KR20130022520A - Pac multiple expression gene increasing carotenoid content of plant and uses thereof - Google Patents

Abstract

PURPOSE: PAC multiple expression gene which enhances the carotenoid content in a plant is provided to enhance the carotenoid content in a transgenic bean. CONSTITUTION: PAC multiple expression gene which enhances the carotenoid content in a plant comprises a promoter, Capsicum annuum-derived Psy(phytoene synthase) gene, foot-and-mouth disease virus-derived 2A nucleotide sequence, Oryza sativa-derived TP(transpeptide) gene, and Erwinia uredovora-derived CrtI(carotene desaturase) gene from 5' to 3'. PAC multiple expression gene has a base sequence of sequence number 1.

Description

PAC multiple expression gene increasing carotenoid content of plant and uses according to the present invention.

The present invention relates to a PAC multi-expression gene for increasing the carotenoid content of a plant and its use, and more particularly, to a plant cell with a recombinant vector comprising a promoter and a PAC ( Psy- 2A- TP - CrtI ) multi-expression gene. Switch to PAC A method of increasing the carotenoid content of a plant comprising overexpressing a multiple expression gene, transforming plant cells with a recombinant vector comprising a promoter and a Psy- 2A- TP - CrtI multiple expression gene, and transducing Method for producing a plant with increased carotenoid content compared to wild type comprising the step of regenerating a plant from converted plant cells, transgenic plant with increased carotenoid content compared to wild type produced by the method and seeds and PACs ( Psy -2A-TP-CrtI ) relates to a composition for increasing the carotenoid content of a plant comprising multiple expression genes.

Soybean is a well-known dicotyledonous crop and is grown all over the world because it can grow under various growth conditions. Beans are rich in grains, 40% protein rich in nutrients, 60% protein and fat, and are the most unique crops with the highest protein and fat content except processed foods such as cheese. Especially, Asian people with rice stocks are involved in economical protein causes and secondary metabolites, so they are constantly exploring varietal development techniques including transformation to improve functional aspects of soybean.

Primary metabolites are carbohydrates, proteins, nucleic acids and lipids, which are indispensable for performing cellular functions. On the other hand, there are many compounds in plants that are not essential for survival, which are called secondary metabolites. Secondary metabolites can be divided into alkaloids, terpenes, phenolic compounds, and other compounds depending on the structure and synthesis process.

Carotenoids are a subfamily of isoprenoids widely distributed in yellow, orange, and red natural pigments synthesized in plants, bacteria, algae, and fungi (Goodwin TW, Britton G: Distribution and analysis). of carotenoids.In: Plant Pigments . Edited by Goodwin TW. London: Academic Press; 1988: 61-132). Carotenoids are a generic term for carotene and xanthophyll. Carotenoids are synthesized and stored in the intracellular plastids of plants. It accumulates in the chloroplasts of chlorophyll-containing tissues such as leaves, and the variegated bodies of chlorophyll-free tissues such as fruits, seeds, and flowers. Chlorophyll carotenoids are involved in light absorption during photosynthesis and protect cells from excessive light energy. Since carotenoids such as fruits, seeds, and flowers have a reddish color, they can be used as a means of attracting animals to breed their own seeds. They are also precursors for the synthesis of plant hormone ABA. Animals that cannot biosynthesize carotenoids get carotenoids from food, which is used as a vitamin A precursor. In addition, it has been proved as an excellent compound for nutrition and function as an antioxidant and a cancer prevention agent (Ha et al., 2003, Korean J. Plant Biotechnology Vol. 30, No. 1, 81 ~ 95).

Research into carotenoid biosynthesis genes has been in full swing since the early 1990s, and to date, nearly all types of carotenoid biosynthesis have been isolated. On the basis of this, golden rice was developed in 2000, and the first successful step for carotenoid metabolic engineering began (Ha et al., 2003, Korean J. Plant Biotechnology Vol. 30, No. 1, 81 ~). 95). Studies attempting to produce beta-carotene and carotenoids in plant tissues that do not produce carotenoids have been studied by linking the Narcissus pseudonarcissus Psy gene to the rice endosperm-specific glutelin promoter Gt1 . Attempts were made to detect phytoene components in (Burkhardt et al., 1997, Plant J 11: 1071-1078). Three years later in a joint study by Professor Peter Beyer of the University of Freiburg, Germany and Professor Ingo Potrykus of the Swiss Federal Institute of Technology, Gt1 :: Psy / 35S :: tp :: CrtI and The so-called GoldenRice (GR), in which beta-carotene was produced from rice seeds by double transformation of Gt1 :: Lcy and turned rice golden, was published in Science (Ye et al., 2000, Science 287: 303- 305). In Korea, Dr. Sun Hwa Ha, National Institute of Agricultural Science, RDA, presented Glb :: P sy :: 2 A :: tp :: C rtI Glb :: P sy :: I RES :: tp :: C rtI was used to produce beta-carotene-producing rice and published in Plant Biotechnology (Ha et al., 2003, Korean J. Plant Biotechnology Vol. 30, No. 1, 81-95).

At least 26 countries, including Asia, Africa and Latin America, have rice as their staple food. In Southwest Asia, in particular, 70% of children under five suffer from vitamin A deficiency (avitaminosis A). A report published by the United Nations Children's Fund (UNICEF) predicted that between 1 and 4 years of age would save between 1 and 4 years of age due to nutritionally increased vitamin A content. Given that the beta-carotene content of golden rice is about 200 ㎍ per 100 grams of rice, the daily intake of provitamin A is about 1/10 of RAD from 300 grams of rice (Giuliano et al., 2000, Trends Plant Sci 5 406-409).

In the meantime, the soybean transformation in Korea has been insufficient in the regeneration technology of soybean plant through tissue culture, and unlike other crops, it is difficult to introduce foreign gene, so a stable transformation technology system has not yet been established. In some laboratories, there were partial successes, but failed to produce sustained results. As a result of visiting and studying the US and Japan, the present inventors produced herbicide-resistant transgenic soybean as domestic varieties in 2006 with its own developed technology, and now it is possible to mass-produce soybean transformants using domestic digestive useful genes.

Soybeans are the most widely used soybean sprouts, 33.2%, tofu 31.2%, tofu 26.6%, and soy milk 9.0% are commonly consumed in the form of soybean processed foods. For globalization of soybean traditional foods and vitalization of soybean-related industries, it is urgently needed to reinforce the functionality of soybeans as raw materials and to develop high quality processing technology. Soy food, which was known only as a traditional food of the East, is a food containing a large amount of health functional ingredients. Thus, we transformed soybeans using a β-conglycinin promoter and a PAC ( Psy- 2A- TP - CrtI ) multiple expression gene to increase the carotenoid content of soybean.

Korean Patent Publication No. 2011-0052865 discloses a beta-carotene hydroxylase gene that increases the beta-carotene content of a plant, and Korean Patent Publication No. 2011-0052862 discloses lycopene ε-between increasing the content of a beta-carotene of a plant. Claase genes are disclosed.

The present invention is derived from the above requirements, and the present inventors transformed using a recombinant vector comprising a β-conglycinin promoter and a PAC ( Psy- 2A- TP - CrtI ) multiple expression gene. The present invention was completed by confirming that the carotenoid content was increased in the prepared soybeans.

In order to solve the above problems, the present invention promoter and capsicum ( Capsicum annuum) derived Psy (phytoene synthase) gene, the FMD virus (foot-and-mouth disease virus ) 2A-derived nucleotide sequence, rice (Oryza sativa ) -derived TP (transpeptide) gene and Erwinia uredovora ) transfected plant cells with a recombinant vector comprising a PAC ( Psy- 2A- TP - CrtI ) multi-expressing gene in which the carotene desaturase ( CrtI ) gene is sequentially linked in the 5 'to 3' direction. Provided is a method of increasing the carotenoid content of a plant comprising overexpressing multiple expression genes.

In addition, the present invention promoter and capsicum ( Capsicum annuum) derived Psy (phytoene synthase) gene, the FMD virus (foot-and-mouth disease virus ) 2A-derived nucleotide sequence, rice (Oryza sativa ) -derived TP (transpeptide) gene and Erwinia uredovora ) Wild type comprising transforming plant cells with a recombinant vector comprising a PAC ( Psy- 2A- TP - CrtI ) multi-expressing gene in which the carotene desaturase ( CrtI ) gene is sequentially linked in the 5 'to 3' direction It provides a method for producing a plant with increased carotenoid content as compared to.

In addition, the present invention provides a transgenic plant and its seed having increased carotenoid content compared to the wild type produced by the above method.

In addition, the present invention provides a composition for increasing the carotenoid content of plants, including PAC ( Psy- 2A- TP - CrtI ) multiple expression genes.

According to the present invention, using a β-conglycinin promoter and a PAC ( Psy- 2A-TP-CrtI ) multi-expression gene, soybean plants with an increased carotenoid content of 20 to 36 times compared to wild type can be prepared. Therefore, it is expected to be useful not only for enhancing the functionality and quality of soybeans but also for activating the soy food industry.

1 shows two recombinant vectors (β- PAC) using the β-conglycine promoter and the 35S promoter. And 35S- PAC ) is shown.
2 shows recombinant vector β- PAC And it shows the growth process of soybean plants transformed using 35S- PAC .
Figure 3 β- PAC T ₀ And 35S-PAC T ₀ Transformant T ₁ Seed (A), T ₂ Seed (B) and cross section (C) of wild type and line 13 seeds. WT: wild type, EV: empty vector.
4 is β- PAC T ₀ Transformants and 35S-PAC T ₀ PCR results performed to confirm the insertion gene in the transformants are shown. NC: negative control, PC: positive control.
5 is β- PAC T ₀ Transformants and 35S-PAC T ₀ RT-PCR results were performed to confirm the insertion gene in the transformant. NC: negative control.
Figure 6 β- PAC T ₀ Transformants and 35S-PAC T ₀ Southern blotting was performed to confirm the insertion gene in the transformant. NC: negative control, EV: empty vector.
Figure 7 β- PAC RT-PCR results were performed to confirm the expression of the insert gene in the leaves and seeds of the transformants and 35S-PAC transformants. NC: negative control, EV: empty vector.

In order to achieve the object of the present invention, the present invention is a promoter and Psy (phytoene synthase) gene derived from Capsicum annuum , 2A nucleotide sequence derived from foot-and-mouth disease virus, rice ( Oryza) sativa ) -derived TP (transpeptide) gene and Erwinia uredovora ) transfected plant cells with a recombinant vector comprising a Psy-2A-TP-CrtI (PAC) multiple-expressing gene whose CrtI (carotene desaturase) genes are sequentially linked in the 5 'to 3' direction. Provided is a method of increasing the carotenoid content of a plant comprising overexpressing multiple expression genes.

The recombinant vector of the present invention may include a promoter and PAC ( Psy- 2A- TP - CrtI ) multiple expression gene, and may also include a terminator, a selection marker, and the like.

The promoter may preferably be a β-conglycinin promoter, but is not limited thereto. Preferably, the carotenoid may be α-carotene or β-carotene, but is not limited thereto.

Preferably, PAC multiple expression of the present invention The gene may include a nucleotide sequence represented by SEQ ID NO: 1. In addition, variants of the above nucleotide sequences are included within the scope of the present invention. Specifically, the gene has a base sequence having a sequence homology of at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% with the nucleotide sequence of SEQ ID NO: 1, respectively. It may include. "% Of sequence homology to polynucleotides" is ascertained by comparing the comparison region with two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region is the reference sequence for the optimal alignment of the two sequences (I. E., A gap) relative to the < / RTI >

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, heterologous peptide or heterologous nucleic acid. The recombinant cell can express a gene or a gene fragment that is not found in the natural form of the cell in one of the sense or antisense form. In addition, the recombinant cell can express a gene found in a cell in its natural state, but the gene has been modified and reintroduced intracellularly by an artificial means.

In the present invention, the PAC multiple expression gene sequence can be inserted into a recombinant expression vector. The term "recombinant expression vector" means a bacterial plasmid, a phage, a yeast plasmid, a plant cell virus, a mammalian cell virus, or other vector. In principle, any plasmid and vector can be used if it can replicate and stabilize within the host. An important characteristic of the expression vector is that it has a replication origin, a promoter, a marker gene and a translation control element.

Expression vectors comprising PAC multiple expression gene sequences and appropriate transcriptional / translational control signals can be constructed by methods well known to those of skill in the art. Such methods include in vitro recombinant DNA technology, DNA synthesis techniques, and in vivo recombination techniques. The DNA sequence can be effectively linked to appropriate promoters in the expression vector to drive mRNA synthesis. The expression vector may also include a ribosome binding site and a transcription terminator as a translation initiation site.

Preferred examples of recombinant vectors of the invention are Ti-plasmid vectors capable of transferring part of themselves, the so-called T-region, to plant cells when present in a suitable host such as Agrobacterium tumefaciens. Another type of Ti-plasmid vector (see EP 0 116 718 B1) is currently used to transfer hybrid DNA sequences to protoplasts from which 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 a so-called binary vector as claimed in EP 0 120 516 B1 and U.S. Patent No. 4,940,838. Other suitable vectors that can be used to introduce the DNA according to the invention into a plant host are viral vectors, such as those which can be derived from double stranded plant viruses (eg CaMV) and single stranded viruses, gemini viruses, etc. For example, it may be selected from an incomplete plant viral vector. The use of such vectors may be particularly advantageous when it is difficult to transform the plant host properly. Preferably, the recombinant vector may be a β- PAC vector, but is not limited thereto.

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

In the recombinant vector of the present invention, conventional terminators can be used. Examples thereof include nopaline synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, Agrobacterium tumefaciens (Agrobacterium tumefaciens ) Octopine gene terminator, but the present invention is not limited thereto. Regarding 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 a terminator is highly desirable in the context of the present invention.

Any host cell known in the art may be used as the host cell capable of continuously cloning and expressing the vector of the present invention in a stable and prokaryotic cell, for example, E. coli JM109, E. coli BL21, E. coli RR1 , E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus tulifene, and Salmonella typhimurium, Serratia marcesensus and various Pseudomonas species And enterobacteria and strains such as the species.

When the vector of the present invention is transformed into eukaryotic cells, yeast ( Saccharomyce cerevisiae), and the like insect cells, human cells (e.g., CHO cells (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cell lines) and plant cell may be used. The host cell is preferably a plant cell.

The method of carrying the vector of the present invention into a host cell is performed by using the CaCl ₂ method or one method (Hanahan, D., J. Mol. Biol., 166: 557-580 (1983)) when the host cell is a prokaryotic cell. And the electroporation method. When the host cell is a eukaryotic cell, the vector is injected into the host cell by microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, and gene bombardment .

Transformation of a plant means any method of transferring DNA to a plant. Such transformation methods do not necessarily have a regeneration and / or tissue culture period. Transformation of plant species is now common for plant species, including both terminal plants as well as dicotyledonous plants. In principle, any transformation method can be used to introduce the hybrid DNA according to the present 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 tumefaci 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. A preferred method according to the present invention comprises Agrobacterium mediated DNA delivery. Particularly preferred is the use of so-called binary vector techniques as described in EP A 120 516 and U.S. Pat. No. 4,940,838.

In addition, the present invention promoter and capsicum ( Capsicum annuum) derived Psy (phytoene synthase) gene, the FMD virus (foot-and-mouth disease virus ) 2A-derived nucleotide sequence, rice (Oryza sativa ) -derived TP (transpeptide) gene and Erwinia uredovora ) transforming plant cells with a recombinant vector comprising a PAC ( Psy- 2A- TP - CrtI ) multi-expressing gene in which a carotene desaturase ( CrtI ) gene is sequentially linked in a 5 'to 3'direction; And

It provides a method for producing a plant with increased carotenoid content compared to the wild type comprising the step of regenerating the plant from the transformed plant cells.

The method of the invention comprises the step of transforming a plant cell with a recombinant vector according to the present invention, the transformant is, for example, Agrobacterium tumefaciens may be mediated by (Agrobacterium tumefiaciens). In addition, the method of the present invention comprises regenerating a transgenic plant from the transformed plant cell. Any of the methods known in the art can be used for regeneration of transgenic plants from transgenic plant cells. In the methods of the invention, the PAC multiple expression genes are as described above.

Transformed plant cells must be regenerated into whole plants. Techniques for regeneration of mature plants from callus or protoplast cultures are well known in the art for many different species.

In addition, the present invention provides a transgenic plant and its seed having increased carotenoid content compared to the wild type produced by the above method. Preferably, the plant may be a dicotyledonous plant, but is not limited thereto. Preferably, the dicotyledonous plants are soybean ( Glycine) max ).

The dicotyledonous plants are Legumes (Leguminosae), Asteraceae (Diaphleaceae, Diapensiaceae), Cacthraceae, Pyrolaceae, Ericaceae, Myrsinaceae, Primaceae, Primaceae ), Ebenaceae, Styracaceae, Stink bug, Symplocaceae, Ash (Olanaceae), Loganiaceae, Gentianaceae, Menyanthaceae, Oleaceae (Apocynaceae, Apocynaceae), Asclepiadaceae, Rubisaceae, Polemoniaceae, Convolvulaceae, Boraginaceae, Verbenaceae, Lapiaceae, Labiatae, Solanaceae , Scrophulariaceae, Bignoniaceae, Acanthaceae, Sesame (Pedaliaceae), Fructose (Orobanchaceae). Gesneriaceae, Lentibulariaceae, Phrymaceae, Plantaginaceae, Caprifoliaceae, (Perox Adoxaceae), Valerianaceae, Dipsacaceae, Campanaceae ( Campanulaceae, Compositae, Myricaceae, Sapaceae, Juglandaceae, Salicaceae, Birchaceae, Beechaceae, Fagaceae, Elmaceae, Moraceae , Urticaceae, Santalaceae, Mistletoe, Lothanthaceae, Polygonaceae, Landaceae, Phytolaccaceae, Nyctaginaceae, Pomegranate, Azizaceae (Portulacaceae), Caryophyllaceae, Chinopodiaceae, Amaranthaceae, Cactaceae, Magnoliaceae, Illiciaceae, Lauraceae, Cassia family, Cecidiphyllaceae, Ranunculus eae), Berberidaceae, Lardizabalaceae, Bird breeze (Mentaceae, Menispermaceae), Nymphaeaceae, Ceratophyllaceae, Cabombaceae, Saururaceae , Piperaceae, Chloranthaceae, Aristolochiaceae, Actinidiaceae, Camellia, Theaceae, Guttaiferae, Droseraceae, Papaveraceae ), Capparidaceae, Cruciferaceae (Mustaceae, Cruciferae), Planeaceae (Plataceae, Platanaceae), Verruaceae, Hamamelidaceae, Pheasant (Snaphaceae, Crassulaceae), Panaxaceae (Saxifragaceae) Eucommiaceae, Pittosporaceae, Rosaceae, Oxalidaceae, Geraniaceae, Tropaeolaceae, Zygophyllaceae, Linaceae, Euphorbiaceae, Alliaceae ), Rutaceae, Simaroubaceae, Meliaceae, Polygalaceae, Anacardiaceae, Mapleaceae, Aceraceae, Sapindaceae, Hippocastanaceae, Nado Sabiaceae, Balsam (Bamsamaceae), Aquifoliaceae, Nova (Archiaceae, Celastraceae), Staphyleaceae, Buxaceae, Empyaceae, Rhamnaceae ), Vitiaceae, Elaeocarpaceae, Tiliaceae, Malvaceae, Walliaaceae, Sterculiaceae, Adenaceae, Thymelaeaceae, Ellaeagnaceae, Flacourtiaceae, Violaceae, Passifloraceae, Tamaricaceae, Elatinaceae, Begoniaceae, Cucurbitaceae, Budaceae (Lythraceae), Pomeaceae , Needle flower (O nagraceae), Haloragaceae, Bataceaeaceae (Alangiaceae), Dogwood (Asteraceae, Cornaceae), Arboraceae (Agaraceae, Araliaceae) or Umbelliferae (Apiaceae), but not limited thereto. It doesn't work.

In addition, the present invention provides a composition for increasing the carotenoid content of plants, including PAC ( Psy- 2A- TP - CrtI ) multiple expression genes. In the composition of the present invention, the PAC multiple expression gene may be composed of the nucleotide sequence of SEQ ID NO: 1. The composition of the present invention includes a PAC multi-expression gene as an active ingredient, and the carotenoid content of the plant can be increased by transforming the plant with the PAC multi-expression gene. The plant is as described above.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Experimental Method

Production of soybean transformation vector

Entry- PAC from Dr. Sun-Hha Ha, RDA, RDA (Psy-2A-CrtI). Invitrogen's Gateway ^® to insert pre-populated Entry- PACs into the destination vectors pB2GW7.0 and pB2GW7.0-β (the vector replacing the 35S promoter with the β-conglycinin promoter) ^. LR Clonase ^TM Enzyme mix Kit was used. Next, Invitrogen's DH5α was transformed using competent cells. PB2GW7.0 - PAC prepared above pB2GW7.0-β - PAC Vector is Agrobacterium tumefaciens tumefaciens ) EHA105.

Soybean transformant production and T _One  Seed production

Seed disinfection and Immersion

Seeds were sterilized for 20 hours in chlorine gas generated by mixing strong hydrochloric acid (5 ml of 12N HCl) and lacx (100 ml of 12% sodium hypochlorite), followed by secondary sterilization with stirring for 1 minute in 1% sodium hypochlorite. Was done. And washed three times with sterilized water every 10 minutes. The sterilized seeds were placed in a 50 ml tube, and sterilized water was poured therein, followed by soaking at room temperature for 20 hours.

Agrobacterium tumefaciens ( Agrobacterium tumefaciens ) Preparation

pB2GW7.0 - PAC And pB2GW7.0-β - PAC Agrobacterium tumefaciens EHA105 strain with the vector inserted was used. The strain was treated with solid YEP medium (50 mg / l of spectinomycin, 25 mg / l of rifampicin, 10 g / l of peptone, 5 g / l of sodium chloride, 5 g / l of yeast extract, 1.5% (w / v) of agar, pH). 7.0), streaking and cultured at 25 ℃ single colony was added to 10 ml of liquid YEP medium containing the same antibiotics and stirred at 250 rpm at 25 ℃ until OD ₆₅₀ 0.8. 10 ml of 30% glycerol stock solution was added to the mature culture solution, and vortexed. 1 ml of each 1.5 ml tube was rapidly cooled with liquid nitrogen and stored at -70 ° C. One day prior to inoculation, 1 ml of the stored Agrobacterium tumefaciens (competence cells) was placed in 200 ml of liquid YEP medium containing antibiotics and stirred at 250 rpm at 25 ° C. until OD ₆₅₀ reached 0.8-1.0. It was.

On the day of the inoculation, 200 ml of liquid YEP medium was divided into 50 ml each, and centrifugation was carried out at 20 ° C and 3,270 x g for 10 minutes. Agrobacterium tumefaciens pellets in each tube were transferred to a liquid coculture medium (B2 salt 0.32 g / L, benzyl adenine 1.67 mg / L, N-morpholinoethane sulphonic acid (MES) 20 mM, gibberellic acid 0.25 Mg / l, acetosyringone (0.2 mM), L-cysteine (3.3 mM), sodium thiosulfate (1.0 mM), DTT (1.0 mM), sucrose (3% and pH 5.4) were suspended in high and low concentrations. The high concentration concentrate was prepared by adding 5 ml of liquid coculture medium and 15 ml of liquid coculture medium.

Inoculation and Co-culture

The scalpel was inserted between the cotyledons of the soaked seeds, and the seeds were cut vertically until the hypocotyl was removed. After cutting the hypocotyl from about 1 cm below the cotyledon, one side of the hypocotyl was wound 8-10 times with a surgical knife (＃ 11 blade). At this time, 5 ml concentrated solution was applied to the surgical knife, and then the target site was wounded. Approximately 50 explants were placed in a tube containing 15 ml concentrate and inoculated for 30 minutes after 20 seconds of sonication, 30 seconds of vacuum treatment using a desiccator and diaphragm pump.

The explants were removed from the tube and placed on sterile filter paper to remove water, and then a sheet of filter paper was also placed on a solid co-culture medium (same as liquid co-culture medium, agar 0.5%) and the axial side down 10 The object has been placed. After sealing with a micropore, the cells were incubated for 5 days at 25 ° C for 18 hours.

Washing and Shinshu  Judo

Liquid 1/2 shoot induction medium (B5 salt 1.6 g / l, benzyl adenine 835 μl / l, MES 1.5 mM, sucrose 1.5%, Cytoxim 250 mg / l, vancomycin 50 mg /) for sterilization after 5 days coculture. L, 100 mg / l of tickarcillin and pH 5.6) was briefly washed for 10 minutes. The explants were placed on the filter paper and drained, and then the shoot-derived medium without antibiotics was selected. ① (B5 salt 3.2 g / l, benzyldenine 1.67 mg / l, MES 3 mM, agar 0.6%, sucrose 3%, cytotaxime 250 Mg / l, vancomycin 50 mg / l, ticacillin 100 mg / l and pH 5.6), with five individuals per plate fixed to the medium and the part to be redifferentiated with the flat side facing up at an angle of 30 ° C. (flat side up) It was wound. Each plate was sealed with micropores and incubated at 25 ° C for 18 hours.

Two weeks later, the explants from which shoots appeared were selected as shoot-derived medium containing antibiotics-② (B5 salt 3.16 g / L, MES 3 mM, benzyl adenine 1.67 mg / L, sucrose 3%, agar 0.6%, cytotaxime 500 Mg / l, DL-phosphinothricin 10 mg / l and pH 5.6), at which time the rest except the shoots were cut off and the axillary axis was abutted down.

Shinshu  kidney

Two weeks later, the browned shoot / paddle pad was cut with a surgical knife (＃ 15 blade) and the shoot kidney medium (4.4 g / l MS salt, MES 3 mM, 0.5 mg / l MS salt) , 50 mg / l asparagine, 100 mg / l pyroglutamic acid, 0.1 mg / l indoleacetic acid, 1 mg / l zeatin-riboside, 3% sucrose, 0.8% agar, cells Taxi 250 mg / L, vancomycin 50 mg / L, ticasillin 100 mg / L, DL-phosphinothricin 5 mg / L, pH 5.6). Every two weeks, the new shoot kidney medium was transferred to a new shoot medium, and the browning part of the shoot was removed with the top of a surgical knife (＃ 15 blade), and the shoot pad was scraped off little by little to allow the medium to be absorbed well.

Root Formation and Leaf Painting leaf painting )

Rooting medium (4.4 g / l MS salt, MES 3 mM, Asparagine 25 mg / l, Pyroglutamic acid 25) Mg / l, sucrose 3%, agar 0.8%, cytotaxime 50 mg / l, vancomycin 50 mg / l, ticasillin 50 mg / l, pH 5.6). At this time, the lower part of the elongated shoots, which were cut and separated, was soaked in 1 mg / ml indolebutyric acid for 3 minutes, removed, and placed in a test tube containing rooting medium. After 1 ~ 2 weeks, if more than 2 roots come out, wash the medium with tertiary distilled water, and then put small soil (Bio Plug No. 2, Heungryong Seedling) and Vermiculite 2: 1 in small pots (6 cm × 6 cm × 5.6). Cm). The pots were again placed in a magenta box and grown at 25 ° C for 18 hours. After 10 days, leaf painting was performed with 100 mg / L DL-phosphinothricin.

Seed harvest

Plants whose genes were identified by leaf painting were transferred to large pots (20 cm × 20 cm × 20 cm), planted, and covered with 10 small holes in a transparent plastic cover (12 cm × 12 cm × 18 cm). When the cover was removed the seeds were harvested.

Confirmation of introduction of the inserted gene

PCR  And Southern analysis

The leaves of the transformants were isolated from total genomic DNA by CTAB (certyltrimethyl-ammonium bromide) method. PCR was performed using the sequences of PAC , the transgene and BAR, the select antibiotic gene. The primers used are as follows. PAC -F (5'-AAT CCC TTC GCC ACC TCA TCC ATT-3 '; SEQ ID NO: 2) and PAC -R (5'-CAT GTA ATG CTG CTC AAG GTA CGC-3'; SEQ ID NO: 3), BAR - F (5'-ATG AGC CCA GAA CGA CGC CC-3 '; SEQ ID NO: 4) and BAR - R (5'-TCA GAT TTC GGT GAC GGG CA-3 '; SEQ ID NO: 5). In addition, PCR was performed on the left board (LB) and RB (right board) parts to determine whether T-DNA was inserted. The primers used are as follows. The LB portion is left board-F (5'-TGG CTG GTG GCA GGA TAT ATT GTG-3 '; SEQ ID NO: 6) and BAR -R (5'-AGA CAA GCA CGG TCA ACT TCC GTA-3'; SEQ ID NO: 7 ) And the RB portion is PAC -F (5'-AAT CCC TTC GCC ACC TCA TCC ATT-3 '; SEQ ID NO: 2) and right board-R (5'-TTA AAC TGA AGG CGG GAA ACG ACA-3' SEQ ID NO: 8) was used.

About 30 μg genomic DNA was digested overnight with Hind III for light bean and each transformant line. The cleaved DNA was electrophoresed on a 1% agarose gel. The separated DNA was transferred to a nylon membrane (Hybond-N ⁺ , Amersham, UK) using capillary action. The nylon membrane to which the DNA was attached was a probe labeled with [32P] dCTP and Ladderman ^™. Hybridization at 55 ° C. for 24 hours using a labeling kit (Takara, Japan) with a solution containing 0.25 M sodium chloride, 0.1 M sodium phosphate (pH 7.2), 0.25 mM EDTA, 7% SDS and 50% formamide ) The membrane was washed for 10 minutes at 55 ° C. with 2 × SSC / 0.1% SDS and exposed to X-ray film at −80 ° C. for 3 days.

RT - PCR

Total RNA was extracted from leaves and seeds of light beans and transformants using Plant RNA Purification Reagent (Invitrogen, Carlsbad, CA). RT-PCR was performed using Maxime RT-PCR PreMix (iNtRON, KOREA). Introduction Gene Semi-quantitative RT-PCR was performed to determine the transcription level of PAC and BAR , a selection gene.

Carotenoid extraction and HPLC  analysis

Bean seeds were ground finely by the methods of Ye et al. (Science, 287, 303-305 (2000)) and Paine et al. (Nat. Biotechnol, 23, 482-487 (2005)). Carotenoids were extracted from the 0.1 g sample using acetone containing 0.1% butyl hydroxytoluene (BHT). After hydrolysis, the organic layer of the extract containing diethyl ether and 0.1% BHT was vacuum dried with liquid nitrogen. The remainder was dissolved in acetone and filtered through a 0.45-μm Teflon PTFE membrane filter before analysis by HPLC. Extraction of all extracts was performed under gentle light to avoid degradation of protein pigments. HPLC systems are auto-sampler, column oven, binary pump (Agilent, Santa Clara, CA, USA), YMC ODS C-30 column (3 lm, 4.6 mm × 250 mm; YMC Europe, Germany). UV-Visible detectors are used to measure lycopene at 472 nm and α-carotene, β-carotene, β-cryptoxanthin, lutein and zeaxanthin at 450 nm. It became. Chemstation software (Agilent) was used to run the HPLC system and calculate the data. The total amount of carotenoids calculated the sum of lycopene, α-carotene, β-carotene, β-cryptoxanthin, lutein and zeaxanthin.

Example  1: Transformant Vector and Transformant Production

Two transgenic vectors were constructed using a β-conglycine promoter that specifically reacts with seeds and a 35S promoter that is overexpressed in all plants (FIG. 1). The transformants inserted with the PAC gene produced 21 transformants (β- PAC ) and 19 transformants (35S- PAC ), respectively, through differentiation of plants as shown in FIG. 2.

Example  2: T _One And T ₂ Color check of seeds

β- PAC T ₁ seeds were harvested from T ₀ transformants. Four lines of harvested T ₁ seeds were able to obtain red colored seeds. On the other hand, T ₁ seed was harvested from the 35S- PAC T ₀ transformant, but no red seed was obtained in a single line. T ₂ seeds were harvested by sowing the secured T ₁ seeds. As a result, the T ₂ seed also exhibited a reddish color (FIG. 3).

Example  3: Confirmation of insertion gene

β- PAC T ₀ Transformants and 35S-PAC T ₀ PCR, RT-PCR and Southern blotting were performed to identify the insert genes in the transformants (FIGS. 4-6). RT-PCR (FIG. 7) was also performed to confirm the expression of the transgene in the seed.

As a result of PCR, insertion of PAC, which is a transgene, and Bar, a selection antibiotic, were confirmed in most transformants. In addition, it was confirmed that the left board (LB) and RB (right board) portion was also amplified. Among the transformants, transformants showing red strains and non-transformants were selected for Southern blotting. As a result, most of the transformants showed two or three copies. Finally, RT-PCR was performed on the leaves and seeds using the same transformants tested for Southern blotting. The expression of PAC, which is the transgene, was higher in the leaves than in the seeds.

Example  4: β- PAC and 35S- PAC Carotenoid Components of Transformants

The carotenoid analysis of seeds obtained from two kinds of transformants showed β- PAC compared to Gwangan (WT, wild type) and empty vector (EV). The total carotenoid content of T ₂ seeds was found to increase by 20 times and as much as 36 times. In contrast, the total carotenoid content of 35S- PAC T ₂ seeds increased 1.5- or 2.2-fold. It is interesting to note that the results increase not only the β-carotene content but also the α-carotene content (Table 1).

<110> Dong-A University Research Foundation For Industry-Academy Cooperation <120> PAC multiple expression gene increasing carotenoid content of          plant and uses according <130> PN11139 <160> 8 <170> Kopatentin 1.71 <210> 1 <211> 2952 <212> DNA <213> Artificial Sequence <220> <223> PAC <400> 1 atgtctgttg ccttgttatg ggttgtttct ccttgtgacg tctcaaacgg gacaggattc 60 ttggtatccg ttcgtgaggg aaaccggatt tttgattcgt cggggcgtag gaatttggcg 120 tgcaatgaga gaatcaagag aggaggtgga aaacaaaggt ggagttttgg ttcttacttg 180 ggaggagcac aaactggaag tggacggaaa ttttctgtac gttctgctat cgtggctact 240 ccggctggag aaatgacgat gtcatcagaa cggatggtat atgatgtggt tttgaggcag 300 gcagccttgg tgaagagaca gctgagatcg accgatgagt tagatgtgaa gaaggatata 360 cctattccgg ggactttggg cttgttgagt gaagcatatg ataggtgtag tgaagtatgt 420 gcagagtacg caaagacgtt ttacttagga acgatgctaa tgactccgga gagaagaaag 480 gctatctggg caatatacgt atggtgcagg agaacagacg aacttgttga tggtccgaat 540 gcatcacaca ttactccggc ggccttagat aggtgggaag acaggctaga agatgttttc 600 agtggacggc catttgacat gctcgatgct gctttgtccg acacagtttc caaatttcca 660 gttgatattc agccattcag agatatgatt gaaggaatgc gtatggactt gaggaagtca 720 agatacagaa actttgacga actataccta tattgttatt acgttgctgg tacggttggg 780 ttgatgagtg ttccaattat gggcatcgca cctgaatcaa aggcaacaac ggagagcgta 840 tataatgctg ctttggcttt ggggatcgca aatcagctga ccaacatact tagagatgtt 900 ggagaagatg ccagaagagg aagagtctat ttgcctcaag atgaattagc acaggcaggt 960 ctatccgacg aagacatatt tgctggaaga gtgaccgata aatggagaat cttcatgaag 1020 aaacaaattc agagggcaag aaagttcttt gacgaggcag agaaaggagt gaccgaattg 1080 agcgcagcta gtagatggcc tgtgttggca tctctgctgt tgtaccgcag gatactggac 1140 gagatcgaag ccaatgacta caacaacttc acaaagagag cttatgtgag caaaccaaag 1200 aagttgattg cattacctat tgcatatgca aaatctcttg tgccttctac aagaacactg 1260 cagctcctca acttcgacct cctcaagctc gccggcgacg tcgagagcaa cgacggcccg 1320 ggcatggccc cctccgtgat ggcgtcgtcg gccaccaccg tcgctccctt ccaggggctc 1380 aagtccaccg ccggcatgcc cgtcgcccgc cgctccggca actccagctt cggcaacgtc 1440 agcaatggcg gcaggatcag gtgcatgcag gccatggaac caactacggt aattggtgca 1500 ggcttcggtg gcctggcact ggcaattcgt ctacaagctg cggggatccc cgtcttactg 1560 cttgaacaac gtgataaacc cggcggtcgg gcttatgtct acgaggatca ggggtttacc 1620 tttgatgcag gcccgacggt tatcaccgat cccagtgcca ttgaagaact gtttgcactg 1680 gcaggaaaac agttaaaaga gtatgtcgaa ctgctgccgg ttacgccgtt ttaccgcctg 1740 tgttgggagt cagggaaggt ctttaattac gataacgatc aaacccggct cgaagcgcag 1800 attcagcagt ttaatccccg cgatgtcgaa ggttatcgtc agtttctgga ctattcacgc 1860 gcggtgttta aagaaggcta tctaaagctc ggtactgtcc cttttttatc gttcagagac 1920 atgcttcgcg ccgcacctca actggcgaaa ctgcaggcat ggagaagcgt ttacagtaag 1980 gttgccagtt acatcgaaga tgaacatctg cgccaggcgt tttctttcca ctcgctgttg 2040 gtgggcggca atcccttcgc cacctcatcc atttatacgt tgatacacgc gctggagcgt 2100 gagtggggcg tctggtttcc gcgtggcggc accggcgcat tagttcaggg gatgataaag 2160 ctgtttcagg atctgggtgg cgaagtcgtg ttaaacgcca gagtcagcca tatggaaacg 2220 acaggaaaca agattgaagc cgtgcattta gaggacggtc gcaggttcct gacgcaagcc 2280 gtcgcgtcaa atgcagatgt ggttcatacc tatcgcgacc tgttaagcca gcaccctgcc 2340 gcggttaagc agtccaacaa actgcagact aagcgcatga gtaactctct gtttgtgctc 2400 tattttggtt tgaatcacca tcatgatcag ctcgcgcatc acacggtttg tttcggcccg 2460 cgttaccgcg agctgattga cgaaattttt aatcatgatg gcctcgcaga ggacttctca 2520 ctttatctgc acgcgccctg tgtcacggat tcgtcactgg cgcctgaagg ttgcggcagt 2580 tactatgtgt tggcgccggt gccgcattta ggcaccgcga acctcgactg gacggttgag 2640 gggccaaaac tacgcgaccg tatttttgcg taccttgagc agcattacat gcctggctta 2700 cggagtcagc tggtcacgca ccggatgttt acgccgtttg attttcgcga ccagcttaat 2760 gcctatcatg gctcagcctt ttctgtggag cccgttctta cccagagcgc ctggtttcgg 2820 ccgcataacc gcgataaaac cattactaat ctctacctgg tcggcgcagg cacgcatccc 2880 ggcgcaggca ttcctggcgt catcggctcg gcaaaagcga cagcaggttt gatgctggag 2940 gatctgattt ga 2952 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PAC-F primer <400> 2 aatcccttcg ccacctcatc catt 24 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PAC-R primer <400> 3 catgtaatgc tgctcaaggt acgc 24 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> BAR-F primer <400> 4 atgagcccag aacgacgccc 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> BAR-R primer <400> 5 tcagatttcg gtgacgggca 20 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> left board-F primer <400> 6 tggctggtgg caggatatat tgtg 24 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> BAR-R primer <400> 7 agacaagcac ggtcaacttc cgta 24 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> right board-R primer <400> 8 ttaaactgaa ggcgggaaac gaca 24

Claims (10)

Promoter and pepper (Capsicum annuum) derived Psy (phytoene synthase) gene, the FMD virus (foot-and-mouth disease virus ) 2A-derived nucleotide sequence, rice (Oryza sativa ) -derived TP (transpeptide) gene and Erwinia uredovora ) transfected plant cells with a recombinant vector comprising a PAC ( Psy- 2A- TP - CrtI ) multi-expressing gene in which the carotene desaturase ( CrtI ) gene is sequentially linked in the 5 'to 3' direction. A method of increasing the carotenoid content of a plant comprising overexpressing multiple expression genes. The method of claim 1, wherein the PAC multiple expression Gene is a method of increasing the carotenoid content of a plant, characterized in that consisting of the nucleotide sequence of SEQ ID NO: 1. The method of claim 1, wherein the promoter is a β-conglycinin promoter. The method of claim 1, wherein the carotenoid is α-carotene or β-carotene. Promoter and pepper (Capsicum annuum) derived Psy (phytoene synthase) gene, the FMD virus (foot-and-mouth disease virus ) 2A-derived nucleotide sequence, rice (Oryza sativa ) -derived TP (transpeptide) gene and Erwinia uredovora ) transforming plant cells with a recombinant vector comprising a PAC ( Psy- 2A- TP - CrtI ) multi-expressing gene in which a carotene desaturase ( CrtI ) gene is sequentially linked in a 5 'to 3'direction; And
Method for producing a plant carotenoid content is increased compared to the wild type comprising the step of regenerating the plant from the transformed plant cells. Transgenic plant with increased carotenoid content compared to wild type produced by the method of claim 5. The transformed plant according to claim 6, wherein the plant is a dicotyledonous plant. The method of claim 7, wherein the dicotyledonous plant Glycine max ) transgenic plant, characterized in that. Seeds of plants according to claim 6. A composition for increasing the carotenoid content of plants, comprising PAC ( Psy- 2A- TP - CrtI ) multiple expression genes.

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