KR101630837B1 - Antibiotic marker-free transgenic rice with TaGlu-Dy10 gene from Triticum asetivum - Google Patents

Abstract

TECHNICAL FIELD The present invention relates to a technique of transgenic plants, and specifically relates to rice plants transformed with TaGlu-Dy10, a glutenin gene of wheat.
The transgenic rice of the present invention has a property of accumulating a large amount of wheat glutenin protein in seeds, and thus it is possible to produce rice containing a large amount of glutenin, thereby improving the processing aptitude of rice, Commercialization of safe transgenic crops is possible by removing the selection marker gene.

Description

{Antibiotic marker-free transgenic rice with TaGlu-Dy10 gene from Triticum asetivum) with glutinin protein gene (TaGlu-Dy10) derived from wheat (Triticum asetivum)

The present invention relates to a method of transforming a plant, and more particularly, to a rice seed-specific recombinant expression vector comprising TaGlu-Dy10, a wheat glutenin gene, and a wheat-derived gene promoter, a rice transformed with the expression vector, .

Rice, one of the world's four major food crops, is one of the most economically viable crops in Asia, including Korea. However, the market size of domestic processed rice is estimated at around W1tn, It is only about 2%. Among the factors limiting the expansion of the rice processed food market are the poor quality of rice processed products compared to flour products and competitiveness in distribution. In other words, the processed food of rice does not have specific physical properties such as wheat glutenin and the like, and the processability is further complicated and the cost is increased because the processing aptitude such as dough, molding, and puffing is lower than that of flour. In addition, rice bran and rice cakes have a hardening hardening speed, so that their shelf life is shorter than that of flour products, and thus there is a difficulty in efficient inventory management. Therefore, although the research for improving the processing aptitude of rice has been continued, there is no solution to this problem.

On the other hand, in order to improve the yield and quality of rice, various breeding methods such as cross breeding, molecular breeding and transformation methods have been mobilized. Among these methods, The introduction of new genetically modified varieties has the advantage of being able to develop. In other words, the transformation technology in the agricultural field increases the useful components of crops such as isoflavone, vitamin A, and iron, as well as the yield related traits of crops such as resistance to various biological and non-biological stresses such as pests, droughts, This is a breeding method that can be utilized more directly and positively to improve the quality related traits that can be made.

However, most of the existing transgenic techniques are not suitable for the selection of cells, callus, and plant tissues with gene transfer, for the production of Basta herbicide resistant gene, hpt (hygromycin phosphotransferase) and NPT II (neomycin phosphotransferase) Herbicide and antibiotic resistance genes are used as selectable markers. The selection marker gene is inserted into the same vector together with the target gene, transformed into plant tissue, and then selected and cultured in a medium containing antibiotics such as phosphinotricin or hygromycin B to produce transgenic plants effectively. However, commercial consumers have a very negative view of transgenic crops and processed foods made of them as a result of concern about the risk of selection marker genes, making commercialization of transgenic crops very difficult.

Under these circumstances, the present inventors have made efforts to develop a plant that is safe from the harmfulness of the selection marker gene, while being able to produce rice containing glutenin in order to improve the processing aptitude of rice. As a result, The promoter of TaGlu-Bx7, which is a wheat-derived gene capable of enhancing the safety of the transgenic plant by removing the selection marker gene, and the TaGlu-Dy10 gene, which are capable of expressing the protein in rice seeds by transformation, So that the present invention has been completed.

The object of the present invention is to provide a process for the production of wheat ( Triticum specific promoter for expression of a rice seed-specific TaGlu-Dy10 gene, which comprises a glutinin gene TaGlu-Bx7 promoter and TaGlu-Dy10 gene derived from Aspergillus oryzae asetivum.

Another object of the present invention is to provide transgenic rice plants transformed with the recombinant expression vectors or seeds thereof.

Yet another object of the present invention is to provide a recombinant expression vector comprising: (a) preparing said recombinant expression vector; (b) transforming the recombinant expression vector into rice; And (c) selecting the transformed rice. The present invention also provides a method for producing a marker-free transgenic rice plant transformed with the TaGlu-Bx7 promoter and the TaGlu-Dy10 gene.

In one aspect of the present invention, there is provided a marker-free recombinant expression vector for expressing a rice seed-specific TaGlu-Dy10 gene comprising a wheat-derived glutenin gene TaGlu-Bx7 promoter and a TaGlu-Dy10 gene do.

Although there is no glutenin in rice, the processing ability of rice can be improved by transforming the TaGlu-Dy10 gene related to the milling suitability in rice with the TaGlu-Bx7 promoter in the present invention. In particular, the recombinant expression vector introduced into the transgenic rice of the present invention can express the gene specifically in rice seeds and at the same time, the antibiotic resistance selection marker gene can be removed, thereby enhancing the safety of the transgenic crop.

Specifically, the recombinant expression vector of the present invention can be obtained by introducing wheat-derived glutenin gene TaGlu-Bx7 promoter and TaGlu-Dy10 gene into a vector and removing the antibiotic resistance gene.

In the present invention, the term "wheat ( Triticum Asetivum "is a monocotyledonous plant distributed mainly in the temperate region. It is also called as wheat. It is the world's fourth largest food crop with rice and more than 90% can be milled to be used for noodles, baking, confectionery etc. Varieties such as Suwon 85, Suwon 86, Yeonggwang, Jangwang, Jeonggwon, Jinwol, Landscape, Landscape, Dwelling, Confectionery, Dahongmil, Cheongyeolmil, Grumil, Olmil, Suwon 215 are widely available But it is not limited thereto.

In the present invention, the term "glutenin" is a protein contained in wheat. It is one of the spherical proteins forming a ball shape due to the bridge formation caused by the interaction between side chains. It is a solution of water, neutral salt, dilute ethanol It does not dissolve and can be easily dissolved in dilute acid or dilute alkali solution. It can be a component of gluten, a relatively simple protein contained in grains, which is a protein mixture that greatly affects the texture of bread. Glutenin can be composed of a subunit of a polymer (96 to 136 kDa) and a low molecule (36 to 44 kDa). The high molecular glutenin subunit -A1 Glu, Glu and Glu -B1 - may be encrypted by the D1 gene, according to amino acid sequence can be divided into type x- and y- type. On the other hand, low molecular weight glutenin subunits is -A3 Glu, Glu and Glu -B3 - may be encoded by a gene D3.

The term "TaGlu-Dy10" in the present invention is one of the polymeric glutenin gene subunits present in wheat and is known as a gene related to the dough strength of wheat flour. Information on the gene can be obtained from a known database such as NCBI GenBank. For example, the accession number may be X12929.2, and its nucleotide sequence is shown in SEQ ID NO: 1, but the same information as TaGlu-Dy10 of the present invention As long as they have activity. The "gene" of the present invention may include DNA, RNA, and the like. DNA may include gemonic DNA, cDNA, etc., and RNA may include mRNA and the like. The gene of the present invention may include, but is not limited to, an open reading frame as well as a non-translated region (UTR) such as a TATA box or a poly-A tail sequence.

In the present invention, the term "promoter" is generally a gene site located upstream of an encoding site including a site for initiating transcription, and is usually a TATA box for regulating gene expression, a CAAT box site, And an enhancer which promotes the expression of almost all genes regardless of the position and direction. Preferably, the sequence of the TaGlu-Bx7 promoter may be the sequence of SEQ ID NO: 2, wherein TaGlu-Bx7 also corresponds to one of the polymeric glutenin gene subunits present in the wheat.

Specifically, in the present invention, a virus-derived CaMV35S promoter, which is most commonly used in the production of genetically modified plants (GMOs), has been generally used. In the present invention, a TaGlu-Bx7 promoter is introduced in place of the promoter Whereby transgenic rice that is specifically expressed in rice seeds can be obtained.

That is, among the promoters, there are a promoter that induces expression at all times in tissues and a promoter that induces expression in a time or position specific manner. Preferably, the promoter according to the present invention is a promoter that induces expression locally, Specifically, it may be a promoter of TaGlu-Bx7 specifically expressed in rice seeds. The term "rice seed-specific promoter" referred to in the present invention means a promoter that specifically expresses a gene whose expression is regulated by being linked to the 3 'position of the promoter in rice seeds. In plant tissues other than rice seeds, Which means that the expression of the gene is hardly achieved. That is, a seed-specific expression transformation vector of the TaGlu-Dy10 gene can be prepared by introducing a TaGlu-Bx7 promoter having characteristics of rice seed-specific expression into a recombinant expression vector.

The term "recombinant expression vector" in the present invention means a plasmid, virus or other medium known in the art into which a promoter of the present invention and a gene sequence encoding the target protein operably linked to the promoter can be inserted or introduced . Specifically, the expression promoter of the wheat glutenin gene TaGlu-Dy10 and the TaGlu-Dy10 gene sequence encoding the wheat glutenin protein operably linked to the promoter may be operably linked to an expression control sequence, Linked gene sequences and expression control sequences can be contained within a recombinant expression vector containing both a selection marker and a replication origin. As used herein, the term "operably linked" means that the appropriate molecule is linked in such a way as to enable gene expression when bound to expression control sequences. The term "expression control sequence" also refers to a DNA sequence that regulates the expression of a polynucleotide sequence operably linked to a particular host cell. The regulatory sequence may include a promoter to effect transcription, an optional operator sequence to regulate transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence to control the termination of transcription and translation.

The recombinant expression vector of the present invention can be obtained by introducing a promoter of TaGlu-Bx7 of the present invention using a conventional vector used for protein expression as a basic framework and encoding a base sequence encoding TaGlu-Dy10 downstream of the promoter Or by insertion. Therefore, the vectors that can be used in the present invention include plant virus vectors, and specific examples include binary vectors such as pCHF3, pPZP, pGA, pCAMBIA, and pBTEX series. However, those skilled in the art can use any vector as long as it can introduce the promoter of the present invention and the nucleotide sequence encoding the target protein operably linked to the promoter into the host cell. Preferably, the pBTEX vector can be used. In the present invention, the MCS part in the pCAMBIA1300 vector is treated with restriction enzymes EcoRI and HindIII to remove the MCS part, and the 35S promoter and the restriction enzyme site and the OPC supplier (Octopine synthase terminator ) Can be inserted.

On the other hand, the antibiotic resistance gene and / or its promoter existing in the expression vector is removed, and the remaining part is ligated to produce the marker-free vector without the antibiotic resistance gene and / or its promoter.

The term "marker-free" in the present invention can mean, but not be limited to, the absence of an antibiotic resistance gene or the removal of an antibiotic resistance gene. The term antibiotic is a metabolite produced by a microorganism or the like, and refers to a substance that suppresses or kills the growth of other microorganisms in a small amount. Examples of the antibiotics include penicillins, cephalosporins, aminoglycosides, tetracyclines, chloramphenicol, polypeptides, quinolones and the like, preferably aminoglycosides, and more preferably, But is not limited thereto.

The recombinant expression vector of the present invention does not contain an antibiotic resistance gene and its promoter. When transgenic plants are transfected with foreign genes, selectable markers can be used so that only the foreign gene-introduced cells can be selected without analyzing each gene product. Generally, antibiotic resistance The gene can be selected as a selection marker and cultured in an antibiotic-containing medium so that only the transformant can be selected. However, in order to enhance biosafety, in order to provide a marker-free transformed rice in which the antibiotic resistance gene was removed, the antibiotic resistance gene and its promoter were removed from the recombinant expression vector.

In one embodiment of the present invention, the pBTEX vector was treated with restriction enzymes EcoRI and KpnI to remove the CaMV35S promoter and insert the promoter of TaGlu-Bx7, which is a glutenin gene derived from wheat, and then treated with restriction enzymes XhoI and EcoRI The hygromycin resistance gene (HPT II) and the hygromycin expression promoter were removed and the DNA exonuclease was treated to remove Xho I and EcoRI (SEQ ID NO: 2) residues at the two ends of the truncated vector (N-terminal, C-terminal) The marker-free vector was prepared by blunt-ending the site and then self-ligation at both ends. Then, a seed-specific expression transformation vector of the wheat TaGlu-Dy10 gene was prepared by inserting the TaGlu-Dy10 gene into the marker-free vector in accordance with the restriction enzymes KpnI and XbaI (Examples 1-1 to 1-3) .

In another aspect, the present invention provides transgenic rice plants transformed with the above recombinant expression vectors or seeds thereof.

The term "transformation" as used herein in the context of the present invention may generally refer to the introduction of DNA into a host, such that the DNA is replicable as an extrachromosomal element or by chromosomal integration. The transformation can be carried out by selecting a suitable standard technique depending on the host cell as known in the art, and preferably, a plant pathogen Agrobacterium can be used, but is not limited thereto. The Agrobacterium has the ability to transduce some of its Ti (tumor-inducing) or Ri (root-inducing) plasmid DNA (T-DNA) into infected plant cells. Therefore, when the target gene is recombined in T-DNA and co-cultured with the bacteria and the plant having the gene, the transformation of the plant cell becomes possible.

In the present invention, the term "rice (Oryza sativa) Is a perennial herbaceous crop of the unicorn plant lime blossom, and the fruit is called "rice." About 40% of the world's population is made up of rice as its main food source. It is well known that the plant is actually grown in rice (O. sativa). As the transgenic object of the present invention, there may be mentioned pung oak, water, palpop, palm, promotion, reconstruction, palm, rice paddy, nakdong, gwanak, pearl, It could be Dong Jinbin.

The transgenic rice of the present invention may be a rice transgenic cell in which the wheat glutenin TaGlu-Dy10 gene is expressed or can be expressed in the cell. In addition, the transgenic rice of the present invention may include all or part of rice plants including transgenic cells into which the gene is introduced, and may be obtained by ovarian reproduction or asexual reproduction of the plant into which the gene is introduced Seeds, plants and parts of tissues or organs thereof. Preferably, the transgenic rice of the present invention is the transgenic rice or its seed, which is the accession number KACC98037P, but is not limited thereto.

In one embodiment of the present invention, Agrobacterium-mediated co-transformation was used to introduce the recombinant vector, specifically Agrobacterium containing the marker-free vector and only the vector Agrobacterium was inoculated simultaneously to the calli at a ratio of 3: 1 (Example 2).

In another embodiment, the present invention provides a method for producing a recombinant expression vector, comprising: (a) preparing the recombinant expression vector; (b) transforming the recombinant expression vector into rice; And (c) selecting the transgenic rice, wherein the TaGlu-Bx7 promoter and TaGlu-Dy10 gene are transformed with the antibiotic marker-free transgenic rice.

The step (a) of the present invention is the step of preparing the recombinant expression vector, as described above.

The step (b) of the present invention is a step of transforming the recombinant expression vector into rice, and more particularly, an antibiotic marker-free expression vector comprising the TaGlu-Bx7 promoter and TaGlu-Dy10 gene prepared by the step (a) Is introduced into the Agrobacterium strain, and the expression vector, Agrobacterium and the transformation using the expression vector are as described above. In step (b) of the present invention, a variety of Agrobacterium such as LBA4404, AGL1 and EHA105 may be used without limitation for the production of transgenic rice, and the conventional methods for introducing Agrobacterium can also be adopted.

In one embodiment of the present invention, Agrobacterium containing only the marker-free vector and Agrobacterium containing only the empty vector were inoculated simultaneously to the calli at a ratio of 3: 1 (Example 3).

The step (c) of the present invention is a step of selecting the transgenic rice, and specifically refers to a step of selecting transgenic rice by culturing Agrobacterium having the recombinant expression vector introduced therein with rice or rice tissue.

More specifically, the Agrobacterium containing the expression vector prepared in the step (a) may be transformed into a tissue of rice by culturing the Agrobacterium with the tissue of rice or rice. At that time, But is not limited thereto. Thereafter, the transformed rice can be selected using the antibiotic-containing medium and the target gene-specific primer.

In one embodiment of the present invention, a medium containing antibiotics (hygromycin) was first used to select the rice plants transformed with TaGlu-Dy10 (Example 3-1), and then the first selected (Example 3-2). The primers specific for the TaGlu-Dy10 gene were used for the secondary selection through PCR (polymerase chain reaction).

The transgenic rice of the present invention has a property of accumulating a large amount of wheat glutenin protein in seeds, and thus it is possible to produce rice containing a large amount of glutenin, thereby improving the processing aptitude of rice, Commercialization of safe transgenic crops is possible by removing the selection marker gene.

1A is a diagram showing a cleavage map of the pCAMBIA1300.
1B shows an MCS portion of pCAMBIA1300.
1C is a diagram showing a gene expression portion of a pBTEX vector prepared by modifying pCAMBIA1300.
2A is a schematic diagram of a promoter replacement and TaGlu-Dy10 gene insertion marker-free transformation vector.
2B is a schematic diagram of a transformed vector into which a hygromycin gene is inserted.
FIG. 3 is a photograph showing the process from selection of rice callus organism to selection, regeneration and positive transformation of transgenic rice (T ₀ ).
FIG. 4 is a diagram showing electrophoresis results in a second selection process using PCR of TaGlu-Dy10 transgenic plants. FIG.
Fig. 5 shows the expression of the TaGlu-Dy10 gene in the seed.
FIG. 6 is a diagram showing the number of genes inserted into transgenic plants (T ₀ ) of 29 individuals by secondary selection.
FIG. 7 is a graph showing the expression amount of glutenin protein in the selected transformed seed (T ₁ ).
FIG. 8 is a diagram showing the separation of genes of TaGlu-Dy10 and HPTII to select marker-free transgenic rice through generational development.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the invention.

Example  1: Replacement of gene expression promoter and Marker-free  Vector production

Example  1-1: Mill Glutenin  Replacement with a gene promoter

For better expression of the wheat glutenin gene TaGlu-Dy10, the CaMV35S promoter was replaced with a wheat-derived TaGlu-Bx7 promoter.

Specifically, the pBTEX vector modified in pCAMBIA1300 was used. The pBTEX vector was obtained by treating the MCS portion in the pCAMBIA1300 vector with the restriction enzymes EcoRI and HindIII to remove the MCS portion and newly introducing the 35S promoter, the restriction enzyme site and the OCS provider (Octopine synthase terminator) was inserted to construct a pBTEX vector (Figs. 1A to 1C).

The CaMV35S promoter was removed by treating the pBTEX vector with 1 U (restriction enzyme, EcoRI and KpnI (New England Biolab, UK) each in an amount of 1 U (at which 1 U of DNA was cleaved at 37 캜) The TaGlu-Bx7 gene promoter was inserted.

Example  1-2: Marker-free  Manufacture of vector

In order to improve the safety of transgenic rice plants, antibiotic resistant genes and their promoter - free marker - free vectors were prepared.

Specifically, the vector in which the promoter was replaced according to the above-mentioned Example 1-1 was treated with 1 U of restriction enzymes XhoI and EcoRI (New England Biolab, UK), respectively, to obtain a hygromycin resistance gene (HPTII) and a hygromycin expression promoter . Thereafter, XhoI and EcoRI sites remaining in the truncated vector N-terminal and C-terminal regions were treated with a DNA exonuclease (TAKARA, Japan) Were self-ligated to each other.

As a result, a marker-free vector in which the hygromycin resistance gene (HPTII) gene and the hygromycin expression promoter were removed was prepared.

Example  1-3: Wheat-derived TaGlu - Dy10  Preparation of vector for gene transformation

To produce transgenic rice plants containing the TaGlu-Dy10 gene derived from wheat and the TaGlu-Bx7 promoter, recombinant vectors for transformation were prepared.

Specifically, TaGlu-Dy10 gene was amplified by attaching cleavage sites of restriction enzymes KpnI and XbaI (New England Biolab, UK) to both ends from wheat Seri82 varieties. Then, the amplified gene was cloned into pGEM T easy (Promega) vector.

forward primer reverse primer TAGlu-Dy10 5'-AGGGTACCGAGATGGCTAAGCGCCTGG-3 '
(SEQ ID NO: 3) 5'-GATCTAGATCACTGCCTGGTCGACAATG-3 '
(SEQ ID NO: 4)

The forward primer of Table 1 contains the KpnI site and the reverse primer contains the XbaI site.

Thereafter, seed-specific expression transformation of the wheat TaGlu-Dy10 gene was performed by inserting the gene into the marker-free vector prepared in Example 1-2 in accordance with the positions of restriction enzymes KpnI and XbaI (New England Biolab, UK) Vector.

The schematic diagram of the vector for transformation of the TaGlu-Dy10 gene derived from wheat as described above is as shown in Fig. 2A.

Example  2: wheat-derived TaGlu - Dy10  Transformation of genes

Using the transfection vector prepared in Example 1, transgenic rice plants were produced by transfecting rice plants, which are heterogeneous plants that do not express wheat glutenin.

Specifically, in order to introduce the recombinant vector, Agrobacterium-mediated co-transformation was used.

A blank vector consisting of the marker-free vector and the hygromycin (HPTII) resistance gene prepared in Example 1 alone was introduced into Agrobacterium EHA105 independently, and the marker-free vector containing the TaGlu-Dy10 gene inserted therein Agrobacterium with a vector and a ball vector was grown in YEP media containing kanamycin antibiotics at 28 ° C for 3 days. Dongjinbyeon callus was cultivated in Dongjinbyeon brown rice with callus organic medium (N6 salts with vitamins, 2.5 g / ℓ proline, 2 mg / ℓ 2,4-D, 0.5 g / ℓ casamino acid, 30 g / ℓ sucrose and 2 g / pH 5.7) and incubated at 25 ° C for 21 days in a dark state.

Agrobacterium grown for 3 days was suspended in suspension medium (N6 salts with vitamins, 2 mg / l 2,4-D, 0.5 g / l casamino acid, 30 g / l sucrose, and 10% g / ℓ glucose, pH 5.7) to TaGlu-Dy10 gene is inserted, marker-free vector and hygromycin (HPTⅡ) the Agrobacterium concentration, respectively of 0.01 containing the empty vector composed only of resistance genes diluted in (OD ₆₀₀ = 0.01) (N6 salts with vitamins, 2 mg / ℓ 2,4-D, 0.5 g / ℓ casaminoacid, 30 g / ℓ) were inoculated into rice callus and mixed with each EHA105 Agrobacterium in a ratio of 3: The co-cultivation of rice callus and Agrobacterium was carried out for 3 days in a final concentration of 10 μg / l sucrose, 10 g / l glucose, and 2 g / l gelrite, pH 5.2 with 200 μM acetosyringone as a final concentration. After 3 days, the callus was washed with medium containing 250 mg / l cefotaxime and 150 mg / l timentin, and the embryos were placed on the selection medium.

Example  3: Transgenic rice ( T ₀ ) Selection

Example  3-1: First selection with antibiotic-containing medium

A medium containing an antibiotic (hygromycin) was used in order to select the rice in which the TaGlu-Dy10 gene was actually transformed in the rice from the rice to be transformed in the above Example 2.

Specifically, transgenic rice plants were selected by culturing the rice calli inoculated with Agrobacterium in a medium containing hygromycin, an antibiotic. Cultured in a selective medium containing 2 mg / l 2,4-D, 50 mg / l hygromycin and 250 mg / l cell cefotaxime for 4 weeks to induce hygromycin-resistant callus in a dark condition The selected and selected calli were regenerated in regeneration medium in light regeneration medium (NAA 0.1 mg / ℓ, Kinetin 2 mg / ℓ, Cefotaxime 250 mg / ℓ, 50 mg / ℓ Hygromycin) for 3-4 weeks.

As a result, the total number of plants transformed with TaGlu-Dy10 was 290. This corresponds to the number of plants having both a ball vector having only a ball vector carrying hygromycin and a marker vector and a marker free vector.

Example  3-2: Glutenin  Gene-specific Primer  2nd selection using

In order to select only the plants containing the TaGlu-Dy10 gene and the co-vector, the TaGlu-Dy10 gene-specific primers were used to remove the plants having only the empty vector among the 290 individuals selected from the first selection, The rice containing the wheat glutenin gene was secondarily selected through polymerase chain reaction (PCR).

For extraction of genomic DNA, 0.2 g of the nursery leaves selected in the regeneration medium were placed in a mortar and shaken with liquid nitrogen, and then transferred to QuickPap buffer (200 mM Tris-HCl, 250 mM NaCl, 25 mM EDTA, 0.5% SDS), and reacted at 60 ° C for 1 hour. After centrifugation at 10,000 × g for 10 minutes, 400 μl of the supernatant was placed in a new tube, 300 μl of isopropanol was added, mixed well, and reacted at -80 ° C. for 30 minutes. The supernatant was removed by centrifugation at 20,000 × g for 10 minutes, and 70% ethanol was added, followed by gently rinsing and then drying for 30 minutes. 150 μl of DNA extraction buffer (10 mM NH ₄ OAC, 0.25 mM EDTA) was added and mixed well. After reacting at 60 ° C for 1 hour, the mixture was centrifuged at 10,000 × g for 10 minutes, and 100 μl of the supernatant was placed in a new tube. Experiments related to the present invention were carried out using DNA thus extracted.

GeneAmp system 9700 (Applied Biosystems USA) was used for the PCR, and 50 ng of genomic DNA of each strain was used for amplification, and 30 μl of each primer (100 μM of each primer, 200 μM At 94.5 ° C for 5 min, at 94 ° C for 1 min, at 56 ° C for 1 min in conditions of each dNTPs, 10 mM Tris-HCl pH 9.0, 2 mM MgCl ₂ , 50 mM KCl, 0.1% Triton X- , 72 ° C for 1 minute and 30 seconds, followed by 35 cycles of reaction. Finally, reaction was carried out at 72 ° C for 10 minutes. The amplified product was confirmed by UV with 0.5% EtBr / ml in 1% agarose gel.

forward primer reverse primer Dy10 5'-CGCAAGACAATATGAGCAAAC-3 '
(SEQ ID NO: 5) Gt;
(SEQ ID NO: 6) HPTⅡ 5'-CGCTTCTGCGGGCGATTT-3 '
(SEQ ID NO: 7) 5'-CCCATTCGGACCGCAAGGA-3 '
(SEQ ID NO: 8)

As a result, the total number of plants transformed with TaGlu-Dy10 was 29.

Example  4: Seed ( T _One ) Confirm my gene expression

In order to test whether the transgenic rice of the wheat TaGlu-Dy10 gene selected in Example 3 can be inherited up to the next generation, the stable expression of the TaGlu-Dy10 gene in the transgenic plant T ₁ seed was confirmed.

Specifically, selection was made through gene insertion number and phenotypic analysis of transgenic plants (T ₀ ) in each gene group, and the amount of protein expression in the selected transformed T ₁ seeds was determined by Western blotting (using x-type specific antibody) Respectively.

Specifically, the rice seeds 20 were ripped together with nitrogen and the total proteins were dissolved in extraction buffer (0.1 ml of 50% (v / v) 1-propanol, 0.05 M Tris-HCl, pH 8.0, containing 1% DTT) After separation (13,000 g), the supernatant was quantitated and 40 g of total protein was electrophoresed on 10% acrylamide gel. The electrophoretic gel was transferred to PVDF membrane, treated with primary antibody and secondary antibody, and exposed to a film (AGFA, Belglum) using ECL kit (GE healthcare, USA).

As described above, the amount of protein expression according to the number of gene insertions was analyzed, and finally 18 lines in which the protein expression was confirmed were selected.

The expression level of the wheat glutenin TaGlu-Dy10 protein in the transgenic rice plants of the 18 lines selected finally is shown in Fig. As shown in FIG. 7, it was confirmed that wheat glutenin TaGlu-Dy10 protein was expressed in most of the transgenic rice plants. Especially, in the case of # 13, # 16, # 29, # 21, # 19, # 23 and # 25 It was confirmed that the expression of protein in transgenic rice was remarkable.

8, marker-free transgenic rice plants were selected through generation progression in line # 23 as shown in FIG. 8, and as a result, These marker-free transgenic rice plants were selected by confirming that no antibiotic resistance gene (HPTII) bands were formed in lines 4, 7, 10, 14, 15 and 21.

Thus, the present inventors named TaGlu-Dy10 as the # 23 transgenic rice seed which expresses most of the wheat glutenin Dy10 protein without forming the antibiotic resistance gene (HPTII) band as described above, And deposited with the National Institute of Agricultural Science and Technology, RDA, under accession number KACC98037P.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are illustrative in all aspects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the spirit or scope of the present invention as defined by the appended claims.

National Institute of Agricultural Science KACC98037P 20140410

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Antibiotic marker-free transgenic rice with TaGlu-Dy10 gene from          Triticum acetine <130> KPA140348-KR <160> 8 <170> Kopatentin 2.0 <210> 1 <211> 1947 <212> DNA <213> Triticum acetylum Glu-Dy10 <400> 1 atggctaagc ggctggtcct ctttgcggca gtagtcatcg ccctcgtggc tctcaccact 60 gctgaaggtg aggcctctag gcaactacag tgtgagcgcg agctccagga gagctcgctt 120 gaggcatgcc ggcaggttgt ggaccaacag ttggccggtc ggctgccatg gagcacgggg 180 ctccagatgc gatgctgcca gcagctccga gatgttagcg ccaagtgccg ctccgtcgcc 240 gtcagccaag tcgcaagaca atatgagcaa actgtggtgc cgcccaaggg cggatccttc 300 taccctggtg agaccacgcc actgcagcaa ctccaacaag gaatattttg gggaacatct 360 tcacaaacag tacaagggta ttacccaggc gtaacttctc ctcggcaggg gtcatattat 420 ccaggccaag cttctccaca acagccagga caagggcaac agcctggcaa atggcaagaa 480 ccaggacaag ggcaacaatg gtactaccca acttctttgc agcagccagg acaagggcaa 540 cagataggaa aagggcaaca agggtactac ccaacttctc tgcagcagcc aggacaaggg 600 caacaagggt actacccaac ttctctgcag cacacaggac aaaggcaaca accagtacaa 660 gggcaacaac cagaacaagg gcaacaacca ggacaatggc aacaagggta ctatccaact 720 tctccacaac agctaggaca agggcaacaa ccaagacaat ggcaacaatc aggacaaggg 780 caacaagggc actacccaac ttctctacaa cagccaggac aagggcaaca agggcattac 840 ctagcttctc agcagcagcc aggacaaggg caacaagggc actacccagc ttctcagcag 900 cagccaggac aagggcaaca agggcactac ccagcttctc agcagcagcc aggacaaggg 960 caacaagggc actacccagc ttctcagcaa gagccaggac aagggcaaca agggcaaatc 1020 ccagcttctc agcagcagcc aggacaaggg caacaagggc actacccagc ttctctgcag 1080 caaccaggac aagggcaaca agggcattac ccaacttctc tacagcagct aggacaaggg 1140 caacaaacag gacagccagg acaaaagcaa caaccaggac aagggcaaca aacaggacaa 1200 gggcaacagc cagaacaaga gcaacaacca ggacaagggc aacaaggata ctatccaact 1260 tctctgcagc agccaggaca agggcaacag caaggacaag ggcaacaagg gtactaccca 1320 acttctctcc agcagccagg acaagggcaa caagggcact acccagcttc tctgcagcag 1380 ccaggacaag gacagccagg acaaaggcaa caaccaggac aagggcaaca tccagaacaa 1440 gggaaacaac caggacaagg gcaacaaggg tactatccaa cttctccaca gcagccagga 1500 caagggcaac aactaggaca agggcaacaa gggtactacc caacttctcc gcagcagcca 1560 ggacaagggc aacaaccagg acaagggcaa caagggcact gcccaacgtc cccgcagcag 1620 tcaggacaag cgcaacaacc aggacaaggc caacaaatag gacaagtgca acaaccagga 1680 caagggcaac aagggtacta cccaacttct gtgcagcagc ctggacaagg gcaacaatca 1740 ggacaagggc aacagtcagg acaaggacac caaccaggac aagggcagca atcaggacaa 1800 gagcaacaag gctacgacag cccataccat gttagcgcag agcagcaagc ggccagccca 1860 atggtggcaa aggcgcagca gcccgcgaca cagctgccga cagtgtgtcg gatggagggg 1920 ggcgacgcat tgtcggctag ccagtga 1947 <210> 2 <211> 1358 <212> DNA <213> Triticum asetivum Glu-Bx7 promoter <400> 2 ctgcccagca aagcgcgctc aactcttcta gtctaaataa ctagcatcca ctaacacatt 60 tctcccgaca tgcaagcacg tcacctatga aaatgcccac ctcaatatgc aaccatgcat 120 agaagaaagc tcacctcagc atgcaaacat gcagcataat ttccatttta cttggctatt 180 tatgtttgat aaatatttca caaatataca ataatcaaaa acaataaatt atatgtgttt 240 ttagttttag ttctcatatc caaatataca tgtttcatac aaccaaatct catttaaata 300 tattgtaaaa tattccggca acaacttgtg gggtacatct agttacagtg gaatattagt 360 gatggcgtga gcaagcgata aggccaacga gagaagaagt gcgtcgtcta tggaggccag 420 ggaaagacaa tggacatgca aagaggtagg ggcagggaag aaacacttgg agatcataga 480 agaacataag aggttaaaca taggagggca taatggacaa ttaaatctac attaattgaa 540 ctcatttggg aagtaaacaa aatccatatt ctggtgtaaa tcaaactatt tgacgcggat 600 ttactaagat cctatgttaa ttttagacat gactggccaa aggtttcagt tagttcattt 660 gtcacggaaa ggtgttttca taagtccaaa actctaccaa cttttttgca cgtcatagca 720 tagatagatg ttgtgagtca ttggatagat attgtgagtc agcatggatt tgtgttgcct 780 ggaaatccaa ctaaatgaca agcatcaaaa cctgaaatgg gctttaggag agatggttta 840 tcaatttaca tgttccatgc aggctacctt ccactactcg acatggttag aagttttgag 900 tgccgcatat ttgcggaagc aatggcacta ctcgacatgg ttagaagttt tgagtgccgc 960 atatttgcgg aagcaatggc taacagatac atattctgcc aaaccccaag agggataatc 1020 actcctctta gataaaaaga acagaccaat gtacaaacat ccacacttct gcaaacaata 1080 caccagaact aggattaagc ccattacgtg gctttagcag accgtccaaa aatctgtttt 1140 gcaagcacca attgctcctt acttatccag cttcttttgt gttggcaaac tgcccttttc 1200 caaccgattt tgttcttctc acgctttctt cataggctaa actaacctcg gcgtgcacac 1260 aaccatgtcc tgaaccttca cctcgtccct ataaaagccc atccaacctt cacaatctca 1320 tcatcaccca caacaccgag caccccaatc tacagatc 1358 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> TaGlu-Dy10 forward primer <400> 3 agggtaccga gatggctaag cggctgg 27 <210> 4 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> TaGlu-Dy10 reverse primer <400> 4 gatctagatc actggctagc cgacaatg 28 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Dy10 forward primer <400> 5 cgcaagacaa tatgagcaaa c 21 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Dy10 reverse primer <400> 6 gttgcctttg tcctgtgtgc t 21 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> HPTII forward primer <400> 7 cgcttctgcg ggcgattt 18 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> HPTII reverse primer <400> 8 cccattcgga ccgcaagga 19

Claims (10)

A marker gene for expressing a rice seed-specific TaGlu-Dy10 gene comprising a Taqlu-Bx7 promoter derived from Triticum asetivum having a nucleotide sequence of SEQ ID NO: 2 and a TaGlu-Dy10 gene comprising a nucleotide sequence of SEQ ID NO: Transformed seed deposited with Accession No. KACC98037P, containing a recombinant expression vector.
delete delete 3. The transformed seed according to claim 1, wherein the expression vector is free of an antibiotic resistance gene and its promoter, deposited at Accession No. KACC98037P.
5. The transformed seed according to claim 4, wherein the antibiotic is hygromycin, deposited with Accession No. KACC98037P.
delete 2. The transformed seed of claim 1, wherein said transformation is mediated by Agrobacterium.
The transformed seed deposited with the deposit No. KACC98037P according to claim 1, wherein the rice is Dongjin rice.
Transgenic rice germinated with the transformed seed deposited at accession number KACC98037P of any one of claims 1, 4, 5, 7 and 8.
(a) Expression of a rice seed-specific TaGlu-Dy10 gene comprising the glutinin gene TaGlu-Bx7 promoter derived from Triticum asetivum consisting of the nucleotide sequence of SEQ ID NO: 2 and the TaGlu-Dy10 gene consisting of the nucleotide sequence of SEQ ID NO: Producing a marker-free recombinant expression vector;
(b) transforming the recombinant expression vector into rice; And
(c) selecting said transgenic rice plants.

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