KR101306678B1 - Promoter of gene specifically expressed in early flower stage in rice and uses thereof - Google Patents

Abstract

PURPOSE: A tissue-specific promoter sequence is provided to function as an important marker gene and to be used as useful information and a tool for manipulating an artificial gene which regulates development stages and expression at a specific stage. CONSTITUTION: An early stage-specific promoter has a base sequence of sequence number 1. A recombinant plant expression vector contains the promoter. A method for producing a transgenic plant in which a foreign gene is specifically expressed at the early stage of flower development comprises the steps of: transforming plant cells with the vector; and redifferentiating a transgenic plant from the transformed plant cells.

Description

Promoter of gene specifically expressed in early flower stage in rice and uses approximately}

The present invention relates to a promoter of a gene that is specifically expressed in the early stages of the rice flower development stage and its use, and more specifically to a promoter of the gene specifically expressed in the early stages of the rice flower development stage, the promoter Recombinant plant expression vector comprising, a method for producing a transformed plant using the recombinant plant expression vector, transformed plants and seeds thereof produced by the method, rice flower development using a rice 3 'Tiling 300k microarray It relates to a method for separating the early stage specific promoter of the step and the rice 3 'Tiling 300k microarray.

During the early stages of rice flower development, many genes involved in differentiation are expressed, and the regulation of these genes is a major factor in determining yield. In particular, panicle branch initiation, organ primordia formation, meristem organization, cell specialization in anther and determination of fire number and type genes involved in floral organ number and shape are expressed (R. Sharma et al., Funct Integr Gemonics, 2012). Understanding the regulation of these genes can identify potential factors that can improve yields.

DNA-topoisomerase, on the other hand, separates phosphodiester bonds of DNA. The enzyme then connects the tyrosine to the enzyme via a phosphate bridge. Topoisomerase type II requires ATP in the cleavage of the double helix and subsequent recombination. Topoisomerase type II has one small subunit with ATPase activity. Although DNA is classified as topoisomerase VI in archaea or plants, DNA topoisomerase VI isolated from rice (Oryza sativa) encodes subunits A and B, and is susceptible to archaea topoisomerase VI and eukaryotes It has a structure similar to the domain of SPO11, a cleavage recombination factor. It is also highly expressed in flowers and callus a few minutes ago, and is regulated by the plant hormones auxin, cytokinin and ABA (M. Jain et al., FEBS Journal, 273; 5245-5260, 2006). Arabidopsis is known to regulate plant growth, development, and endogenous replication.

A large number of promoters have been reported for each tissue of various plant-derived plants for various purposes, and are still under development, but in particular, we have developed promoters specifically expressed in early rice plants, which contribute to yield determination. Organ differentiation can be controlled to improve yield.

Meanwhile, Korean Patent No. 0892904 discloses a gene for regulating flowering time, a transformed plant using the same, and a method for regulating flowering time, and Korean Patent No. 0996667 discloses a rice callus or a callus under differentiation. Promoters and transcription factors of genes that are specifically expressed are disclosed. However, as in the present invention, there is no disclosure about the promoter of the gene specifically expressed in the early stage of the rice flower development stage.

The present invention is derived from the above requirements, the present inventors select a gene specifically expressed in the early stages of the rice flower development stage through a rice 3 'Tiling 300k DNA microarray, the selected The promoter was separated from the gene, and by confirming that the target gene linked to the promoter was specifically expressed in the early stages of flower development, the present invention was completed.

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

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

The present invention also provides a method for producing a transformed plant using the recombinant plant expression vector.

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

In addition,

Designing 10 probes of 60 bp in length moving 30 bp in the 5 'direction at the 3'-end of each gene derived from rice and fixing them to a microarray to prepare a rice 3' tiling 300k microarray;

Synthesizing cDNA using RNA isolated as a template for each rice plant, and then hybridizing the synthesized cDNA with the prepared microarray;

Identifying genes that are specifically expressed in the early stages of flower development compared to other tissues based on the hybridization results; And

It provides a method of separating the flower tissue specific promoter of the early stage of the flower development stage comprising the step of separating the promoter of the identified gene.

In addition, the present invention is immobilized 300,000 probes including 10 designed probes of 60bp length and selection marker gene probes moving 30bp in the 5 'direction at the 3'-end of each gene derived from rice, and the rice flower development Rice 3 'Tiling 300k microarrays used for isolation of early specific promoters during the phases are provided.

According to the present invention, tissue-specific promoter sequences not only play an important marker gene role that researchers can use as important factors for determining each stage of development, but also regulate genes or regulate gene expression at specific stages. It can also be used as useful information and tools for controlling artificial genetic manipulation. In addition, the method for separating tissue-specific promoters using the rice 3 ′ tiling 300k microarray of the present invention may be utilized as a useful tool for further exploring early stage-specific promoters during flower development.

1 shows a method of making a probe of a rice 3 'tiling 300k microarray.
FIG. 2 shows Hierarchical Clustering of 11,840 genes that are either two-fold or two-fold reduced compared to rice leaves.
Figure 3 shows RT-PCR results for gene expression in the early stages of the development of leaves, roots and flowers before fertilization (P1cm: flower size 0-3cm, P8cm: flower size 8-15cm, P20cm: flower size 20-22cm , P22cm: flower size 22 cm or more).
4 shows a recombinant vector linking GUS to the promoter of DNA topoisomerase II.
5 shows the results of investigating the expression of GUS under the control of the Topo promoter in transgenic rice by histochemical analysis. pTopo: early stage flower of plant transformed with recombinant vector linking GUS to DNA topoisomerase II promoter, NT: early stage flower of control plant.

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

The CaMV35S promoter derived from Cauliflower mosaic virus, which is widely used in the past, expresses a gene introduced from all tissues, whereas the promoter from the early stage of the rice flower development stage of the present invention is a rice flower development stage transformed with a foreign gene. It can be expressed specifically in the early stages.

The "early stage" of the stages of rice flower development is the pre-fertilization stage where the size of the rice flower is 0-3cm, when the size of the rice flower (0-3cm, 8-15cm, 20-22cm and 22cm or more) is divided by the growth period. Means. Rice is usually drained after about 60-70 days after planting seeds, and the blossoms of the rice plant begin to bloom, 3 days for the flowering of an ear, 7 days for the flowering of an abandonment, and 10 to 14 days for the flowering of a package. It is known to take.

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

Also, variants of the promoter sequence are included within the scope of the present invention. The mutant is a base sequence having a functional characteristic similar to that of the nucleotide sequence of SEQ ID NO: 1, although the nucleotide sequence thereof 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 >

In addition, the present invention provides a recombinant plant expression vector comprising a specific expression promoter early in the flowering stage of the present invention.

The early stage specific expression vector of the flower development stage of the present invention is a transient expression vector that can be temporarily expressed in a plant into which a foreign gene is introduced and a plant expression that can be permanently expressed in a plant to which a foreign gene is introduced. 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. Another type of Ti-plasmid vector (see EP 0 116 718 B1) is currently used to transfer hybrid DNA sequences to protoplasts from which plant cells or new plants can be produced that properly insert hybrid DNA into the plant's genome. have. A particularly preferred form of the Ti-plasmid vector is the so-called binary vector as claimed in EP 0 120 516 B1 and US Pat. No. 4,940,838. Binary vectors that can be used in the present invention are Agrobacterium tumerfaciens ( Agrobacterium) It can be any binary vector containing the right border (RB) and left border (LB) of T-DNA, which can be transformed when present with the Ti plasmid of tumefaciens ), but is preferably used frequently in the art. PBI101 (Cat #: 6018-1, Clontech, USA), pBIN19 (Genbank Accession No. U09365), pBI121, pCAMBIA vectors, and the like, are preferably used. Other suitable vectors that can be used to introduce the genes according to the invention into a plant host are viral vectors, such as those which can be derived from double stranded plant viruses (eg CaMV) and single stranded viruses, gemini viruses, etc. For example, it may be selected from an incomplete plant viral vector. 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, and chloramphenicol. However, the present invention is not limited thereto.

In the plant expression vector according to an embodiment of the present invention, the terminator may use a conventional terminator, such as nopalin synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, agro Terminators of the octopine gene of Bacterium tumerfaciens, but are 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 protein of interest may be any kind of protein, for example, a protein that has a medical utility such as enzymes, hormones, antibodies, cytokines, or the like, and may accumulate a large amount of nutrients that may enhance the health of animals including humans. And enzymes capable of decomposing cellulose, but are not limited thereto.

In addition, the present invention is E. coli or Agrobacterium tumerfaciens ( Agrobacterium ) transformed with a specific plant expression vector in the early stage of the flower development stage of the present invention tumefaciens .

In addition, the present invention comprises the steps of transforming plant cells with the recombinant plant expression vector; And

It provides a method for producing a transgenic plant that specifically expresses the foreign gene, including the step of re-differentiating the transgenic plant from the transformed plant cell early in the flower development stage.

The method of the present invention comprises the step of transforming plant cells with the recombinant plant expression vector according to the present invention, wherein the transformation of the plant means any method of transferring 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 seeds 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 of the flower development stage of the present invention may be any gene desired to be expressed in the early stage of the rice flower development stage, the early stage specific of the flower development stage of the present invention It is located behind the promoter in an expression vector and may be expressed by fusion with a reporter gene as necessary. The method of transforming a plant with an early stage specific expression vector among the recombinant flower development stages may be carried out as described above.

The plant of the present invention is a food crop selected from the group consisting of rice, wheat, barley, corn, soybeans, potatoes, wheat, red beans, oats and sorghum; 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 fodder crops selected from the group consisting of lygragrass, redclover, orchardgrass, alfalfa, tolsfeque and perennial lygragrass. Preferably, the plant may be a monocotyledonous plant selected from the group consisting of rice, barley, wheat, rye, corn, sugar cane, oats and onions, and more preferably, the plant may be rice, but is not limited thereto.

In addition, the present invention designed 10 probes of 60bp length moving 30bp in the 5 'direction at the 3'- end of each gene derived from rice and fixed to a microarray to prepare a rice 3' Tiling 300k microarray Making;

Synthesizing cDNA using RNA isolated as a template for each rice plant, and then hybridizing the synthesized cDNA with the prepared microarray;

Identifying genes that are specifically expressed in the early stages of flower development compared to other tissues based on the hybridization results; And

Provided is a method for separating tissue-specific promoters at an early stage of flower development, including separating the promoters of the identified genes.

In the rice 3 ′ tiling 300k microarray of the present invention, “tiling 300k microarray” refers to a microarray in which 300,000 oligonucleotide probes of 60 bp in length are immobilized on a substrate. A "tiling microarray" is a microarray method that includes overlapping oligonucleotides designed to provide a complete or nearly complete presentation of the entire region of interest dielectric. In the case of a tiling microarray, it is possible to deduce the identification and structure of a novel gene by using a method in which probes for both the sense strand and the antisense strand on the genome are arranged on the tiles of equal intervals, Structural changes can be analyzed, and analysis of antisense transcription products and selective splicing is also possible.

In the microarray of the present invention, a "microarray" is a group of oligonucleotides (or probes of the present invention) immobilized at a high density on a substrate, and each of the oligonucleotide groups is immobilized in a predetermined region. Means. Microarrays can be used interchangeably with chips. Such microarrays are well known in the art. Microarrays are described, for example, in U.S. Patent Nos. 5,445,934 and 5,744,305. The oligonucleotide probe set is as described above.

The term "substrate" as defined in the present invention refers to any substrate that has hybridization properties and under which oligonucleotide probes can be coupled under conditions where the background level of hybridization is kept low. Typically, the substrate can be a microtiter plate, a film (e.g., nylon or nitrocellulose) or a microsphere (bead) or chip. Before application or fixation to the membrane, the nucleic acid probe may be modified to promote immobilization or improve hybridization efficiency. Such modifications may include homopolymer tailing, coupling with different reactive functional groups such as aliphatic groups, NH 2 groups, SH groups and carboxyl groups, or coupling with biotin, hapten or protein.

The term "hybridization", as defined herein, is the process by which complementary nucleotide sequences that are substantially homologous anneal to each other. The hybridization process can occur entirely in solution, ie when two complementary nucleic acids are in solution. The hybridization process can also occur when one of the complementary nucleic acids is immobilized on a substrate such as magnetic beads, Sepharose beads or some other resin. The hybridization process may also occur when one of the complementary nucleic acids is immobilized on a solid support such as a nitrocellulose or nylon membrane or fixed on a siliceous glass support (known as a nucleic acid array, microarray or nucleic acid chip) by photolithography . To allow hybridization to occur, nucleic acid molecules are generally thermally or chemically modified to dissolve the double strand into two single strands and / or to remove hairpins or other secondary structures from the single stranded nucleic acid. Those skilled in the art are aware of various parameters that can vary during hybridization and maintain or change the conditions under which hybridization can occur.

Also, the specificity of the hybridization typically depends on the post-hybridization wash function. To remove the background caused by nonspecific hybridization, the samples are washed with dilute salt solution. A crucial factor in such cleaning includes the ionic strength and temperature of the final wash. Those skilled in the art are aware of various parameters that can change during cleaning and maintain or change the conditions under which hybridization occurs.

In order to determine the level of hybridization conditions [Sambrook et al. (2001) Molecular Cloning: laboratory manual, 3 ^rd Edition, Cold Spring Harbor Laboratory Press, CSH, New York or to Current Protocols in Molecular Biology, John Wiley & (1989, revised annually)].

In the method of the present invention, a synthesized nucleic acid sample such as cDNA to be hybridized to the microarray can be labeled with a detectable labeling substance. In an embodiment of the present invention, the labeling material may be a fluorescent, phosphorescent or radioactive substance, but is not limited thereto. Preferably, the labeling material may be Cy3 or Cy5, and most preferably Cy3. When the first strand cDNA is synthesized by labeling Cy3 or Cy5 at the 5'-end of the primer, the target sequence can be labeled with a detectable fluorescent labeling substance. When the radioactive isotope such as ³² P or ³⁵ S is added to the reaction solution during the synthesis of the first strand cDNA, the synthetic product is synthesized and radioactive is incorporated into the synthesized product so that the synthesized product can be labeled with radioactivity have.

In the method according to one embodiment of the present invention, the promoter may consist of the nucleotide sequence of SEQ ID NO: 1, but is not limited thereto.

In addition, the present invention is immobilized 300,000 probes including 10 designed probes of 60bp length and selection marker gene probes moving 30bp in the 5 'direction at the 3'-end of each gene derived from rice, and the rice flower development Rice 3 'Tiling 300k microarrays used for isolation of early specific promoters during the phases are provided. The microarray is immobilized with 10 probes for each gene derived from rice, so that approximately 280,000 probes are immobilized, and further, mitochondria (123), chloroplasts (74), and markers of transformants, Gus and GFP. Three to six probes were constructed for five genes, Bar, Kan, and Hyg, to verify the gene expression changes in organs and whether the transformant expressed the gene directly. Therefore, a total of about 300,000 probes are immobilized, which is called 300k microarray.

The microarray may be used for separation of a rice tissue specific promoter, preferably an early specific promoter of a rice flower development stage, and more preferably a promoter consisting of a nucleotide sequence of SEQ ID NO: 1, but is not limited thereto.

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. Preparing for the early stages of flower development

About 60 days after planting the seeds, the flowers of the rice were distinguished by the growth period. The flowers before fertilization were wrapped in leaf sheaths and sampled by the size of the flowers divided into 0-3cm (P1cm), 8-15cm (P8cm), 20-22cm (P20cm), and 22cm or more (P22cm). As a control, the leaves and roots grown for 2 weeks were sampled separately.

2. Microarray  Experiment

1) chip target  design

By synthesizing DNA directly on the slide, 60 - 100mer 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 and 5180 pieces of information based on the prediction program, totaling 29,389 pieces. These genes were cut by 60 bp and examined for similarity between genes by NCBI's BLAST algorithm. The results showed the least similarity and the highest uniqness between the genes in 3 'direction and UTR (untranslated region). (Data not shown). Based on these results, 274,664 probes were prepared by designing 10 probes having a length of 60 bp, starting at 60 bp in the ORF 3 'direction and shifting by 30 bp for each rice-derived gene (27,647). 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. After mixing the sample and TRI reagent completely at room temperature and reacted for 5 minutes. After centrifugation at 2,800 g at 4 ° C for 5 minutes, 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 added with 1/5 of 5M NaCl and mixed slowly. The same amount of isopropyl alcohol was added to the supernatant previously taken, then slowly mixed and reacted at room temperature for 10 minutes. After centrifugation at 7,600 g at 4 DEG C for 10 minutes, the supernatant was removed, 10 mL of 75% ethyl alcohol stored at -20 DEG C was added, centrifuged at 10,000 g for 5 minutes at 4 DEG C, and RNA pellet was taken. After allowing to stand at room temperature for 5 minutes to evaporate the remaining ethyl alcohol, RNA 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 of total RNA (10 μg) aqueous solution, reacted at 70 ° C for 10 minutes, and reacted on ice for 5 minutes. To each sample, 4 μl of 5X First strand buffer (Invitrogen, USA), 1 μl of 10 mM dNTP mix, 2 μl of 0.1 M dithiothreitol (DTT) and 2 μl Superscript II RT enzyme (Invitogen, USA) Lt; 0 > C for 1 hour. 3 μl of 10 mM dNTP mix, 1 μl of 10 U / μl DNA ligase (Invitrogen, USA), 4 μl of 10 U / μl DNA polymer (Invitrogen, USA) (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 쨉 l of 5 U / T T4 DNA polymerase was added and reacted at 16 째 C for 2 hours, and 10 쨉 l 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 minutes, 90 페 phenol and 90 클로 chloroform were added and mixed thoroughly for 1 minute. 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 7.5 M ammonium acetate was added and mixed well. 326 μl of stored at -20 ° C. 100% ethyl alcohol was added, centrifuged at 15,800 g for 20 minutes at 4 ° C., and the supernatant was removed. Next, 500 μl of 70% ethyl alcohol (10 ml) stored at -20 ° C was added and centrifuged at 15,800 g for 5 minutes at 4 ° C. The supernatant was removed to obtain cDNA pellet. The remaining 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. In order to fluorescently label the cDNA, 40 μl Cy3 9-mer Random Primer (1 OD / 42 μl, Sigma, USA) was added to 10 μl cDNA (1 μg) in water and reacted at 98 ° C for 10 minutes. . 10 μl of 10 × Klenow Fragment buffer (Takara, Japan), 10 μl of 3.5 U / μl Klenow and 10 μl of 50 × dNTP mix (10 mM) were added to each sample, followed by reaction at 37 ° C. for 2 hours. 10 μl of 0.5 M EDTA was added to stop the reaction, and 11.5 μl of 5 M NaCl was added and mixed well. 110 μl of 100% isopropyl alcohol was added, and the reaction was performed in a dark room for 10 minutes. Then, the supernatant was removed by centrifugation at 15,800 g for 10 minutes at room temperature. 500 μl of -20 ° C storage 70% ethyl alcohol was added, centrifuged at 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 (eppendoff, USA) for 5 minutes at room temperature, 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. 20 μl of 2 × hybridization buffer (Genisphere Inc., Hatfield, USA), 8 μl of RNase free water and 0.5 μl of 100 μM of Cy3 CPK6 Oligo (Sigma, USA) were added to the Cy3 labeled cDNA solution (15 μg). After reacting at 95 ° C. for 5 minutes, it was immediately placed in a 42 ° C. MAUI hybridization chamber for 30 seconds, and then hybridized in a microarray and reacted for 16-18 hours. After removing the MAUI Mixer SL type from the microarray, react with 0.2% SDS, 0.2X SSC solution for 2 minutes, 0.2X SSC for 1 minute, and 0.05X SSC for 30 seconds.The microarray was 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. 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

About 60 days after transgenic seed germination, flowers with a size of 0-3 cm before the pollination were collected for GUS histochemical analysis. Untransformed seeds were also collected as flowers of the size described above and used as controls.

Flowers at the early stage of development (experimental and control) were maintained in 90% acetone for 30 minutes at room temperature. After washing with stain buffer, it was immersed in 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 then reacted overnight at 37 ° C. The staining solution was washed several times with 50% ethanol until the tissue was clear, then 70% ethanol was added and stored for a long time at 4 ℃.

Example  One. Microarray  analysis

First, flowers were sampled by size before fertilization to find genes with high expression in the early stages of flower development, and microarray experiments were performed using a rice 3 'tiling 300k microarray (2 repetitions). In addition, experimental results were added to the leaf and root tissues in order to compare gene expression with other tissues (FIG. 2). In microarray experiments, six genes overexpressed at a intensity of 20,000 or more compared to other tissues were selected from a sample of size 0-3 cm during the stage of flower development (Table 1).

List of genes expressing at least 20,000 intensity in flower size 0-3cm Rap2_no P1cm P8cm P20cm P22cm leaf Root Rap2_no_desc Os03g0672400 23088 5344 3690 4498 1301 8155 Preserved Putative Protein Os05g0476200 24623 6594 4064 5127 1778 7103 DNA replication permitting factor MCM3 homolog Os02g0699700 22614 6059 4460 7332 2242 12067 DNA Topoisomerase Type II Os12g0636100 36292 8651 6306 7082 2861 3672 Tetratricopeptide-like Spiral Domain Containing Proteins Os05g0459400 28964 7232 4892 6372 3918 9370 Kinesin, Molecular Motor Protein Os08g0538700 20579 6171 4773 5448 3928 7215 Retinoblastoma Associated Proteins

RT-PCR was carried out by preparing primers from genes expressing high specificity (Os02g0699700; DNA topoisomerase II) during the early stages of flower development, and the same experimental results as the microarray experiments were obtained.

There are six DNA topoisomerase II genes in rice. Genes such as Os02g0699700, Os04g0482800, Os09g0279600, and Os12g0622500 were overexpressed in the early stages of the flower development stage, Os04g0482800 was expressed higher in the whole tissue, and Os12g0622500 was expressed more in the flower stage than in the leaves and roots. In particular, Os02g0699700 was shown to be specifically expressed in the early stages of flower development.

Tissue Expression of Rice DNA Topoisomerase II Gene Rap2_no P1cm P8cm P20cm P22cm Leaf Root Rap2_no_desc Os02g0699700 22614 6059 4460 7332 2242 12067 DNA Topoisomerase Type II Os03g0222100 1547 1258 2416 11290 8265 1313 Proteins similar to topoisomerase Os03g0284800 6896 4622 4104 2884 2924 4310 Spo11 / DNA Topoisomerase VI, Subunit A Family Proteins Os03g0812000 9563 4354 5161 6058 7232 4331 DNA topoisomerase, type IIA, subunit A Os04g0482800 12926 18139 15291 14636 15818 20040 Proteins similar to topoisomerase Os08g0156900 4628 3293 2244 2686 1964 3770 Spo11 / DNA Topoisomerase VI, Subunit A Family Proteins Os09g0279600 12469 5568 4612 6163 5607 4429 Similar to topoisomerase VI subunit B Os09g0500600 8433 4924 4201 5137 4127 4619 DNA Topoisomerase III Beta-1 Os12g0622500 23788 17760 14654 13624 3346 6795 Spo11 / DNA Topoisomerase VI, Subunit A Family Proteins

Example  2. In the early stages of flower development DNA Topoisomerase  Using a promoter GUS  Expression

2 kb of PCR was isolated upstream from the ATG of Os02g0699700 DNA topoisomerase II gene, and a vector linking the GUS to the promoter was constructed to confirm gene expression by this promoter. As a result of histochemical analysis of the expression of GUS in the transformed rice using a recombinant plant expression vector linked to the GUS gene as shown in FIG. 4, unlike in the early stage flowers of the control plants, the early stage flowers of the transformed rice plants were GUS. It was confirmed that is expressed (Fig. 5).

Attach an electronic file to a sequence list

Claims (10)

Early stage specific promoter of 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. The recombinant plant expression vector of claim 2, wherein the recombinant plant expression vector is prepared by operably linking a foreign gene encoding a protein of interest downstream of the promoter. Transforming a plant cell with a vector according to claim 3; And
Method for producing a transgenic plant that specifically expresses the foreign gene, including the step of re-differentiating the transformed plant from the transformed plant cells early in the flower development stage. A transgenic plant, wherein the foreign gene produced by the method of claim 4 is specifically expressed at an early stage of the flower development stage. A seed of the transgenic plant of claim 5. Designing 10 probes of 60 bp in length moving 30 bp in the 5 'direction at the 3'-end of each gene derived from rice and fixing them to a microarray to prepare a rice 3' tiling 300k microarray;
Synthesizing cDNA using RNA isolated by rice tissue as a template and hybridizing the prepared microarray;
Identifying genes that are specifically expressed in the early stages of flower development compared to other tissues based on the hybridization results; And
Separation method of the flower tissue specific promoter in the early stage of the flower development step comprising the step of separating the promoter of the identified gene. 8. The method of claim 7, wherein the promoter is a promoter consisting of the nucleotide sequence of SEQ ID NO: 1. 300,000 probes including 10 designed probes of 60bp length and selection marker gene probes moving 30bp in the 5 'direction at the 3'-end of each gene derived from rice are immobilized and specific for the early stages of the rice flower development stage. Rice 3 'Tiling 300k microarray for separation of enemy promoters. The rice 3 'tiling 300k microarray of claim 9, wherein the promoter is a promoter consisting of a nucleotide sequence of SEQ ID NO: 1.

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