KR20200000018A - OsNFY16 promoter specific for plant seed embryo and uses thereof - Google Patents

Abstract

The present invention relates to an embryo-specific Oryza sativa nuclear factor-Y16 (OsNFY16) promoter of plant seeds, and a usage thereof. More specifically, the present invention relates to: an OsNFY16 promoter inducing a target gene to be specifically expressed in an embryo of plant seeds; a recombinant vector containing the promoter; a method for manufacturing a transformant plant body using the recombinant vector; a transformant plant body transformed by the method; and a method for inducing a foreign gene to be specifically expressed in an embryo of the transformant plant body by using the promoter. The OsNFY16 promoter specifically expressed in an embryo of a rice mature seed of the present invention is used to specifically express a target gene in an embryo of mature seeds, and can be used in adjusting types of embryos such as giant embryo and the like and functional characteristics.

Description

OsNFY16 promoter specific for plant seed embryo and uses according to plant seed.

The present invention relates to an embryo-specific OsNFY16 ( Oryza sativa Nuclear Factor-Y16) promoter of plant seeds, and more particularly, to an OsNFY16 promoter that specifically induces expression of a target gene in the embryo of a plant seed. , A recombinant vector comprising the promoter, a method of producing a transformed plant using the recombinant vector, a plant transformed by the method and a promoter using the promoter to induce expression of a foreign gene specifically in the embryo of the transformed plant It is about how to.

Recently, with the development of genetic engineering technology, many researches to improve the traits of plants have been conducted. In particular, much attention has been paid to techniques for obtaining a target useful gene from a transgenic plant, and a promoter involved in gene expression is required when the target useful gene is to be expressed in a transgenic plant.

In general, a promoter is a type of regulatory region having a function of controlling the expression of a gene, that is, a base sequence that determines when and how much the gene is expressed. In the meantime, numerous studies have revealed many kinds of promoters. Conventionally reported promoters may be classified according to their characteristics and functions, such as a promoter that induces expression at all times and a promoter that induces expression in a time or position-specific manner.

Looking more specifically, firstly there is a strong promoter capable of maximizing the amount of expression. It was initially developed as a promoter for maximizing the expression level of the gene of interest, and the 35S RNA gene promoter of Cauliflower mosaic virus (P35S) and the actin gene promoter of rice are representative. These are mainly used for the purpose of antibiotic resistance genes used as selection markers when transforming plants, plant pest suppression genes or the production of substances using plants.

Second, there is a promoter that limits the timing of expression. Many examples have been reported as promoters that limit the expression of the gene of interest to only a particular stage of plant development. For example, promoters of genes involved in flowering express the gene only in the flowering period, and promoters of genes expressed by various biological or abiotic stimuli may be included in the timing controlling promoter.

Third, tissue-specific promoters that limit the expression tissue can be cited. These are promoters that are used to accomplish the transformation goal by confining the expression of the gene of interest to specific tissues of the plant. Promoters that induce such tissue specific expression have been reported for each tissue of the plant according to the various purposes to be used.

In particular, among the promoters classified according to functions and features as described above, promoters that induce tissue-specific expression are classified as follows according to tissues expressed in plants.

First, a systemic expression induction promoter can be mentioned. As a promoter for inducing expression throughout the whole plant, a promoter of 35S RNA gene of cauliflower mosaic virus (CaMV), which is a promoter used to maximize expression, is used as a representative dicotyledonous plant promoter. As a systemic expression induction promoter for monocotyledonous plants, promoters of rice actin and corn ubiquithin genes are mainly used, and recently, a promoter of rice cytochrome C gene (OsOc1) has been developed (Korea Patent No. 10-0429335). They are also used to induce the expression of antibiotic or herbicide resistance genes and reporter genes used as selection markers when transforming, as in the promoters that maximize the expression amount. Promoters are the first things to consider when trying to uncover them.

Second, seed specific promoters can be mentioned. A representative example is the rice gluten promoter used to develop golden rice as a promoter of the major storage protein gene of rice, which has been widely used to induce seed-specific expression of monocotyledonous plants. Promoters mainly used to induce seed specific expression include soybean-derived lectin promoters, cabbage-derived napin promoters, and gamma-tocopherol methyl transferase (γ-TMT) in seedlings of Arabidopsis. There are carrot-derived DC-3 promoters and perilla-derived oleosin promoters used in studies to enhance vitamin E production by inducing gene expression.

Third, as a root-specific expression promoter, this has not been commercialized yet, but Arabidopsis peroxidase (peroxidase, prxEa) was isolated to confirm root-specific expression, and recently, sweet potato-derived maz gene (ibMADS) and sugar induction. It has been reported that the ADP-glucose pyrophosphatase (AGPase) gene is isolated to induce the expression of the promoter in the roots and to the transient expression of the roots in carrots and radishes. .

Fourth, other tissue-specific promoters such as leaves, and the related promoters include rice and corn-derived albics (rbcS) ribulose bisphosphate carboxylase / oxygenase small subunit that induces strong gene expression only in photosynthetic tissues such as leaves. Promoter), tomato-derived fruit maturation-specific expression-induced phytoene synthase (PDS) promoter, and oleosin (oleosin) promoter from pollen-specific cabbage of Chinese cabbage flowers.

These seed, root or leaf specific expression promoters are expected to be used for the purpose of improving agricultural traits, accumulating useful proteins and producing useful substances in major crops used as food, food or food ingredients.

However, the known promoters, in particular, conventional promoters known to induce tissue-specific expression in plants, have the advantage of expressing a target gene in a tissue-specific manner, but the amount of expression thereof is insignificant. Therefore, there is an urgent need to discover useful new promoters capable of expressing the genes of interest in tissues and expressing them in large quantities.

Under this background, the present inventors have been eager to develop new promoters capable of tissue-specific expression of genes of interest in plants, specifically useful promoters capable of specifically expressing genes of interest in the embryos of plant seeds. As a result of the effort, the present invention has been completed by separating an OsNFY16 promoter capable of specifically expressing a target gene in seed embryos in rice.

Korea Patent Registration No. 10-0429335

An object of the present invention is an embryo-specific OsNFY16 ( Oryza sativa Nuclear Factor-Y16) promoter of a plant seed, a primer set for amplifying the promoter, a recombinant vector comprising the promoter, a transformed transformed with the recombinant vector Providing a transformant, a method for producing a transformed plant using the transformant, a plant transformed by the method, and a method for inducing expression of a foreign gene specifically in the embryo of the transformed plant using the promoter It is for.

As one aspect for achieving the above object, the present invention is an embryo specific ( Oryza sativa Nuclear Factor-Y16) promoter of plant seeds, a primer set for amplifying the promoter, a recombinant vector comprising the promoter , A transformant transformed with the recombinant vector, a method for producing a transformed plant using the transformant, a plant transformed by the method and a foreign gene using the promoter are specific for the embryo of the transformed plant. It provides a method of inducing expression.

By using the OsNFY16 promoter specifically expressed in the embryo of rice mature seed of the present invention, the target gene can be specifically expressed in the embryo of mature seed, and using this, the shape of embryo, such as a giant embryo, and the like This can be useful for adjusting functional characteristics.

1 is a view showing the analysis results derived for the purpose of seed expression transcription factor selection of the present invention through transcript analysis according to the seed development stage after rice extraction. Specifically, (A) the main characteristics of the stages of rice seed development, (B) a graph showing the expression level according to the number of days (0, 3-6, 25, 40 days) after the rice emergence of the gene group that increased expression in the seed development process And (C) is a diagram showing the results of microarray analysis (microarray) showing the expression pattern of OsNFY16 gene for each seed development stage.
Figure 2 is a diagram showing the results of RT-PCR analysis of the expression level of OsNFY16 gene of the present invention according to the seed development stage after rice extraction.
Figure 3 is a diagram showing the results of analyzing the expression pattern of each tissue of the OsNFY16 gene through in silico expression analysis using a public database. Specifically, (A) RiceXPRO data analysis results and (B) Rice Oligonucleotide Array data analysis results.
4 is a diagram showing the nucleotide sequence of the OsNFY16 promoter of the present invention.
5 is a diagram showing a schematic diagram of an OsNFY16 promoter-GUS expression vector according to an embodiment of the present invention.
Figure 6 is a diagram showing the results of PCR analysis confirming the introduction of the vector in the transformed rice using the OsNFY16 promoter-GUS expression vector according to an embodiment of the present invention. Here, DJ is a control rice Dongjinbyeo, TJ60-14 is a transgenic rice inserted with the OsNFY16 promoter.
Figure 7 is a diagram showing the results of GUS staining analysis of the transgenic rice inserted OsNFY16 promoter of the present invention. Here Em is Emryo, En is Endosperm, AL is Aleurone layer, An is Anther, OV is Ovary, FL is First leaf, R Is the root.

The present invention relates to an embryo-specific OsNFY16 ( Oryza sativa Nuclear Factor-Y16) promoter of plant seed and its use. More specifically, the OsNFY16 promoter to induce expression of the gene of interest in the embryo of the plant seed, a primer set for amplifying the promoter, a recombinant vector comprising the promoter, transformation transformed by the recombinant vector Sieve, the transgenic plant prepared using the transformant and the seed obtained therefrom, the method of producing the transgenic plant, and the promoter to induce the expression of a foreign gene specifically in the embryo of the transgenic plant. It is about how to.

In one embodiment, the present invention provides an embryo-specific OsNFY16 ( Oryza sativa Nuclear Factor-Y16) promoter of plant seed, consisting of the nucleotide sequence of SEQ ID NO: 1.

As used herein, the term “embryo” is also referred to as an embryo or seed eye, and refers to a part of a seed plant that is located in a dormant seed and later becomes a plant.

As used herein, the term "promoter" refers to a genome region that is linked to an upper side of a gene, and controls a structural gene linked thereto to be transcribed into mRNA. The promoter is activated by the binding of several general transcription factors.TATA boxes, CAAT box sites that regulate gene expression, sites, locations, and orientations that affect gene expression in response to external stimuli. Regardless of whether it consists of an enhancer that promotes the expression of almost all genes. Proteins necessary for the basic metabolism of the living body must maintain a constant concentration in the cell, so the promoters linked to these genes are always activated by the action of the general transcription factor. In contrast, proteins that do not play a role in normal life and require function only under special circumstances are linked with a tissue specific promoter or inducible promoter that induces expression of the structural gene. That is, tissue-specific promoters are activated in the development of organisms, and inducible promoters are activated by binding of specific transcription factors activated by external stimuli from environmental factors from the surroundings.

The promoter of the present invention is a promoter that specifically induces expression at a specific site of a plant, particularly in embryos of mature seeds of a plant, and has a nucleotide sequence of SEQ ID NO: 1 but a sequence having at least 80% homology with the sequence. , Preferably at least 90% homology, more preferably at least 95% homology, even more preferably at least 98% homology, most preferably at least 99% homology, substantially identical or corresponding It may include a base having a biological activity. In addition, as long as the sequence having substantially the same or corresponding biological activity as a sequence having such homology, it is obvious that a promoter having a nucleotide sequence in which some sequences are deleted, modified, substituted or added is also included in the scope of the present invention.

In the present invention, "homologous" is intended to indicate a degree of similarity with a wild type base sequence, and may include a sequence having the same sequence as the percentage of the base sequence of the present invention. This homology comparison can be performed by using a comparison program that is easy to see with the naked eye. Commercially available computer programs can calculate the percent homology between two or more sequences, and the percent homology can be calculated for adjacent sequences.

The promoter of the present invention can express the target protein to the target gene RNA in the embryo of the mature seed of the plant, more preferably can express the target protein specifically in the embryo of the mature seed of the plant.

Specifically, in one embodiment of the present invention, as a result of transforming rice with a recombinant expression vector comprising the OsNFY16 promoter of the present invention, the target protein operably linked to the promoter is specific only in the embryo region of rice mature seed. It can be confirmed that the expression is ever (Figure 7).

Tissue-specific promoters of the invention can act as promoters of conventional recombinant expression vectors.

In the present invention, the OsNFY16 ( Oryza sativa Nuclear Factor-Y16) gene may be an NF-Y family transcription factor.

The OsNFY16 promoter of the present invention may be isolated from rice ( Oryza sativa L. ), specifically may be separated from Dongjin rice. In one embodiment of the present invention, the translation start upper nucleotide sequence of the OsNFY16 gene whose gene expression is increased by at least about 5 times between 3-6 days and 25 days after rice extraction was isolated.

The promoter may consist of a nucleotide sequence of SEQ ID NO: 1 of 2,009 bp, and the nucleotide sequence of SEQ ID NO: 1 is 100% of the promoter sequence of the LOC_Os07g41720 gene extracted from the genome database of Nipponbarre. It may be a homologous base sequence, but is not limited thereto.

The promoter may include a variety of regulators involved in seed germination and dormancy control and growth, and specifically involved in motif and gibberellin (GA) hormone signaling involved in seed or embryo specific expression. Motifs, motifs involved in abscisic acid (ABA) hormone signaling, and motifs involved in auxin (auxin) hormone signaling may be included, but are not limited thereto.

The base sequence of SEQ ID NO: 1 may be modified to some extent through various recent research techniques, for example, directed evolution or site-directed mutagenesis. That is, the OsNFY16 promoter nucleic acid molecule of the present invention includes a functional equivalent thereof that performs the same function as the functional equivalent, wherein the functional equivalent is partially deleted, substituted by a nucleotide sequence of the nucleotide by artificial modification. , Variants modified by insertion or combinations thereof or functional fragments of the polynucleotides that perform the same function.

The term "specific" in the present invention 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 can be assessed by comparing the expression level of the promoter in tissues previously measured using methods known in the art to those in other tissues. In general, the expression level of a promoter is measured by the amount of gene product expressed under the control of the promoter, such as proteins and RNA.

Thus, the term "embryo specific of plant seeds" means that the expression activity of the promoter is higher in the germ tissue of the plant seed than other tissues in the same plant.

In another aspect, the present invention provides a primer set represented by SEQ ID NO: 4 and SEQ ID NO: 5 for amplifying the OsNFY16 promoter.

As used herein, the term "primer" refers to a single stranded oligonucleotide sequence that is complementary to the nucleic acid strand to be copied and may serve as a starting point for the synthesis of a primer extension product. The length and sequence of the primers should allow to start the synthesis of extension products. The specific length and sequence of the primer will depend on the primer utilization conditions such as temperature and ionic strength as well as the complexity of the DNA or RNA target required. Oligonucleotides used as primers may also include nucleotide analogues such as phosphorothioate, alkylphosphothioate or peptide nucleic acid, or intercalating agents ) May be included. Preferably, the primer is deoxyribonucleotide and single chain. Primers used in the present invention can include naturally occurring dNMPs (ie, dAMP, dGMP, dCMP and dTMP), modified nucleotides or non-natural nucleotides. In addition, the primer may also include ribonucleotides.

Using the primer set as a sample to perform the PCR amplification reaction of the OsNFY16 promoter of the present invention can be isolated. Specifically, the OsNFY16 promoter of the present invention can be isolated by PCR amplification reaction using genomic DNA of Dongjin rice as a sample using the primer set.

The term "amplification reaction" in the present invention means a reaction for amplifying a nucleic acid molecule. Various amplification reactions have been reported in the art, which include polymerase chain reaction (PCR) (US Pat. Nos. 4,683,195, 4,683,202, and 4,800,159), reverse transcriptase-polymerase chain reaction (RT-PCR) (Sambrook et al. , Molecular Cloning.A Laboratory Manual, 3rd ed.Cold Spring Harbor Press (2001)), Miller, HI (WO 89/06700) and Davey, C. et al. (EP 329,822), ligase chain reaction LCR), Gap-LCR (WO 90/01069), repair chain reaction (EP 439,182), transcription-mediated amplification (TMA) (WO 88/10315), self-sustaining sequence replication self sustained sequence replication (WO 90/06995), selective amplification of target polynucleotide sequences (US Pat. No. 6,410,276), consensus sequence primed polymerase chain reaction; CPPCR) (US Pat. No. 4,437,975), optionally Liberally primed polymerase chain reaction (AP-PCR) (US Pat. Nos. 5,413,909 and 5,861,245), nucleic acid sequence based amplification (NASBA) (US Pat. No. 5,130,238, 1) 5,409,818, 5,554,517, and 6,063,603), strand displacement amplification and loop-mediated isothermal amplification; LAMP), but is not limited thereto. Other amplification methods that can be used are described in US Pat. Nos. 5,242,794, 5,494,810, 4,988,617 and US Pat. No. 09 / 854,317.

As another aspect, the present invention provides a recombinant vector for embryo specific expression of plant seeds comprising the OsNFY16 promoter having the nucleotide sequence represented by SEQ ID NO: 1.

The recombinant vector may additionally include a foreign gene encoding a target protein operably linked to the OsNFY16 promoter, but is not limited thereto.

As used herein, 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, a heterologous peptide, or a heterologous nucleic acid. Recombinant cells can express genes or gene fragments that are not found in their natural form in either the sense or antisense form. Recombinant cells can also express genes found in natural cells, but the genes are modified and reintroduced into cells by artificial means.

The term "vector" is used in the present invention to refer to a DNA fragment (s), nucleic acid molecules to be delivered into the cell. Vectors can replicate DNA and be reproduced independently in host cells. The term "expression vector" is often used interchangeably with "recombinant vector." The term "recombinant vector" refers to a recombinant DNA molecule comprising a coding sequence of interest and an appropriate nucleic acid sequence necessary to express the coding sequence operably linked in a particular host organism. Promoters, enhancers, termination signals and polyadenylation signals available in eukaryotic cells are known.

The expression vector will preferably comprise one or more selectable markers. The marker is typically a nucleic acid sequence having properties that can be selected by chemical methods, and all genes that can distinguish transformed cells from non-transformed cells. Examples include herbicide resistance genes such as glyphosate, glufosinate ammonium, or phosphinothricin, kanamycin, G418, bleomycin, hygromycin Antibiotic resistance genes such as, but not limited to, chloramphenicol.

As used herein, the term “operably linked” means that the coding sequence and the promoter are functionally linked such that the promoter sequence initiates and mediates the transcription of the coding sequence.

In one embodiment of the present invention, the production of the vector used a Gateway vector system. Specifically, both sequences of the gene sequence to be expressed were amplified by PCR (Polymerase chain reaction) using a primer sequence. To insert into the pENTR / Directional TOPO vector (Invitrogen) having att L1 and att L2 sites, the amplified gene can enter the pENTR ™ Directional TOPO vector with direction by inserting CACC in front of the forward primer. (PENTR ™ Directional TOPO Cloning Kits, Invitrogen). And again, the target gene was inserted into the target vector using the LR Clonase II Enzyme Mix in the target gateway vector system having att R1 and att R2. In this way, a vector was produced which overexpresses the gene of interest. It uses site-specific recombination (SSR) through a specific sequence called att .

The recombinant vector may be linked to the reporter gene downstream of the promoter to measure the activity of the promoter, for example, GUS can be used as a reporter gene.

One embodiment of the present invention illustrates a recombinant vector OsNFY16 promoter-GUS expression vector prepared by inserting an OsNFY16 promoter into a pBGWFS7 vector containing a GUS reporter gene, but the present invention is not limited to this specific vector. The GUS reporter gene is for measuring the degree of activity of the promoter, and it is obvious that a recombinant gene of the present invention can introduce a foreign gene of interest for inducing specific expression in the embryo of plant seed instead of the GUS reporter gene. .

Recombinant vector according to the embodiment can specifically express the foreign gene in the embryo (embryo) of the plant seed.

As another aspect, the present invention provides a transformant transformed by a recombinant vector comprising the OsNFY16 promoter of the present invention.

In the present invention, the term "transformation" means that the trait of the individual or cell is genetically changed by DNA, which is a genetic material given from the outside, and in the present invention, by introducing a promoter represented by SEQ ID NO: 1 This means that the gene of interest is specifically expressed in tissue-specifically, particularly in the embryos of mature seeds of the plant.

In the present invention, transformation with the recombinant vector of the present invention may be performed by transformation techniques known to those skilled in the art. For example, microprojectile bombardment, particle gun bombardment, silicon carbide whiskers, sonication, electroporation, PEG-mediated fusion ( PEG-mediated fusion, microinjection, liposome-mediated method, In planta transformation, Vacuum infiltration method, floral meristem dipping method, or Agrobacterium sp. mediated method, and the like, but are not limited thereto.

As used herein, the term "transformer" refers to a host cell transformed with a recombinant vector comprising a promoter of the present invention and a base sequence encoding a protein of interest linked to the promoter.

The transformants, and microorganisms are preferred, but are not limited to, Escherichia coli (Escherichia coli), Bacillus subtilis (Bacillus subtilis), Streptomyces (Streptomyces), Pseudomonas (Pseudomonas), Proteus Mira Billy's (Proteus mirabilis ), Staphylococcus and Agrobacterium tumefaciens , and more preferably Agrobacterium tumefaciens .

Specifically, in one embodiment of the present invention, Agrobacterium tumefaciens LBA4404 was used (Example 4-2).

As another aspect, the present invention provides a plant transformed by introducing into the plant a recombinant vector comprising the OsNFY16 promoter of the present invention and a nucleotide sequence encoding the target protein operably linked to the promoter.

The transformed plant according to the present invention can be obtained through a sexual propagation method or an asexual propagation method which is a conventional method in the art. More specifically, the plant of the present invention may be obtained through oily breeding, which is a process of producing seeds from the pollination process of flowers and breeding from the seeds. In addition, after transforming a plant with a recombinant vector according to the present invention it can be obtained through the asexual propagation method which is a process of induction, rooting and soil purification of callus according to a conventional method. That is, the explants of plants transformed with the recombinant vector of the present invention are inoculated in a suitable medium known in the art and then cultured under appropriate conditions to induce the formation of callus, and when shoots are formed, transfer to a hormone-free medium to culture. do. After about 2 weeks, the shoots are transferred to rooting medium to induce roots. The transformed plant according to the invention can be obtained by inducing roots and then transplanting them into the soil to purify them. Transgenic plants in the present invention may include whole plants as well as tissues, cells or seeds that can be obtained therefrom.

In the present invention, the transgenic plant may be a monocotyledonous plant, but is not limited thereto. Preferably, the transgenic plant may be rice, wheat, barley or corn, and more preferably rice ( Oryza sativa ).

Specifically, in one embodiment of the present invention switch the transformed microorganism transformed by the recombinant vector containing the OsNFY16 promoter chain, Agrobacterium tumefaciens (Agrobacterium tumefaciens) Transgenic Rice Plants transformed by inoculation of LBA4404 in rice (Oryza sativa) Was prepared (Example 5-1).

As another aspect, the present invention provides a transformed seed obtained from the transgenic plant.

In one embodiment of the present invention, OsNFY16 promoter insertion was confirmed according to the development stage of the seed of the transformed rice and analyzed by GUS staining, so that the expression of GUS only in the embryo (embryo) of mature seeds , It was confirmed that the embryo is specifically expressed.

In another embodiment, the present invention provides a recombinant vector prepared by operatively linked an OsNFY16 promoter (Oyza sativa Nuclear Factor-Y16) promoter consisting of the nucleotide sequence of SEQ ID NO: 1 and a foreign gene encoding a target protein. It provides a method for producing a transgenic plant, the target protein is specifically expressed in the embryo (embryo) of the transgenic plant, comprising the step of transforming the plant.

In the present invention, the transgenic plant may be a monocotyledonous plant, but is not limited thereto. Preferably, the transgenic plant may be rice, wheat, barley or corn, and more preferably rice ( Oryza sativa ).

In one embodiment of the present invention, a variety of tissues of the transgenic rice confirmed OsNFY16 promoter insertion was analyzed by GUS staining, GUS expression was strongly induced only in the embryo (embryo) of the seed, most of the other areas are stained Since it was not observed, it was confirmed that the OsNFY16 promoter could specifically express the gene of interest in the embryo of the plant seed.

In another aspect, the present invention provides a method of inducing expression of a protein of interest in the embryo of a mature seed of a plant.

Prepared by operatively linked (Operationally linked) OsNFY16 promoter for the expression of the target protein in the embryo of rice mature seed comprising the nucleotide sequence represented by SEQ ID NO: 1 and the structural gene encoding the target protein By transforming rice with a recombinant vector, a method of inducing expression of a target protein in embryos of rice mature seeds can be provided.

The target protein may be any kind of protein, for example, a protein that has a medical utility such as enzymes, hormones, antibodies, cytokines, or the like, which may accumulate a large amount of nutrients that may improve the health of animals including humans It is not limited to this. Examples of the protein of interest include interleukin, interferon, platelet-induced growth factor, hemoglobin, elastin, collagen, insulin, fibroblast growth factor, human serum albumin, erythropoietin, and the like.

If the promoter of the present invention is used, in the case of crops that ingest certain parts of the plant, such as seeds, leaves, roots, or stems, the desired protein may be expressed along with the seed, leaf or stem by expressing a useful target protein in specific tissues of the plant. You can develop a crop that can be eaten with.

Hereinafter, the configuration and effects of the present invention through the embodiments will be described in more detail. These examples are only for illustrating the present invention, but the scope of the present invention is not limited by these examples.

Example  One: Rice seed development Transcript  Analysis and transcription factor selection

In order to select genes that are specifically expressed in plant seeds, the genes whose expression is rapidly increased during the development process are selected through microarray analysis according to the development stage of rice seeds, and the selected genes are analyzed through public data analysis. The degree of expression of each stage of seed development was analyzed.

Specifically, RNA was isolated according to the seed development stage after extraction from high-quality rice and microarray analysis was performed, and 134 species of transcription factors were selected in which the expression of the gene was increased more than five times between 3 and 6 to 25 days after the extraction. (A of FIG. 1 and B of FIG. 1). Functional group classification was performed for selected transcription factors, and OsNFY16 ( Oryza sativa Nuclear Factor-Y16) gene (LOC_Os07g41720) was selected among NF-Y family transcription factors whose gene expression was greatly increased during seed development. C).

Then, the degree of OsNFY16 gene expression according to seed development stage was analyzed using RT-PCR method. Specifically, RNA was isolated from seeds 0, 3-6, 25, and 40 days after extraction. Total RNA (total RNA) as a template oligo (dT18) primer (primer) and reverse transcriptase (Superscript II reverse transcriptase, Invitrogen) using a single cDNA chain was synthesized. PCR was performed using the synthesized cDNA as a template using a primer sequence specific primer set of OsNFY16 shown in Table 1 below. At this time, the PCR reaction conditions were denatured at 95 ° C for 5 minutes, repeated 30 times at 95 ° C, 30 seconds at 61 ° C, 30 seconds at 72 ° C for a total of 35 cycles, and last step 5 minutes at 72 ° C PCR was performed.

As a result, it was confirmed that the expression of OsNFY16 gene is maximized in the 25-day-old seed after extraction (Fig. 2).

SEQ ID NO: primer  designation primer  direction Sequence (5 '→ 3') 2 OsNFY16-250F Forward direction 5'-AAGTCAGTATCATCGTTGGGGAA-3 ' 3 OsNFY16-580R Reverse 5'-ACGAGCTCGCTTCATGGCATG-3 '

Example  2: OsNFY16  Gene expression analysis

Expression patterns of the gene OsNFY16 (LOC_Os07g41720) whose expression is specifically increased according to the seed development stage selected in Example 1 are described in two public databases (RiceXPro; http://ricexpro.dna.affrc.go.jp/). Rice Oligonucleotide Array; using http://www.ricearray.org/) persons were Rico (in silico).

As a result, the expression of OsNFY16 (LOC_Os07g41720) gene was predicted to be particularly high in the embryo or endosperm of rice seeds (FIG. 3).

Example  3: genomic DNA PCR  by OsNFY16  In the promoter area Cloning  And sequencing

In order to select a useful promoter capable of specifically expressing a gene of interest in the embryo of a plant seed, in Example 1, a gene whose expression is increased according to the development stage of rice seed, an OsNFY16 gene (LOC_Os07g41720), is selected. Based on this, embryo-specific expression promoter, OsNFY16 promoter of seeds, especially mature seeds, was isolated.

Example  3-1: OsNFY16  In the promoter area PCR  Amplification

In order to isolate the promoter region of the OsNFY16 gene (LOC_Os07g41720) expressed in rice seeds, base sequence information of 2,009 bp of the base sequence of translation start was extracted from the Riceponbarre genome database, and the fragment was amplified. The primer sets in Table 2 below were designed and synthesized. Using the synthesized primer set, genomic DNA isolated from the leaves of Dongjin rice was used as a template and amplified by using Applied Biosystem 9700 in a PCR reaction solution with Primestar polymerase (Takara). It was. In this case, PCR reaction conditions were denatured at 95 ° C. for 5 minutes, followed by a total of 35 cycles of 30 seconds at 95 ° C., 30 seconds at 54 ° C., 90 seconds at 72 ° C., and finally at 5 ° 72 ° C. PCR was performed for a minute. After the PCR reaction, the amplified gene was electrophoresed in 1% agarose, and the band estimated as OsNFY16 promoter was cut out and purified using a QIAquick gel extraction kit, and then used for cloning.

SEQ ID NO: primer  designation primer  direction Sequence (5 '→ 3') 4 OsNFY16P-F Forward direction 5'-gccgcccccttcaccGCTTTACTCCTTATTTAACTGTTAGC-3 ' 5 OsNFY16P-R Reverse 5'-tcggcgcgcccacccttTATGAGATGGTCTCACTTTGGTCC-3 '

Example  3-2: OsNFY16  In the promoter area Cloning cloning and sequencing

The PCR product of the OsNFY16 promoter region amplified in Example 3-1 was cloned into a gateway donor vector (pENTER / D-TOPO, Invitrogen) and the entire nucleotide sequence was determined. OsNFY16 promoter gene having a nucleotide sequence was obtained (Fig. 4).

On the other hand, when compared with the promoter sequence of the OsNFY16 gene (LOC_Os07g41720) extracted from the genome database of Nipponbarre (Nipponbarre), it was confirmed that the promoter of 100% concordant size 100%.

Example  3-3: OsNFY16  Motif search in promoter area

In order to identify reaction regulators present in the OsNFY16 promoter gene obtained in Example 3-2, New PLACE (database of plant cis-acting regulatory DNA elements: http://www.dna.affrc.go.jp/ Gene expression motifs were analyzed using the PLACE /) program.

As a result, as shown in Table 3, within the OsNFY16 promoter gene, the motif (DPBFCOREDCDC3), gibberellin (GA), which indicates seed or embryo-specific expression, motifs involved in hormone signaling (GAREAT) and abscisic acid ( Absortic acid (ABA) hormones involved in hormonal signaling (ABRELATERD1) and auxin (auxin) hormones involved in hormonal signaling (ARFAT) were found to exist a number of.

These results indicate that the OsNFY16 promoter may be involved in the seed development process by confirming the presence of motifs involved in seed germination, dormancy control and growth.

Example  4: Transformation vector and transformant construction

Example  4-1: OsNFY16  Promoter Carrier Fabrication

A recombinant expression vector was constructed to express the reporter gene GUS using the promoter of OsNFY16 gene isolated in Example 3-1.

Specifically, a recombinant vector (OsNFY16 promoter-GUS expression vector) in which the promoter was inserted into a pBGWFS7 vector for expressing a gateway promoter (PSB, Plant System Biology) was prepared (FIG. 5).

Example  4-2: Agrobacterium Agrobacterium tumefaciens ) Transformant production

In order to transform the OsNFY16 promoter expression vector prepared in Example 4-1 to Agrobacterium tumefaciens (LBA4404), it was transformed using a freeze-thaw method.

Specifically, freeze and thaw was repeated two or three times, and then transformed by a heat shock method at 37 ° C., followed by YEP medium (Yeast extract 10g, NaCl 5g, peptone 10g, Agar 15g / 1L) overnight colonies (overnight) confirmed colonies. Colonies confirmed to be transformed by colony PCR were prepared using AB medium (AB buffer (K ₂ HPO ₄ 60 g, NaH ₂ PO ₄ 20 g / 1 L), AB Salts (NH ₄ Cl 60 g, MgSO ₄ 7H _2). O 6g, were cultured in _{KCl 3g, CaCl 2 2H 2 O} 0.265g, FeSO 4 .7H 2 O 50mg / 1L), Glucose 5g / 1L).

Example  5: Preparation and Analysis of Rice Transformant Using Agrobacterium

Example  5-1: Preparation of Rice Transformant Using Agrobacterium

In order to study the function of the OsNFY16 promoter, the Agrobacterium LBA4404 into which the OsNFY16 promoter expression vector prepared in Example 4-2 was introduced, was inoculated with a callus of Dongjin rice, and then infected with PPT (phosphinothricin). , Phosphinothricin) 6 ㎍ / L transformants were selected, and finally the selected transformants were grown in a greenhouse to prepare a transformed rice.

Specifically, in order to induce the transformation of rice by Agrobacterium, in order to induce the callus of dongjinbyeon in 2N6 medium, the seed was washed in lax and then dried appropriately in 2N6 medium (Duchefa vitamin-containing medium). It was wound in the medium added mg / L. After induction of callus by cancer culture at 27 ° C. for 3 to 4 weeks, embryogenic callus was sub-cultured in 2N6 fresh medium. Agrobacterium transformed genes containing the transgene were cultured in AB liquid medium, co-cultured with embryogenic callus for 20 minutes, and then cultured for 3 days. After adding cefotaxime to sterile water, wash it until complete removal of Agrobacterium, and then add 2 μg of cefotaxime and PPT (phosphinothricin) at 6 μg / L. Cancer culture was carried out for 3 weeks. Callus that survived unchanged in 2N6 medium containing cefotaxime and PPT was transferred to 2N6 medium containing cefotaxime and PPT again and cultured for 2 weeks. To induce shooting in callus, transfer to MSR medium with cefotaxime and PPT (Maltose and Sorbitol added to Duchefa MS medium), incubate for 4 weeks, and shoot before rooting. Treatment was carried out.

Example  5-2: OsNFY16  Promoter Inserted Rice Transformant Analysis

In order to confirm whether the OsNFY16 promoter expression vector is properly inserted into the transformed rice prepared using the Agrobacterium transformant into which the OsNFY16 promoter expression vector was introduced in Example 5-1, the preparation was performed in Example 5-1. PCR analysis was performed by genomic DNA of rice transformants into which an OsNFY16 promoter was introduced.

Specifically, the genomic DNA was extracted from the leaves of the transgenic rice into which the OsNFY16 promoter was inserted using the Inclone Genomic DNA prep kit (Inclone Cat # IN1003-0200), and the beta − present in the introduction vector for amplifying the fragment. The primer sets in Table 4 below for the glucoronidase A (GUSA) and OsNFY16 promoter portions were designed and synthesized. PCR was performed using the extracted genomic DNA as a template using the synthesized primer set. At this time, PCR was heated for 5 minutes at 95 ° C under PCR amplification conditions, followed by a total of 35 cycles for a total of 35 cycles consisting of 30 seconds at 95 ° C, 30 seconds at 53 ° C, and 1 minute at 72 ° C. After that, the mixture was further heated at 72 ° C. for 5 minutes. After completion of the PCR reaction, the amplified PCR product was developed on a 1% agarose gel using an electrophoresis apparatus and the band of the reaction product developed under UV was confirmed.

As a result, as shown in FIG. 6, the PCR amplification product did not appear in the Dongjin rice (DJ), the wild type used as a control, whereas in the transformed rice (T60-14) into which the OsNFY16 promoter expression vector was inserted. PCR amplification product bands were clearly seen.

SEQ ID NO: primer  designation Sequence (5 '→ 3') 6 GUSA-Anti 5'-CGCGATCCAGACTGAATGCCC-3 ' 7 NFY16P-1704F 5'-CCTACTGTCTCTATTCAGTATTAC-3 '

These results indicate that the OsNFY16 promoter expression vector was correctly inserted into the transformed rice produced using the Agrobacterium transformant into which the OsNFY16 promoter expression vector was introduced.

Example  6: OsNFY16  GUS Staining Analysis of Transgenic Rice for Tissue Expression Assay of Promoter

According to Example 5-2, the seed of the transformed rice in which the OsNFY16 promoter insertion was confirmed was collected according to the development stage and analyzed by GUS protein staining. At this time, the tissue was cleared and the staining site was observed, and the site where the OsNFY16 promoter was operated was stained blue by GUS staining.

As a result of comparative analysis of GUS expression, it was confirmed that GUS expression was specifically induced only in the embryos of mature seeds (FIG. 7A).

In addition, GUS staining of the transformed rice seedling 5 days old after germination, it was not expressed in the stem and leaves (Fig. 7 B).

From these results, it can be seen that the OsNFY16 promoter can specifically express the target gene in the embryos of rice seeds, particularly mature seeds, which are representative monocotyledonous plants.

SmSpR: spectinomycin and streptomycin resistant
LB: left T-DNA border
Bar: bialaphos resistance gene
attR1: gateway attR1
OsNFY16P: OsNFY16 promoter
attR2: gateway attR2
Egfp: enhanced green fluorescence protein
gus: beta-glucuronidase A
T35S: CaMV 35S terminator
RB: right T-DNA border

<110> Republic of Korea <120> OsNFY16 promoter specific for plant seed embryo and uses <130> P18R12C0431 <160> 7 <170> KoPatentIn 3.0 <210> 1 <211> 2009 <212> DNA <213> Artificial Sequence <220> <223> OsNFY16 promoter <400> 1 gctttactcc ttatttaact gttagctcac aggaatctcc ataagccttg gaaaatagag 60 caacctatcc ataattagtt ctacccttct caaaaaacat aataaccttt ggcatacaaa 120 ataaatccta ttaatatatc ttcacttact ctacatacaa ctaattacaa ccatctcgca 180 tttaatttct ccacatattt tttcttaatt aataaataat tgcctccatt ttattttgcc 240 tactttcttt atatttataa tacttgtgcc aacttcatga atcgctaggc aattattttg 300 ggataaaaca gcagaagtag aattttagca tagccatatg cctctatgca ccttctgact 360 tttcttggtg catctatcaa ttttattgaa gatgtgacgt gtgagtgtgg aaagaaaggc 420 caaacatttc ttttccttta ttatatacca aaacaataag aaagcttgat ttatacctac 480 aagtttatat agcaggtcgc atatttggca gcaaagagag catagagcac agtcctctcc 540 tcttccccgt gctccccatt tctgcttctt ttcccacaaa tttctctctc tatctcggaa 600 aggattcggg agacttgaag agaggagctc gaagcgggac aggagagaga ggtacatctc 660 tatgcgcgcc gctgcgctac tccctcctcg gagaaatccc caaattgtgt tcttgcatgc 720 gtcgtcctag tagcagagag ctgtcccttc cattttcctt tcatggagtg gattcttggt 780 tcttgattac gacatgctgt gtttggtcca gttcttatcc cctgcgattt tttcccattt 840 cttttctctc ccttgttcca cctcggaaat ttgggttctt tttttcttct tcttttgatt 900 cttggttggt ttgtgttgca taatttcagt tctttgttag gcttgcttcg gagttgtagt 960 tagacatcgc aagatgcatc ctttcgagga agtcaatgca tcccctgttg ttagctgacc 1020 actgtatatc gaatgcactg tgagttgtgt tttaggccgt ggagtgggtg gaaatttgac 1080 ttccccattg caggatttgg taacttgaag ctaaattccg tgtgaatttg tgacggtatc 1140 aagcgattgt gtctctgata agattctgaa tgtgtgaact tgctgcgata tgttctgatt 1200 tactagtggt ttagcttttc aatcgttatt ctgattaaga caagggactt cttctgtgca 1260 tatggattgg caatttggac tcgagaacaa tccactaaaa gttgtgtagg ttatctcttt 1320 atgatctgca tgtaataaat gtaaacctga gactgatgta ggctacctct tattcgagaa 1380 aaagaaaaag aggctgatgt aagctaccta atgtgcattt tgtgcttatt cctcttgatc 1440 aggttgtttc tatataagaa ttttctgtct gtctacttag ggtaggatgc tgtgtgttat 1500 ttgtagtggt actcttttta ggttgattta tttgtttttt tattcagtta tttcctactc 1560 ttgcaacaag cacttaacaa cctactctgc ctactctgtt cctactgaac agtaccacta 1620 ttaagtatta ccttattatg atattaaggt aatatcataa tttatttgct tttatggttc 1680 agaatctcat gattcagtta tttcctactg tctctattca gtattacaac atctattgtc 1740 gtttttttta gataattcaa cacctgaaag taactatgca taatcatatt ccagttaatg 1800 agtagaaacc tttaataggc atcttttgat agaaaggatt agagctaatc ctttgatggg 1860 caacaaaatc cccaccaatc tgaagaggtc aggttaaaag ccctatcagt tcaatttgct 1920 taagttaatg ttatcaactt gtcttgaagg ctaattatag aagtacacga agaaaaatgg 1980 tgcctggacc aaagtgagac catctcata 2009 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> OsNFY16-250F <400> 2 aagtcagtat catcgttggg gaa 23 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> OsNFY16-580R <400> 3 acgagctcgc ttcatggcat g 21 <210> 4 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> OsNFY16P-F <400> 4 gccgccccct tcaccgcttt actccttatt taactgttag c 41 <210> 5 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> OsNFY16P-R <400> 5 tcggcgcgcc caccctttat gagatggtct cactttggtc c 41 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GUSA-Anti <400> 6 cgcgatccag actgaatgcc c 21 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> NFY16P-1704F <400> 7 cctactgtct ctattcagta ttac 24

Claims (12)

Embryo specific OsNFY16 ( Oryza sativa Nuclear Factor-Y16) promoter of the plant seed which consists of a nucleotide sequence of SEQ ID NO: 1.
The OsNFY16 promoter according to claim 1, wherein the promoter is isolated from rice ( Oryza sativa L. ).
A primer set represented by SEQ ID NO: 4 and SEQ ID NO: 5 for amplifying the OsNFY16 promoter of claim 1.
A recombinant vector for embryo specific expression of plant seeds, comprising the OsNFY16 promoter of claim 1.
A transformant transformed by the recombinant vector of claim 4.
The transformant of claim 5, wherein the transformant is Agrobacterium tumefaciens .

A transformed plant transformed with the recombinant vector according to claim 4 or the transformant according to claim 5.
The transgenic plant of claim 7, wherein the transgenic plant is a monocotyledonous plant.
The transgenic plant of claim 8, wherein the monocotyledonous plant is Oryza sativa .
A transformed seed obtained from the transformed plant according to claim 7.
Connecting a foreign gene coding for SEQ ID NO: 1 OsNFY16 (Oryza sativa Nuclear Factor- Y16) promoter and a target protein comprising the nucleotide sequence of operatively by comprising the step of transforming a plant by (operatively linked) prepared recombinant vector , A method for producing a transgenic plant, wherein the protein of interest is specifically expressed in the embryo of the transgenic plant.
By transforming the plant with by a foreign gene coding for SEQ ID NO: 1 OsNFY16 (Oryza sativa Nuclear Factor- Y16) promoter and a target protein comprising the nucleotide sequence of the operably linked (operatively linked) producing a recombinant vector, transformed plant A method of inducing the expression of a protein of interest in the embryo.

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