KR20090053450A - Method for producing strawberry plant producing Monelin protein and strawberry plant produced by the method - Google Patents

Abstract

The present invention includes a recombinant strawberry plant expression vector for increasing or improving the sweetness of a strawberry, including a monellin coding gene, a strawberry plant transformed with the vector, and transforming the strawberry plant with the vector. A method for producing a transgenic strawberry plant, a method of increasing or improving the sweetness of a strawberry by transforming the strawberry plant with the vector, a strawberry plant produced by the method, a strawberry produced by the strawberry plant and the strawberry as raw materials It is about food to make.

By utilizing the plant-containing plant produced by the present invention can not only improve the added value of strawberries, but also develop as a functional food.

Monelin, Strawberry, Sweetness, Vector, Food

Description

Method for producing strawberry plant which produces monellin protein, and strawberry plant produced by the same method}

The present invention relates to a method for producing a strawberry plant and a strawberry plant produced by the method, more specifically to increase or improve the sweetness of the strawberry, including a monellin coding gene Recombinant strawberry plant expression vector for, a strawberry plant transformed with the vector, a method for producing a transformed strawberry plant comprising the step of transforming the strawberry plant with the vector, transforming the strawberry plant with the vector to the sweetness of the strawberry The present invention relates to a method of increasing or improving, a strawberry plant produced by the method, a strawberry produced by the strawberry plant, and a food based on the strawberry.

Strawberries are a fruit group belonging to the Rosaceae family. According to the statistics, the world's strawberry area is about 220,000 hectares to 3 million tons, of which the United States is the largest, from 20,000 hectares to 780,000 tons, and Korea produces more than 154,000 tons of 65,000 hectares a year. It is the world's sixth-largest strawberry producer after Japan, and the third-largest crop after watermelons and melons.

The total demand of strawberries is expected to increase gradually from 140,000 tons in 2001 to 150,000 tons in 2004 and to increase the consumption per capita.However, the required area is expected to decrease due to the development of cultivation technology and the increase of yields. do. In addition, the production of high-quality strawberries is urgently needed due to the change in consumer patterns due to the income improvement due to economic growth.

Strawberry cultivation relies on pesticide spraying to control pests such as spotted mite, gray mold, powdery mildew, wilted disease, snow blight and leaf nematodes. have.

Strawberry production, fruit size and quality have been greatly improved by strawberry breeders, but many problems such as resistance to viruses and insects, weakening of defense against environmental stress, and overspreading of herbicides have emerged. Due to genetic characteristics such as drainage and high heterozygotes, the superior breeds by traditional breeding methods are limited.

In order to solve the shortcomings of the traditional breeding method, since 1990, biotechnologies have been applied to stably maintain useful traits for varieties of economic value by introducing genetic propagation through clonal propagation.

Accordingly, the transfection system by introducing reporter genes such as GUS is established by foreign research groups, but the transformation rate is very low by about 6%.

Monellin is an African plant Discorephyllum A sweet protein isolated from cucuminsii that binds specifically to taste receptors and is known to be about 100,000 times sweeter than sugar on a molar basis. The increase in sugar content of edible plants by breeding has been mainly achieved by increasing the sugar content, but has reached its limit due to complex carbohydrate metabolism.

Therefore, the sweetness of the gene (sweet protein), such as Monelin will be able to increase or improve the sweet taste. Monelin protein is a weak covalent bond between A-chain of 44 amino acids and B-chain of 50 amino acids, so both chains must be introduced into a plant.

The present invention has been made by the above-mentioned demands, and in the present invention, it is intended to increase or improve the sweetness of strawberries by introducing a Monelin gene in which A and B-chain are expressed as a single peptide.

In order to solve the above problems, the present invention provides a recombinant strawberry plant expression vector for increasing or improving the sweetness of strawberries, including Monellin coding gene.

The present invention also provides a strawberry plant transformed with the vector.

In addition, the present invention provides a method for producing a transformed strawberry plant comprising the step of transforming the strawberry plant with the vector.

The present invention also provides a method for increasing or improving the sweetness of strawberries by transforming strawberry plants with the vector.

The present invention also provides a strawberry plant produced by the above method.

The present invention also provides a strawberry produced by the strawberry plant.

Moreover, this invention provides the foodstuff which uses the said strawberry as a raw material.

According to the present invention, utilizing the plant-containing plant produced by the present invention can not only improve the added value of strawberries, but also develop as a functional food.

In order to achieve the object of the present invention, the present invention provides a recombinant strawberry plant expression vector for increasing or improving the sweetness of the strawberry, including the monellin coding gene.

Recombinant plant expression vector according to an embodiment of the present invention is a vector for increasing or improving the sweetness of strawberries, Monelin coding gene according to the invention may be any gene encoding Monelin , preferably Discorephyllum It may consist of the nucleotide sequence represented by SEQ ID NO: 1 isolated from cucuminsii . In addition, variants of such sequences are included within the scope of the present invention. A variant is a nucleotide sequence that changes in base sequence but has similar functional properties to that of SEQ ID NO: 1. Specifically, the Monelin gene has a nucleotide sequence having at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% homology with the nucleotide sequence of SEQ ID NO: 1. It may include.

The "% sequence homology" for a polynucleotide is identified by comparing two optimally arranged sequences with a comparison region, wherein part of the polynucleotide sequence in the comparison region is the reference sequence (addition or deletion) for the optimal alignment of the two sequences. It may include the addition or deletion (ie, gap) compared to).

The recombinant plant expression vector according to one embodiment of the present invention is a pGA482-PS1D vector, which is described in FIG. 1. However, the vector of the present invention is not limited to the vector described in FIG. 1, and any vector useful for plant transformation can be used.

The term “recombinant” refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, a heterologous peptide, or a heterologous nucleic acid. Recombinant cells can express genes or gene fragments that are not found in their natural form in either the sense or antisense form. Recombinant cells can also express genes found in natural cells, but the genes have been modified and reintroduced into cells by artificial means.

The term “vector” is used to refer to a DNA fragment (s), a nucleic acid molecule, that is delivered into a cell. Vectors can replicate DNA and be reproduced independently in host cells. The term "carrier" is often used interchangeably with "vector". The term “expression vector” refers to a recombinant DNA molecule comprising a coding sequence of interest and a suitable nucleic acid sequence necessary to express a coding sequence operably linked in a particular host organism.

Preferred examples of plant expression vectors are Ti-plasmid vectors which, when present in a suitable host such as Agrobacterium tumerfaciens, can transfer part of themselves, the so-called T-region, into plant cells. Another type of Ti-plasmid vector (see EP 0 116 718 B1) is used to transfer hybrid DNA sequences to protoplasts from which current plant cells or new plants can be produced that properly insert hybrid DNA into the plant's genome. have. A particularly preferred form of the Ti-plasmid vector is the so-called binary vector as claimed in EP 0 120 516 B1 and US Pat. No. 4,940,838. Other suitable vectors that can be used to introduce Monelin DNA according to the invention into a plant host are viral vectors such as those derived from double stranded plant viruses (eg CaMV) and single stranded viruses, gemini viruses and the like. For example, from an incomplete plant viral vector. The use of such vectors can be advantageous, especially when it is difficult to properly transform a plant host.

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

In the plant expression vector according to an embodiment of the present invention, the promoter may be CaMV 35S, actin, ubiquitin, pEMU, MAS or histone promoter, but is not limited thereto. The term "promoter" refers to a region of DNA upstream from a structural gene and refers to a DNA molecule to which an RNA polymerase binds to initiate transcription. A "plant promoter" is a promoter capable of initiating transcription in plant cells. A "constitutive promoter" is a promoter that is active under most environmental conditions and developmental conditions or cell differentiation. Constitutive promoters may be preferred in the present invention because selection of the transformants may be made by various tissues at various stages. Thus, the constitutive promoter does not limit the selection possibilities.

The terminator may use a conventional terminator, and examples thereof include nopaline synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, Agrobacterium tumefaciens ( Ogrobacterium tumefaciens ) Terminator of the Fine (Octopine) gene, etc., but is not limited thereto. With regard to the need for terminators, it is generally known that such regions increase the certainty and efficiency of transcription in plant cells. Therefore, the use of terminators is highly desirable in the context of the present invention.

The present invention also provides a plant transformed with the recombinant vector according to the present invention. The plant according to the invention expresses the monelin protein. Plants according to the present invention is rice, wheat, barley, corn, soybeans, potatoes, wheat, red beans, oats and sorghum food crops selected from the group consisting of; Vegetable crops selected from the group consisting of Arabidopsis, Chinese cabbage, radish, red pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, green onion, onion, and carrot; Special crops selected from the group consisting of ginseng, tobacco, cotton, sesame, sugar cane, sugar beet, perilla, peanut and rapeseed; Fruit trees selected from the group consisting of apple trees, pear trees, jujube trees, peaches, lambs, grapes, citrus fruits, persimmons, plums, apricots and bananas; Flowers selected from the group consisting of roses, gladiolus, gerberas, carnations, chrysanthemums, lilies and tulips; And fodder crops selected from the group consisting of lysis, redclover, orchardgrass, alphaalpha, tolsquescue and perennial lygragrass. Preferably, the plant is strawberry, Arabidopsis, potato, eggplant, tobacco, pepper, tomato, burdock, garland chrysanthemum, lettuce, bellflower, spinach, beetroot, sweet potato, celery, carrot, buttercup, parsley, cabbage, cabbage, mustard , Watermelon, melon, cucumber pumpkin, gourd, soybean, mung beans, kidney beans, or peas can be a dicotyledonous plant, most preferably, the plant is a strawberry.

The present invention also provides seed of the plant. Preferably, said seed is a seed of a strawberry.

The invention also comprises the steps of transforming strawberry plant cells with the recombinant vector according to the invention; And

It provides a method for producing a transformed strawberry plant comprising the step of re-differentiating the transformed plant from the transformed strawberry plant cells.

Plant transformation refers to any method of transferring DNA to a plant. Such transformation methods do not necessarily have a period of regeneration and / or tissue culture. Transformation of plant species is now common for plant species, including both dicotyledonous plants as well as monocotyledonous plants. In principle, any transformation method can be used to introduce hybrid DNA according to the invention into suitable progenitor cells. Method is calcium / polyethylene glycol method for protoplasts (Krens, FA et al., 1982, Nature 296, 72-74; Negrutiu I. et al., June 1987, Plant Mol. Biol. 8, 363-373), protoplasts Electroporation (Shillito RD et al., 1985 Bio / Technol. 3, 1099-1102), microscopic injection into plant elements (Crossway A. et al., 1986, Mol. Gen. Genet. 202, 179-185 ), (DNA or RNA-coated) particle bombardment of various plant elements (Klein TM et al., 1987, Nature 327, 70), Agrobacterium tumulopasis by infiltration of plants or transformation of mature pollen or vesicles And infection with (incomplete) virus (EP 0 301 316) in en mediated gene transfer. Preferred methods according to the invention include Agrobacterium mediated DNA delivery. Especially preferred is the use of the so-called binary vector technology as described in EP A 120 516 and US Pat. No. 4,940,838.

The method of the present invention comprises the step of transforming strawberry plant cells with the recombinant vector according to the present invention, the transformation can be mediated by Agrobacterium tumefaciens ( Agrobacterium tumefaciens ). The method also includes the step of regenerating the transgenic plant from the transformed strawberry plant cells. The method for regenerating the transformed plant from the transformed plant cell may use any method known in the art.

The present invention also provides a method of increasing or improving the sweetness of a strawberry, comprising the step of transforming strawberry plant cells with the recombinant vector according to the present invention, overexpressing the monelin coding gene. Monellin is a sweet-tasting protein and is known to be about 100,000 times sweeter than sugar on a molar basis, so overexpressing the Monelin gene in strawberries can increase or improve the sweetness of strawberries. In the present invention, it was confirmed that the Monel transcript is stably expressed in the strawberry transformant through the northern blot (see FIG. 4).

Plants used in the method is not limited to strawberries, food crops selected from the group consisting of wheat, barley, corn, soybeans, potatoes, wheat, red beans, oats and sorghum; Vegetable crops selected from the group consisting of Arabidopsis, Chinese cabbage, radish, pepper, tomato, watermelon, cucumber, cabbage, melon, pumpkin, green onion, onion, and carrot; Special crops selected from the group consisting of ginseng, tobacco, cotton, sesame, sugar cane, sugar beet, perilla, peanut and rapeseed; Fruit trees selected from the group consisting of apple trees, pear trees, jujube trees, peaches, lambs, grapes, citrus fruits, persimmons, plums, apricots and bananas; Flowers selected from the group consisting of roses, gladiolus, gerberas, carnations, chrysanthemums, lilies and tulips; And fodder crops selected from the group consisting of lysis, redclover, orchardgrass, alphaalpha, tolsquescue and perennial lygragrass.

The present invention also provides a strawberry plant in which the sweetness of the strawberry produced by the method is increased or improved. In the present invention, it was confirmed that the Monelin transcript is stably expressed in the strawberry transformant through the northern blot. Thus, strawberry transformants may be increased or improved in sweetness.

The present invention also provides a strawberry containing monelin produced by a strawberry plant in which the sweetness of the strawberry produced by the method of the present invention is increased or improved. Since the strawberry plant according to the present invention produces the monelin protein, it can be inferred that the monelin protein may be produced even in the strawberry which is a fruit produced by the strawberry plant. Accordingly, the present invention is to provide a strawberry containing Monelin protein.

This invention also provides the foodstuff which uses the strawberry of this invention as a raw material. Strawberries containing Monelin protein may be used by itself or as appropriately processed or extracted to be used as a food, health food or dietary supplement, or as a component of various medicines.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example

Example  One: Monelin  Preparation of Plant Transformation Vectors Containing Genes

Plasmid pGA482 was used as a plant nucleotransformation vector containing the monelin gene. As shown in FIG. 1, plasmid pGA482 has a kanamycin resistance gene (NPTII), a 35S promoter of CaMV (P35S), a nos terminator (Tnos), and various restriction enzyme cleavage sites. Discorephyllum in the vector above Monelin cDNA isolated from cucuminsii was inserted to prepare a recombinant plant expression vector pGA482-PS1D (FIG. 1). pGA482-PS1D was introduced into EHA105 Agrobacterium by freeze-thaw method.

Example  2: transformation of strawberry plants with the recombinant vector of the present invention

Strawberry plant material was used as a transgenic material by making a slice of approximately 5 x 7 mm sized slices of domestic varieties 'Mayang' strawberry leaves of 1-2 months passage culture in 1 / 2MS medium.

Specific strawberry transformation method is as follows.

(1) Agrobacterium single colony containing Monelin gene was added to YEP (Yeast Extract-Phosphate) medium for 2 days in 10 mL of liquid medium with 100 mg / L Rif (Rifampicin) and 50 mg / L km. Agrobacterium in cultured logarithmic phase was centrifuged (3,000 rpm, 10 min) to discard supernatant and liquid coculture medium (MS salts + B5 vit + 2.2 mg / L TDZ (Thidiazuron) + 0.3 mg / L IBA Agrobacterium was suspended in 10 mL of Indole-3-Butyric Acid) + 3% sucrose, pH 5.6). 200 μM of acetosyringone (AS) was added thereto, and then, strawberry leaf slices were added and co-cultured with occasional shaking for 20 minutes.

(2) After coculture, excess moisture of the leaf sections was removed with sterile paper towels and then solid coculture medium (MS salts + B5 vit + 2.2 mg / L TDZ + 0.3 mg / L IBA + 3% sucrose + 0.6% Phyto Agar, pH 5.6) was co-cultured at 25 ° C. and dark for 3 days after adaxial (non-axial-pore) surface contacted the media.

(3) After the coculture, the sections were washed three times with sterile distilled water and then drained, and then the callus selection medium (MS salts + B5 vit + 2.2 mg / L TDZ + 0.3 mg / L IBA + 300 mg / L carbenicillin + 50 mg / L Km + 3% sucrose + 0.6% Phyto Agar, pH 5.6) was incubated at 25 ℃, dim light. Four weeks later, the same medium was passaged and cultured for four more weeks.

(4) The callus formed from the leaf sections was separated and incubated at 25 ° C. under light conditions (16 h light, 8 dark) and subcultured every 3-4 weeks.

(5) After about five months of culture, malformation began to develop from callus formed in the selection medium.

(6) Negative infants from selection medium were plant derived medium (MS salts + B5 vit + 3.2 mg / L Zeatin (or 3 mg / L BA) + 0.5 mg / L IBA + 300 mg / L carbenicillin + 50 mg / L Km + 3% sucrose + 0.6% Phyto Agar, pH 5.6) to differentiate into the media.

(7) The news-derived roots were derived from rooting medium (1/2 MS + 3% sucrose + 300 mg / L carbenicillin + 50 mg / L Km) and transferred to the soil and grown in growth through the purification step (FIG. 2). ).

Example  3: from regenerating strawberry plants Monelin  Gene expression confirmation

It was confirmed whether the actual monelin gene was introduced into the plant cell obtained in the above process and whether the gene was transcribed.

(One) PCR  analysis

From leaves of potential strawberry transformants transferred to soil, Edwards et al. PCR was performed by separating gDNA by the method (1991) (Nucleic Acids Research 19 (6): 1349). The primer sequences of the kanamycin resistance gene (NPTII) and the monelin gene are as follows.

Monelin primer (PCR product about 300 bp)

Mon F: 5'-ggg aat ggg aaa tta tcg at-3 '(SEQ ID NO: 2)

Mon R: 5'-cta tta tgg tgg tgg aac tgg-3 '(SEQ ID NO: 3)

NPTII primer (PCR product about 700 bp)

NPTII F: 5'-gag gct att cgg cta tga ctg-3 '(SEQ ID NO: 4)

NPTII R: 5'-atc ggg agc ggc gat acc gta-3 '(SEQ ID NO: 5)

PCR was performed by performing preliminary denaturation at 94 ° C. for 5 minutes with each primer followed by 30 cycles of 94 ° C., 30 seconds, 55 ° C., 30 seconds, and 72 ° C., 30 seconds, and then finally extending 72 ° C. for 5 minutes. PCR was performed using the monelin primer, and the PCR product was about 300 bp and NPTII primer was obtained. The PCR product was about 700 bp in size. However, no control bands were identified in the non-transformed control. From this result, it was confirmed that the Monelin gene was introduced into the chromosomal genome of these transgenic strawberry plants (FIG. 3).

(2) Northern Blot  analysis

Total RNA was isolated from the plants whose gene introduction was confirmed by PCR, and Northern blot analysis was performed. As a method of separating total RNA from a plant, a general method known in the art was used. About 10 μg of total RNA extracted was electrophoresed on a 1% agarose gel containing 5.1% (v / v) formaldehyde and then transferred to Zeta-Probe GT blotting membrane (Bio-Rad) in 20X SSC solution. Specific portions of the Monelin gene were synthesized by PCR and used as probes. Prehybridization and hybridization were monelin specific labeled with isotope [α- ³² P] dCTP using 0.25 M sodium phosphate buffer (pH 7.2) containing 7% (w / v) SDS. DNA was added and performed overnight at 65 ° C. After completion of the reaction, the membrane was washed with 20 mM sodium phosphate buffer (pH 7.2) containing 5% and 1% (w / v) SDS for 10 minutes at 65 ° C, and exposed to an image plate (Fuji film, Japan) film.

As a result, it was confirmed that all of the transformants were strongly expressed in the Monelin gene, but not at all in the control plants (Fig. 4).

Figure 1 is a schematic diagram showing the pGA482-PS1D plasmid vector with the Monelin gene inserted. NPTII (kanamycin resistance gene), P35S (cauliflower mosaic virus 35S promoter), Pnos (Nos promoter), Tnos (Nos terminator).

Figure 2 is a picture of the regeneration of plants from the 'falcon' strawberry leaf fragment transformed with the Monellin gene. A: organogenic callus formation in medium supplemented with 50 mg / L kanamycin; B: Shoot differentiation from organogenic callus; C: development into news media; D: rooting news object; E: Plants growing in the soil.

Figure 3 shows the results of the introduction of Monelin and NPTII gene using PCR. M: molecular weight size marker; PC: pS1D vector control; 1-4: Monelin transgenic plant; NC: untransformed strawberry plant.

Figure 4 shows the northern analysis of the monelin transformed strawberry plant. WT: untransformed strawberry plant; 1-3: Monelin transgenic plant.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Method for producing strawberry plant which produces monellin          protein, and strawberry plant produced by the same method <130> PN07102 <160> 5 <170> KopatentIn 1.71 <210> 1 <211> 280 <212> DNA <213> Dioscoreophyllum cumminsii <220> <221> gene (222) (1) .. (280) <223> Monellin gene <400> 1 atgggaaatt atcgatattg gaccattcac tcaaaacttg ggtaagttcg ctgttgacga 60 agaaaacaag attggtcaat atggtagatt gactttcaac aaggttatta gaccatgtat 120 gaagaagact atttacgaaa acgaaagaga aattaagggg tacgaatacc aattgtatgt 180 ttacgcttct gacaagcttt tcagagctga catttctgaa gactacaaga cccgcggtag 240 aaagttgttg agattcaacg gtccagttcc accaccataa 280 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Mon F primer <400> 2 gggaatggga aattatcgat 20 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Mon R primer <400> 3 ctattatggt ggtggaactg g 21 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> NPTII F primer <400> 4 gaggctattc ggctatgact g 21 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> NPTII R primer <400> 5 atcgggagcg gcgataccgt a 21  

Claims (9)

A recombinant strawberry plant expression vector for increasing or improving the sweetness of a strawberry, comprising a monellin coding gene. According to claim 1, wherein the gene is a vector, characterized in that consisting of the nucleotide sequence represented by SEQ ID NO: 1. The vector of claim 1, wherein the vector is a pGA482-PS1D vector described in FIG. 1. Strawberry plant transformed with the vector of claim 1. Transforming the strawberry plant cells with the vector according to claim 1; And Regenerating the transformed plant from the transformed strawberry plant cells method of producing a transformed strawberry plant. A method of increasing or improving the sweetness of a strawberry, comprising the step of transforming the strawberry plant cells with the vector according to claim 1 overexpressing the monelin coding gene. Strawberry plant, the sweetness of the strawberry produced by the method of claim 6 is increased or improved. A strawberry containing monelin produced by the strawberry plant of claim 7. Food which uses strawberry of Claim 8 as raw material.

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