KR101134004B1 - Lycopene ?-cyclase gene increasing ?-carotene content of plants and uses thereof - Google Patents

Abstract

The present invention provides a lycopene ε-cyclase protein derived from sweet potato, a gene encoding the protein, a recombinant vector including the gene, a host cell transformed with the recombinant vector, and the gene. The present invention relates to a recombinant plant RNAi vector, a method for increasing the beta carotene content of a plant using the recombinant plant RNAi vector, a plant transformed with the recombinant plant RNAi vector, and an increase in the beta carotene content, and a seed thereof.

Sweet potato, lycopene ε-cyclase, beta carotene, RNAi vector

Description

Lycopene ε-cyclase gene increasing β-carotene content of plants and uses approximately}

The present invention relates to a lycopene ε-cyclase gene derived from sweet potato varieties of sweet yellow rice varieties having a high carotenoid content, and specifically, expression of lycopene ε-cyclase, an enzyme that produces alpha carotene from lycopene. It relates to a method of increasing the content of beta carotene in the plant by inhibiting.

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 lycopene ε-cyclase 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 lycopene ε-cyclase protein derived from sweet potatoes.

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 which inhibits the expression of the gene encoding the lycopene ε-cyclase 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 ) to provide a lycopene ε-cyclase protein.

The range of lycopene ε-cyclase proteins according to the present invention includes proteins having an amino acid sequence represented by SEQ ID NO: 2 isolated from sweet potatoes ( 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 lycopene ε-cyclase protein. Genes of the invention are involved in increasing the beta-carotene content of sweet potatoes and include both genomic DNA and cDNA encoding lycopene ε-cyclase proteins. 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 the lycopene ε-cyclase 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 CaMV 35S, actin, ubiquitin, pEMU, MAS, or histone promoter, but is not limited thereto. 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 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.

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

The present invention also provides a recombinant plant RNAi vector comprising the lycopene ε-cyclase gene according to the present invention. RNA interfernce is a target gene mRNA 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 the expression of proteins by inducing degradation. 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 beta carotene content of a plant comprising the step of transforming a plant with a recombinant plant RNAi vector comprising the lycopene ε-cyclase gene of the present invention, thereby inhibiting the expression of lycopene ε-cyclase. 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 the 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 lycopene ε-cyclase 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 lycopene ε-cyclase partial cDNA derived from sweet potato. The partial length of the lycopene ε-cyclase gene of the present invention is that 439 bp of cDNA encodes 146 amino acids (FIG. 1). The blasting of the genes in the database showed high homology with lycopene ε-cyclase of various plants (FIG. 2). In addition, when the amino acid sequence of the lycopene ε-cyclase of the present invention was examined by blast (BlastX), it showed the highest homology of 97% with the putative gene of Ipomoea nil. Daucus carota, barley (Hordeum vulgare), rice (Oryza sativa), corn (Zea mays), banana (Musa acuminate) and the like also showed a high homology of more than 80% (Fig. 3).

It was confirmed that the lycopene ε-cyclase gene of the present invention is a gene specifically expressed in leaves in plants (FIG. 4).

RNAi vectors were prepared to inhibit the expression of the lycopene ε-cyclase 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 lycopene ε-cyclase gene of the present invention and subjected to PCR analysis. For lycopene ε-cyclase RNAi transformants, PCR products were identified at 11, 12, 13, 17, 18, 19, 28, and 29 callus (FIG. 6).

As a result of comparing the lycopene ε-cyclase 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, the biosynthesis of beta-carotene was increased by the introduction of RNAi vector of the lycopene ε-cyclase gene produced by cloning in the present invention.

It was confirmed that the expression of the lycopene ε-cyclase gene was actually inhibited through RT-PCR in the transformed callus of the lycopene ε-cyclase gene of the present invention. In the case of transgenic callus 11, 12, 17, the expression of the gene was significantly suppressed compared to the control of Yumi callus, and in the case of the transgenic callus 13, the expression of the gene was reduced to less than half (Fig. 8).

As a result of investigating DPPH radical scavenging activity showing low molecular weight antioxidant activity in the transformed callus of the lycopene ε-cyclase gene of the present invention, it was confirmed that the activity was at least 2-4 times higher than that of the control, Yumi. It was found that the level was similar to the activity of the high yellow rice varieties (Hm). 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. 9).

The transgenic callus of the lycopene ε-cyclase gene of the present invention was treated with 150 mM and 200 mM NaCl, respectively, and the degree of oxidation in cells was measured by DAB staining. As a result, it was confirmed that the control group, Yumi callus, browning of DAB due to the increase of reactive oxygen species in the cell proceeded much more than the transformed callus (FIG. 10A). Specifically, as a result of measuring the amount of DAB oxidized, the overall degree of oxidation of DAB due to oxidative stress in cells was higher 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. 10B). Therefore, it was confirmed that the transforming callus inhibiting the expression of the lycopene ε-cyclase gene increased the antioxidant activity as the content of beta-carotene increased, thereby increasing the ability to remove active oxygen species in the cells.

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 Lycopene  ε- Cyclase  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 lycopene ε-cyclase gene, blasted with the lycopene ε-cyclase 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 the lycopene ε-cyclase gene was found from the EST clone of the morning glory (Ipomoea nil) close to sweet potato, and a PCR primer for cloning these genes from sweet potato was prepared. In order to use the gateway expression system of Invitrogen, primer sequences (in uppercase letters) were added to the primer sequences. Nucleotide sequence of lycopene ε- cyclase forward primer (5′- CAAAAAAGCAGGCTNN gaacaaactaatgttaagactggagaca-3 '; SEQ ID NO: 3) and reverse primer (5'- CAAGAAAGCTGGGTN gatagagttgatcccagaaatcct-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 lycopene ε-cyclase gene is 439 bp of cDNA encoding 146 amino acids (FIG. 1). The blasting of the genes in the database showed high homology with lycopene ε-cyclase of various plants (FIG. 2). In addition, the amino acid sequence of the coding portion of the lycopene ε-cyclase of the present invention was examined by BlastX and ClustalW program (http://align.genome.jp/), and the putative gene of Ipomoea nil and 97 The highest degree of homology was found in%, as well as over 80% in carrot (Daucus carota), barley (Hordeum vulgare), rice (Oryza sativa), corn (Zea mays) and banana (Musa acuminate). (FIG. 3).

Example  2: sweet potato organization Lycopene  ε- Cyclase  Gene expression analysis

RT-PCR was performed to analyze tissue-specific expression patterns of sweet potato lycopene ε-cyclase 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 lycopene ε-cyclase forward primer (5'-gaacaaactaatgttaagactggagaca-3 '; SEQ ID NO: 5) and reverse primer (5'-gatagagttgatcccagaaatcct-3'; SEQ ID NO: 6).

As a result, it was confirmed that the lycopene ε-cyclase gene of the present invention specifically expressed in leaves in plants (FIG. 4).

Example  3: sweet potato Lycopene  ε- Cyclase  Gene expression of plant expression vector and development of transformant

RNAi vectors were prepared to inhibit the expression of the lycopene ε-cyclase gene of the present invention (FIG. 5). First, the gene was cloned into the pDONR207 vector by BP reaction of the pGEMeasy vector in which the lycopene ε-cyclase gene was cloned. Thereafter, RNAi vectors of the lycopene ε-cyclase 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 lycopene ε-cyclase RNAi transformants, PCR products were identified in 11, 12, 13, 17, 18, 19, 28, 29 transgenic callus (FIG. 6). Therefore, it was confirmed that the lycopene ε-cyclase RNAi vector was introduced into the callus to be transformed.

Example  4: Lycopene  ε- Cyclase  Increased beta carotene content due to inhibition of gene expression

In order to confirm whether the inhibition of the expression of the lycopene ε-cyclase gene of the present invention substantially increased the content of beta-carotene, the color of the transformed callus confirmed by genomic DNA PCR as a result of comparing the phenotype with the control callus Yumi It can be seen that this dark yellow (Fig. 7). In addition, to confirm whether the expression of the lycopene ε-cyclase gene was actually inhibited, RNA was extracted from the callus transformed by the method of Example 1 and RT-PCR was performed. As a result, in the case of transgenic callus 11, 12, 17, expression of the gene was significantly suppressed compared to the control of Yumi callus, and in the case of transgenic callus 13, the expression of the gene was reduced to less than half (Fig. 8).

It was confirmed that the expression of the lycopene ε-cyclase gene was suppressed by the introduction of the lycopene ε-cyclase RNAi vector of the present invention.

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

The transgenic callus of the lycopene ε-cyclase gene of the present invention was investigated for DPPH radical scavenging activity showing low molecular antioxidant activity. In order to check whether beta-carotene has high antioxidant activity and transformed callus actually increased antioxidant activity due to increased beta-carotene content, we measured DPPH radical scavenging activity in Yuly callus and transformed callus. The low molecular weight antioxidant activity was analyzed and the antioxidant activity was 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 Yumi, a control, which is similar to the activity of Cinwangmi variety with high beta-carotene content (Fig. 9). 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, NaCl-treated callus was reacted with reactive oxygen species and stained with browning DAB when oxidized to compare phenotypes. 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. 10A). 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. 10B). Therefore, it was confirmed that the transforming callus inhibiting the expression of the lycopene ε-cyclase gene increased the antioxidant activity as the content of beta-carotene increased, thereby increasing the ability to remove active oxygen species in the cell.

1 is a partial sequence of the lycopene ε-cyclase 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 lycopene ε-cyclase gene of the present invention and various plants (morning glory, beans, grapes, coffee, gentian, potatoes, tomatoes, cotton, Arabidopsis, carrot, cabbage, corn, A comparison of the amino acid sequences of lycopene ε-cyclase genes of rice, barley, radishes, and other 11 species).

FIG. 3 is a diagram showing a flexible relationship between lycopene ε-cyclase genes compared with FIG. 2.

Figure 4 is a photograph of the lycopene ε-cyclase gene of the present invention in a sweet potato plant by electrophoresis by performing RT-PCR. L, leaf; S, stem; FR, fibrous roots (root); SR, storage roots.

5 is a diagram illustrating the construction of a transformation vector (RNAi) for inhibiting the expression of the lycopene ε-cyclase gene in plants.

FIG. 6 is a diagram illustrating a RNAi vector transformed callus of the lycopene ε-cyclase 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 examining the expression of the lycopene ε-cyclase gene in the transformed callus through RT-PCR.

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

10 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> Lycopene epsilon-cyclase gene increasing beta-carotene content of          plants and uses according <130> PN09221 <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 439 <212> DNA <213> Ipomoea batatas <400> 1 gaacaaacta atgttaagac tggagacaat gggtatccga ataaagaaaa tttatgaaga 60 ggaatggtct tatataccag ttggaggatc attgccaaat actgatcaaa gaaaccttgc 120 ttttggtgct gctgctagta tggttcatcc cgctacaggg tattcagtgg taagatcatt 180 gtcggaggct ccaagatatg catctgtcat agcaaatatt ttgaaacgaa gtcctggtat 240 gggtgatatg cttgtcagtt caagaagtat ggaaaatatt tcaacacaag cttgggaaac 300 gctttggccg caagaaagga aacgacaaag atcatttttc ctatttggtt tggcacttat 360 attgcagctt gatattgaag gaatacggac atttttccac acattcttcc gcttaccaaa 420 ctggatgtgg cgaggattc 439 <210> 2 <211> 146 <212> PRT <213> Ipomoea batatas <400> 2 Asn Lys Leu Met Leu Arg Leu Glu Thr Met Gly Ile Arg Ile Lys Lys   1 5 10 15 Ile Tyr Glu Glu Glu Trp Ser Tyr Ile Pro Val Gly Gly Ser Leu Pro              20 25 30 Asn Thr Asp Gln Arg Asn Leu Ala Phe Gly Ala Ala Ala Ser Met Val          35 40 45 His Pro Ala Thr Gly Tyr Ser Val Val Arg Ser Leu Ser Glu Ala Pro      50 55 60 Arg Tyr Ala Ser Val Ile Ala Asn Ile Leu Lys Arg Ser Pro Gly Met  65 70 75 80 Gly Asp Met Leu Val Ser Ser Arg Ser Met Glu Asn Ile Ser Thr Gln                  85 90 95 Ala Trp Glu Thr Leu Trp Pro Gln Glu Arg Lys Arg Gln Arg Ser Phe             100 105 110 Phe Leu Phe Gly Leu Ala Leu Ile Leu Gln Leu Asp Ile Glu Gly Ile         115 120 125 Arg Thr Phe Phe His Thr Phe Phe Arg Leu Pro Asn Trp Met Trp Arg     130 135 140 Gly phe 145 <210> 3 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 3 caaaaaagca ggctnngaac aaactaatgt taagactgga gaca 44 <210> 4 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 4 caagaaagct gggtngatag agttgatccc agaaatcct 39 <210> 5 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 5 gaacaaacta atgttaagac tggagaca 28 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 6 gatagagttg atcccagaaa tcct 24  

Claims (11)

Ipomoea , consisting of the amino acid sequence of SEQ ID NO: 2 lycopene ε-cyclase protein from batatas ). A gene encoding the lycopene ε-cyclase protein of claim 1. 3. The gene according to claim 2, wherein the gene consists 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 lycopene ε-cyclase. 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.

Images