expressing a heterologous protein. For example, a promoter operably linked to a heterologous DNA encoding a protein promotes the production of a functional mRNA corresponding to a heterologous DNA.

The target protein may be any kind of protein. For example, the target protein may be a protein capable of accumulating a large amount of nutrients that can be used for medical purposes such as enzymes, hormones, antibodies, cytokines, And enzymes capable of degrading cellulose, but are not limited thereto.

The present invention also provides a plant transformed with an early stage specific plant expression vector during the flower development stage of the present invention. Wherein the plant is selected from the group consisting of rice, wheat, barley, corn, soybean, potato, wheat, red bean, oats and millet; Vegetable crops selected from the group consisting of Arabidopsis, cabbage, radish, red pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, squash, onions, onions and carrots; Ginseng, tobacco, cotton, sesame, sugar cane, sugar beet, perilla, peanut and rapeseed; Apple trees, pears, jujubes, peaches, sheep grapes, grapes, citrus fruits, persimmons, plums, apricots and banana; Roses, gladiolus, gerberas, carnations, chrysanthemums, lilies and tulips; And feed crops selected from the group consisting of ragras, red clover, orchardgrass, alfalfa, tall fescue, and perennialla grass. Preferably, the plant may be a monocot plant selected from the group consisting of rice, barley, wheat, rye, maize, sorghum, oats and onion, more preferably the plant may be rice, but is not limited thereto.

The present invention also provides a method for producing a plant cell, comprising the steps of: transforming a plant cell with the recombinant plant expression vector; And

And regenerating a transgenic plant from the transgenic plant cell. The present invention also provides a method for producing a transgenic plant which expresses the transgenic plant at an early stage during the flower development stage.

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

In addition, the present invention provides a transgenic plant and a seed thereof, wherein the foreign gene produced by the method for producing a transgenic plant according to the present invention is specifically expressed at an early stage of the flower development stage.

The foreign gene recombined in the early stage specific plant expression vector during the flower development stage of the present invention may be any gene desired to be expressed at the early stage of the rice flower development stage, It is located after the promoter in an expression vector and may be expressed by fusion with a reporter gene as necessary. A method for transforming an early stage specific expression vector into a plant during the recombinant flower development step can be carried out as described above.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Materials and Methods

1. Prepare early stage of flower development

About 60 days after planting the seeds in the pots, the rice flowers were distinguished on the basis of their size by the growing season. The flowers before the fertilization were wrapped in sheaths and sampled by 0-3cm (P1cm), 8-15cm (P8cm), 20-22cm (P20cm) and 22cm (P22cm) according to the size of flowers. The leaves and roots grown for 2 weeks as a control were separated and sampled.

2. Microarray  Experiment

1) chip target  design

By synthesizing DNA directly on the slide, 60-100mers can be produced. To date, probes have been constructed using IRGSP Pseudomolecule v.5 (v.5), which has the most accurate annotation information on the chromosome of rice. This v.5 has 24,209 total length cDNA libraries, 5,180 information based on the prediction program, and a total of 29,389 information. As a result of the similarity between each gene in the BLAST algorithm of NCBI by breaking these genes into units of 60bp, the sequence similarity in the 3'-direction and the UTR (untranslated region) was found to be lowest and the uniqueness was high (No data). Based on these results, 10 of the 60 bp probes were designed starting from 60 bp in ORF 3 'direction for each 27,448 genes of rice origin and shifted by 30 bp, and 274,449 probes were constructed. This chip was named Rice 3-Prime Tiling 300k MicroArray (Fig. 1). In addition, 3 to 6 probes were constructed for 5 genes including mitochondria (123), chloroplasts (74), and Gus, GFP, Bar, and Hyg as markers of transformants. And that the transgenes directly express the genes.

2) Microarray  Experimental course

The sample obtained from calli of rice was frozen with liquid nitrogen, ground with a mortar and then TRI reagent (MRC, USA) corresponding to 2/3 of the volume was added. The sample and TRI reagent were mixed thoroughly at room temperature and reacted for 5 minutes. After centrifugation for 5 minutes at a rate of 2,800 g at 4 ° C, the supernatant was taken, chloroform was added in an amount corresponding to 3/5 of the supernatant obtained, and the mixture was mixed well. After centrifugation at 7,600 g at 4 ° C for 15 minutes, the supernatant was taken, and 5M sodium chloride corresponding to 1/5 was added and mixed slowly. The same amount of isopropyl alcohol was added to the supernatant previously taken, and the mixture was slowly mixed and reacted at room temperature for 10 minutes. After centrifugation at 7,600 g at 4 ° C for 10 minutes, the supernatant was removed, 10 ml of 75% ethyl alcohol stored at -20 ° C was added, centrifuged at 4 ° C for 5 minutes at a rate of 10,000 g, I took a pellet. After incubation at room temperature for 5 minutes, the remaining ethyl alcohol was evaporated, and RNA pellet was dissolved in 200 μl of water treated with 1% DEPC.

To synthesize single-stranded cDNA, 1 μl of oligo dT-primer (Invitrogen, USA) was added to 10 μl total RNA (10 μg) in water, reacted at 70 ° C for 10 minutes, and reacted on ice for 5 minutes. To each sample was added 4 μl 5X First strand buffer (Invitrogen, USA), 1 μl 10 mM dNTP mix, 2 μl 0.1M dithiothreitol (DTT) and 2 μl Superscript II RT enzyme (Invitogen, USA) For 1 hour. (Invitrogen, USA), 4 쨉 l 10 U / ㎕ DNA polymerase I (Invitrogen, USA), 3 쨉 l 10 mM dNTP mix, 1 쨉 l 10 袖 l DNA ligase (Invitrogen, USA) and 1 μl of 2 U / μl of RNase H (Invitrogen, USA) were added to each sample and reacted at 16 ° C for 2 hours. Subsequently, 2 μl of 5 U / μl T4 DNA polymerase was added thereto, followed by reaction at 16 ° C. for 2 hours, and 10 μl of 0.5 M EDTA was added to stop the reaction. 1 μl of 10 mg / ml RNase A was added to the synthesized double-stranded cDNA solution, followed by reaction at 37 ° C for 10 minutes. During the reaction, Maxtract HighDensity tubes (QIAGEN, USA) were centrifuged at 15,800 g for 2 min, 90 페 phenol and 90 클로 chloroform, and mixed thoroughly for 1 min. Each sample was transferred to a Maxtract HighDensity tube and centrifuged at 15,800 g for 5 minutes. The supernatant was transferred to a new 1.5 ml tube and 16.5 μl of 7.5 M ammonium acetate was added and mixed well. 326 占 퐇 of -20 占 폚 storage 100% ethyl alcohol was added and the mixture was centrifuged at 4 占 폚 and 15,800 g for 20 minutes, and the supernatant was removed. Subsequently, 500 μl of 70% ethyl alcohol (10 ml) kept at -20 ° C. was added and centrifuged at 4 ° C. at a rate of 15,800 g for 5 minutes, and the supernatant was removed to obtain cDNA pellet. Residual ethyl alcohol was evaporated using a Speed Vac (eppendoff, USA) for 5 minutes at room temperature, and then 10 μl of RNase-free water was added. 40 μl of Cy3 9-mer random primer (1 OD / 42 μl, Sigma, USA) was added to 10 μl of cDNA (1 μg) in order to fluorescently label the cDNA. After reacting at 98 ° C for 10 minutes, . 10 μl of Klenow Fragment buffer (Takara, Japan), 10 μl of 3.5 U / μl of Klenow and 10 μl of 50 × dNTP mix (10 mM) were added to each sample, followed by reaction at 37 ° C. for 2 hours. The reaction was stopped by adding 10 μl of 0.5 M EDTA, and 11.5 μl of 5 M NaCl was added thereto and mixed well. 110 μl of 100% isopropyl alcohol was added and reacted in a dark room for 10 minutes. Then, the supernatant was removed by centrifugation at room temperature for 15 minutes at a rate of 15,800 g. 500 의 of 70% ethyl alcohol stored at -20 캜 was added, centrifuged at a rate of 15,800 g for 2 minutes at room temperature, and the supernatant was removed to take the DNA pellet. The remaining ethyl alcohol was evaporated using a Speed Vac at room temperature for 5 minutes, and then 13 μl of RNase-free water was added.

The microarray was covered with MAUI Mixer SL type (BioMicro Systems Inc. USA) and transferred to a MAUI hybridization chamber (BioMicro Systems Inc. USA) set at 42 ° C. (Genisphere Inc., Hatfield, USA), 8 占 퐇 of RNase-free water, and 0.5 占 퐇 of 100 占 Cy Cy3 CPK6 oligo (Sigma, USA) were added to the Cy3-labeled cDNA solution (15 占 퐂). After reacting at 95 ° C for 5 minutes, it was immediately placed in a 42 ° C MAUI hybridization chamber for 30 seconds, hybridized to the microarray, and reacted for 16-18 hours. After the MAUI Mixer SL type was removed from the microarray, the microarray was reacted with 0.2% SDS, 0.2X SSC solution for 2 minutes, 0.2X SSC for 1 minute, 0.05X SSC for 30 seconds, and the microarray was subjected to Array-Go-Round (NimbleGen , USA) for 1 minute. Microarrays with excess fluorescence removed were scanned at 5 μm resolution using Genepix 4000B (Axon Instruments, USA) and data were extracted using NimbleScan 4.0. The normal distribution of Cy3 brightness was verified by qqline. The data were normalized, cubic spline normalization was performed, and the signal changes between the microarrays were adjusted using Rubid multi-chip analysis (RMA) and median polish algorithm using NimbleScan 4.0 Irizarry, 2003 Biostatistics, 4: 249-264).

3) GUS  Histochemical analysis

Approximately 60 days after transgenic seed germination, flowers with a diameter of 0-3 cm before hydration were collected for histochemical analysis of GUS protein. The non-transformed seeds were also used as a control by collecting flowers of the sizes described above.

Flowers in the early development stage (experimental group and control) were kept in 90% acetone for 30 minutes at room temperature. After washing with a staining buffer, the cells were immersed in a staining solution (sodium phosphate buffer 0.1 mM, pH 7.0, EDTA 10 mM, Triton X-100 0.1%, K ₃ Fe (CN) ₆ 1 mM, KFeCN 1 mM and X-Gluc 2 mM). For more effective staining, tissues were vacuum infiltrated for 30 minutes and reacted overnight at 37 ° C. The staining solution was washed several times with 50% ethanol until the tissue was clean, then 70% ethanol was added and stored at 4 ° C for a long time.

Example  One. Microarray  analysis

First, in order to find genes with high expression in early stage of flower development stage, pre-fertilization flowers were sampled by size and microarray experiment was performed using rice 3'-tilting 300k microarray (repeated twice). To compare gene expression with other tissues, experimental results were also added to the analysis of leaf and root tissue (Fig. 2). In the microarray experiment, 6 genes were overexpressed at an intensity of 20,000 or more compared to other tissues in a sample of 0-3 cm in size during the flower development stage (Table 1).

List of genes expressing intensity of flower size 0-3 cm to over 20,000 Rap2_no P1cm P8cm P20cm P22cm leaf Root Rap2_no_desc Os03g0672400 23088 5344 3690 4498 1301 8155 Preserved Estimated Protein Os05g0476200 24623 6594 4064 5127 1778 7103 DNA replication licensing factor MCM3 homologue Os02g0699700 22614 6059 4460 7332 2242 12067 DNA topoisomerase type II Os12g0636100 36292 8651 6306 7082 2861 3672 Tetra-tricopeptide-like helical domain-containing protein Os05g0459400 28964 7232 4892 6372 3918 9370 Kinesin, molecular motor protein Os08g0538700 20579 6171 4773 5448 3928 7215 Plant tumor suppressor associated protein

RT-PCR was performed on each primer from Os08g0538700 (Plant Tumor Suppressor Associated Protein Coding), which is expressed specifically at the early stage of flower development stage, and the same experimental results as the microarray experiment were obtained.

There are three plant inhibitory protein genes in rice, and the expression level of these genes in early stages, leaves and roots during the flower development stage is shown in Table 2. Os03g0207900 And Os08g0538700 gene is overexpressed in the early stages of flower development stage, Os11g0533500 genes have been shown to express a whole lot of organization.

Expression of Rice Tumor Suppressor Associated Protein Gene by Tissue Rap2_no P1cm P8cm P20cm P22cm Leaf Root Rap2_no_desc Os03g0207900 12776 8531 8909 8298 7396 7671 Like plant tumor suppressor protein Os08g0538700 20579 6171 4773 5448 3928 7215 Plant tumor suppressor protein Os11g0533500 14424 10009 10800 11513 11041 9833 Similar plant tumor suppressor protein 2b

Example  2. Plant-tumor suppressor protein promoter was used at early stage of flower development GUS  Protein expression

Os08g0538700 2 kb upstream of the ATG of the plant tumor suppressor protein gene was isolated by PCR. To confirm gene expression by this promoter, a vector was constructed by connecting the GUS gene to the promoter. Histochemical analysis of the expression of GUS protein in the transgenic rice plants using the recombinant plant expression vector with the GUS gene as shown in FIG. 4 revealed that in the early stage flower of the transgenic rice plants, unlike the early stage flower of the control plant GUS protein was expressed (Fig. 5).

<110> GREENGENE BIOTECH INC. <120> Promoter of gene specifically expressed in early flower stage          rice and uses thereof <130> PN13250 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 2006 <212> DNA <213> Oryza sativa <400> 1 cctccacaga taaccccaat caagtccatg agctgtgatc ctgtgaagat gcagctttac 60 ctggaacaac ctagccgcac agaacccagg gatccaccaa ctgaaccctg aatccgtgtt 120 cgtcctgtat attaatttgc ttgtgacctg aacaaacgca gaaacatttt ggtggtagtt 180 tgatacagtc atcagagggt atatttaact aacatatgct ccaaattaca aactaagcta 240 aaagttaatg gttcagaact cttgtgacca tttggattcc atttctgaaa caattactcc 300 tcctacccga catacttgta ctcgtaagga cagcatggtt gaatttcacc cagtggatta 360 ttatcagtag tgagatgtaa tagtacagta ctgatgataa cgtgtgtctg agaatgttct 420 gctgctggtg gtactctatt tggcatcagt gaccgaacta ggactagctt ccattctcct 480 gaagtatcca atggatagag aagagactgg agtactactg gaccaatgaa agcaaaggcg 540 ggcaagtcca gaagagaaga atccgatggc gtgagtcgtg tcgtcgtgta cgcgacgtga 600 cgctgccact gcacgccgta ctggattctc tccttaaggc tgtttctagt tcagcacaaa 660 gtttagattt tggttgaaat taaagatgat gtgactgaaa agttgtgtgt atgataggtt 720 gatgtgatgg aaaagtactg aagtttggat ccaaatttta gatctaaaca cagccttggt 780 tgagtgcacc ggcggcgatc gtcgccggcg gcccgctgcc gtgtccacgt cttgtcgtct 840 ctttcttcct gttccttcta cggcacacga caccttgtta cacgttggtc atatatttat 900 gcaatgaggt acgagtagta ttagaaaaag tccaaacagc catcaaaact tggtttgcct 960 agcactctga gcttaaaatc ggacatccac tcccttaaac tttacaataa acagggttac 1020 tctctccgtc ctaaaatata agcattttta gctatgaatc tagacaagta tatgtccaga 1080 ttcatagcca aaagttgtta tattttgagt ttagggagtt ggatgactat tttaaagttt 1140 agggttgact ttcatataga gtttaggaat gtatgtcaac ttttttcttt tatatttgaa 1200 gcagttagat ttttggacat aagtttaata ttatatttta taatgggagg aaaatcttat 1260 aaaatataat gaaagttacg agtcaattta gataaaattt tcatttaaat ttgtatgaaa 1320 tttctatttt cgtagacaga aaatcttcta tactttgatt atgcatcatt tgttaagtga 1380 aattaattca atataaattg aaaatgtgtt aaaaccgatg tactgtacta cattttaccg 1440 gtgcaaattg ttttttaatc atatcatcat ctttttttac ttggacgcat ctgcactatg 1500 cactgcatcc gtcaaaggcg ccgcacgtgg ctacggatcc cgacaagcca cgccgccctc 1560 ctgcaaagcg ggacccatcc tctgactccc cccgggcccc gccgctttgc cacccaaatc 1620 tcccgccatt tcccacccct ctcccgccaa aaccaaccaa tccccgcccg cccatcgcca 1680 cgtgtcaccc tcatggcccc accgctcagt gacacggcag ttctcgtttt gccgcgcaag 1740 ccacccgcga gagagataaa aaacggcagc gcgcgcgccg cgcggaacta ggattttttt 1800 ttggcgcgca ttccaaccaa acgccgccct cttcgctcca atctccatct ccatctccat 1860 ctccaccgcg caggcgagcg caaccctagc tgcaggcagc gcaggcagga gggctcgttc 1920 gtcgtctcct cccaccgccg ctcgcgggag agactctggt tttttttggg gggagtttgg 1980 ttggttggtg gtgttcttga cggggc 2006

Claims (7)

An early stage specific promoter in the rice flower development stage consisting of the nucleotide sequence of SEQ ID NO: 1. A recombinant plant expression vector comprising the promoter according to claim 1. 3. The recombinant plant expression vector according to claim 2, wherein the recombinant plant expression vector is produced by operatively linking a foreign gene encoding a target protein downstream of the promoter. A plant transformed with a recombinant plant expression vector according to claim 3. Transforming a plant cell with a recombinant plant expression vector according to claim 3; And
And regenerating a transgenic plant from the transgenic plant cell, wherein the transgenic plant is expressed at an early stage of the flower development stage. A transgenic plant wherein the foreign gene produced by the method of claim 5 is expressed specifically at the early stage of the flower development stage. A seed of the transgenic plant of claim 6.

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