KR20110052865A - ß CAROTENE HYDROXYLASE GENE INCREASING ß -CAROTENE CONTENT OF PLANTS AND USES THEREOF - Google Patents

Abstract

PURPOSE: A beta-carotene hydroxylase gene derived form Ipomoea betatas (L.) Lam is provided to selectively enhance beta-carotene content and to develop a plant with antioxidation. CONSTITUTION: A beta-carotene hydroxylase protein derived from Ipomoea betatas (L.) Lam has an amino acid of sequence number 2. A gene encoding the beta-carotene hydroxylase protein contains a base sequence of sequence number 1. A recombinant vector contains the gene encoding the beta-carotene hydroxylase protein. A host cell is transformed by the recombinant vector.

Description

Beta-carotene hydroxylase gene increasing β-carotene content of plants and uses approximately}

The present invention relates to a beta-carotene hydroxylase gene derived from sweet potato varieties of high sulfur cultivars of carotenoids, and specifically, inhibits the expression of beta-carotene hydroxylase, an enzyme that degrades beta-carotene. It relates to a method of increasing the content of beta carotene in the plant.

As the changes and levels of various dietary lifestyles have improved since the economic growth, consumers' desire for functional health foods has increased, and functionalities such as antioxidants, beta-carotene, and dietary fiber have become known.

The nutritional value of sweet potato roots is carbohydrates, which are mostly energy sources except water, which are high calorie foods, especially in comparison with other crops containing beta-carotene, various minerals, vitamins and dietary fiber, and sweet potato leaves and petioles are vegetables. High value for use.

In addition, it contains a large amount of vitamins B and C, Ca, Fe, etc., and has various polyphenols such as flavonoids such as anthocyanin and chlorogenic acid (Bovell-Benjamin 2007, Adv Food Nut Res 52: 1-). 59). Yellow sweet potato varieties contain high carotenoids and play an important role in overcoming vitamin A deficiency (VAD) worldwide. In developing countries, VAD causes temporary and permanent blindness and is particularly fatal to children, pregnant women, and lactating women. Many people around the world suffer from this VAD, which can be overcome by taking Provitamin A (betacarotene or other carotenoids) (Stephenson et al. 2000, Parasitology 121 Suppl: S5-22). Already more than 90 years ago, it was known that sweet potatoes overcome VAD in rats (Steenbock 1919, Science 50: 352-353), and in Kenya, yellow sweet potatoes are the most economical beta carotene available in the year and are widely promoted. (Solomons and Bulux 1997, Eur J Clin Nutr 51 Suppl 4: S39-45).

Sweet potatoes are a much better source of vitamin A than most other plants, since most of the carotenoids are made up of beta-carotene, which has the highest conversion to vitamin A. Thus, Van Jaarsveld (2005, Am J Clin Nutr 81: 1080-1087) concluded that increasing the consumption of yellow sweet potatoes is the most efficient strategy for eliminating vitamin A deficiency through food in developing countries. Carotenoids, which appear in red, yellow and orange colors, are produced through the biosynthesis of isoprenoids in plants and are known to play an important role in eliminating lipid oxidative radicals and reactive oxygen species (Ben-Amotz and Fishler 1998, Radiat Environ Biophys 37). : 187-193).

Carotenoids in plants are essential components of photosynthesis, volatile components of fruits and flowers, precursors of ABA, a plant hormone, and precursors of provitamin A, which are very useful not only for the plant itself but also for humans and animals. Carotenoids, such as beta-carotene, lycopene, and lutein, are potent antioxidants and are important industrial sources of cancer, heart disease, and eye disease.

The present invention is derived from the above requirements, and in the present invention, a novel beta-carotene hydroxylase partial gene derived from sweet potato, and functional activity by selectively increasing the beta-carotene content by controlling the expression of the gene The present invention has been completed by increasing the production of substances and developing plants with increased antioxidant activity.

In order to solve the above problems, the present invention provides a beta-carotene hydroxylase protein derived from sweet potato.

The present invention also provides a gene encoding the protein.

The present invention also provides a recombinant vector comprising the gene.

The present invention also provides a host cell transformed with the recombinant vector.

The present invention also provides a recombinant plant RNAi vector comprising the gene.

The present invention also provides a method for increasing the beta carotene content of a plant using the recombinant plant RNAi vector.

In addition, the present invention is transformed with the recombinant plant RNAi vector, to provide a plant and its seed having increased beta carotene content.

According to the present invention, the transformant that inhibits the expression of the gene encoding the beta-carotene hydroxylase protein derived from the new yellow rice sweet potato of the present invention not only selectively increases the content of beta-carotene, which is a useful bioactive substance, but also has high antioxidant activity. By confirming the activity of the transformed callus of the present invention will be applicable to the development of plants resistant to environmental stress such as high salt.

In order to achieve the object of the present invention, the present invention is sweet potato ( Ipomoea batatas ) beta-carotene hydroxylase protein.

The range of beta carotene hydroxylase proteins according to the present invention includes proteins having an amino acid sequence represented by SEQ ID NO: 2 isolated from sweet potato ( Ipomoea batatas ) and functional equivalents of the proteins. "Functional equivalent" means at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 70% of the amino acid sequence represented by SEQ ID NO: 2 as a result of the addition, substitution, or deletion of the amino acid Is 95% or more of sequence homology, and refers to a protein that exhibits substantially homogeneous physiological activity with the protein represented by SEQ ID NO: 2.

The present invention also provides a gene encoding the beta carotene hydroxylase protein. The gene of the present invention is involved in increasing the beta carotene content of sweet potatoes, and includes both genomic DNA and cDNA encoding beta carotene hydroxylase protein. Preferably, the gene of the present invention may include the nucleotide sequence shown in SEQ ID NO: 1. Variants of the above base sequences are also included within the scope of the present invention. Specifically, the gene is 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, respectively. 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 present invention also provides a recombinant vector comprising a beta carotene hydroxylase gene according to the present invention.

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.

The term "vector" is used to refer to a DNA fragment (s), nucleic acid molecule, which is transferred into a cell. The vector replicates the DNA and can be independently regenerated in the host cell. 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 an appropriate nucleic acid sequence essential for expressing the coding sequence operably linked in a particular host organism. Promoters, enhancers, termination signals and polyadenylation signals available in eukaryotic cells are known.

Preferred examples of recombinant vectors are Ti-plasmid vectors which, when present in a suitable host such as Agrobacterium tumerfaciens, can transfer a portion of themselves, the so-called T-region, to plant cells. 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 a chemical method, which corresponds to all genes capable of distinguishing transformed cells from non-transformed cells. Examples include herbicide resistance genes such as glyphosate or phosphinothricin, kanamycin, G418, bleomycin, hygromycin, chloramphenicol There are antibiotic resistance genes such as, but not limited to.

In the recombinant vector of the present invention, the promoter may be, but is not limited to, CaMV 35S, actin, ubiquitin, pEMU, MAS or histone 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, but 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.

The present invention also provides a host cell transformed with the recombinant vector of the present invention. The host cell capable of continuously cloning and expressing the vector of the present invention in a prokaryotic cell while being stable can be used in any host cell known in the art, for example, E. coli JM109, E. coli BL21, E. coli RR1. , Bacillus genus strains, such as E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus thuringiensis, and Salmonella typhimurium, Serratia marcensons, and various Pseudomonas Enterobacteria such as species and strains.

In addition, when transforming a vector of the present invention to eukaryotic cells, yeast ( Saccharomyce) as a host cell cerevisiae ), insect cells, human cells (eg, CHO cell lines (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cell lines), and plant cells. 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 .

The present invention also provides a recombinant plant RNAi vector comprising the beta carotene hydroxylase gene according to the present invention. RNA interfernce is a target gene by introducing a double strand RNA (dsRNA) composed of a sense RNA having a sequence homologous to an mRNA of a target gene and an antisense RNA having a complementary sequence thereto. It is a phenomenon that can inhibit protein expression by inducing degradation of mRNA. The gene knock-down method using RNAi is a method that has been in the spotlight recently because of its simplicity and excellent effect of inhibiting gene expression. The recombinant plant RNAi vector is preferably described in FIG. 5, but is not limited thereto.

The present invention also relates to a method for transforming a plant into a recombinant plant RNAi vector comprising the beta-carotene hydroxylase gene of the present invention, thereby suppressing the expression of beta-carotene hydroxylase. It provides a way to increase. The plant is preferably sweet potato.

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 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 plant infiltration 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. 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.

"Plant cell" used for transformation of a plant may be any plant cell. Plant cells are cultured cells, cultured tissues, cultured organs or whole plants.

"Plant tissue" refers to tissues of differentiated or undifferentiated plants, such as, but not limited to, roots, stems, leaves, pollen, seeds, cancer tissues and various types of cells used in culture, ie single cells, protoplasts. (protoplast), shoots and callus tissue. Plant tissue is planted in planta ) or in organ culture, tissue culture or cell culture.

The present invention also provides a plant which is transformed with a recombinant plant RNAi vector comprising the beta carotene hydroxylase gene of the present invention, thereby increasing the beta carotene content. The plant is preferably sweet potato.

The present invention also provides seeds obtained from plants with increased beta carotene content.

Hereinafter, the present invention will be described in detail.

The present invention provides sweet potato derived beta-carotene hydroxylase partial cDNA. In the partial length of the beta-carotene hydroxylase gene of the present invention, 371 bp of cDNA encodes 123 amino acids (FIG. 1). Blast of the gene in the database showed high homology with beta-carotene hydroxylase of various plants (Fig. 2). In addition, when the amino acid sequence of the coding portion of the beta-carotene hydroxylase of the present invention was examined by blast (BlastX), it showed the highest homology of 99% with the putative gene of Ipomoea nil, Diospyros kaki , Gentian lutea, coffee (Coffee arabica), citrus (Citrus unshiu) and the like also showed a high homology of more than 80% (Fig. 3).

Beta carotene hydroxylase gene of the present invention was confirmed that the gene is strongly expressed in the leaves and roots excluding the stem (Fig. 4).

RNAi vectors were prepared for inhibiting the expression of the beta-carotene hydroxylase gene of the present invention (FIG. 5).

Genomic DNA was extracted from the callus transformed with the RNAi vector for inhibiting the expression of the beta-carotene hydroxylase gene of the present invention and analyzed by PCR. For beta-carotene hydroxylase RNAi transformants, PCR products were identified at 3, 5, 6, 7, 10, 23, 38 callus (FIG. 6).

As a result of comparing beta-carotene hydroxylase RNAi transformed callus of the present invention with Yuli callus as a control, it was confirmed that the transformed callus showed a dark yellow phenotype due to the production of beta carotene (FIG. 7). Therefore, it was confirmed that the calli showed yellow color due to the increase in the biosynthesis of beta-carotene by introducing the RNAi vector of the beta-carotene hydroxylase gene produced by cloning in the present invention.

It was confirmed that the expression of beta-carotene hydroxylase gene was actually inhibited through RT-PCR of the transformed callus of the beta-carotene hydroxylase gene of the present invention. In the case of beta-carotene hydroxylase transformed callus, it was confirmed that the expression of the gene was completely suppressed in all callus (5, 7, 38) (Fig. 8).

Beta carotene production was analyzed using HPLC on the transformed callus of the beta carotene hydroxylase gene of the present invention. Peaks not seen in the control, Yumi callus, were seen at retention time 12.5 min, and the comparative analysis with authentic beta carotene confirmed that the newly appeared peak was beta carotene. As a result, it was confirmed that the beta-carotene content of the transformed callus that inhibited beta-carotene hydroxylase expression was increased (FIG. 9).

As a result of investigating DPPH radical scavenging activity showing low molecular weight antioxidant activity in the transformed callus of the beta-carotene hydroxylase gene of the present invention, it was confirmed that the activity was more than 2-4 times higher than that of the control, Yumi. It was found to be similar to the activity of the high-yield rice varieties. Therefore, it was confirmed that the transformant having increased beta-carotene content of the present invention actually increased with low molecular weight antioxidant activity (FIG. 10).

After transformation of the beta-carotene hydroxylase gene of the present invention to 150 mM and 200 mM NaCl, respectively, the degree of oxidation in cells was measured by DAB staining. As a result, it was confirmed that the brown rice of DAB caused by the increase of reactive oxygen species in the control, Yumi callus, progressed much more than the transformed callus (FIG. 11A). Specifically, the measured amount of oxidized DAB showed higher overall oxidation of DAB due to intracellular oxidative stress at 200 mM NaCl treatment than at 150 mM NaCl treatment. In addition, as a result of measuring the amount of DAB oxidized it was confirmed that the decrease from 1/2 to 1/5 or more compared to the control (Fig. 11B). Therefore, the transgenic callus inhibiting the expression of the beta-carotene hydroxylase gene increased the antioxidant activity as the content of the beta-carotene increased, thereby increasing the ability to remove the active oxygen species in the cell.

Therefore, the present invention is expected to be very useful for the development of plants that are resistant to environmental stress as well as functional improvement by increasing the beta carotene content through the regulation of gene expression.

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.

Example  1: sweet potato beta-carotene Hydroxylase  Gene Cloning , Sequencing and softness analysis

Total RNA was isolated from leaves of sweet potato (Ipomoea batatas (L.) Lam) varieties of yellow rice plants using QRNGEN's RNeasy Mini Kit. First cDNA was synthesized using Invitrogen's SuperScript III First-Strand Synthesis System for RT-PCR. To isolate the beta-carotene hydroxylase gene, a blast was carried out with the beta-carotene hydroxylase gene of tomato (Lycopersicon esculentum) on the website of the TIGR Plant Transcript Assemblies (http://plantta.tigr.org/). As a result, a clone having a nucleotide sequence similar to that of beta-carotene hydroxylase gene was found from the EST clone of morning glory (Ipomoea nil) close to sweet potato, and a PCR primer was prepared to clone these genes from sweet potato. In order to use the gateway expression system of Invitrogen, primer sequences (in uppercase letters) were added to the primer sequences. Nucleotide sequence is beta-carotene hydroxylase forward primer (5'- CAAAAAAGCAGGCTNN) aagttccgtatactgagatgttc-3 '; SEQ ID NO: 3) and reverse primer (5'- CAAGAAAGCTGGGTN cactctcctaaaataaggcacgtc-3 '; SEQ ID NO: 4). PCR was performed using Clonetech's advantage2 polymerase, and the PCR product of the expected size was cloned using pGEMeasy cloning vector (promega) and sequenced to confirm sequencing. The partial length of the beta-carotene hydroxylase gene is 371 bp of cDNA encoding 123 amino acids (FIG. 1). The blasting of the genes in the database showed high homology with beta-carotene hydroxylase of various plants (FIG. 2). In addition, when the amino acid sequence of the coding portion of the beta-carotene hydroxylase of the present invention was examined by BlastX, it showed the highest homology of 99% with the putative gene of Ipomoea nil, Diospyros kaki, Gentian lutea, coffee (Coffee arabica), citrus (Citrus unshiu) and the like showed high homology of 80% or more (Fig. 3).

Example  2: Beta Carotene by Sweet Potato Tissue Hydroxylase  Gene expression analysis

RT-PCR was performed to analyze tissue expression patterns of sweet potato beta-carotene hydroxylase gene of the present invention. Total RNA was isolated using QIAGEN's RNeasy Mini Kit, and first cDNA was synthesized using Invitrogen's SuperScript III First-Strand Synthesis System for RT-PCR. The base sequences of the primers are beta carotene hydroxylase forward primers (5'-aagttccgtatactgagatgttc-3 '; SEQ ID NO: 5) and reverse primers (5'-cactctcctaaaataaggcacgtc-3'; SEQ ID NO: 6).

As a result, it was confirmed that the beta carotene hydroxylase gene of the present invention is a gene that is strongly expressed in the leaves and roots except the stem (FIG. 4).

Example  3: sweet potato beta-carotene Hydroxylase  Gene expression of plant expression vector and development of transformant

RNAi vectors were prepared for inhibiting the expression of the beta-carotene hydroxylase gene of the present invention (FIG. 5). First, the gene was cloned into the pDONR207 vector by BP reaction of the pDONR207 vector in which the beta-carotene hydroxylase gene was cloned. Thereafter, RNAi vectors of the beta-carotene hydroxylase gene cloned by the LR reaction of pDONR207 and pH7GWIWG2 (I) RNAi vector were prepared. RNAi vectors were transformed into Agrobacterium mediated Yul callus and antibiotic selected. The transformed callus was genomic DNA was extracted using the G-spin ^TM IIp Genomic DNA Extraction Kit (Int.) Of Intron Biotegon Co., Ltd., and then PCR was analyzed using a gene-specific primer. For beta carotene hydroxylase RNAi transformants, PCR products were identified in 3, 5, 6, 7, 10, 23, 38 transgenic callus (FIG. 6). Therefore, it was confirmed that the beta-carotene hydroxylase RNAi vector was introduced into the callus to be transformed.

Example  4: beta-carotene Hydroxylase  Increased beta carotene content due to inhibition of gene expression

In order to confirm whether the inhibition of the expression of the beta-carotene hydroxylase gene of the present invention substantially increased the content of beta-carotene, the color of the transformed callus confirmed by genomic DNA PCR was compared with the control callus, Yumi, and phenotype. It can be seen that this dark yellow (Fig. 7). In addition, RT-PCR was performed by extracting RNA from the callus transformed by the method of Example 1 to confirm whether the beta-carotene hydroxylase gene expression was inhibited. As a result, it was confirmed that the beta carotene hydroxylase transformed callus completely inhibited the expression of genes in all callus (5, 7, 38) (FIG. 8). As a result, the beta-carotene hydroxylase RNAi vector of the present invention was confirmed that the beta-carotene hydroxylase gene expression was suppressed.

In addition, carotenoid analysis of callus was performed using HPLC to confirm whether the change of the transformed callus color is due to the increase in the content of beta carotene. Absorbance at 450 nm was injected into a C18 column (Alltech, Apollo C18 5u column, 250mm x 4.6mm) in a mobile phase of acetonitrile extracted with 100% acetone, after the control of Yumi callus and transformed callus. Obtained and compared with a standard curve using β-carotene. As a result, a peak at retention time 12.5 min, which was not seen in the control Yulim callus, was seen in the extract of the transformed callus, and it was confirmed that the new peak was β-carotene by comparison with authentic β-carotene (FIG. 9).

Example  5: transformation Callus  Increased antioxidant activity due to increased beta carotene content

Transgenic callus of the beta-carotene hydroxylase gene of the present invention was investigated for DPPH radical scavenging activity showing low molecular antioxidant activity. In order to confirm that beta-carotene has high antioxidant activity, the transformed callus actually increased the antioxidant activity due to the increase of beta-carotene content. Antioxidant activity was analyzed and antioxidant activities were compared after 150 mM and 200 mM NaCl treatment, respectively. Yulmi and the transformed callus were extracted with 100% methanol, and then reacted with 10 mM DPPH solution for 30 minutes to calculate the remaining amount of DPPH. The low molecular weight antioxidant activity was measured and the activity was expressed as a value corresponding to ascorbic acid. As a result, it was confirmed that the activity is more than 2-4 times higher than Yulmi, which is a level similar to the activity of Cinwangmi variety (Hm) having high beta-carotene content (Fig. 10). Therefore, it was confirmed that the transformant having increased beta-carotene content of the present invention actually increased with low molecular weight antioxidant activity.

In addition, the NaCl-treated callus was stained with DAB, which reacts with reactive oxygen species, and then changed to brown. As a result, it was confirmed that the brown rice of DAB caused by the increase of reactive oxygen species in the control, Yumi callus, progressed much more than the transformed callus (FIG. 11A). Specifically, as a result of measuring the amount of DAB oxidized, the overall oxidation of DAB due to oxidative stress in cells was higher in NaCl treatment of 200 mM than in NaCl treatment of 150 Mm. In addition, as a result of measuring the amount of DAB oxidized it was confirmed that the decrease from 1/2 to 1/5 or more compared to the control (Fig. 11B). Therefore, it was confirmed that the transgenic callus inhibiting the expression of the beta carotene hydroxylase gene increased the antioxidant activity as the beta carotene content increased and the ability to remove the active oxygen species in the cell increased.

1 is a partial sequence of the beta-carotene hydroxylase gene derived from sweet potato (breed yellow rice) of the present invention.

Figure 2 shows the amino acid sequence of the deduced protein of the sweet potato beta-carotene hydroxylase gene of the present invention and various plants (morning glory, tomato, grapes, beans, poplar, coffee, carrots, potatoes, citrus, Arabidopsis, cabbage, corn, Figure 9 shows the comparison of amino acid sequences of beta-carotene hydroxylase genes of 9 species).

FIG. 3 is a diagram showing a flexible relationship between beta carotene hydroxylase genes compared with FIG. 2.

Figure 4 is a picture of the beta-carotene hydroxylase gene of the present invention in sweet potato plants by electrophoresis by performing RT-PCR. L, leaf; S, stem; FR, fibrous roots (root); SR, storage roots.

5 is a diagram of the production of a transforming vector (RNAi) for inhibiting the expression of beta carotene hydroxylase gene in plants.

FIG. 6 is a diagram illustrating a selection of RNAi vector transformed callus of beta carotene hydroxylase gene by genomic DNA PCR.

Figure 7 is a diagram showing the phenotype of the transformed callus due to the increase in beta carotene content of yellow.

8 shows the results of investigating the expression of beta carotene hydroxylase gene in the transformed callus through RT-PCR.

9 is a result of analyzing the production of beta carotene through the HPLC analysis of the transformed callus.

10 is a result of analyzing the low molecular weight antioxidant activity through the investigation of DPPH radical scavenging activity in the transformed callus.

FIG. 11 shows the results of analyzing the phenotype (A) after treating 150 mM and 200 mM NaCl in the transformed callus, respectively, and analyzing the oxidized DAB content (B).

<110> Korea Research Institute of Bioscience and Biotechnology <120> Beta-carotene hydroxylase gene increasing beta-carotene content          of plants and uses according <130> PN09247 <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 371 <212> DNA <213> Ipomoea batatas <400> 1 aagttccgta tactgagatg ttcgggacat ttgctctctc cgtcggagcc gcggtgggaa 60 tggaattctg ggccagatgg gctcacagag cgctatggca cgcttcgttg tggcacatgc 120 acgagtctca ccacaaacca agagaaggac cgttcgagct caacgatgtt ttcgccataa 180 ctaacgctgt ccctgccatc gctctcctct cctacggttt cttccacaaa ggtctcgttc 240 ctggcctctg cttcggagct gggcttggaa tcacagtgtt cgggatggcc tacatgttcg 300 tccacgatgg cctcgttcac aagcgattcc ccgtggggcc catcgccgac gtgccttatt 360 ttaggagagt g 371 <210> 2 <211> 123 <212> PRT <213> Ipomoea batatas <400> 2 Val Pro Tyr Thr Glu Met Phe Gly Thr Phe Ala Leu Ser Val Gly Ala   1 5 10 15 Ala Val Gly Met Glu Phe Trp Ala Arg Trp Ala His Arg Ala Leu Trp              20 25 30 His Ala Ser Leu Trp His Met His Glu Ser His His Lys Pro Arg Glu          35 40 45 Gly Pro Phe Glu Leu Asn Asp Val Phe Ala Ile Thr Asn Ala Val Pro      50 55 60 Ala Ile Ala Leu Leu Ser Tyr Gly Phe Phe His Lys Gly Leu Val Pro  65 70 75 80 Gly Leu Cys Phe Gly Ala Gly Leu Gly Ile Thr Val Phe Gly Met Ala                  85 90 95 Tyr Met Phe Val His Asp Gly Leu Val His Lys Arg Phe Pro Val Gly             100 105 110 Pro Ile Ala Asp Val Pro Tyr Phe Arg Arg Val         115 120 <210> 3 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 3 caaaaaagca ggctnnaagt tccgtatact gagatgttc 39 <210> 4 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 4 caagaaagct gggtncactc tcctaaaata aggcacgtc 39 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 5 aagttccgta tactgagatg ttc 23 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 6 cactctccta aaataaggca cgtc 24  

Claims (11)

Ipomoea , consisting of the amino acid sequence of SEQ ID NO: 2 beta-carotene hydroxylase protein from batatas ). Gene encoding the beta-carotene hydroxylase protein of claim 1. According to claim 2, wherein the gene is a gene, characterized in that consisting of the nucleotide sequence of SEQ ID NO: 1. A recombinant vector comprising the gene of claim 2. A host cell transformed with the recombinant vector of claim 4. Recombinant plant RNAi vector comprising the gene of claim 2. A method of increasing the beta carotene content of a plant comprising the step of transforming a plant with the recombinant plant RNAi vector of claim 6, thereby inhibiting the expression of beta carotene hydroxylase. 8. The method of claim 7, wherein the plant is sweet potato. A plant transformed with the recombinant plant RNAi vector of claim 6, wherein the plant has increased beta carotene content. The plant of claim 9, wherein the plant is sweet potato. Seeds of plants according to claim 9.

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