KR100990330B1 - IbOrange gene involved in carotenoid accumulation from Ipomoea batatas - Google Patents

Abstract

The present invention relates to a sweet potato-derived IbOrange protein related to carotenoid accumulation, a gene encoding the protein, a recombinant vector comprising the gene, a host cell transformed with the recombinant vector, and a primer set for amplifying the IbOrange gene.

According to the present invention, by increasing the carotenoid content through the IbOrange gene is expected to be able to develop plants that are resistant to environmental stress as well as improving the functionality.

Carotenoids, sweet potatoes, orange genes, primers

Description

IbOrange gene involved in carotenoid accumulation from Ipomoea batatas}

The present invention relates to an orange gene derived from sweet potato varieties of high sulfur cultivars of high carotenoid content, and more particularly, to regulate the expression of genes related to carotenoid biosynthesis, thereby increasing the production of carotenoids and accumulating carotenoids in chromoplasts (chromosomes or chromosomes). It relates to a protein involved in and a gene encoding the same.

Sweet Potato ( Ipomoea batatas L. Lam) can be cultivated on relatively poor lands and has a high yield of about 30 tons per hectare, which is a representative root crop for food and livestock feed. In particular, about 70% of the buildings are made of starch, which has been used as a raw material for alcoholic beverages for a long time, and has recently been re-examined as an environmentally friendly alternative energy crop for bioethanol production.

Recently, due to the rapid industrialization and population increase, environmental and food problems on a global scale have been raised, and the development of environmentally resistant crops that grow well in unfavorable areas such as deserted areas, high seas and cold areas is being actively developed. In particular, in order to develop industrial sweet potatoes suitable for the conditionally disadvantaged area, research on environmentally resistant transgenic sweet potatoes by molecular breeding has been attempted (Lim et al. Mol Breeding 19, 227-239, 2007).

The World Health Organization (WHO) has announced that more than 100 million children worldwide suffer from a lack of vitamin A, which causes more than half a million children to lose sight each year. Beta-carotene is a precursor of vitamin A, and has the physiologically active functions of nutrient enhancers and food supplements. Therefore, metabolic engineering studies on carotenoid accumulation in foods are essential subjects for enhancing nutritional value. Beta-carotene is a substance with high antioxidant activity and is known to play an important role not only in physiological activity but also in the defense mechanism against oxidative stress of the plant itself. Recently, carotenoid biosynthesis is affected by ABA, one of plant hormones. As a result, research on these antioxidants and environmental stresses has emerged.

Meanwhile, Lu et al., Cornell University, announced that the Orange gene, which was mutated in cauliflower, was involved in the accumulation of carotenoids in chromoplasts (Lu S et al. 2006 Plant Cell. 18: 3594-3605, 2006).

The present invention has been made in accordance with the above requirements, the present inventors have cloned the orange gene from the new wangwang sweet potato varieties with high carotenoid content, and completed the present invention by confirming the production of recombinant protein in E. coli.

In order to solve the above problems, the present invention provides a sweet potato-derived IbOrange protein related to carotenoid accumulation.

The present invention also provides a gene encoding the IbOrange protein.

The invention also relates to IbOrange A recombinant vector comprising a gene is provided.

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

The present invention also provides a primer set for IbOrange gene amplification.

According to the present invention, the orange protein and genes derived from sweet yellow rice of the present invention are confirmed that expression is strongly induced by plant hormones such as ABA, ethephon, methyl jasmonate, and salicylic acid related to dry stress and plant defense mechanisms. It was. Therefore, by increasing the carotenoid content through the IbOrange gene is expected to be able to develop a plant that is resistant to environmental stress as well as functional enhancement.

In order to achieve the object of the present invention, the present invention provides IbOrange protein derived from sweet potato (Ipomoea batatas) related to carotenoid accumulation.

The range of IbOrange protein according to the present invention includes a protein having an amino acid sequence represented by SEQ ID NO: 2 isolated from Cinchu rice varieties of sweet potato and a functional equivalent of the protein. "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 IbOrange protein. Genes of the invention include both genomic DNA and cDNA encoding IbOrange proteins. Preferably, the gene of the present invention may include the nucleotide sequence shown in SEQ ID NO: 1. In addition, the IbOrange gene is a multigene family, and there are many homologous genes in sweet potatoes. The IbOrange gene of the present invention may include not only an IbOrange gene having a nucleotide sequence of SEQ ID NO: 1, but also a multigene family of IbOrange genes. .

Variants of the above base sequences are also 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. 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).

In the overall length of the IbOrange cDNA gene of the present invention, 942 bp cDNA encodes 313 amino acids (see FIG. 1). The isoelectric point (pI) and molecular weight (Mw) predicted by the amino acid sequence are 8.46 and 34.4 kDa, respectively. As a result of blasting the gene from the database, it is a type of DNA J protein known as chaperone, which has a repeat motif of CxxCxGxG, which is a cysteine that is well conserved in other Orange proteins.

IbOrange gene of the present invention was found to be expressed in all varieties of off-white sweet potato, Yumi, purple sweet potato, Shinjami, yellow sweet potato, Xinhuangmi (see Fig. 4). IbOrange gene was expressed evenly throughout the plant, especially strong in the leaf (leaf) and fibrous root (fibrous root) (see Figure 5).

The IbOrange gene of the present invention can be strongly induced by plant hormones such as ABA, ethephon, methyl jasmonate, or salicylic acid. The IbOrange gene of the present invention showed no significant change in leaves when ABA was treated, but showed strong expression of IbOrange gene after 36 hours in the root. In the case of treatment with ethephone, the expression of IbOrange gene was induced at 36 hours after treatment in leaves and roots, particularly in leaves. In the case of methyl jasmonate treatment, IbOrange gene expression was strongly induced from 12 hours after treatment and lasted up to 36 hours, and the root increased slightly from 24 hours and then strongly expressed at 36 hours. . When salicylic acid was treated, there was no significant change across the leaves and roots, but the expression of IbOrange gene was weakly increased from 12 hours to 36 hours after treatment in the leaves (see FIG. 6).

The present invention also provides a recombinant vector comprising the IbOrange gene according to the present invention. The recombinant vector is preferably a recombinant E. coli expression vector.

The term "recombinant" refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, a heterologous peptide, or a heterologous nucleic acid. 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. Recombinant cells can also express genes found in natural cells, but the genes have been modified and reintroduced into cells by artificial means.

The term "vector" is used to refer to a DNA fragment (s), 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 a suitable nucleic acid sequence necessary to express a coding sequence operably linked in a particular host organism.

Vectors of the invention can typically be constructed as vectors for cloning or expression. In addition, the vector of the present invention can be constructed using prokaryotic or eukaryotic cells as hosts. For example, when the vector of the present invention is an expression vector and the prokaryotic cell is a host, a strong promoter (for example, a pLλ promoter, a trp promoter, a lac promoter, a T7 promoter, a tac promoter, etc.) capable of promoting transcription, It is common to include ribosomal binding sites and transcription / detox termination sequences for initiation of translation. When E. coli is used as a host cell, a promoter and an operator site of the E. coli tryptophan biosynthetic pathway, and a phage λ left promoter (pLλ promoter) can be used as regulatory sites.

On the other hand, vectors that can be used in the present invention are plasmids (eg, pSC101, ColE1, pBR322, pUC8 / 9, pHC79, pGEX series, pET series and pUC19, etc.) which are often used in the art, phage (e.g. λgt4.λB , λ-Charon, λΔz1 and M13, etc.) or viruses (eg SV40, etc.).

On the other hand, when the vector of the present invention is an expression vector and the eukaryotic cell is a host, a promoter derived from the mammalian cell genome (for example, metallothionine promoter) or a promoter derived from a mammalian virus (for example, adeno) Late viral promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter and tk promoter of HSV) can be used and generally have a polyadenylation sequence as a transcription termination sequence.

Vectors of the present invention may include antibiotic resistance genes commonly used in the art as optional markers, for example ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin And resistance genes for tetracycline.

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 stable manner can be used in any host cell known in the art, for example, E. coli JM109, E. coli BL21, E. coli RR1, E. strains of the genus Bacillus, such as coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus thuringiensis, and Salmonella typhimurium, Serratia marcensons, and various Pseudomonas species. Enterobacteria and strains. The host cell is preferably Escherichia coli.

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 line (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cell lines) and the like.

The method of carrying the vector of the present invention into a host cell is performed by the CaCl ₂ method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9: 2110-2114 (1973), when the host cell is a prokaryotic cell). ), Hanahan method (Hanahan, D., J. Mol. Biol., 166: 557-580 (1983)) and electroporation method (Dower, WJ et al., Nucleic. Acids Res., 16: 6127-6145 (1988)) and the like. In addition, when the host cell is a eukaryotic cell, microinjection method (Capecchi, MR, Cell, 22: 479 (1980)), calcium phosphate precipitation method (Graham, FL et al., Virology, 52: 456 (1973)), Electroporation (Neumann, E. et al., EMBO J., 1: 841 (1982)), liposome-mediated transfection (Wong, TK et al., Gene, 10:87 (1980)), DEAE- Dextran treatment (Gopal, Mol. Cell Biol., 5: 1188-1190 (1985)), and gene balm (Yang et al., Proc. Natl. Acad. Sci., 87: 9568-9572 (1990)) The vector can be injected into the host cell.

The present invention also provides a primer set for IbOrange gene amplification. The primer set may consist of oligonucleotides of SEQ ID NOs: 3 and 4.

The primer set is at least one oligonucleotide selected from the group consisting of fragments of at least 20 contiguous nucleotides in the sequence of SEQ ID NO: 3 and oligonucleotides consisting of fragments of at least 20 contiguous nucleotides in the sequence of SEQ ID NO: 4 It may consist of a primer set comprising one or more oligonucleotides selected from the group consisting of.

The primer set is preferably 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, in the sequence of SEQ ID NO: 3, At least 21 oligonucleotides selected from the group consisting of oligonucleotides consisting of fragments of 30 consecutive nucleotides and at least 21, at least 22, at least 23, at least 24, at least 25, at least 26 in the sequence of SEQ ID NO: 4 Or at least 27, at least 28, at least 29, at least 30 oligonucleotides consisting of a sequence of oligonucleotides consisting of a set of primers comprising at least one oligonucleotide selected from the group consisting of.

The primer set may most preferably consist of a primer set of SEQ ID NO: 3 and SEQ ID NO: 4.

In the present invention, "primer" refers to a single stranded oligonucleotide sequence that is complementary to the nucleic acid strand to be copied and may serve as a starting point for the synthesis of the primer extension product. The length and sequence of the primer should allow to start the synthesis of the extension product. As used herein, oligonucleotides used as primers may also include nucleotide analogues such as phosphorothioate, alkylphosphothioate or peptide nucleic acid, or It may comprise an intercalating agent.

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

< Example  1> sweet potato IbOrange  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 Orange gene, blast the cabbage with the Orange gene of cauliflower on the website of the TIGR Plant Transcript Assemblies (http://plantta.tigr.org/) to find the Orange from the EST clone of Ipomoea nil, which is close to sweet potato. A clone with a nucleotide sequence similar to that of the gene was found and PCR primers for cloning the orange gene in sweet potato were prepared from this gene. The primer sequence is as follows: forward primer (5'-atggtatattcaggtagaatcttgtcgctc-3 '; SEQ ID NO: 3) and reverse primer (5'-ttaatcaaatgggtcaattcgtgggtcatg-3'; SEQ ID NO: 4). In the base sequence of the primer, an adapter sequence (uppercase) was added to the 5 'end of the primer to use the gateway expression system of Invitrogen. The base sequence is a forward primer (5'- CAAAAAAGCAGGCTNN atggtatattcaggtagaatcttgtcgctc-3 '; SEQ ID NO: 5) and a reverse primer (5'- CAAGAAAGCTGGGTN ttaatcaaatgggtcaattcgtgggtcatg-3'; SEQ ID NO: 6). PCR was performed using Clonetech's advantage2 polymerase, and the PCR product of the expected size was cloned using pGEMeasy cloning vector (Promega), followed by sequencing to confirm the entire nucleotide sequence. The cDNA gene was named IbOrange cDNA.

The overall length of the IbOrange cDNA gene is 942 bp cDNA codes for 313 amino acids, and has a signal target of the plastid target at the N-terminus (FIG. 1). The result of blasting the gene from the database is a type of DNA J protein known as chaperone, which has a repeat motif of CxxCxGxG that repeats cysteines that are well conserved in other Orange proteins. Using the Blast X and ClustalW program (http://plant.pdrc.re.kr/gene/align/ClustalW.html), the flexible relationship between the amino acid sequence of the coding portion of IbOrange of the present invention and Orange of various plant species The results showed the highest homology of 97% to the putative orange gene of morning glory (Ipomoea nil), as well as the putative orange gene of tomato (Lycopersicon esculentum), grape (Vitis vinifera) gene and Arabidopsis thaliana. At5g61670 gene, cabbage (Brassica oleracea var. Botrytis) showed a homology of 73 ~ 80% (Fig. 2 and 3).

< Example  2> IbOrange  Analysis of Gene Expression by Sweet Potato Varieties and Tissues

RT-PCR was performed to analyze the expression patterns of sweet potato varieties and tissues of the IbOrange 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 sequence of the primer used is (forward: 5'-atcttgtcgctctcgtcctccacgacgccg-3 '(SEQ ID NO: 7), reverse: 5'-cgtgggtcatgctcgcttgccatagccatc-3' (SEQ ID NO: 8)).

As a result, it was confirmed that IbOrange gene is expressed in all varieties of off-white sweet potato, Yumi (Ym), purple sweet potato, Shinjami (Jm), and yellow sweet potato, Xinhuangmi (Hm) (FIG. 4). In addition, in the tissue-specific analysis of plants, the IbOrange gene was expressed evenly throughout the plant, particularly in the leaf and fibrous root (FIG. 5). Therefore, IbOrange gene was found to be a gene that is expressed evenly throughout the plant regardless of breed.

< Example  3> By various plant hormone treatment IbOrange  Gene expression analysis

In order to investigate the expression patterns of plant hormones such as ABA, ethephon, methyl jasmonate, and salicylic acid related to dry stress and plant defense mechanism of the IbOrange gene of the present invention, the time period after treatment of each hormone (0, 12, 24, 36, 48 hours) RNA was isolated by the method described in Examples 1 and 2 and cDNA was synthesized, respectively. RT-PCR was performed with the IbOrange gene specific primer used in Example 2 above. As a result, the IbOrange gene showed the following expression pattern. ABA; There is no obvious change in the leaves, but IbOrange genes are strongly expressed after 36 hours in the roots. Ethephon; It is shown that the expression of IbOrange gene is induced at 36 hours after treatment in leaves and roots, particularly in leaves. Methyl jasmonate; In the case of leaves, the expression of IbOrange gene is strongly induced from 12 hours after treatment and lasts up to 36 hours, and the root is weakly increased from 24 hours and then strongly expressed at 36 hours. Salicylic acid; There was no significant change throughout the leaves and roots, but IbOrange gene expression was slightly increased from 12 hours to 36 hours after treatment in the leaves (Fig. 6).

In general, under the dry stress, the amount of ABA, a plant hormone, is increased, and ABA is known to regulate the pore opening and closing of plants in response to drying. On the other hand, carotenoids are precursors of ABA, and the increase of ABA due to dry stress is thought to be directly related to the increase of carotenoids. Therefore, the increase of IbOrange gene expression by ABA treatment in this experiment shows the correlation between dry stress and IbOrange.

< Example  4> IbOrange  Confirmation of protein production by expression of E. coli gene

To confirm that the IbOrange gene actually produces a protein of the desired size, the coding portion of the IbOrange gene was cloned into the pDONR207 vector, which contains a histidine affinity tag and a glutathione-S-transferase (GST) fusion protein. The expression vectors pDEST 17 and pDEST 15 were introduced. This expression vector was transformed into E. coli expression strain (BL21 DE3) to induce the expression of orange protein by IPTG. As a result of extracting the E. coli protein and confirming it through SDS-PAGE gel analysis, it was confirmed that the recombinant protein tagged with 34 kDa histidine and the GST-fusion protein of 57 kDa (FIG. 7). Therefore, it was confirmed once again that the cloned IbOrange gene in the present invention is a gene encoding an orange protein.

Therefore, by increasing the carotenoid content through the IbOrange gene of the present invention it is expected that it can be very useful for the development of plants resistant to environmental stress as well as functional enhancement.

1 is a diagram showing the nucleotide sequence of the sweet potato (breed yellow rice) derived IbOrange gene of the present invention and the amino acid sequence inferred therefrom. The underlined portion is the plastid localization signal, with a total of 942 bp cDNA encoding 313 amino acids.

Figure 2 is the amino acid sequence of the deduced protein of the sweet potato (Ipomoea batatas) IbOrange gene of the present invention and various plants (morning flower, tomato, grapes, beans, soybeans, cotton, Arabidopsis, cauliflower, singular, corn, rice, barley Figure shows a comparison of the amino acid sequence of orange genes.

Figure 3 is between the amino acid sequence of the deduced protein of the IbOrange gene of the present invention and the orange gene of various plants (morning glory, tomato, grapes, beans, soybeans, cotton, Arabidopsis, cauliflower, singular, corn, rice, barley) This figure shows the flexible relationship of.

Figure 4 is a picture of the IbOrange gene expression of the present invention for each sweet potato varieties electrophoresis by performing RT-PCR. Ym; Yulmi, Jm; Shinjami, Hm; Xinhuangmi.

Figure 5 is a photograph of electrophoresis by performing RT-PCR on the expression of the IbOrange gene of the present invention for each portion of the sweet potato. L, leaf; S, stem; storage roots; fibrous roots (roots); thick pigmented roots.

6 is sweet potato leaves and roots of the present invention, 0, 12, 24, 36, 48 hours after treatment with plant hormones ABA, ethylene precursors etepon, methyl jasmonate (MeJA), salicylic acid (SA) The expression pattern of IbOrange gene is electrophoresed by RT-PCR.

Figure 7 is a protein electrophoresis picture showing recombinant protein production of IbOrange gene using E. coli. Lane 1; Soluble IbOrange with His-tag, lane 2; Insoluble IbOrange with His-tag, lane 3; Soluble IbOrange with GST-tag, lane 4; Insoluble IbOrange with GST-tag.

<110> Korea Research Institute of Bioscience and Biotechnology <120> IbOrange gene involved in carotenoid accumulation from Ipomoea          batatas <130> PN08125 <160> 8 <170> KopatentIn 1.71 <210> 1 <211> 942 <212> DNA <213> Ipomoea batatas <400> 1 atggtatatt caggtagaat cttgtcgctc tcgtcctcca cgacgccgtt tcatctctcc 60 acttcgccgt tccacagttc ccggtatcat ctccatggaa ggctcaaatc cagagtcaga 120 ttgcgtccta tggccgccga tgccgattcc tcctcctttt cttcatccgt cgacaccgaa 180 gcacccgata aaaacgcagc cgggttttgt attatagaag ggcctgagac agtgcaggac 240 tttgctcaaa tggaattgaa agaaattcaa gacaatattc ggagtcgccg gaataaaata 300 tttttgcata tggaagaggt tcgtaggctg agaatacagc aacgaattaa gaatgctgag 360 cttgggaatc ttaatgaaaa gcaagaaaat aaacttccga attttccttc gttcattcct 420 tttttgcctc ctctgacatc cgcaaatctt aaacaatatt atgccacttg tttctctctc 480 atagccggag ttatgctttt tggcggactg ctagcaccta ctttggaact aaaattgggc 540 ttaggaggta catcgtacgc tgatttcatt cgcagcatgc accttccgat gcaattaagt 600 gatgtggacc ccattgtggc gtccttctcc ggtggagcag tcggggtaat ctctgccttg 660 atggtagttg aaataaacaa tgtgaaacag caggagcata agaggtgcaa gtactgttta 720 ggaacagggt atcttgcatg tgctcgctgt tcaagcactg gatcactagt ccttatcgaa 780 cctgtctcca cagttaatcg tggagatcag ccactatcac cacctaaaac agaaagatgc 840 acaaactgct cgggttcagg gaaggtcatg tgccctacat gtctttgtac tgggatggct 900 atggcaagcg agcatgaccc acgaattgac ccatttgatt aa 942 <210> 2 <211> 313 <212> PRT <213> Ipomoea batatas <400> 2 Met Val Tyr Ser Gly Arg Ile Leu Ser Leu Ser Ser Ser Thr Thr Pro   1 5 10 15 Phe His Leu Ser Thr Ser Pro Phe His Ser Ser Arg Tyr His Leu His              20 25 30 Gly Arg Leu Lys Ser Arg Val Arg Leu Arg Pro Met Ala Ala Asp Ala          35 40 45 Asp Ser Ser Ser Phe Ser Ser Ser Val Asp Thr Glu Ala Pro Asp Lys      50 55 60 Asn Ala Ala Gly Phe Cys Ile Ile Glu Gly Pro Glu Thr Val Gln Asp  65 70 75 80 Phe Ala Gln Met Glu Leu Lys Glu Ile Gln Asp Asn Ile Arg Ser Arg                  85 90 95 Arg Asn Lys Ile Phe Leu His Met Glu Glu Val Arg Arg Leu Arg Ile             100 105 110 Gln Gln Arg Ile Lys Asn Ala Glu Leu Gly Asn Leu Asn Glu Lys Gln         115 120 125 Glu Asn Lys Leu Pro Asn Phe Pro Ser Phe Ile Pro Phe Leu Pro Pro     130 135 140 Leu Thr Ser Ala Asn Leu Lys Gln Tyr Tyr Ala Thr Cys Phe Ser Leu 145 150 155 160 Ile Ala Gly Val Met Leu Phe Gly Gly Leu Leu Ala Pro Thr Leu Glu                 165 170 175 Leu Lys Leu Gly Leu Gly Gly Thr Ser Tyr Ala Asp Phe Ile Arg Ser             180 185 190 Met His Leu Pro Met Gln Leu Ser Asp Val Asp Pro Ile Val Ala Ser         195 200 205 Phe Ser Gly Gly Ala Val Gly Val Ile Ser Ala Leu Met Val Val Glu     210 215 220 Ile Asn Asn Val Lys Gln Gln Glu His Lys Arg Cys Lys Tyr Cys Leu 225 230 235 240 Gly Thr Gly Tyr Leu Ala Cys Ala Arg Cys Ser Ser Thr Gly Ser Leu                 245 250 255 Val Leu Ile Glu Pro Val Ser Thr Val Asn Arg Gly Asp Gln Pro Leu             260 265 270 Ser Pro Pro Lys Thr Glu Arg Cys Thr Asn Cys Ser Gly Ser Gly Lys         275 280 285 Val Met Cys Pro Thr Cys Leu Cys Thr Gly Met Ala Met Ala Ser Glu     290 295 300 His Asp Pro Arg Ile Asp Pro Phe Asp 305 310 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 3 atggtatatt caggtagaat cttgtcgctc 30 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer <400> 4 ttaatcaaat gggtcaattc gtgggtcatg 30 <210> 5 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 5 caaaaaagca ggctnnatgg tatattcagg tagaatcttg tcgctc 46 <210> 6 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer <400> 6 caagaaagct gggtnttaat caaatgggtc aattcgtggg tcatg 45 <210> 7 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 7 atcttgtcgc tctcgtcctc cacgacgccg 30 <210> 8 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer <400> 8 cgtgggtcat gctcgcttgc catagccatc 30  

Claims (8)

IbOrange protein from sweet potato (Ipomoea batatas) consisting of the amino acid sequence of SEQ ID NO. The gene encoding the IbOrange 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. The gene of claim 2, wherein the gene is increased in expression by ABA, etepon, methyl jasmonate, or salicylic acid. Recombinant vector comprising the gene of claim 2. A host cell transformed with the recombinant vector of claim 5. The host cell according to claim 6, which is E. coli. delete

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