KR20130054479A - Method for preparing transgenic alfalfa plant with increased anthocyanin content and the plant thereof - Google Patents

Abstract

PURPOSE: A system for preparing a large amount of anthocyanin is provided to easily obtain natural pigments and to develop a health functional food industry, a cosmetic industry, a medical product industry, and a dye industry. CONSTITUTION: IbMYB1a gene has a base sequence in sequence number 1. A method for preparing a transgenic alfalfa plant with enhanced anthocyanin content comprises: a step of transforming an alfalfa plant cell with a recombinant vector containing Ipomoea batatas-derived IbMYB1a gene; and a step of redifferentiating an alfalfa plant from the transformed plant cells. A feed composition contains the transgenic alfalfa plant. A composition for enhancing anthocyanin in the alfalfa plant contains Ipomoea batatas-derived IbMYB1a gene.

Description

Method for preparing transgenic alfalfa plants with increased anthocyanin content and resulting alfalfa plants {Method for preparing transgenic alfalfa plant with increased anthocyanin content and the plant}

The present invention for transforming a recombinant vector comprising a method for producing a transgenic alfalfa plant of the anthocyanin content is increased and that, more specifically, IbMYB1a gene derived from the sweet potato (Ipomoea batatas) according to alfalfa plants hence the alfalfa plant cells Method of increasing the anthocyanin content of the alfalfa plant comprising the step, transformed alfalfa plants of which the anthocyanin content is increased compared to the control plant comprising transforming the alfalfa plant cells with a recombinant vector comprising the sweet potato-derived IbMYB1a gene A method of preparation, a transformed alfalfa plant with increased anthocyanin content compared to the control plant produced by the method and its seeds, a feed composition comprising the transformed alfalfa plant, and extracting anthocyanin from the transformed alfalfa plant Ha It relates to a composition for promoting anthocyanin content of the alfalfa plant containing the gene of IbMYB1a method of producing anthocyanins from alfalfa plants and potato derived.

Anthocyanins are water-soluble pigment glycosides (glucosides), which are plant-based natural pigments contained in plants, fruits, stems, leaves, and roots. Anthocyanins are usually present in the vacuoles of plants, and have colors such as purple, red, blue, etc., due to the acidic concentration of the cell solution, the chemical structure of the pigment compound, and the bonding state with various metal ions.

The physiological activity of anthocyanin is known to be anti-aging activity, antibacterial activity, antimutagenic activity, anti-cholesterol activity, anti-ulcer activity, antioxidant activity, eye protection activity. It is known to exhibit strong antioxidant activity. Anthocyanins are currently being used in health functional foods, cosmetics, medicines, dyes, etc. due to their physiological activity.

Through the development of biotechnology, researches on new physiological activity of anthocyanin, isolation and function of genes related to anthocyanin biosynthesis, and method of separating anthocyanin from plants have been actively conducted. As a result, it will be possible to produce a large amount of the desired pigment in the plant through the identification of the mechanism of regulation of pigment synthesis of the plant.

In particular, with the development of molecular agriculture, research is being conducted on the mass production of secondary metabolites through tissue culture of various plant gene sources. Studies on natural pigments have been actively studied in anthocyanin, betacyanin, shikonin, and carthamin in Europe and Japan. However, in order to mass-produce pigments, further research on production process, pigment extraction, and purification should be conducted (Mapari et al. 2005 Curr. Opin. Biotechnol. 16, 231-238).

Meanwhile, Korean Patent No. 10-0990842 discloses an anthocyanin mass production system, and Korean Patent No. 10-1018079 discloses a polypeptide, a polynucleotide, and a use thereof having anthocyanin biosynthesis promoting function. Patent No. 2006-0037150 discloses an anthocyanin biosynthesis related gene isolated from a melon, but the method for producing a transformed alfalfa plant having an anthocyanin content increased using a sweet potato-derived transcription factor IbMYB1a gene as described in the present invention. There is no bar.

The present invention has been derived by the above-mentioned demands, and the present inventors have found IbMYB1a derived from sweet potato ( Ipomoea batatas ). It was confirmed that the alfalfa plant transformed with a recombinant vector containing a gene significantly increased the anthocyanin content, thereby enabling the construction of an anthocyanin mass production system and the provision of an anthocyanin content-enhanced feed, thereby completing the present invention. It was.

In order to solve the above problems, the present invention is sweet potato ( Ipomoea It provides a method for increasing the anthocyanin content of alfalfa plants comprising the step of transforming alfalfa plant cells with a recombinant vector comprising the IbMYB1a gene from batatas ).

The present invention also provides a method for producing a transformed alfalfa plant having an anthocyanin content as compared to a control plant comprising transforming alfalfa plant cells with a recombinant vector comprising a sweet potato-derived IbMYB1a gene.

In addition, the present invention provides a transformed alfalfa plant and its seed having increased anthocyanin content compared to the control plant produced by the above method.

The present invention also provides a feed composition comprising the transformed alfalfa plant.

The present invention also provides a method for producing anthocyanins from an alfalfa plant comprising extracting anthocyanins from the transgenic alfalfa plants.

The present invention also provides a composition for improving anthocyanin content of alfalfa plants comprising IbMYB1a gene derived from sweet potato.

The anthocyanin mass production system of the present invention can be easily used to secure natural pigments, and can greatly contribute to the development of functional food industry, cosmetics industry, pharmaceutical industry, dye industry using natural pigments, and to provide feeds with enhanced functionality. .

Figure 1 shows an anthocyanin biosynthesis related transcription factor gene IbMYB1a expression vector schematic diagram.
2 shows alfalfa plants transformed with anthocyanin biosynthesis related transcription factor gene IbMYB1a .
3 shows expression analysis of anthocyanin biosynthesis related structural genes in alfalfa plants transformed with anthocyanin biosynthesis related transcription factor gene IbMYB1a .
Figure 4 shows the results of anthocyanin quantitative analysis of transformed alfalfa plants.
Figure 5 shows the results of examining the production of anthocyanin in tobacco leaves by overexpression of various plant-derived R2R3 type MYB transcription factors.

In order to achieve the object of the present invention, the present invention is a sweet potato ( Ipomoea It provides a method for increasing the anthocyanin content of alfalfa plants comprising the step of transforming alfalfa plant cells with a recombinant vector comprising the IbMYB1a gene from batatas ).

The IbMYB1a protein and gene of the present invention are registered in the National Center for Biotechnology Information (NCBI) (Genebank Accession No. AB444398), but the correlation between the IbMYB1a gene and the anthocyanin content of alfalfa plants is not known at all.

IbMYB1a Gene may be composed of the nucleotide sequence of SEQ ID NO: 1, but is not limited thereto. 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 >

"% 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.

Expression vectors comprising the IbMYB1a gene sequence 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.

A preferred example of the recombinant vector of the present invention is a Ti-plasmid vector capable of transferring a so-called T-region to a plant cell when present in a suitable host, such as Agrobacterium tumefaciens. Other types of Ti-plasmid vectors (see EP 0 116 718 B1) are currently used to transfer hybrid DNA sequences to plant cells or protoplasts in which new plants capable of properly inserting hybrid DNA into the plant's genome can be produced 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 the plant host include viral vectors such as those that can be derived from double-stranded plant viruses (e. G., CaMV) and single- For example, from non -complete plant virus vectors. The use of such vectors may be particularly advantageous when it is difficult to transform the plant host properly.

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, kanamycin, G418, bleomycin, hygromycin, and chloramphenicol. Resistance gene, aadA gene, and the like, but are not limited thereto.

In the recombinant vector of the present invention, the promoter may be, but is not limited to, CaMV 35S, actin, ubiquitin, pEMU, MAS, histone promoter, Clp promoter. The term "promoter " refers to the region of DNA upstream from the 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. Constructive promoters may be preferred in the present invention because the choice of transformants can be made by various tissues at various stages. Thus, the constitutive promoter does not limit the selection possibilities.

In the recombinant vectors of the present invention, conventional terminators can be used, for example nopalin synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, Agrobacterium tumefaciens ( Agrobacterium tumefaciens) Terminator of the octopine gene, and the rrnB1 / B2 terminator of E. coli, but are 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.

In addition, the present invention is sweet potato ( Ipomoea transforming alfalfa plant cells with a recombinant vector comprising the IbMYB1a gene from batatas ); And it provides a method for producing a transformed alfalfa plant with increased anthocyanin content compared to the control plant comprising the step of regenerating the alfalfa plant from the transformed alfalfa plant cells. The control plant refers to an alfalfa plant not transfected with the IbMYB1a gene.

In the production method according to an embodiment of the present invention, the IbMYB1a gene may be composed of the nucleotide sequence of SEQ ID NO: 1, but is not limited thereto.

The method of the present invention comprises the step of transforming plant cells with the recombinant vector according to the present invention, for example, Agrobacterium tumerfaciens ( 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 addition, the present invention provides a transformed alfalfa plant and its seed having increased anthocyanin content compared to the control plant produced by the above method.

The present invention also provides a feed composition comprising the transformed alfalfa plant.

The 'feed composition' comprising the transformed alfalfa plant of the present invention corresponds to the use as a general feed additive, except that the transformed alfalfa plant produces and contains anthocyanins.

The present invention also provides a method for producing anthocyanins from an alfalfa plant comprising extracting anthocyanins from the transgenic alfalfa plants.

In order to extract anthocyanin from the transformed alfalfa plant of the present invention, water or an organic solvent, or a mixed solvent thereof may be used. The organic solvent may be any solvent that can be commonly used, and the extraction method may also be a method commonly used in the art.

In another aspect, the present invention provides a composition for enhancing the anthocyanin content of alfalfa plants comprising the IbMYB1a gene derived from sweet potato ( Ipomoea batatas ) consisting of the nucleotide sequence of SEQ ID NO: 1. The composition of the present invention comprises an IbMYB1a gene consisting of the nucleotide sequence of SEQ ID NO: 1 as an active ingredient, by converting the gene into an alfalfa plant to increase the anthocyanin content of the alfalfa plant.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and the scope of the present invention is not to be construed as being limited by these examples.

Example  1. Anthocyanin Pigmentation Transcription Factors IbMYB1a  Gene Cloning

The gene used to develop the anthocyanin mass production system was used to isolate anthocyanin biosynthesis transcription factor gene IbMYB1a from purple sweet potato (cv. Sinzami Shinjami variety) (Kim CY et al. 2010, Physiologia Plantarum 139: 259-261). ). Total RNA was isolated from tubers of purple sweet potatoes. Total RNA isolated was synthesized cDNA as described using the First-Strand cDNA Synthesis Kit (Fermantas, Canada). Using the synthesized cDNA as a template, the sweet potato IbMYB1a transcription factor was amplified by PCR using an Adavantage 2 polymerase mix (Clontech, Tokyo, Japan). Primer sequences used in this experiment are as follows: IbMYB1a-F: 5'-AGCTAAGAATTTCCGACACCCTTCAATA-3 '(SEQ ID NO: 2), IbMYB1a-R: 5'-GTGAATTTAACGCTTAGCTTAACAGTTCT-3' (SEQ ID NO: 3). PCR amplification was performed by denaturation at 95 ° C. for 2 minutes, 15 seconds at 95 ° C., 30 seconds at 55 ° C., and 2 minutes at 72 ° C. for 30 minutes and final extension at 72 ° C. for 10 minutes. The amplified PCR product was cloned into pGEM-T Easy vector (Promega, USA) and analyzed for sequencing. As a result of sequencing, the cloned IbMYB1a gene was composed of a total of 797bp, ORF (open readin frame) was composed of 750 bp.

Example  2. Preparation of plant overexpression carrier of anthocyanin pigment synthesis transcription factor

In order to determine whether anthocyanin pigment synthesis can be enhanced when the IbMYB1a cloned in Example 1 is overexpressed in a plant, a plant expression vector of pCAMBIA 2300 having kanamycin resistance (Center for Application of Molecular Biology to International Agriculture, Australia) ) A vector was constructed in which the entire open reading frame of the IbMYB1a gene was inserted between the duplicated 35S promoter and the 35S terminator. The resulting overexpressing carrier was named pCam-d35s-IbMYB1a (FIG. 1).

Example  3. Overexpression of Anthocyanin Pigmentation Transcription Factors Alfalfa  making

The pCam-d35s-IbMYB1a plant expression vector prepared in Example 2 was freeze-thaw in Agrobacterium tumefaciens GV3101 (H, R. and Willmitzer L. (1988) Nucleic Acids Res 16: 9877). Transformation method was used. In the present invention, the agrobacterium tumefaciens GV3101 strain was transformed into alfalfa, which is a feed crop, to produce an alfalfa plant transformed with the plant carrier comprising the IbMYB1a gene.

As plant material, 'Vernal' variety was used. Seed sterilization was performed for 30 seconds in 70% ethanol, and then surface sterilization with 30% sodium hypo-chloride solution for 30 minutes with stirring. The sterilized seeds were washed three times with sterile water, transferred to sterilized filter paper to remove water and sterilely sown in 1/2 MS medium and incubated for 7-10 days in a growing room at 24 ± 2 ℃ for 16 hours. Plants were used as plant material for transformation. Basic callus induction medium to induce callus from the hypocotyl of aseptically grown alfalfa seedlings was 1 mg / L 2,4-D (2,4-dichlorophenoxyacetic acid), 0.1 mg / L kinetin, 0.5 g / L L-proline Gamborg B-5 medium with 0.5 g / L MgSO ₄ , 0.5 g / L KNO ₃ , 30 g / L sucrose, and 0.8% agar was used. Tissues were dense and bright green, and the cells were cultured for two weeks with a rapid growth rate and a glossy cell. mg / L 2,4-D, 0.1 mg / L kinetin, 0.5 g / L L-proline, 0.5 g / L MgSO ₄ , 0.5 g / L KNO ₃ , 30 g / L sucrose, 0.8% agar and 100 uM aceto Co-culture in a dark state for 3-5 days in Shiling One (AS).

Cocultured plant tissues were 2 mg / L 2,4-D, 0.2 mg / L kinetin, 0.5 g / L L-proline, 0.5 g / L MgSO ₄ , 0.5 g / L KNO ₃ , 30 g / L sucrose Gamborg B-5 medium to which 0.8% agar was added was selected to induce regeneration by using a selection medium containing 100 mg / L kanamycin and 500 mg / L celltaxime. Shoots formed in regeneration medium were Gamborg B- without added 0.5 g / L L-proline, 0.5 g / L MgSO ₄ , 0.5 g / L KNO ₃ , 30 g / L sucrose, and 0.8% agar plant growth regulator. Regeneration in 5 medium was induced and subcultured every 2 weeks until regeneration plants emerge. The regenerated plants are transplanted into 1/2 MS medium to grow as seedlings, induce root development, to differentiate into complete plants, and then transplanted into soil after being purified in a humidified environment in sterilized horticultural soils. Was grown in a greenhouse (Fig. 2).

Example  4. Transformation Alfalfa Phenotypic  Mutation observation

In each of the transformed alfalfa plants selected in Example 3, the phenotypic difference was significantly higher in the seedling plants than in the non-transformed control group. In the alfalfa plants transformed with IbMYB1a gene, the accumulation of anthocyanin pigments in leaves, stems, and roots was visually confirmed (FIG. 2).

Example 5 Introduction and Expression Pattern Analysis of Genes Related to Anthocyanin Biosynthesis

To confirm the introduction of the anthocyanin pigment synthesis gene, genomic DNA was isolated from the fresh leaf tissues of the transformed alfalfa plants using CTAB. PCR analysis revealed specific primers ( IbMYB1a gene: forward primer 5'-AGCTAAGAATTTCCGACACCCTTCAATA-3 '(SEQ ID NO: 2)) and reverse primers 5'-GTGAATTTAACGCTTAGCTTAACAGTTCT-3' (sequence number 3), kanamycin Gene: PCR analysis was performed using forward primer 5'-AGGACTCTCTGTCATCTCACCTT-3 '(SEQ ID NO: 4) and reverse primer 5'-CTCTTCAGCAATATCACGGGTAG-3' (SEQ ID NO: 5.) PCR amplification was denatured at 95 ° C for 2 minutes, The final extension reaction was carried out at 30 cycles and 10 minutes at 72 ° C. with 1 cycle of 15 seconds at 95 ° C., 30 seconds at 55 ° C. and 1 minute at 72 ° C. As a result, 759 bp of IbMYB1a and 359 bp of kanamycin resistance gene The band size of (Fig. 3A) was confirmed that the T-DNA region of the expression vector was introduced into the genome of the transgenic plant. Treating the genomic DNA (20㎍) of alfalfa as a result of restriction enzyme Hin dⅢ Southern analysis confirmed the one or first or second number of copies in all transgenic plants (Fig. 3B). In the anthocyanins introduced in alfalfa RNA was isolated and Northern blot analysis was performed to confirm that the pigment synthesis transcription factor IbMYB1a gene was transcribed, and as a result, the expression level of the transgene was not found in non-transgenic plants, but in transgenic plants. However, it was observed that the introduced gene was stably expressed (Fig. 3C), which means that the IbMYB1a gene was normally introduced into the transformed plant and is always expressed by the duplicated 35S promoter. Taken together, the above results, IbMYB1a gene is switched stably transfected to express alfalfa For it was found.

Example  6. Mass production of anthocyanins Alfalfa  Anthocyanin Content Analysis

As described by Mehrtens F et al. (2005) Plant Physiol. 138: 1083-1096 for nontransgenic controls and transformed alfalfa plants (alfalfa transformed with a pCam-d35S-IbMYB1a expression vector). Total anthocyanin content was analyzed by the method. Anthocyanins were extracted by sampling plants of three non-transformed control and three transformed alfalfa plants, and the anthocyanin contents were absorbed at 530 nm and 657 nm using an absorbance spectrophotometer, and the following formula was substituted for the total anthocyanin content of each sample. The mean value was measured and expressed as the mean value: ( Q _Anthocyanins = (A ₅₃₀ -0.25 xA ₆₅₇ ) xM ^-1 , M is the g weight of the sample used). As a result, as shown in Figure 4, in the d35S-IbMYB1a-OX alfalfa individuals into which the anthocyanin pigment synthesis transcription factor gene is introduced, it was confirmed that mass production of anthocyanins is 74-129 times more than the control group. 4).

Example  7. Derived from various plant R2R3  type MYB  Investigation of Anthocyanin Production in Tobacco Leaves by Overexpression of Transcription Factors

Anthocyanin production in tobacco leaves (Nicotiana benthamiana) due to overexpression of various plant-derived R2R3 type MYB transcription factors was investigated. As a result, MYB transcription factors derived from sweet potatoes and tomatoes increased anthocyanin synthesis, while MYB transcription factors derived from potatoes, Arabidopsis and grape did not increase anthocyanin synthesis (FIG. 5).

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION)          Korea Research Institute of Bioscience and Biotechnology <120> Method for preparing transgenic alfalfa plant with increased          anthocyanin content and the plant <130> PN11197 <160> 5 <170> Kopatentin 2.0 <210> 1 <211> 750 <212> DNA <213> Ipomoea batatas <400> 1 atggttattt catctgtatg gtcgggatcg tcttcgagag tgagaaaagg ttcatggtcc 60 gaagaagaag accaactttt gagggagtgc attcagaaat atggtgaagg aaaatggcat 120 ctaattcccc ttagagctgg attgaatagg tgcagaaaaa gttgtagatt aagatggttg 180 aattatctcc gtcccgatat aaagagaggc gaatttagtc ccgatgaaat tgatctcatt 240 ctgcgcctcc ataggctctt aggcaacagg tggtcgctta ttgctggaag aattccggga 300 agaacagcaa acgatgtgaa gaatttatgg aacacccatc ttcagaagaa ggtgtctgcc 360 atggcttctt caaggcaaga taattattgg aagggcaaag ccccagaaat cacggaaaac 420 accgtcgtta ggcctcgacc tcggagattc ttaaaggcct catcatctcc gacgactcta 480 ttgaccggaa atgctaccat ggttgcctat gatggtcaac tccaagaaca tatgacgaca 540 caaccggaaa caacgtcgga cttgctaatg gaaaatgtcc aacaaaaaaa cttaacaacc 600 actttgcctt cagcactaga aacaacgcca cacgacaatg tgaagtggtg ggaagatgta 660 ctctccgaca aggaactcaa tgaggaagga caaatctgtt ggagtgagtt tccaactgat 720 atagacctac tgtcagaact gttaagctaa 750 <210> 2 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 agctaagaat ttccgacacc cttcaata 28 <210> 3 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 gtgaatttaa cgcttagctt aacagttct 29 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 aggactctct gtcatctcac ctt 23 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 ctcttcagca atatcacggg tag 23

Claims (9)

Sweet Potato ( Ipomoea A method for increasing the anthocyanin content of an alfalfa plant, comprising transforming alfalfa plant cells with a recombinant vector comprising the IbMYB1a gene from batatas ). The method of claim 1, wherein the IbMYB1a gene, characterized in that consisting of the nucleotide sequence of SEQ ID NO: 1. Sweet Potato ( Ipomoea transforming alfalfa plant cells with a recombinant vector comprising the IbMYB1a gene from batatas ); And
Method for producing a transformed alfalfa plant having an anthocyanin content increased compared to the control plant comprising the step of regenerating the alfalfa plant from the transformed alfalfa plant cells. The method of claim 3, wherein the IbMYB1a gene comprises a nucleotide sequence of SEQ ID NO: 1. Transgenic alfalfa plants with increased anthocyanin content compared to the control plants produced by the method of claim 3. Seeds of alfalfa plants according to claim 5. Feed composition comprising the transformed alfalfa plant of claim 5. A method for producing anthocyanin from an alfalfa plant, comprising extracting anthocyanin from the transformed alfalfa plant of claim 5. Sweet potato ( Ipomoea , consisting of the nucleotide sequence of SEQ ID NO: 1) batatas) a composition for the promotion of, alfalfa plant containing the gene derived from IbMYB1a anthocyanin content.

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