KR101642797B1 - Endosperm-specific expression promoter derived from oryza sativa and use thereof - Google Patents
Endosperm-specific expression promoter derived from oryza sativa and use thereof Download PDFInfo
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Abstract
[0001] The present invention relates to a seed-specific expression promoter derived from rice ( Oryza sativa ) and a use thereof, and more particularly, to a method for promoting expression of a target gene in the endosperm and aleurone layer tissues of mature seeds An OsNFY26 ( Oryza sativa Nuclear Factor Y 26) promoter, a recombinant expression vector containing the promoter, a method for producing a transformed plant using the recombinant expression vector, and a plant transformed by the method.
The OsNFY26 promoter specifically expressed in the rice seeds of the present invention can be used to regulate the expression of the desired gene in the endosperm of the mature seed and in the phyllosphere of the seed, thereby improving the production of seed specific substances, dormancy, germination, Not only can it be usefully used for trait improvement, but it can also be useful for analysis of target gene function in mature seeds.
Description
The present invention is rice (Oryza sativa) derived from seed endosperm-specific expression promoter and relates to their use, more particularly OsNFY26 (Oryza sativa Nuclear Factor inducing specific expression in the target gene in the endosperm (endosperm) and hobuncheung (aleurone layer) structure of mature seeds Y 26) promoter, a recombinant expression vector containing the promoter, a method for producing a transformed plant using the recombinant expression vector, and a plant transformed by the method.
Expression of a gene refers to a series of processes for synthesizing a protein according to a code entered into the gene through transcription and translation occurring within the cell. In particular, the transcription process is the initial stage of gene expression, in which the RNA polymerase is initiated by binding to a promoter sequence located on top of the gene with the aid of several cofactors, and the transcription factor (TF) One such cofactor is known to bind directly to the promoter sequence.
Crop molecular breeding can use all kinds of genes as a material and it is possible to control the effect of breeding infinitely by dragging the breeding technique which was possible only by the existing genome unit as a gene unit, Technology. In order to maximize the effect of these crop molecular breeding techniques, it is necessary to 1) accumulate a large amount of gene data that can represent various plant genes, 2) construct a transformation system for various crops, 3) The development of various promoters capable of regulating the expression of the inserted gene should be preceded.
Known promoters used to achieve the transfection purpose by limiting the expression of foreign genes to specific tissues of plants can be classified as follows according to their functions.
First, seed-specific promoters can be mentioned. As a representative example, the rice glutelin promoter used for the development of golden rice as promoters of rice major storage protein gene has been widely used to induce seed-specific expression of monocotyledonous plants so far. Promoters that are mainly used to induce seed-specific expression include soybean-derived lectin promoter, cabbage-derived napin promoter and γ-tocopherol methyl transferase (γ-TMT) in Arabidopsis seeds. A DC-3 promoter derived from carrot used in the study of promoting the production of vitamin E by inducing gene expression, and the case of confirming the induction of seed-specific expression of an oleosin promoter derived from perilla. The seed-specific promoters are mainly used for the purpose of accumulating useful proteins and producing beneficial substances in major crops in which seed itself is used as a food, a food, or a raw material for food.
Second, it is a root tissue specific expression promoter. Although there are no commercially available examples, it has been confirmed that the peroxidase (prxEa) is isolated and specific expression of root center main tissues has been confirmed. Recently, the expression of maz genes (ibMADS) derived from sweet potatoes and the sugar-induced ADP-glucan pyrophosphatase ADP-glucose pyrophosphatase, AGPase) gene has been isolated and it has been confirmed that the promoter induces specific expression in roots and induces root-specific transient expression in carrot and radish (Korean Patent Publication No. 10-18771 and 10-604191). These promoters can be expected to be mainly used for the purpose of improving agricultural traits, accumulating useful proteins, and producing useful substances in major root crops used for food, food, or food.
Third, a systemic expression-inducible promoter can be mentioned. As a promoter for plant systemic expression induction, 35S RNA gene promoter of cauliflower mosaic virus (CaMV) has been used as a typical promoter for dicotyledonous plants. As a promoter inducing systemic expression for rice plants such as rice, rice actin ) And maize ubiquitin gene promoters have been mainly used. Recently, a promoter of rice cytochrome C gene (OsOc1) has been developed and used by domestic researchers (Korean Patent Publication No. 10-429335). They are already inherent in inducing the expression of antibiotics, herbicide resistance genes and reporter genes used as selection markers in the plant transgenic carrier. From a research point of view, Promoters considered.
Fourth, other tissue-specific promoters such as leaves may be mentioned. RbcS (ribulose bisphosphate carboxylase / oxygenase small subunit) promoter derived from rice and maize, which induces expression of a strong gene only in photosynthetic tissues such as leaves, RolD promoter inducing expression of plant roots derived from Agrobacterium, induction of specific expression of potato-derived tuber A phattoin promoter, a PDS (phytoene synthase) promoter derived from tomato-derived fruit-specific expression, and a cabbage-derived oleosin promoter whose function is confirmed to be a pollen-specific plant of a Chinese cabbage. A male sterile plant has been developed by inducing the expression of a Bt protein gene having toxicity to cells in a carpet-specific tissue-specific BcA9 promoter (Korean Patent Registration No. 10-435143).
In addition, a large number of promoters have been reported for various plant-derived plant tissues for various purposes and are still under development. However, the types of promoters commercialized or commonly used among plant researchers are not limited to those mentioned above And new promoters that can regulate gene expression sites more precisely according to the intention of the developer need to be developed in the future.
Recently, as the genetic engineering technology has developed, many researches have been carried out to improve the characteristics of plants. In particular, there is much interest in techniques for obtaining a useful gene from a transgenic plant. A promoter involved in expression of the gene is required when the desired gene is expressed in a transgenic plant. For this purpose, a promoter derived from cauliflower mosaic virus (CaMV35S), in which expression is induced in whole tissues of plants, is widely used. However, in the case of producing a useful substance only in seeds, or when a foreign gene is expressed in tissues other than seeds and harms the plant, a promoter which is specifically expressed in the seed is required. Korean Patent Publication No. 2010-0043820 discloses a method for producing a target protein in a seed-seeded plant using a seed-expression-specific expression promoter, a recombinant plant expression vector and a recombinant plant expression vector for transformation of a monocotyledonous plant Korean Patent No. 1166239 discloses a promoter capable of inducing expression of a gene located at the 3 'end only in seeds of a paddy plant and a method capable of accumulating a large amount of a target protein in plant seeds using the promoter Japanese Patent No. 5168511 discloses a promoter that specifically expresses a specific site such as a seed of a seed and an endosperm, but is different from the promoter of the present invention.
The known 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 raw materials.
However, conventional promoters known to induce tissue-specific expression in plants have the advantage of being able to express a target gene in a tissue-specific manner, but have a disadvantage in that the expression amount thereof is insignificant. Therefore, it is urgent to find useful new promoters capable of expressing a gene of interest in a plant in a tissue-specific and large-scale expression.
Under these circumstances, the present inventors have made intensive efforts to develop a novel promoter capable of expressing a target gene in a tissue-specific manner in a plant, and as a result, the seed expression transcription factor was selected through analysis of the transcript according to the developmental stage of the seed, The present invention has been accomplished by isolating the OsNFY26 promoter which is capable of expressing the target gene mainly in the endosperm of the mature seed and in the phyllosphere tissue.
It is an object of the present invention to provide a promoter which specifically expresses a target protein in the endosperm of the mature seed of rice and the phloem structure of the rice seed, an expression vector containing the promoter, and a transformant transformed with the expression vector.
Another object of the present invention is to provide a method for producing the transformant.
It is still another object of the present invention to provide a method for inducing expression of a target protein using the promoter and a vector.
In order to achieve the above object, the present invention provides a method for producing a promoter which specifically induces expression in an endosperm or aleurone layer tissue of a rice seedling having the nucleotide sequence of SEQ ID NO: 1 to provide.
The term "endosperm " in the present invention refers to a tissue that develops in the embryo sac of a plant, stores most of the nutrients the seed has, and supplies the nutrients at the time of embryo development.
The term "aleurone layer" in the present invention refers to a cell layer containing a large amount of aleurone grain, which is differentiated from cells in the periphery of endosperm and secretes amylase and other enzymes in addition to nutrient storage, Into a soluble component and supplying it to the abdomen.
The term "promoter" in the present invention is a genome region linked to the upper side of a structural gene, and regulates the transcription of the structural gene linked thereto to mRNA. Promoters are activated by the binding of several common transcription factors, such as the TATA box that controls gene expression, the CAAT box site, the site that affects gene expression in response to external stimuli, And an enhancer that promotes the expression of almost all of the genes regardless of the gene expression. Since proteins necessary for basic metabolism of living organisms must maintain a constant concentration in cells, the promoter linked to these genes is always activated by the action of common transcription factors. On the other hand, proteins that do not have a role in normal times and require functions only under specific circumstances are connected to a tissue specific promoter or an inducible promoter that induces the expression of the structural gene. In other words, tissue specific promoters are activated by the binding of specific transcription factors activated by external stimuli from environmental factors from the environment in the developmental process of the organism.
The promoter of the present invention is a promoter that specifically induces expression in specific parts of plants, particularly in endosperm and aleurone layer tissues of mature seeds, and has a nucleotide sequence of SEQ ID NO: 1, but has a sequence of 80% , Preferably 90% or more homology, more preferably 95% or more homology, even more preferably 98% or more homology, and most preferably, 99% or more homology with a sequence having homology, And may comprise bases having substantially the same or corresponding biological activity. In addition, it is obvious that the promoter having a base sequence in which some sequences are deleted, modified, substituted or added is also included in the scope of the present invention if it is a base sequence having substantially the same or corresponding biological activity as a sequence having such homology.
In the present invention, the term " homology "refers to a degree of similarity to a wild-type nucleotide sequence, and may include a nucleotide sequence of the present invention having the same sequence as the above-mentioned nucleotide sequence. A commercially available computer program can calculate the homology between two or more sequences as a percentage (%), and the homology (%) is the number of sequences in an adjacent sequence / RTI >
The promoter of the present invention can express the target protein or the target gene RNA in the endosperm of the rice seed or in the phyllosphere of the rice seed, and more preferably, can express the target protein specifically in the endosperm of the rice seed or the phloem structure.
Specifically, in one embodiment of the present invention, rice transformed with a recombinant expression vector containing the OsNFY26 promoter of the present invention showed that the target protein operably linked to the promoter was specifically expressed in the endosperm of rice seeds or in the hornbone tissue (Fig. 6A).
The tissue specific promoter of the present invention may serve as a promoter of a conventional recombinant expression vector.
In another embodiment, the present invention provides a recombinant expression vector comprising a foreign gene encoding a target protein operably linked to the OsNFY26 promoter having the nucleotide sequence shown in SEQ ID NO: 1.
As used herein, the term "recombinant expression vector" refers to a gene construct that contains an essential regulatory element operably linked to express a desired protein or RNA of interest in a host cell, such that the gene insert is expressed.
The recombinant expression vector of the present invention may contain a promoter of the present invention and a plasmid, virus, and the like known in the art into which a nucleotide sequence encoding a target protein operably linked to the promoter can be inserted or introduced Or other media. The nucleotide sequence encoding the plant-specific expression promoter of the plant according to the invention and the target protein operably linked to the promoter may be operably linked to an expression control sequence, wherein the operably linked gene sequence and the expression control sequence May be included in an expression vector that also contains a selection marker and a replication origin.
Said "operably linked" may be a gene and expression control sequence linked in such a way as to enable gene expression when a suitable molecule is coupled to an expression control sequence. "Expression control sequence" means a DNA sequence that regulates the expression of a polynucleotide sequence operably linked to a particular host cell. Such regulatory sequences may include a promoter for carrying out transcription, any operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence controlling the termination of transcription and translation.
The expression vector according to the present invention may be prepared by inserting a promoter of the present invention using a conventional vector used for protein expression as a basic framework and inserting a nucleotide sequence encoding a target protein downstream of the promoter have. Therefore, plasmids derived from Escherichia coli (pBR322, pBR325, pUC118 and pUC119), Bacillus subtilis -derived plasmids (pUB110 and pTP5), yeast-derived plasmids (YEp13, YEp24 and YCp50 ) And a Ti plasmid, and may be a plant virus vector, and binary vectors such as pCHF3, pPZP, pGA and pCAMBIA sequences may be used. However, any person skilled in the art can use any vector as long as it can introduce the promoter of the present invention and the base sequence encoding the target protein operably linked to the promoter into the host cell.
The target protein that can be used in the present invention may be any target protein derived from the outside, that is, an exogenous protein or an endogenous protein and a reporter protein, which is desired to be expressed specifically by a plant tissue by the promoter of the present invention. The exogenous protein refers to a protein that does not naturally exist in a specific tissue or cell, and the endogenous protein refers to a protein that is expressed by a gene naturally present in a specific tissue or cell. In addition, the reporter protein is a protein expressed by a reporter gene, and refers to a marker protein that shows its activity in a cell by its presence.
In addition, the expression vector of the present invention may preferably comprise one or more selectable markers. The marker may be any gene capable of distinguishing a transfected cell from a non-transfected cell, typically a nucleic acid sequence having a selectable characteristic by a chemical method. Examples include herbicide resistance genes such as glyphosate or phosphinotricin, antibiotics such as G418, kanamycin, Bleomycin, hygromycin, cholramphenicol, Resistant genes, but are not limited thereto.
Specifically, in one embodiment of the present invention, the OsNFY26 promoter of the present invention having the nucleotide sequence of SEQ ID NO: 1 is inserted into a known vector having a reporter system in which a blue coloring gene, Gus, The recombinant expression vector pBGWFS7-pOsNFY26, which was linked as much as possible, was prepared (Example 3-1 and Fig. 4).
In another embodiment, the present invention provides a transformant transformed with a recombinant expression vector comprising the OsNFY26 promoter of the present invention.
The term "transformation" in the present invention means genetically changing the trait of an individual or a cell by DNA, which is a genetic material given from the outside. In the present invention, by the introduction of the promoter shown in SEQ ID NO: 1 Means that the gene of interest is expressed specifically in tissue-specific, especially in the endocrine or colonic tissues of mature seed.
Transformation with the recombinant expression vector of the present invention in the present invention can be carried out by a transformation technique known to a person 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, Agrobacterium sp. mediated method, and the like, but the present invention is not limited thereto.
The term "transformant" in the present invention means a host cell transformed with a recombinant expression vector comprising a promoter of the present invention and a base sequence encoding a target protein operably linked to the promoter.
The transformant is preferably a microorganism, including, but not limited to Escherichia coli), Bacillus subtilis (Bacillus subtilis), may be a Streptomyces (Streptomyces), Pseudomonas (Pseudomonas), Proteus Mira Billy's (Proteus mirabilis), Staphylococcus (Staphylococcus) and Agrobacterium Tome Pacific Enschede (Agrobacterium tumefaciens), and more preferably Agrobacterium tumefaciens . ≪ / RTI >
Specifically, in one embodiment of the present invention Agrobacterium tumefaciens (Agrobacterium tumefaciens ) LBA4404 (Example 3-2).
In another embodiment, the present invention provides a transgenic plant by introducing into a plant a recombinant expression vector comprising a nucleotide sequence encoding an OsNFY26 promoter and a promoter operably linked to the promoter of the present invention.
The transformed plant according to the present invention can be obtained by a conventional method in the art, such as an ovine reproduction method or an aseptic reproduction method. More specifically, the plant of the present invention can be obtained through reproductive process, which is a process of producing seeds through the moisture process of flowers and propagating from the seeds. Also, the plant can be transformed with the recombinant expression vector according to the present invention, and then obtained by a conventional method, such as induction, rooting and soil purification of the callus. That is, a fragment of a plant transformed with the recombinant expression vector of the present invention is pelleted in a suitable medium known in the art, and cultured under appropriate conditions to induce callus formation. When shoots are formed, the plant is transferred to a hormone-free medium Lt; / RTI > After about two weeks, the shoots are transferred to rooting medium to induce roots. The transformed plants according to the invention can be obtained by roots being induced and then transplanted into the soil for purification. Transgenic plants in the present invention may include whole plants as well as tissues, cells or seeds obtainable therefrom.
Specifically, in one embodiment of the present invention, Agrobacterium ( Agrobacterium tumefaciens), which is a microorganism transformation transformed with a recombinant expression vector containing OsNFY26 promoter tumefaciens ) LBA4404 in rice ( Oryza sativa ) to produce transgenic plants (Example 4-1).
In yet another aspect, the present invention provides a method for producing a transgenic plant in which a target protein is expressed specifically in the endosperm or hornbreak tissue of rice seed mature seed, comprising the following steps.
a) preparing a recombinant vector comprising a promoter consisting of the nucleotide sequence of SEQ ID NO: 1 and a foreign gene encoding a target protein operably linked thereto;
b) As the recombinant expression vector, Agrobacterium tumefaciens ) to produce transformed Agrobacterium; And
c) infecting the plant with the transformed Agrobacterium.
In the present invention, the plant may be a dicotyledonous plant or a monocotyledonous plant. It may be, but not limited to, a monocotyledonous plant, more preferably rice, wheat, barley or corn, Rice ( Oryza sativa .
In another aspect, the present invention provides a method for inducing the expression of a target protein in an endosperm or hornbreak tissue of a rice seedling.
The OsNFY26 promoter for expression of the target protein in the endosperm of the rice seeds or the phloem structure of the rice seeds containing the nucleotide sequence of SEQ ID NO: 1 according to the present invention, and the structural gene encoding the target protein are operatively linked, And transforming rice with the recombinant expression vector thus produced to induce the expression of the target protein in the endosperm of rice seeds or in the phyllotaxis tissue.
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, etc., , But is not limited thereto. Examples of the target protein include interleukin, interferon, platelet-derived growth factor, hemoglobin, elastin, collagen, insulin, fibroblast growth factor, human serum albumin and erythropoietin.
When the promoter of the present invention is used, in the case of a crop consuming a specific part of the plant, for example, a seed, a leaf or a stem, a useful protein can be expressed in a seed of a plant, Can be developed.
The OsNFY26 promoter specifically expressed in the rice seeds of the present invention can be used to regulate the expression of the desired gene in the endosperm of the mature seed and in the phyllosphere of the seed, thereby improving the production of seed specific substances, dormancy, germination, Not only can it be usefully used for trait improvement, but it can also be useful for analysis of target gene function in mature seeds.
FIG. 1 shows the result of selection of the OsNFY26 gene of the present invention expressed in the seed maturation stage. (A) is a result of gene expression analysis (microarray analysis) according to the seed development stage, DAH means day after heading, (B) indicates a transcription factor The results of the functional group classification are as follows.
FIG. 2 is a graph showing the results of RT-PCR analysis of the OsNFY26 gene of the present invention according to the seed development stage. Here, DAH means day after heading.
3 is a diagram showing the nucleotide sequence of the OsNFY26 promoter of the present invention.
FIG. 4 is a schematic diagram showing an expression vector of the OsNFY26 promoter for rice transformation prepared through the gateway system of the present invention.
5 is a diagram showing the result of genomic PCR analysis of the transgenic rice of the present invention. Here, WT represents a control group, Dong Jinbun, and T53-12 series and T54-22 series represent transgenic rice plants.
FIG. 6 is a graph showing the results of a comparison between the mature seeds of the transgenic rice plants transfected with the OsNFY26 promoter of the present invention, (B) seedlings grown 7 days after germination, (C) matured leaves and stems grown 2 months after germination, and (D) The results of the GUS staining analysis are shown in Fig. Here, Em is an embryo, En is an endosperm, AL is an Aleurone Layer, S is a shoot, R is a root, LB is a Leaf (Leaf) Blade), AU is auricles and LS is Leaf Sheath.
Hereinafter, the constitution and effects of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.
Example
1: specifically expressed in seeds
OsNFY26
Gene selection and expression analysis
Example 1-1: OsNFY26 Gene selection
In order to obtain a gene specifically expressed in the rice seed development stage, RNA was extracted according to the seed development stage after heading in high quality rice and microarry analysis was performed to obtain OsNFY26 ( Oryza sativa Nuclear Factor Y 26) genes were selected.
Specifically, the rice seed development process was divided into four stages according to the number of days of
Example 1-2: OsNFY26 Gene expression analysis
RT-PCR was performed to confirm the expression pattern of the OsNFY26 gene selected in Example 1-1 according to the seed development stage.
Specifically, for the RT-PCR analysis, a single cDNA chain was synthesized using dT 18 primer and reverse transcriptase (Superscript II reverse transcriptase, Invitrogen). (SEQ ID NO: 2) and OsNFY26R: 5'-GGC CTC ATC AAG AGT ACG AA-3 '(SEQ ID NO: 2) (SEQ ID NO: 3)). At this time, the reaction condition of PCR was repeated 25 times (total cycle) consisting of heating at 94 ° C for 5 minutes and then heating at 94 ° C for 40 seconds, 58 ° C for 30 seconds, and 72 ° C for 30 seconds Then, it was heated at 72 캜 for 7 minutes. After the PCR reaction, the amplified PCR products were 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. 2, it was confirmed that the expression of OsNFY26 gene was maximized at 25 days after emergence.
Example
2: The genome
DNA
of
PCR
On by
OsNFY26
Of the promoter region
Cloning
And base sequence analysis
Example 2-1: OsNFY26 Of the promoter region PCR Amplification
In order to isolate the promoter region of the OsNFY26 gene selected in Example 1-1, nucleotide sequence information of 2,000 bp at the start of translation was extracted from a rice genome database, and primers for amplifying the fragment (Forward primer) OsNFY26P-F: 5'-CTA GTC TAG AAT GCC GAT CCC GGA AAA GG-3 '(SEQ ID NO: 4) and reverse primer OsNFY26P-R: 5'-ACG CGG ATC CTC ACT CAG GAA GAT CTT TGT CAG-3 '(SEQ ID NO: 5) was synthesized. Using the primer set, the genomic DNA of Dongjin rice was used as a template, and the gene was amplified using a Applied Biosystem 9700 machine in a PCR reaction solution together with a Primestar polymerase (Takara). At this time, the reaction conditions for PCR were denaturation at 94 ° C for 5 minutes, followed by 40 cycles of 94 ° C for 30 seconds, 58 ° C for 40 seconds, and 72 ° C for 5 minutes, Min. After completion of the PCR reaction, the amplified gene was electrophoresed on 1% agarose, and the band assumed to be the OsNFY26 promoter was cut out and purified using a QIAquick gel extraction kit and then used for cloning.
Example 2-2: OsNFY26 Of the promoter region Cloning ( cloning ) And base sequence analysis
The PCR product of the OsNFY26 promoter region amplified in Example 2-1 was cloned into a gateway donor vector (pENTER / D-TOPO, Invitrogen) and the entire nucleotide sequence was determined. Thus, the nucleotide sequence of SEQ ID NO: 1 OsNFY26 promoter gene (Fig. 3).
Example 2-3: OsNFY26 Motif of promoter region ( motif ) Search
In order to confirm the regulatory factors present in the OsNFY26 promoter gene obtained in Example 2-2, a PLACE (Plant Cis-acting Elements database: http://www.dna.affrc.go.jp/PLACE/) program Were used to analyze gene expression motifs. As a result, as shown in Table 1, it was confirmed that seed or papillary reaction motif (CANBNNAPA or DPBFCOREDCDC3) was present and involved in seed germination and dormancy control such as GARE (GA responsive element) and ABRE (ABA responsive element) There is also a motif that responds to the hormone. Through this, it was found that the OsNFY26 promoter could induce expression specifically in seeds and could be involved in seed germination, dormancy and growth regulation.
Example
3: Transformation vector (
vector
) And transformant production
Example 3-1: OsNFY26 Production of Promoter Carrier
A recombinant expression vector was constructed to express the reporter gene GUS using the OsNFY26 gene promoter isolated in Example 2-1 .
Specifically, a recombinant vector in which the promoter was inserted was constructed in pBGWFS7 vector (PSB company, Plant System Biology) for expression of a gateway promoter (Fig. 4).
Example 3-2: Agrobacterium ( Agrobacterium tumefaciens ) Production of Transformants
The OsNFY26 promoter expression vector prepared in Example 3-1 was transformed into Agrobacterium tumefaciens (LBA4404) using the freeze-thaw method.
Specifically, YEP medium (10 g of Yeast, 5 g of NaCl, 10 g of peptone, 15 g of Agar, and 10 g of Agar) was added to the medium after transfection by freeze and thaw two to three times, followed by heat shock at 37 캜. 1 L) overnight to identify colonies. Colony transformed by colony PCR was transformed into 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, KCl 3g, CaCl 2 2H 2 O 0.265g, FeSO 4 .7H 2 O 50mg / 1L), Glucose 5g / 1L).
Example
4: Production and analysis of rice plant transformants using Agrobacterium
Example 4-1: Construction of transgenic rice plants using Agrobacterium
Agrobacterium LBA4404 into which the OsNFY26 promoter expression vector prepared in Example 3-2 was introduced was inoculated into callus of Dongjin-jinja and infected. Then, 6 쨉 g / ml of PPT (phosphinothricin, phosphinotricin) L, and finally the selected transformants were grown in a greenhouse to produce transformed rice.
Specifically, transformation of rice with Agrobacterium was carried out by washing the seeds in a lactose to induce the callus of Dongjinbyeong in 2N6 medium, drying it appropriately, and allowing the 2,4-
Example 4-2: OsNFY26 Analysis of promoter-inserted rice plant transformants
In order to confirm whether the OsNFY26 promoter expression vector was properly inserted into the transgenic rice produced using the Agrobacterium transformant into which the OsNFY26 promoter expression vector was introduced in Example 4-1, A genomic DNA of an OsNFY26 promoter-introduced rice plant transformant was isolated and subjected to PCR analysis.
Specifically, the genomic DNA was extracted from the leaves of transgenic rice plants into which the OsNFY26 promoter was inserted using an Inclone Genomic DNA prep kit, and a portion (GUSA- anti) and the promoter part (OsNFY26P-273F) were designed and synthesized. The primer sets GUSA-Anti: 5'-CGC GAT CCA GAC TGA ATG CCC-3 '(SEQ ID NO: 6) and OsNFY26P-273F: 5'-AAT CAT CCT ATA GTT GCT CCC C- No. 7) was used to perform PCR using the extracted genomic DNA as a template. At this time, the PCR was performed by heating the PCR reaction at 94 DEG C for 5 minutes and then heating the reaction mixture at 94 DEG C for 40 seconds, 55 DEG C for 40 seconds, and 72 DEG C for 40 seconds. Then, it was heated at 72 캜 for 7 minutes. After the PCR reaction, the amplified PCR products were 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. 5, in the wild type (WT) used as a control, the PCR amplification product was not observed in Dongjinbyeo, whereas in the transgenic rice transfected with the OsNFY26 promoter expression vector, I could confirm that it appeared. As a result, it was found that the OsNFY26 promoter expression vector was correctly inserted into the transgenic rice plants produced using the Agrobacterium transformant into which the OsNFY26 promoter expression vector was introduced.
Example 5: OsNFY26 Promoter GUS Analysis of Expression by Dyeing
In order to test the expression of the OsNFY26 promoter, the transgenic rice in which OsNFY26 promoter insertion was confirmed in Example 4-2 was analyzed by GUS protein staining. At this time, the site where the OsNFY26 promoter was activated was stained blue by GUS staining .
As a result of analysis of the expression of the promoter, it was confirmed that GUS staining was strongly stained in mature seeds as shown in Fig. 6A. In particular, it was confirmed that the embryo was hardly stained in the seeds, and that it was stained specifically in the endosperm and the aleurone layer.
On the other hand, as shown in Fig. 6B, GUS staining was not observed in other organs such as roots, stems, and leaves of transgenic rice seedlings in which the OsNFY26 promoter inserted 7 days after germination was inserted. Also, as shown in Fig. 6C, GUS staining was not observed in mature leaves and stems of transgenic rice grown for 2 months after germination. In addition, it was confirmed that GUS expression was not induced by external stimuli such as wound, ABA, abscisic acid hormone, and drought stress in the mature leaf slice (FIG. 6D). These results indicate that the OsNFY26 promoter induces specific expression in the endosperm of the seed and in the hornbone tissue.
Therefore, it has been proved that the OsNFY26 promoter can express the target gene mainly in the endosperm of the mature seed and in the phyllotaxis tissue in the representative monocotyledonous rice.
SmSpR: streptomycin / spectinomycin (Sm / Sp) resistance
LB: left T-DNA border
Bar: bialaphos resistance gene
attR1: gateway attR1
OsNFY26P: OsNFY26 ( Oryza sativa Nuclear Factor Y 26) promoter
attR2: gateway attR2
eGFP: enhanced green fluorescence protein
gus: beta-glucuronidase
T35S: CaMV 35S terminator
RB: right T-DNA border
<110> Republic of Korea <120> Endosperm-specific expression promoter derived from Oryza sativa and use thereof <130> P15R12D0376 <160> 7 <170> Kopatentin 2.0 <210> 1 <211> 2000 <212> DNA <213> Artificial Sequence <220> <223> OsNFY26 promoter sequence <400> 1 agaaggagca ctgtagcacg ggggagataa acagactttc ggccgggctg agaggaatgg 60 agggaggctt tggggcccaa agggaggagg gggagaaaga gagggggggg ggaagagagg 120 actttgggct gggcttggcc caaaggagga agaggattta tttttaggtt ttctttttaa 180 taaactttga taattgtttt tgttgcttaa taattatttc cggtgctctg aaaattcaag 240 taaaatttga gggctccttt tagaccaagg agaatttaac aaaaattctc cgggccacat 300 tcgaattttt ctggtacgta ttttagtgtt tgtcaatttc ttttcgaatt ttaattaatt 360 ctattattcc ttttagggaa tgatttttaa ttcgggatga atttatcagg acgtgacagt 420 ttcatattta atttgttaac ttagtagaga gtgaggggaa gttcaggaag gagtgccctg 480 aaatatcgga gttccttcga gaattttacg gaggagtgct ggaatgtcgg cattctacca 540 acggaggcta cggaggcgac agtgtaggcg cggcaaccag gaaggtgcag aaagagaggg 600 aaatatcctg gaatctgata caatgtagaa ggaattagag gaacaccaca cacccaatgt 660 tgtgggatga ccatgtattg ttatagtata gctaaggtac aagatcctca aatggaggag 720 aatacaagga atacaatgat acaatatgat ctatacatat atggttatac attcctggta 780 tgcattgcga caaacagagg aatagtgcat ctctctctgg tgtttttttg tttttaacga 840 tgatgtgggt gtgggtgagt tgggcttttt gcaggagttt agctgggctg ttggcaaggt 900 cttagaattg ggccagtact gagaaagctg ttaaaaaatt gaaagcgtgt ttttgaggta 960 gaagtgaatc cgacattttt tatttgatgc ttacgtggaa gactgtggtg caagatagaa 1020 aatatttaat gctaattagt aggctgcgat tgtatagtaa tagaggatta caaataaaac 1080 tcttgcgcca caattacaca gcaagattat cagcattttg gcttatccaa atagtcctta 1140 ctggaaacaa atataacata taacaaagac aaattaaagt aggaaagggg aaaatgcaat 1200 agttttatat ttcttttgtc ttataacaag gtttatcata tgacacaaac atttgaatta 1260 tagcacacta ctttattaac tatgttgtta cacaacacga catagataat cccctactat 1320 gcttaactta cagcggacta tccgtttccc gagccttcca gaaggctctt tctggcatgc 1380 aggcaaaatc catctgttag gatgtatcca tccattggta aggatgagct ggaagagtca 1440 tcaaagcatg gcacggaggg ttagatcacc aggggcgggt tgatgcaaaa aattagtatt 1500 ccgccattag atcaatgtac aagtgtctag atatgcagtg gtataagagg aaagatgcaa 1560 agaaggctaa caaaacacat gcgacaaacc cgcaaggaaa caattagaga caactaacct 1620 ttatactttt tttcatcaat taaagtgcat ctagttggca cccacaatat cttcactacc 1680 ttgtagttct atataaaccg gttttcctgg ttcccattct ctcatcaatc atcctatagt 1740 tgctccccta tttcagcatc tcaatttaag aaaggtcact catctctatt agtatgataa 1800 agttattcta attcctaagt agatgcatta ttagttattc tattttagtg tacatattgc 1860 tattatctaa gtttgcaaga tgagaaaatc tagtttctat tcaatttaga tatttttgtg 1920 gcgtgtaata acatgttgat tcgatgaagt acttaacaat gttctgttct tgccatactt 1980 caaaatatag gtgtgaaggc 2000 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> OsNFY26-forward primer <400> 2 gcacacaaca tctacacatg 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> OsNFY26-reverse primer <400> 3 ggcctcatca agagtacgaa 20 <210> 4 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> OsNFY26P-forward primer <400> 4 ctagtctaga atgccgatcc cggaaaagg 29 <210> 5 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> OsNFY26P-reverse primer <400> 5 acgcggatcc tcactcagga agatctttgt cag 33 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GUSA-Anti <400> 6 cgcgatccag actgaatgcc c 21 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> OsNFY26P-273F <400> 7 aatcatccta tagttgctcc cc 22
Claims (8)
b) As the recombinant expression vector, Agrobacterium tumefaciens ) to produce transformed Agrobacterium; And
c) infecting the plant with the transformed Agrobacterium;
Wherein the target protein is expressed specifically in the endosperm or the phloem tissue of rice seed mature seeds.
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| CN109097364B (en) * | 2018-09-03 | 2022-02-11 | 深圳广三系农业科技有限公司 | Identification and application of plant endosperm specific expression promoter pOsEnS100 |
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