WO2009154334A1 - Iborange gene involved in carotenoid accumulation from ipomoea batatas - Google Patents

Iborange gene involved in carotenoid accumulation from ipomoea batatas Download PDF

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WO2009154334A1
WO2009154334A1 PCT/KR2008/006813 KR2008006813W WO2009154334A1 WO 2009154334 A1 WO2009154334 A1 WO 2009154334A1 KR 2008006813 W KR2008006813 W KR 2008006813W WO 2009154334 A1 WO2009154334 A1 WO 2009154334A1
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gene
iborange
protein
present
seq
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PCT/KR2008/006813
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French (fr)
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Sang Soo Kwak
Haeng Soon Lee
Young Ock Ahn
Cha Young Kim
Sun Ha Kim
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Korea Research Institute Of Bioscience And Biotechnology
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Priority to CN2008801298504A priority Critical patent/CN102083985B/en
Publication of WO2009154334A1 publication Critical patent/WO2009154334A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine

Definitions

  • the present invention relates to the Orange gene originating from "Shinwhangmi
  • the pr esent invention relates to a protein which can increase the production of carotenoid by controlling the expression of a gene that is related to biosynthesis of carotenoid and inv olved with the carotenoid accumulation in chromoplast and a gene which encodes said protein.
  • Sweet potato ⁇ Ipomoea batatas L. Lam is a root crop that can be cultivated in a relatively barren soil and has a productivity as high as about 30 tons per one ha. As s uch, it is commonly consumed both as a food for humans and a feed for livestocks. In particular, consisting of about 70% of starch in dry matter, the sweet potato has long be en used as a source material for obtaining alcohols and also is now believed as an envi ronmentally-friendly crop which can be used as an alternative source for obtaining ener gy, i.e., for producing bioethanol. Due to dramatic industrialization and population increase in recent years, environ ment and food problems are raised all over the world.
  • Beta-carotene is a precursor compound of vitamin A.
  • ⁇ -carotene has a physiological activity as a nutrition fortifying agent and a food ai d
  • a metabolism study regarding the accumulation of carotenoid in food products is an e ssential research subject to improve nutritional value of them.
  • Beta-carotene is known as a substance which has a high anti-oxidative activity and it is known not only for its p hysiological activity but also for its key role in a defense mechanism of a plant itself agai nst oxidative stress.
  • biosynthesis of carotenoid is affected by ABA, which is one type of plant hormones. As such, a further research is needed t o determine the relationship between these anti-oxidative substances and environment al stress.
  • the present invention is devised in view of the above-described needs. Invent ors of the present invention cloned the Orange gene from "Shinwhangmi" sweet potato which has a high amount of carotenoid. Consequently, with the confirmation of the pro duction of recombinant protein in Escherichia coli, the present invention was completed.
  • the present invention provi des IbOrange protein originating from a sweet potato which is related to the accumulati on of carotenoid.
  • the present invention provides a gene encoding IbOrange protein.
  • the present invention provides a recombinant vector which comprises th e IbOrange gene.
  • the present invention provides a host cell that is transformed with said re combinant vector.
  • the present invention provides a primer set for the amplification of th e IbOrange gene.
  • Fig. 1 represents the nucleotide sequence of the Orange gene originating from a sweet potato ("Shinwhangmi") and the amino acid sequence deduced therefrom.
  • the underlined region indicates a plastid localization signal, and cDNA consisting of 942 bp encodes the protein consisting of 313 amino acids.
  • Fig. 2 represents the amino acid sequence of the orange protein that is deduced from the IbOrange gene of a sweet potato (Ipomoea batatas) according to the present i nvention, wherein the amino acid sequences of the Orange gene from other various pla nts are also described for comparison (morning glory, tomato, grape, bean, soybean, co tton, Arabidopis, cauliflower, sweet sorghum, maize, rice, and barley).
  • Fig. 3 represents the genetic similarity relation between the amino acid sequence that is deduced from the IbOrange of a sweet potato (Ipomoea batatas) according to t he present invention and the Orange gene from other various plants (morning glory, to mato, grape, bean, soybean, cotton, Arabidopis, cauliflower, sweet sorghum, maize, ric e, and barley).
  • Fig. 4 represents the expression mode of the IbOrange gene according to the pr esent invention that is measured by RT-PCR and electrophoresis for various breeds of sweet potato.
  • Ym indicates “Yoolmi”
  • Jm indicates “Shinjami”
  • Fig. 5 represents the expression mode of the IbOrange gene according to the pr esent invention that is measured by RT-PCR and electrophoresis for various parts of sw eet potato. Specifically, L indicates a leaf, S indicates a stem, and others include stora ge roots, fibrous roots and thick pigmented roots.
  • Fig. 6 represents the expression mode of the IbOrange gene according to the pr esent invention that is measured by RT-PCR and electrophoresis in which leaves and r oots of the sweet potato were treated with plant hormones such as ABA, ethephon (i.e., precursor of ethylene), methyl jasmonate (MeJA), and salicylic acid (SA) and the RT-P CR was carried out 0, 12, 24, 36 or 48 hours after the treatment.
  • plant hormones such as ABA, ethephon (i.e., precursor of ethylene), methyl jasmonate (MeJA), and salicylic acid (SA) and the RT-P CR was carried out 0, 12, 24, 36 or 48 hours after the treatment.
  • Fig. 7 represents the result of electrophoresis of the protein, indicating the produ ction of the recombinant protein of the IbOrange gene in E.coli.
  • Lane 1 soluble IbOra nge having His-tag, Lane 2; insoluble IbOrange having His-tag, Lane 3; soluble IbOrang e having GST-tag, and Lane 4; insoluble IbOrange having GST-tag.
  • the pres ent invention provides IbOrange protein which originates from a sweet potato (Ipomoea batatas) that is related to the accumulation of carotenoid.
  • IbOrange protein includes a protein having an amino acid sequence described in SEQ ID NO: 2, that is isolated fr om "Shinwhangmi" sweet potato, and functional equivalents of said protein.
  • the term "functional equivalent” means that, as a result of addition, substitution or deletion of ami no acid residues, it has an amino acid sequence with at least 70%, preferably at least 8 0%, more preferably at least 90%, still more preferably at least 95% homology with the amino acid sequence of SEQ ID NO: 2, thus representing a protein which has substanti ally the same physiological activity as the protein expressed by SEQ ID NO: 2.
  • the present invention provides a gene which encodes the above-describ ed IbOrange protein.
  • the gene according to the present invention includes both geno mic DNA and cDNA which encode IbOrange protein.
  • the gene according t o the present invention may comprise a nucleotide sequence that is represented by SE Q ID NO: 1.
  • the IbOrange g ene according to the present invention may comprise not only the IbOrange gene which has a nucleotide sequence represented by SEQ ID NO: 1 but also a multigene family of the IbOrange gene.
  • the above described gene may comprise a nucleotide sequence which has preferably at least 70%, more preferably at least 80%, still more p referably at least 90%, and most preferably at least 95% homology with the nucleotide s equence of SEQ ID NO: 1.
  • a part of the polynucleotide in comparative region may comprise an addition or a deletion (i.e., a gap) compared to a reference sequence (without any a ddition or deletion) relative to the optimized alignment of the two sequences.
  • Full length IbOrange cDNA of the present invention consists of 942bp and it enc odes 313 amino acid residues (see, Fig. 1).
  • the isoelectric point (pi) and molecular w eight (Mw), that are calculated from the amino acid sequence, are 8.46 and 34.4 kDa, r espectively.
  • pi isoelectric point
  • Mw molecular w eight
  • the IbOrange gene of the present invention is expressed in various breeds of a sweet potato including Yulmi (off-white colored sweet potato), Shinj ami (purple colored sweet potato), and Shinwhangmi (yellow colored sweet potato) (see , Fig. 4). Specifically, the IbOrange gene is generally expressed over the entire part of the plant while it is more strongly expressed in a leaf and a fibrous root (see, Fig. 5).
  • IbOrange gene can be stro ngly induced by plant hormones such as ABA, ethephon, methyl jasmonate, salicylic aci d and the like. Although there was no significant change occurred in the leaf that had been treated with ABA, the IbOrange gene was strongly expressed in the root 36 hours after the treatment. In the case of the treatment with ethephone, expression of the IbO range gene was induced in the leaf and the root 36 hours after the treatment. In partic ular, the expression was stronger in the leaf. In the case of the treatment with methyl j asmonate, expression of the IbOrange gene was strongly induced in the leaf 12 hours a fter the treatment, maintaining the high level until the 36 hour.
  • plant hormones such as ABA, ethephon, methyl jasmonate, salicylic aci d and the like.
  • the present invention provides a recombinant vector comprising the IbOr ange gene according to the present invention.
  • Said recombinant vector is preferably a recombinant E. coli expression vector.
  • the term "recombinant” indicates a cell which replicates a heterogeneous nucleo tide or expresses the nucleotide, a peptide, a heterogeneous peptide, or a protein enco ded by a heterogeneous nucleotide.
  • Recombinant cell can express a gene or a gene f ragment, that are not found in natural state of cell, in a form of a sense or antisense.
  • a recombinant cell can express a gene that is found in natural state, provide d that said gene is modified and re-introduced into the cell by an artificial means.
  • vector is used herein to refer DNA fragment (s) and nucleotide molec ules that are delivered to a cell.
  • Vector can be used for the replication of DNA and be i ndependently reproduced in a host cell.
  • delivery system and “vector” are often interchangeably used.
  • expression vector means a recombinant DNA molecule comprising a desired coding sequence and other appropriate nucleotide sequ ences that are essential for the expression of the operatively-linked coding sequence in a specific host organism.
  • the vector of the present invention can be constructed to be a vector for cloning or expression in general.
  • the vector of the present invention can be constr ucted to be a vector which has a prokaryotic cell or an eukaryotic cell as a host.
  • the vector of the present invention when it is an expression vector and has a pro karyotic cell as a host, it generally comprises a strong promoter which can promote tran scription (for example, pL ⁇ promoter, trp promoter, lac promoter, T7 promoter, tac prom oter and the like), a ribosome binding site for initiation of translation, and termination se quences for transcription/translation.
  • a strong promoter which can promote tran scription (for example, pL ⁇ promoter, trp promoter, lac promoter, T7 promoter, tac prom oter and the like), a ribosome binding site for initiation of translation, and termination se quences for transcription/translation.
  • coli is employed as a host cell, the pro moter and the operator region involved in tryptophan biosynthesis in E. coli, and left side promoter of phage ⁇ (i.e., pL ⁇ promoter) can be used as a regulatory site.
  • pL ⁇ promoter left side promoter of phage ⁇
  • the vector that can be used in the present invention can be construct ed by using a plasmid (example: pSC101 , CoIEI , pBR322, pUC8/9, pHC79, pGEX seri es, pET series and pUC19 and the like), a phage (example: ⁇ gt4 ⁇ B, ⁇ -Charon, ⁇ z1 an d M13 and the like) or a virus (example: SV40 and the like), that are typically used in th e pertinent art.
  • a plasmid example: pSC101 , CoIEI , pBR322, pUC8/9, pHC79, pGEX seri es, pET series and pUC19 and the like
  • a phage example: ⁇ gt4 ⁇ B, ⁇ -Charon, ⁇ z1 an d M13 and the like
  • a virus example:
  • the vector of the present invention is an expression vector and has an eukaryotic cell as a host
  • a promoter originating from mammalian genome exa mple: metallothionein promoter
  • a promoter originating from mammalian virus exam pie: adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegal ovirus promoter and tk promoter of HSV
  • a transcription termination s equence polyadenylated sequence is generally comprised.
  • the vector of the present invention may comprise an antibiotics-resistant gene, t hat is typically used in the pertinent art, as a selection marker. Examples thereof include de a gene resistant to ampicillin, gentamycin, carbenicillin, chloramphenicol, streptomyc in, kanamycin, geneticin, neomycin or tetracycline, etc.
  • the present invention further provides a host cell that is transformed with the rec ombinant vector of the present invention.
  • a host cell any one known i n the pertinent art to have an ability for stable and continuous cloning and expression of the vector of the present invention can be used. Examples thereof include, Bacillus s p. strain including E. coli JM 109, E. coli BL21 , E. coli RR1 , E. coli LE392, E coli B, E. c o// X 1776, E.
  • the host cell is E. coli.
  • Saccharomyce cerevisiae an insect cell, a human cell (for example, CHO (Ch inese hamster ovary) cell line, W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK c ell line) and the like can be used as a host cell.
  • CHO Cho inese hamster ovary
  • a host cell is a prokaryotic cell
  • delivery of the vector of the present inventi on into a host cell can be carried out by CaC ⁇ method (Cohen, S.N. et al., Proc. Natl. A cac. Sci. USA, 9:2110-2114 (1973)), Hanahan's method (Hanahan, D., J. MoI. Biol., 16 6:557-580 (1983)) or an electroporation method (Dower, W.J. et al., Nucleic. Acids Res. , 16:6127-6145 (1988)) and the like.
  • CaC ⁇ method Cohen, S.N. et al., Proc. Natl. A cac. Sci. USA, 9:2110-2114 (1973)
  • Hanahan's method Hanahan, D., J. MoI. Biol., 16 6:557-580 (1983)
  • an electroporation method Dower, W.J. et al.,
  • the vector when a host cell is an eukaryotic cell, the vector can be introduced to a host cell by a microinjection method (Capecchi, M. R., Cell, 22:479 (1980)), calcium phosphate precipitation method (Graham, F. L. et al., Virol ogy, 52:456 (1973)), an electroporation method (Neumann, E. et al., EMBO J., 1 :841 (1 982)), a liposome-mediated transfection method (Wong, T.K. et al., Gene, 10:87 (1980) ), DEAE-dextran treatment method (Gopal, MoI. Cell Biol., 5:1188-1190 (1985)), or a ge ne bombardment method (Yang et al., Proc. Natl. Acad. ScL, 87:9568-9572 (1990)) and the like.
  • a microinjection method Capecchi, M. R.,
  • the present invention further provides a primer set for the amplification of the Ib Orange gene.
  • This primer set may consist of the oligonucleotide that is represented b y SEQ ID NO: 3 or SEQ ID NO: 4.
  • the primer set may consist of a primer set which comprises at least one oligonuc leotide that is selected from a group consisting of oligonucleotides that consist of a frag ment having 20 or more consecutive nucleotides included in the sequence of SEQ ID N
  • the primer set may consist of a primer set which comprises at least o ne oligonucleotide that is selected from a group consisting of oligonucleotides that consi st of a fragment having 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, or 30 consecutive nucleotides included i n the sequence of SEQ ID NO: 3 and at least one oligonucleotide that is selected from a group consisting of oligonucleotides that consist of a fragment having 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, or 30 consecutive nucleotides included i n the sequence of SEQ ID NO: 3 and at least one oligonucleotide that is selected from a group consisting of oligonucleotides that consist of a fragment having 21 or more, 22 or more,
  • the primer set may consist of the primer set having SEQ ID NO: 3 and SEQ ID NO: 4.
  • the term "primer” indicates a single-stranded oligonucleotide which is complementary to the nucleotide strand to be copied and it can function as an initiation point for the synthesis of primer elongation product.
  • the leng th and the sequence of the above-described primer should satisfy the condition require d for the initiation of the synthesis of an elongation product.
  • an oligonucleotide that is used as a primer may comprise a nucleotide analog ue, for example, phosphorothioate, alkyl phosphorothioate or a peptide nucleic acid, or an intercalating agent.
  • Example 1 Cloning of sweet potato IbOrange gene, analysis of nucleotid e sequence and genetic similarity
  • RNA was isolated from the leaves of a sweet potato (Ipomoea batatas (L.) Lam, Shinwhangmi breed) using RNeasy Mini Kit by QIAGEN. Further, using SuperSc ript III First-Strand Synthesis System for RT-PCR from Invitrogen, first cDNA was synth esized. In order to isolate the Orange gene, BLAST analysis using the Orange gene o f a cauliflower was carried out based on the website provided by TIGR Plant Transcript Assemblies (http://plantta.tiqr.org/).
  • nucleotide sequence of the primers an adapter sequence (bold letters) was attached at the 5'-end of each primers so that the gateway expression system provided by Invitro gen can be used.
  • the resulting nucleotide sequences are as follows: forward primer ( 5'-CAAAAAAGCAGGCTNNatggtatattcaggtagaatcttgtcgctc-3'; SEQ ID NO: 5) and rever se primer (5'-CAAGAAAGCTGGGTNttaatcaaatgggtcaattcgtgggtcatg-3'; SEQ ID NO: 6).
  • PCR was carried out. Aft er cloning the PCR product having a desired size using pGEMeasy cloning vector (Pro mega), entire nucleotide sequence was identified by sequencing. The resulting cDNA was named IbOrange cDNA.
  • cDNA of the IbOrange gene which has total length of 942 bp, encodes 313 amin o acid residues and has a signal peptide targeting plastid at N'-end (Fig. 1).
  • Example 2 Expression analysis of IbOrange gene in different breeds and tissues of a sweet potato
  • RT-PCR was carried out. Sp ecifically, total RNA was isolated by using RNeasy Mini Kit from QIAGEN 1 and first cDN A was synthesized by using Superscript III First-Strand Synthesis System for RT-PCR f rom Invitrogen.
  • nucleotide sequence of the primers used were as follows: (forwar d primer: 5'-atcttgtcgctctcgtcctccacgacgccg-3 * (SEQ ID NO: 7), reverse primer: ⁇ '-cgtgg gtcatgctcgcttgccatagccatc-3 1 (SEQ ID NO: 8)).
  • the IbOrange gene had been expressed in all breed s that were tested, including Yulmi (Ym), i.e., a cream-colored sweet potato, Shinjami (J m), i.e., a purple-colored sweet potato, and Shinwhangmi (Hm), i.e., a yellow-colored s weet potato (Fig. 4). Further, according to the analysis of the different tissues of the pi ant, it was found that the IbOrange gene had been expressed generally all over the pla nt while there was strong expression in the leaves and fibrous roots of the plant (Fig. 5).
  • the IbOrange gene is a gene which is expressed homoge neously in a plant regardless of specific breed.
  • the expression pattern of the IbOrange gene under the influence of various hormones was as follows: ABA; alt hough there was no significant change in the leaves, strong expression of the IbOrange gene was found in the roots 36 hours after the treatment, ethephon; for both the leave s and the roots, strong expression of the IbOrange gene was found 36 hours after the tr eatment, in particular, the expression was stronger in the leaves, methyl jasmonate; for the leaves, expression of the IbOrange gene was strongly induced from 12 hours and m aintained until 36 hours after the treatment, and for the roots, the expression started to slightly increase from 12 hours and was highest 36 hours after the treatment, salicylic a cid; there was no significant change in both the leaves and the roots, but a slight increa se of the expression of the IbOrange gene was found from 12 hours to 36 hours after th e treatment (Fig.
  • the coding region of the IbOrange gene was cloned in pDONR20 7 vector, and then introduced in the expression vectors pDEST 17 and pDEST 15 wher ein a histidine affinity tag and glutathione-S-transferase (GST) fusion protein are compri sed.
  • E.coli BL21 DE3 strain
  • pDEST 17 and pDEST 15 wher ein a histidine affinity tag and glutathione-S-transferase (GST) fusion protein are compri sed.
  • E.coli BL21 DE3 strain
  • the protein s were isolated from the E.coli, and then characterized by using SDS-PAGE analysis.
  • the IbOrange gene cloned according to the present invention is indeed a gene which encodes the orange protein.

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Abstract

The present invention relates to IbOrange protein originating from a sweet potato which is related to accumulation of carotenoid, a gene which encodes the protein, a recombinant vector comprising the gene, a host cell transformed with the recombinant vector and a primer set for the amplification of IbOrange gene. According to the present invention, by increasing the production of carotenoid via the IbOrange gene, it is expected that a plant which has not only an improved function al activity but also strong resistance to environmental stress can be developed.

Description

IBORANGE GENE INVOLVED IN CAROTENOID ACCUMULATION FROM IPOMOEA
BATATAS
TECHNICAL FIELD The present invention relates to the Orange gene originating from "Shinwhangmi
" sweet potato which comprises a high amount of carotenoid. More specifically, the pr esent invention relates to a protein which can increase the production of carotenoid by controlling the expression of a gene that is related to biosynthesis of carotenoid and inv olved with the carotenoid accumulation in chromoplast and a gene which encodes said protein.
BACKGROUND ART Sweet potato {Ipomoea batatas L. Lam) is a root crop that can be cultivated in a relatively barren soil and has a productivity as high as about 30 tons per one ha. As s uch, it is commonly consumed both as a food for humans and a feed for livestocks. In particular, consisting of about 70% of starch in dry matter, the sweet potato has long be en used as a source material for obtaining alcohols and also is now believed as an envi ronmentally-friendly crop which can be used as an alternative source for obtaining ener gy, i.e., for producing bioethanol. Due to dramatic industrialization and population increase in recent years, environ ment and food problems are raised all over the world. As such, development of agricul tural crops which have resistance to extreme environmental condition in desert region, polluted region or cold region and the like is now actively being carried out. In particul ar, in order to develop an industrially favorable sweet potato that can be suitably cultivat ed under an environmentally-challenging region as described above, a transformed swe et potato which has resistance to environmentally disastrous conditions is currently bein g developed based on a molecular breeding method (Lim et al. MoI Breeding 19, 227-2 39, 2007). World Health Organization (WHO) reported that more than one billion children ar e suffering from vitamin A deficiency and as a result more than 500,000 children lose th eir sight every year. Beta-carotene is a precursor compound of vitamin A. Considerin g that β-carotene has a physiological activity as a nutrition fortifying agent and a food ai d, a metabolism study regarding the accumulation of carotenoid in food products is an e ssential research subject to improve nutritional value of them. Beta-carotene is known as a substance which has a high anti-oxidative activity and it is known not only for its p hysiological activity but also for its key role in a defense mechanism of a plant itself agai nst oxidative stress. Recently, it was found that biosynthesis of carotenoid is affected by ABA, which is one type of plant hormones. As such, a further research is needed t o determine the relationship between these anti-oxidative substances and environment al stress.
Meanwhile, Lu et. al. at Cornell University (USA) reported that the mutated Oran ge gene is related to the accumulation of carotenoid in chromoplast of cauliflower (Lu S et al. 2006 Plant Cell. 18: 3594-3605, 2006).
DETAILED DESCRIPTION OF THE INVENTION Technical Problem
The present invention is devised in view of the above-described needs. Invent ors of the present invention cloned the Orange gene from "Shinwhangmi" sweet potato which has a high amount of carotenoid. Consequently, with the confirmation of the pro duction of recombinant protein in Escherichia coli, the present invention was completed.
Technical Solution In order to solve the problems described in the above, the present invention provi des IbOrange protein originating from a sweet potato which is related to the accumulati on of carotenoid.
Further, the present invention provides a gene encoding IbOrange protein.
Further, the present invention provides a recombinant vector which comprises th e IbOrange gene.
Further, the present invention provides a host cell that is transformed with said re combinant vector.
Still further, the present invention provides a primer set for the amplification of th e IbOrange gene.
Effect of the Invention
According to the present invention, it was confirmed that the expression of orang e protein and the Orange gene originating from "Shinwhangmi" sweet potato is strongly induced by plant hormones such as ABA, ethephon, methyl jasmonate, salicylic acid an d the like that are related to drying stress and defense mechanism of a plant. Thus, it i s expected that by increasing the content of carotenoid via IbOrange gene a plant havin g not only the improved function but also the resistance to environmental stress can be developed.
BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 represents the nucleotide sequence of the Orange gene originating from a sweet potato ("Shinwhangmi") and the amino acid sequence deduced therefrom. The underlined region indicates a plastid localization signal, and cDNA consisting of 942 bp encodes the protein consisting of 313 amino acids.
Fig. 2 represents the amino acid sequence of the orange protein that is deduced from the IbOrange gene of a sweet potato (Ipomoea batatas) according to the present i nvention, wherein the amino acid sequences of the Orange gene from other various pla nts are also described for comparison (morning glory, tomato, grape, bean, soybean, co tton, Arabidopis, cauliflower, sweet sorghum, maize, rice, and barley).
Fig. 3 represents the genetic similarity relation between the amino acid sequence that is deduced from the IbOrange of a sweet potato (Ipomoea batatas) according to t he present invention and the Orange gene from other various plants (morning glory, to mato, grape, bean, soybean, cotton, Arabidopis, cauliflower, sweet sorghum, maize, ric e, and barley).
Fig. 4 represents the expression mode of the IbOrange gene according to the pr esent invention that is measured by RT-PCR and electrophoresis for various breeds of sweet potato. In the figure, Ym indicates "Yoolmi", Jm indicates "Shinjami" and Hm ind icates "Shinwhangmi", respectively.
Fig. 5 represents the expression mode of the IbOrange gene according to the pr esent invention that is measured by RT-PCR and electrophoresis for various parts of sw eet potato. Specifically, L indicates a leaf, S indicates a stem, and others include stora ge roots, fibrous roots and thick pigmented roots.
Fig. 6 represents the expression mode of the IbOrange gene according to the pr esent invention that is measured by RT-PCR and electrophoresis in which leaves and r oots of the sweet potato were treated with plant hormones such as ABA, ethephon (i.e., precursor of ethylene), methyl jasmonate (MeJA), and salicylic acid (SA) and the RT-P CR was carried out 0, 12, 24, 36 or 48 hours after the treatment.
Fig. 7 represents the result of electrophoresis of the protein, indicating the produ ction of the recombinant protein of the IbOrange gene in E.coli. Lane 1; soluble IbOra nge having His-tag, Lane 2; insoluble IbOrange having His-tag, Lane 3; soluble IbOrang e having GST-tag, and Lane 4; insoluble IbOrange having GST-tag.
BEST MODE FOR CARRYING OUT THE INVENTION In order to achieve the purpose of the invention described in the above, the pres ent invention provides IbOrange protein which originates from a sweet potato (Ipomoea batatas) that is related to the accumulation of carotenoid. Included in the scope of IbOrange protein according to the present invention are a protein having an amino acid sequence described in SEQ ID NO: 2, that is isolated fr om "Shinwhangmi" sweet potato, and functional equivalents of said protein. The term "functional equivalent" means that, as a result of addition, substitution or deletion of ami no acid residues, it has an amino acid sequence with at least 70%, preferably at least 8 0%, more preferably at least 90%, still more preferably at least 95% homology with the amino acid sequence of SEQ ID NO: 2, thus representing a protein which has substanti ally the same physiological activity as the protein expressed by SEQ ID NO: 2. Further, the present invention provides a gene which encodes the above-describ ed IbOrange protein. The gene according to the present invention includes both geno mic DNA and cDNA which encode IbOrange protein. Preferably, the gene according t o the present invention may comprise a nucleotide sequence that is represented by SE Q ID NO: 1. In addition, considering that IbOrange gene corresponds to a multigene f amily and has various homologous genes comprised in a sweet potato, the IbOrange g ene according to the present invention may comprise not only the IbOrange gene which has a nucleotide sequence represented by SEQ ID NO: 1 but also a multigene family of the IbOrange gene.
Further, a variant of this nucleotide sequence is also included in the scope of the present invention. Specifically, the above described gene may comprise a nucleotide sequence which has preferably at least 70%, more preferably at least 80%, still more p referably at least 90%, and most preferably at least 95% homology with the nucleotide s equence of SEQ ID NO: 1. Said "sequence homology %" for a certain polynucleotide i s identified by comparing a comparative region with two sequences that are optimally ali gned. In this regard, a part of the polynucleotide in comparative region may comprise an addition or a deletion (i.e., a gap) compared to a reference sequence (without any a ddition or deletion) relative to the optimized alignment of the two sequences.
Full length IbOrange cDNA of the present invention consists of 942bp and it enc odes 313 amino acid residues (see, Fig. 1). The isoelectric point (pi) and molecular w eight (Mw), that are calculated from the amino acid sequence, are 8.46 and 34.4 kDa, r espectively. With a BLAST analysis of the gene, it was found that it carries a repeatin g motive of CxxCxGxG comprising repeating cystein residues, that is well preserved in other orange proteins known as a kind of DnaJ protein, which is a molecular chaperone
It was confirmed that the IbOrange gene of the present invention is expressed in various breeds of a sweet potato including Yulmi (off-white colored sweet potato), Shinj ami (purple colored sweet potato), and Shinwhangmi (yellow colored sweet potato) (see , Fig. 4). Specifically, the IbOrange gene is generally expressed over the entire part of the plant while it is more strongly expressed in a leaf and a fibrous root (see, Fig. 5).
The expression of IbOrange gene according to the present invention can be stro ngly induced by plant hormones such as ABA, ethephon, methyl jasmonate, salicylic aci d and the like. Although there was no significant change occurred in the leaf that had been treated with ABA, the IbOrange gene was strongly expressed in the root 36 hours after the treatment. In the case of the treatment with ethephone, expression of the IbO range gene was induced in the leaf and the root 36 hours after the treatment. In partic ular, the expression was stronger in the leaf. In the case of the treatment with methyl j asmonate, expression of the IbOrange gene was strongly induced in the leaf 12 hours a fter the treatment, maintaining the high level until the 36 hour. In the root, the expressi on started to increase slightly 24 hours after the treatment, and reached the highest lev el at the 36 hour. In the case of the treatment with salicylic acid, there was no significa nt change both in the leaf and the root. However, there was slight increase in the expr ession of the IbOrange gene in the leaf from 12 hour to 36 hour period after the treatme nt (see, Fig. 6).
Further, the present invention provides a recombinant vector comprising the IbOr ange gene according to the present invention. Said recombinant vector is preferably a recombinant E. coli expression vector. The term "recombinant" indicates a cell which replicates a heterogeneous nucleo tide or expresses the nucleotide, a peptide, a heterogeneous peptide, or a protein enco ded by a heterogeneous nucleotide. Recombinant cell can express a gene or a gene f ragment, that are not found in natural state of cell, in a form of a sense or antisense. I n addition, a recombinant cell can express a gene that is found in natural state, provide d that said gene is modified and re-introduced into the cell by an artificial means.
The term "vector" is used herein to refer DNA fragment (s) and nucleotide molec ules that are delivered to a cell. Vector can be used for the replication of DNA and be i ndependently reproduced in a host cell. The terms "delivery system" and "vector" are often interchangeably used. The term "expression vector" means a recombinant DNA molecule comprising a desired coding sequence and other appropriate nucleotide sequ ences that are essential for the expression of the operatively-linked coding sequence in a specific host organism.
The vector of the present invention can be constructed to be a vector for cloning or expression in general. In addition, the vector of the present invention can be constr ucted to be a vector which has a prokaryotic cell or an eukaryotic cell as a host. For e xample, when the vector of the present invention is an expression vector and has a pro karyotic cell as a host, it generally comprises a strong promoter which can promote tran scription (for example, pLλ promoter, trp promoter, lac promoter, T7 promoter, tac prom oter and the like), a ribosome binding site for initiation of translation, and termination se quences for transcription/translation. When E. coli is employed as a host cell, the pro moter and the operator region involved in tryptophan biosynthesis in E. coli, and left side promoter of phage λ (i.e., pLλ promoter) can be used as a regulatory site.
Meanwhile, the vector that can be used in the present invention can be construct ed by using a plasmid (example: pSC101 , CoIEI , pBR322, pUC8/9, pHC79, pGEX seri es, pET series and pUC19 and the like), a phage (example: λgt4 λB, λ-Charon, λΔz1 an d M13 and the like) or a virus (example: SV40 and the like), that are typically used in th e pertinent art.
Meanwhile, when the vector of the present invention is an expression vector and has an eukaryotic cell as a host, a promoter originating from mammalian genome (exa mple: metallothionein promoter) or a promoter originating from mammalian virus (exam pie: adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegal ovirus promoter and tk promoter of HSV) can be used. As a transcription termination s equence, polyadenylated sequence is generally comprised. The vector of the present invention may comprise an antibiotics-resistant gene, t hat is typically used in the pertinent art, as a selection marker. Examples thereof inclu de a gene resistant to ampicillin, gentamycin, carbenicillin, chloramphenicol, streptomyc in, kanamycin, geneticin, neomycin or tetracycline, etc.
The present invention further provides a host cell that is transformed with the rec ombinant vector of the present invention. With respect to a host cell, any one known i n the pertinent art to have an ability for stable and continuous cloning and expression of the vector of the present invention can be used. Examples thereof include, Bacillus s p. strain including E. coli JM 109, E. coli BL21 , E. coli RR1 , E. coli LE392, E coli B, E. c o// X 1776, E. coli W3110, Bacillus subtillus, Bacillus thuringiensis and the like, and inte stinal bacteria and strains including Salmonella typhimurium, Serratia marcescens and various Pseudomonas sp. etc. Preferably, the host cell is E. coli.
In addition, when an eukaryotic cell is transformed with the vector of the present i nvention, Saccharomyce cerevisiae, an insect cell, a human cell (for example, CHO (Ch inese hamster ovary) cell line, W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK c ell line) and the like can be used as a host cell.
When a host cell is a prokaryotic cell, delivery of the vector of the present inventi on into a host cell can be carried out by CaC^ method (Cohen, S.N. et al., Proc. Natl. A cac. Sci. USA, 9:2110-2114 (1973)), Hanahan's method (Hanahan, D., J. MoI. Biol., 16 6:557-580 (1983)) or an electroporation method (Dower, W.J. et al., Nucleic. Acids Res. , 16:6127-6145 (1988)) and the like. In addition, when a host cell is an eukaryotic cell, the vector can be introduced to a host cell by a microinjection method (Capecchi, M. R., Cell, 22:479 (1980)), calcium phosphate precipitation method (Graham, F. L. et al., Virol ogy, 52:456 (1973)), an electroporation method (Neumann, E. et al., EMBO J., 1 :841 (1 982)), a liposome-mediated transfection method (Wong, T.K. et al., Gene, 10:87 (1980) ), DEAE-dextran treatment method (Gopal, MoI. Cell Biol., 5:1188-1190 (1985)), or a ge ne bombardment method (Yang et al., Proc. Natl. Acad. ScL, 87:9568-9572 (1990)) and the like.
The present invention further provides a primer set for the amplification of the Ib Orange gene. This primer set may consist of the oligonucleotide that is represented b y SEQ ID NO: 3 or SEQ ID NO: 4.
The primer set may consist of a primer set which comprises at least one oligonuc leotide that is selected from a group consisting of oligonucleotides that consist of a frag ment having 20 or more consecutive nucleotides included in the sequence of SEQ ID N
O: 3 and at least one oligonucleotide that is selected from a group consisting of oligonu cleotides that consist of a fragment having 20 or more consecutive nucleotides included in the sequence of SEQ ID NO: 4. Preferably, the primer set may consist of a primer set which comprises at least o ne oligonucleotide that is selected from a group consisting of oligonucleotides that consi st of a fragment having 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, or 30 consecutive nucleotides included i n the sequence of SEQ ID NO: 3 and at least one oligonucleotide that is selected from a group consisting of oligonucleotides that consist of a fragment having 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, or 30 consecutive nucleotides included in the sequence of SEQ ID NO: 4.
Most preferably, the primer set may consist of the primer set having SEQ ID NO: 3 and SEQ ID NO: 4. According to the present invention, the term "primer" indicates a single-stranded oligonucleotide which is complementary to the nucleotide strand to be copied and it can function as an initiation point for the synthesis of primer elongation product. The leng th and the sequence of the above-described primer should satisfy the condition require d for the initiation of the synthesis of an elongation product. According to the present i nvention, an oligonucleotide that is used as a primer may comprise a nucleotide analog ue, for example, phosphorothioate, alkyl phosphorothioate or a peptide nucleic acid, or an intercalating agent.
The present invention will now be described in greater detail with reference to th e following examples. However, it is only to specifically exemplify the present invention and in no case the scope of the present invention is limited by these examples.
Examples
<Example 1> Cloning of sweet potato IbOrange gene, analysis of nucleotid e sequence and genetic similarity
Total RNA was isolated from the leaves of a sweet potato (Ipomoea batatas (L.) Lam, Shinwhangmi breed) using RNeasy Mini Kit by QIAGEN. Further, using SuperSc ript III First-Strand Synthesis System for RT-PCR from Invitrogen, first cDNA was synth esized. In order to isolate the Orange gene, BLAST analysis using the Orange gene o f a cauliflower was carried out based on the website provided by TIGR Plant Transcript Assemblies (http://plantta.tiqr.org/). As a result, from EST clone of a morning glory flo wer (Ipomoea nil), which has genetic similarity with a sweet potato, a clone having a nu cleotide sequence that is similar to the Orange gene was found. By using this gene, pr imers for cloning the Orange gene from a sweet potato were produced. Sequence of t he primers are as follows: forward primer (5'-atggtatattcaggtagaatcttgtcgctc-3'; SEQ ID NO: 3) and reverse primer (5'-ttaatcaaatgggtcaattcgtgggtcatg-3'; SEQ ID NO: 4). To th e nucleotide sequence of the primers, an adapter sequence (bold letters) was attached at the 5'-end of each primers so that the gateway expression system provided by Invitro gen can be used. The resulting nucleotide sequences are as follows: forward primer ( 5'-CAAAAAAGCAGGCTNNatggtatattcaggtagaatcttgtcgctc-3'; SEQ ID NO: 5) and rever se primer (5'-CAAGAAAGCTGGGTNttaatcaaatgggtcaattcgtgggtcatg-3'; SEQ ID NO: 6).
Using the advantage2 polymerase provided by Clonetech, PCR was carried out. Aft er cloning the PCR product having a desired size using pGEMeasy cloning vector (Pro mega), entire nucleotide sequence was identified by sequencing. The resulting cDNA was named IbOrange cDNA. cDNA of the IbOrange gene, which has total length of 942 bp, encodes 313 amin o acid residues and has a signal peptide targeting plastid at N'-end (Fig. 1). As a resul t of gene BLAST against the Database, it was found that there is a repeat motif of CxxC xGxG having repeating cysteins, which is a kind of DnaJ protein known as a chaperone and well preserved in other orange proteins. The genetic similarity between the amino acid sequence from the coding sequence of the IbOrange of the present invention and the Orange genes of various plant species was determined by using programs of Blast X and ClustalW (http://plant.pdrc.re.kr/qene/aliqn/ClustalW.html). As a result, it was f ound that the highest homology was founds with the putative orange gene of a morning glory flower (Ipomoea nil). In addition, 73% to 80% of sequence homology was found with the putative orange gene of a tomato (Lycopersicon esculentum), a grape gene (Vi tis vinifera), At5g61670 gene of Arabidopsis thaliana, and a cauliflower gene (Brassica oleracea var. botrytis) (Fig. 2 and Fig. 3).
< Example 2> Expression analysis of IbOrange gene in different breeds and tissues of a sweet potato
In order to analyze the expression pattern of the IbOrange gene of the present in vention in different breeds and tissues of a sweet potato, RT-PCR was carried out. Sp ecifically, total RNA was isolated by using RNeasy Mini Kit from QIAGEN1 and first cDN A was synthesized by using Superscript III First-Strand Synthesis System for RT-PCR f rom Invitrogen. The nucleotide sequence of the primers used were as follows: (forwar d primer: 5'-atcttgtcgctctcgtcctccacgacgccg-3* (SEQ ID NO: 7), reverse primer: δ'-cgtgg gtcatgctcgcttgccatagccatc-31 (SEQ ID NO: 8)).
As a result, it was found that the IbOrange gene had been expressed in all breed s that were tested, including Yulmi (Ym), i.e., a cream-colored sweet potato, Shinjami (J m), i.e., a purple-colored sweet potato, and Shinwhangmi (Hm), i.e., a yellow-colored s weet potato (Fig. 4). Further, according to the analysis of the different tissues of the pi ant, it was found that the IbOrange gene had been expressed generally all over the pla nt while there was strong expression in the leaves and fibrous roots of the plant (Fig. 5).
As such, it was learned that the IbOrange gene is a gene which is expressed homoge neously in a plant regardless of specific breed.
<Example 3> Expression analysis of the IbOrange gene after the treatment with various plant hormones
In order to determine the expression pattern of the IbOrange gene of the present invention responding to various plant hormones such as ABA, ethephon, methyl jasmo nate, salicylic acid and the like which are related to defense mechanism and drying stre ss of a plant, the plants were treated with each of said hormones and then at various ti me points (i.e., 0, 12, 24, 36, and 48 hours after the hormone treatment) RNA was isola ted and cDNA was synthesized therefrom in the same manner as Examples 1 and 2. The primer which is specific to the IbOrange gene, which has been used in the above E xample 2, was used for RT-PCR. As a result, it was found that the expression pattern of the IbOrange gene under the influence of various hormones was as follows: ABA; alt hough there was no significant change in the leaves, strong expression of the IbOrange gene was found in the roots 36 hours after the treatment, ethephon; for both the leave s and the roots, strong expression of the IbOrange gene was found 36 hours after the tr eatment, in particular, the expression was stronger in the leaves, methyl jasmonate; for the leaves, expression of the IbOrange gene was strongly induced from 12 hours and m aintained until 36 hours after the treatment, and for the roots, the expression started to slightly increase from 12 hours and was highest 36 hours after the treatment, salicylic a cid; there was no significant change in both the leaves and the roots, but a slight increa se of the expression of the IbOrange gene was found from 12 hours to 36 hours after th e treatment (Fig. 6). In general, it is known that under drying stress the amount of ABA, which is one kind of plant hormones, increases so that ABA can effectively control the opening and c losing of stomata of a plant as a response to drying condition. Meanwhile, since carot enoid is a precursor material of ABA, it is believed that the increase in ABA responding to drying stress has a direct relation with the increase in the amount of carotenoid. Th erefore, the increased expression of the IbOrange gene responding to the ABA treatme nt as shown in this experiment clearly demonstrates the close relationship between dryi ng stress and the IbOrange gene.
<Example 4> Characterization of the protein produced according to the exp ression of the IbOrange gene in E.coli
In order to determine whether or not a protein having desired size is produced fro m the IbOrange gene, the coding region of the IbOrange gene was cloned in pDONR20 7 vector, and then introduced in the expression vectors pDEST 17 and pDEST 15 wher ein a histidine affinity tag and glutathione-S-transferase (GST) fusion protein are compri sed. E.coli (BL21 DE3 strain) for expression was transformed with the resulting expressi on vector and the expression of the orange protein was induced by IPTG. The protein s were isolated from the E.coli, and then characterized by using SDS-PAGE analysis. As a result, it was found that 34kDa recombinant protein having histidine tag and 57kDa GST-fusion protein were successfully expressed (Fig. 7). Consequently, it was again confirmed that the IbOrange gene cloned according to the present invention is indeed a gene which encodes the orange protein. Taken together, by increasing the content of carotenoid via the IbOrange gene, itected that a plant which has not only an improved functional activity but also strostance to environmental stress can be developed.

Claims

1.
IbOrange protein originating from a sweet potato (Ipomoea batatas) wherein the protein consists of the amino acid sequence represented by SEQ ID NO: 2.
2.
A gene encoding IbOrange protein according to Claim 1.
3.
The gene according to Claim 2, characterized in that said gene consists of the n ucleotide sequence represented by SEQ ID NO: 1.
4.
The gene according to Claim 2, characterized in that expression of said gene is i ncreased by ABA, ethephon, methyl jasmonate, or salicylic acid.
5. A recombinant vector which comprises the gene according to Claim 2.
6.
A host cell that is transformed with the recombinant vector according to Claim 5.
7.
The host cell according to Claim 6, characterized in that it is Escherichia coli.
8.
A primer set for the amplification of IbOrange gene, consisting of the oligonucle otide represented by SEQ ID NO: 3 and SEQ ID NO: 4.
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