KR101560745B1 - Antibiotic marker-free transgenic rice with TaGlu-Bx7 gene from Triticum asetivum - Google Patents

Abstract

TECHNICAL FIELD The present invention relates to a technique of transgenic plants, and specifically relates to a rice plant transformed with TaGlu-Bx7, 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-Bx7 gene from Triticum asetivum transformed with TaGlu-Bx7 gene derived from Triticum asetivum}

The present invention relates to a method of transgenic plant transformation, specifically to a rice seed-specific recombinant expression vector comprising TaGlu-Bx7, which is a wheat glutenin gene, and its own promoter, a rice transformed with the above expression vector, will be.

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 to improve the processing aptitude of rice has been continued, until now, no solution has been found so far.

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 intensive efforts to develop a plant that is safe from the harmfulness of the selection marker gene, while being able to produce rice containing a large amount of glutenine in order to improve the processing aptitude of rice. As a result, , And the TaGlu-Bx7 self-promoter and the TaGlu-Bx7 gene, which are capable of promoting the stability of the transgenic plant by removing the selection marker gene in this process, can be introduced into rice seeds by transfection 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 rice seed-specific TaGlu-Bx7 gene comprising TaGlu-Bx7 self-promoter and TaGlu-Bx7 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 TaGlu-Bx7 self-promoter and TaGlu-Bx7 gene.

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

Although there is no glutenin in rice, in the present invention, the processing suitability of rice can be improved by transforming the TaGlu-Bx7 gene involved in the milling suitability into rice with the TaGlu-Bx7 own promoter. 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 TaGlu-Bx7 self-promoter and TaGlu-Bx7 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, also called wheat. It is the world's fourth largest food crop with rice, and more than 90% of it is milled and can be used for noodles, baking, and confectionery. Among domestic varieties, varieties such as Suwon 85, Suwon 86, Yeongwang, Janggwang, Gwangwang, Jinpung, Landscape, Gwanghwamyeong, 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 polymeric glutenin subunit can be encoded by the Glu-A1, Glu-B1 and Glu-D1 genes, and can be classified into x-type and y-type according to the amino acid sequence. On the other hand, the low molecular weight glutenin subunit can be encoded by the Glu-A3, Glu-B3 and Glu-D3 genes.

In the present invention, the term " TaGlu-Bx7 "is one of polymeric glutenin gene subunits present in wheat and is known as a gene related to the dough strength of wheat flour. Preferably, the TaGlu- The "gene" of the present invention may include DNA, RNA, etc. The DNA may include gemonic DNA, cDNA, etc., and the RNA may include, but is not limited to, mRNA, etc. The gene of the present invention may include not only an open reading frame but also a non-translated region (UTR) such as a TATA box or a poly-A tail sequence. It is not limited.

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.

Specifically, virus-derived CaMV35S promoter, which is most commonly used in the production of genetically modified plants (GMOs), has been generally used in the context of the present invention. In the present invention, TaGlu-Bx7 self-promoter To thereby obtain transgenic rice that is specifically expressed in rice seeds.

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 itself expressed specifically 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-Bx7 gene can be prepared by introducing the TaGlu-Bx7 promoter having the characteristic of rice seed specific expression together into the 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-Bx7 and the TaGlu-Bx7 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 according to the present invention can be produced by introducing a promoter of TaGlu-Bx7 of the present invention into a promoter of the present invention using the existing vector used for protein expression as a basic framework and amplifying the base sequence encoding TaGlu-Bx7 toward the downstream of the promoter As shown in FIG. 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 the wheat-derived TaGlu-Bx7 gene itself, followed by treatment with restriction enzymes XhoI and EcoRI, The XhoI and EcoRI sites remaining in the truncated vector terminal (N-terminal, C-terminal) were deleted by treating the DNA exonuclease with removal of the hygromycin expression promoter and the hygromycin expression promoter The marker-free vector was prepared by blunt-ending and then self-ligation of both ends. The marker free vector was then prepared. Then, a TaGlu-Bx7 gene was inserted into the marker-free vector in accordance with the positions of the restriction enzymes KpnI and XbaI to prepare a seed-specific expression transformation vector for the wheat TaGlu-Bx7 gene (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 an annual crop of an annual plant of the alopecia plantus. The fruit is called rice, and about 40% of the world's population is made up of rice. More than 20 species are known to belong to the rice plant, but most of them are cultivated 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 transgenic cell of rice in which the wheat glutenin TaGlu-Bx7 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 KACC98025P, 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. The present invention also provides a method for producing an antibiotic marker-free transgenic rice transgenic plant transformed with TaGlu-Bx7 self-promoter and TaGlu-Bx7 gene.

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. Specifically, the step (b) of the present invention is a step of transforming the recombinant expression vector into rice, which comprises the TaGlu-Bx7 self-promoter and the TaGlu- And introducing the vector into an Agrobacterium strain, and the expression vector, Agrobacterium, and 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 rice plants transformed with TaGlu-Bx7 (Example 3-1), and then the first selected The primers specific to the TaGlu-Bx7 gene were used for secondary selection (Example 3-2) by polymerase chain reaction (PCR).

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-Bx7 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 a TaGlu-Bx7 transgenic plant. FIG.
5 shows the expression of the TaGlu-Bx7 gene in the seed.
FIG. 6 is a diagram showing the number of genes inserted into transgenic plants (T ₀ ) of 24 individuals of the genetic group that have undergone the second 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-Bx7 and HPT II to select marker-free transgenic rice through generational evolution.

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 self-promoter

To better express the wheglutenin gene TaGlu-Bx7, the CaMV35S promoter was replaced with the promoter of the wheat-derived TaGlu-Bx7 gene itself.

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 Example 1-1 was treated with restriction enzymes XhoI and EcoRI to remove the hygromycin resistance gene (HPTII) and the hygromycin expression promoter. The DNA exonuclease was then treated to make the XhoI and EcoRI sites at the two ends of the truncated vector (N-terminal, C-terminal) to be blunt ends, (self-ligation).

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 - Bx7  Preparation of vector for gene transformation

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

Specifically, TaGlu-Bx7 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 Bx7 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-Bx7 gene was carried out 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-Bx7 gene derived from wheat as described above is as shown in Fig. 2A.

Example  2: wheat-derived TaGlu - Bx7  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.

Blank vectors consisting of only the TaGlu-Bx7 gene-inserted marker-free vector and the hygromycin (HPTII) -resistant gene prepared in Example 1 were independently introduced into Agrobacterium EHA105, and the Taqlu-Bx7 gene inserted marker- 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% (OD600 = 0.01), respectively, containing a co-vector consisting of a marker-free vector in which a TaGlu-Bx7 gene was inserted and a hygromycin (HPTII) Each of the EHA105 Agrobacterium was mixed with a callus of EHA105 Agrobacterium in a ratio of 3: 1, and seeded in calli callus and cultured in co-culture medium (N6 salts with vitamins, 2 mg / ℓ 2,4-D, 0.5 g / ℓ casaminoacid, 30 g / The cells were co-cultured with rice callus and Agrobacterium for 3 days in 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 to select the rice in which the TaGlu-Bx7 gene was actually transformed in the rice of the above-mentioned Example 2 to be transformed.

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 the plants transformed with TaGlu-Bx7 was 216. 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

Of the 216 individuals selected for the first selection, only plants having only a blank vector were removed, and in order to select only the plant including the TaGlu-Bx7 gene and the empty vector, TaGlu-Bx7 gene-specific primers were used 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 Bx7 5'-AGGGTACCGAGATGGCTAAGCGCCTGG-3 '
(SEQ ID NO: 3) 5'-GATCTAGATCACTGCCTGGTCGACAATG-3 '
(SEQ ID NO: 4) HPTⅡ 5'-CGCTTCTGCGGGCGATTT-3 '
(SEQ ID NO: 5) 5'-CCCATTCGGACCGCAAGGA-3 '
(SEQ ID NO: 6)

As a result, the number of plants transformed with TaGlu-Bx7 was 24 in total.

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

In order to test whether the transgenic rice of the wheat TaGlu-Bx7 gene selected in Example 3 can be inherited up to the next generation, the stable expression of the TaGlu-Bx7 gene in the transformed 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 wheat glutenin Bx7 protein in the transgenic rice plants of the 18 selected lines was examined and the results are shown in Fig. As shown in FIG. 7, it was confirmed that wheat glutenin Bx7 protein was expressed in most of the transgenic rice plants. Especially, the transgenic rice plants of # 8, # 24 and # 3 showed remarkable protein expression.

8, marker-free transgenic rice plants were selected through generation progression in line # 24 as shown in FIG. 8, and as a result, These marker-free transgenic rice plants were selected by confirming that the antibiotic resistance gene (HPT II) band was not formed at lines 16 and 21.

Thus, the present inventors named the # 24 transgenic rice seed, which expresses the most abundant whey glutenin Bx7 protein, as TaGlu-Bx7 without the antibiotic resistance gene (HPTII) band as described above, And deposited it with the accession number KACC98025P to the Center for Agricultural Genetic Resources of the Rural Development Administration.

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 KACC98025P 20130830

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Antibiotic marker-free transgenic rice with TaGlu-Bx7 gene from          Triticum acetine <130> PA130644 / KR <160> 6 <170> Kopatentin 2.0 <210> 1 <211> 2370 <212> DNA <213> Triticum acetumbium Glu-Bx7 <400> 1 atggctaagc gcctggtcct ctttgcggca gtagtcgtcg ccctcgtggc tctcaccgcc 60 gctgaaggtg aggcctctgg acaactacaa tgtgagcacg agctcgaggc atgccaacag 120 gtggtggacc agcaactccg agacgttagc cccgggtgcc gccccatcac cgtcagcccg 180 ggcacgagac aatacgagca gcaacctgtg gtgccgtcca aggccggatc cttctacccc 240 agcgagacta cgccttcgca gcaactccaa caaatgatat tttggggaat acctgcacta 300 ctaagaaggt attacccaag tgtaacttct tcgcagcagg ggtcatacta tccaggccaa 360 gcttctcccc aacagtcagg acaaggacag cagccaggac aagaacagca accaggacaa 420 gggcaacaag atcagcagcc aggacaaaga caacaaggat actacccaac ttctccgcaa 480 cagccaggac aagggcaaca actgggacaa gggcaaccag ggtactaccc aacttcacag 540 cagccaggac aaaagcagca ggcaggacaa gggcaacaat caggacaagg acaacaaggg 600 tactacccaa cttccccgca acagtcagga caagggcaac aaccgggaca agggcaacca 660 gggtactacc caacttctcc gcagcagtca ggacaatggc agcaaccagg acaagggcaa 720 caaccaggac aagggcagca atcaggacaa gggcaacaag gtcagcagcc aggacaaggg 780 caacgaccag gacaaggaca acaagggtac tacccaattt ctccgcaaca gccgggacaa 840 gggcaacaat caggacaagg gcaaccaggg tactacccaa cttctttgcg gcagccagga 900 caatggcagc aaccaggaca agggcagcaa ccaggacaag ggcaacaagg tcagcagcca 960 ggacaaggac aacaatcagg acaaggacaa caaggatact acccaacttc tctgcaacag 1020 ccaggacaag ggcaacaact gggacaaggg caaccagggt actacccaac ttcgcagcag 1080 tcggaacaag ggcagcagcc aggacaagga aaacaaccag gacaaggaca acaagggtac 1140 tacccaactt ctccgcaaca gtcaggacaa gggcaacaac tgggacaagg gcaaccaggg 1200 tactacccaa cttctccaca gcagtcagga caaggacaac aatcaggaca aggacaacaa 1260 gggtactacc caacttctcc gcaacagtca ggacaagggc aacaaccggg acaagggcaa 1320 tcggggtact tcccaacttc tcggcagcag tcaggacaag ggcagcagcc aggacaagga 1380 caacagtcgg gacaagggca acaaggtcag caaccaggac aaggacaaca agcgtactac 1440 ccaacttctt cgcaacagtc aagacaaagg caacaggcag gacaatggca acgaccggga 1500 caagggcaac cagggtacta cccaacctct ccacagcagc caggacaaga gcaacaatca 1560 ggacaagcgc aacaatcagg acaatggcaa ctagtgtact acccaacttc tccgcaacag 1620 ccaggccaat tgcaacaacc agcacaaggg caacaaccag cacaagggca acaatcagca 1680 caagagcaac agccaggaca agcgcaacaa tcaggacaat ggcaactagt gtactaccca 1740 acttctccgc aacagccagg acaattgcaa caaccagcac aagggcaaca agggtactac 1800 ccaacttctc cacaacagtc aggacaaggg caacaagggt actacccaac ttctccgcaa 1860 cagtcaggac aagggcaaca agggtactac ccaacttctc cgcaacagtc aggacaaggg 1920 cagcagccag gacaaggaca acagccaaga caagggcaac aagggtacta cccaatttct 1980 ccgcagcagt caggacaagg gcaacaacca ggacaagggc aacaaggata ctacccaact 2040 tctccgcagc agtcaggaca agggcaacaa ccaggacatg agcaacagcc aggacaatgg 2100 ctgcaaccag gacaagggca acaagggtac tatccaactt cttcacagca gtcaggacaa 2160 gggcatcaat caggacaagg gcaacaaggg tactacccaa cttctctctg gcaaccagga 2220 caagggcaac aaggctacgc cagcccatac catgttagcg cggagtacca ggcggcccgc 2280 ctaaaggtgg caaaggcgca gcagctcgcg gcacagctgc cggcaatgtg ccggctggag 2340 ggcagcgacg cattgtcgac caggcagtga 2370 <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-Bx7 forward primer <400> 3 agggtaccga gatggctaag cgcctgg 27 <210> 4 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> TaGlu-Bx7 reverse primer <400> 4 gatctagatc actgcctggt cgacaatg 28 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> HPTII forward primer <400> 5 cgcttctgcg ggcgattt 18 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> HPTII reverse primer <400> 6 cccattcgga ccgcaagga 19

Claims (10)

Seeds of transgenic rice deposited with deposit number KACC98025P, which contains a TaGlu-Bx7 self-promoter derived from wheat ( Triticum asetivum ) and a marker-free recombinant expression vector for expression of rice seed-specific TaGlu-Bx7 gene containing TaGlu-Bx7 gene .
The transformed rice seed of claim 1, wherein the TaGlu-Bx7 gene has the nucleotide sequence of SEQ ID NO: 1.
2. The transformed rice seed according to claim 1, wherein the TaGlu-Bx7 self-promoter has the nucleotide sequence of SEQ ID NO: 2.
The transformed rice seed according to claim 1, wherein the expression vector is free of an antibiotic resistance gene and its promoter.
5. The seed of transgenic rice according to claim 4, wherein said antibiotic is hygromycin.
delete The transformed rice seed of claim 1, wherein said transformation is mediated by Agrobacterium.
The seed of transgenic rice according to claim 1, wherein the rice is Dongjin rice.
A transgenic rice germinated seed of transgenic rice of any one of claims 1 to 5, 7, and 8.
(a) preparing a marker-free recombinant expression vector for expression of rice seed-specific TaGlu-Bx7 gene comprising TaGlu-Bx7 self-promoter derived from wheat ( Triticum asetivum ) and TaGlu-Bx7 gene;
(b) transforming the recombinant expression vector into rice; And
(c) selecting said transgenic rice plants.

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