KR20110105969A - Ibmt2 gene from ipomoea batatas and uses thereof - Google Patents

Abstract

The present invention is sweet potato ( Ipomoea batatas IbMT2 ( Ipomoea batatas ) Related to Heavy Metal Resistance from L. cv Yulmi) metallothionein 2) a protein, a gene encoding the protein, a recombinant vector comprising the gene, a host cell transformed with the recombinant vector, a method for enhancing heavy metal resistance of a plant by transforming the recombinant vector into a plant cell, the The present invention relates to a transformed plant having enhanced heavy metal resistance and a seed produced by the method, a method for producing a heavy metal resistant transformed plant using the recombinant vector, and a composition for enhancing heavy metal resistance of a plant comprising the gene.

Description

IbMT2 gene from Ipomoea batatas and uses approximately}

The present invention is IbMT2 derived from sweet potatoes The present invention relates to a protein and a gene encoding the same, and more particularly, to sweet potato ( Ipomoea) batatas IbMT2 ( Ipomoea ) Related to Heavy Metal Resistance from L. cv Yulmi) batatas metallothionein 2) a protein, a gene encoding the protein, a recombinant vector comprising the gene, a host cell transformed with the recombinant vector, a method for enhancing heavy metal resistance of a plant by transforming the recombinant vector into a plant cell, the The present invention relates to a transformed plant having enhanced heavy metal resistance and a seed produced by the method, a method for producing a heavy metal resistant transformed plant using the recombinant vector, and a composition for enhancing heavy metal resistance of a plant comprising the gene.

Plants are subjected to oxidative stress not only when exposed to various stresses such as infections or wounds of heavy metals or pathogens, but also through normal respiration and photosynthetic mechanisms (Apel et al, 2004, Annu Rev Plant Biol 53, 373-399). Oxidative stress is a common process in almost all stress responses. Oxygen is an essential molecule for the aerobic survival, but depending on the reaction, it can turn into a deadly harmful molecule. Molecules in which the electrons are added to the excited oxygen to speed up the chemical reaction are called reactive oxygen species. These include singlet oxygen, superoxide, hydrogen peroxide, and hydroxyl radicals. radicals). In particular, chloroplasts are intracellular organelles in which photosynthesis occurs in plants and is always considered to be a high oxidative stress condition due to the presence of a high energy electron transport system for energy acquisition and mass production of free radicals.

Therefore, plants have various antioxidant systems to combat this oxidative stress. Plant antioxidants include tocopherol, ascorbic acid, carotenoids and flavonoids, and antioxidant enzymes include superoxide dismutase (SOD), ascorbrate peroxidase (APX), and catalase ( Catalase) and many other reductases are known. However, despite its own antioxidant mechanisms, there is a limit in detoxifying oxidative stresses introduced under abnormal conditions (Lee et al, 2004, Plant Mol Biol 54, 805-815).

Metallothioneins (MTs) are low molecular weight (<10 kDa) cysteine (Cys) -rich proteins that can bind heavy metals (Hamer, 1986, Annu Rev Biochem 55, 913-951; Robinson et al, 1993, Biochem J 295, 1-10). This protein is widely distributed in a wide variety of organisms, from fungi, plants, cyanobacteria and mammals, and is involved in homeostasis of metal ions and detoxification of heavy metals in cells (Cobbett and Goldsbrough, 2002, Annu Rev). Plant Biol 53, 159-182). Expression of the plant metallothionein gene is primarily regulated by metal ions, and other factors include stress such as salinity stress, abscis acid, sucrose starvation, heat shock, wounds and viral infections. (Haq et al, 2003, Mutation Research 533, 211-226). Plant metallothionein is classified into three classes, Class I, Class II and Class III, based on the amino acid sequence (Rauser, 1999, Cell Biochem Biophys 31, 19-48).

Class I metallothionein has two Cys-rich domains separated by a central Cys-free spacer, and is divided into four types depending on the arrangement of cysteine residues in amino acids and COOH domains. (Yu et al, 1998, Gene 206, 29-35). Type 1 metallothionein has a Cys-Xaa-Cys motif specific to two terminal domains. Type 2 metallothionein has eight conserved cysteine residues through Cys-Cys and two Cys-X-Cys, Cys-XX-Cys motifs inside the N-terminal domain, and the C-terminal domain also has six Cys-X-Cys is highly conserved. Type 3 metallothionein has typical features with four conserved cysteine residues at the N-terminus. Type 4 metallothionein is specifically expressed in seeds.

Class II metallothionein is an Ec protein in wheat with cysteine residues scattered throughout the amino acid sequence. Finally, class III metallothionein is a phytochelatins, a peptide that is enzymatically synthesized in a poly-glycine structure.

Another function of metallothionein is its antioxidant function. Wong (2004, Plant Physiol 135, 1447-1456) reported that the superoxide and hydrogen peroxide activity as a scavenger increased when overexpressed rice type 2 metallothionein (OsMT2b). Andrew (2000, Biochem Phyrmacol 59, 95-104) also reported that the expression of the metallothionein gene is induced by oxidative stress and is active as an antioxidant.

Recently, the environmental pollution caused by the rapid industrialization has reached a serious state that can threaten the environmental resistance of plants. Therefore, efforts have recently been made to create plants that are resistant to oxidative stress as well as to various environmental stresses. In order to solve these problems, an over-expression of antioxidant system-related enzymes has been introduced (Kwon et al, 2002, Plant Cell). and Environm 25, 873-882) Research into the introduction of chelators or transporters of various heavy metals (Song et al, 2004, Plant Physiol 135, 1-13). Based on these recent studies, the team is conducting basic research to develop environmentally resistant crops that are resistant to heavy metal ions and to oxidative stress.

In this study, three types of metallothionein genes (IbMT) were isolated from sweet potato plants to determine their resistance to heavy metals and various oxidative stresses. To this end, E. coli transformation vector was prepared and transformed into E. coli recombinant protein expression strain BL21 (DE3) species to induce the expression of metallothionein protein. In addition, we examined the expression of three metallothionein genes (IbMT) by treating various plant hormones, heavy metals, and abiotic oxidative stress in sweet potato cells.

Korean Patent Publication No. 2010-0022236 discloses a CeMT2 gene that exhibits heavy metal resistance derived from taro, and Korean Patent Publication No. 2005-0023044 discloses a Pcr gene showing heavy metal resistance and accumulation.

The present invention is derived from the above demands, and the present inventors have found IbMT2 derived from sweet potato. The present invention was completed by confirming that E. coli transformed with a gene exhibits resistance to various heavy metals.

In order to solve the above problems, the present invention is sweet potato ( Ipomoea batatas IbMT2 ( Ipomoea ) Related to Heavy Metal Resistance from L. cv Yulmi) batatas metallothionein 2) provides a protein.

The present invention also provides a gene encoding the IbMT2 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 method of enhancing the heavy metal resistance of plants by transforming the recombinant vector into plant cells.

In addition, the present invention provides a transgenic plant with enhanced heavy metal resistance produced by the above method and its seed.

The present invention also provides a method for producing a heavy metal resistant transformed plant using the recombinant vector.

The present invention also provides a composition for enhancing heavy metal resistance of a plant comprising the gene.

Yuli sweet potato-derived metallothionein protein and gene of the present invention was increased in expression by a variety of plant hormones, heavy metals and various abiotic external stress, it was confirmed that the expression in E. coli shows resistance in the medium containing heavy metals. Therefore, the IbMT gene is expected to be able to develop transgenic plants that grow not only in heavy metal contaminated regions but also resistant to environmental stress.

Figure 1 shows the nucleotide sequence of the sweet potato (breed Yumi) derived IbMT gene of the present invention (Fig. 1A) and the amino acid sequence deduced therefrom (Fig. 1B).
Figure 2 shows the amino acid sequence of the deduced protein of the sweet potato (Ipomoea batatas) IbMT gene of the present invention and various plants (mulmullia, ginseng, cephalopod, birch, tea tree, buttercup, tobacco, potato, cotton, peanut, wild Rice, barley and bamboo metallothionein gene amino acid sequence comparison results.
FIG. 3 is a diagram showing a flexible relationship between the IbMT gene and other metallothionein genes compared to those of FIG. 2.
4A is a photograph of electrophoresis by RT-PCR of the expression of the IbMT gene of the present invention for each portion of sweet potato, and FIG. 4B shows the expression of the IbMT gene in sweet potato calli callus. L: leaf, S: stem, Storage roots: Fibrous roots.
Figure 5 is treated with sweet potato callus plant hormones (apsis acid, etepon, methyl jasmonate and salicylic acid), heavy metals (Fe, Cu, Zn and Mn), and MV (methyl viologen), sodium chloride, hydrogen peroxide and low temperature, respectively RT-PCR was performed after 3 and 18 hours to show the expression of IbMT1 gene. Con: control, SA: salicylic acid, ABA: absic acid, MJ: methyl jasmonate, ETE: etepon, Cold: low temperature.
6 shows the results of SDS-PAGE confirming that IbMT genes of the present invention are expressed in E. coli to produce proteins. A: IbMT1 (Ni-column tablets), B: IbMT2 (crude extract, insoluble), C: IbMT3 (crude extract, soluble).

In order to achieve the object of the present invention, the present invention consists of an amino acid sequence represented by SEQ ID NO: 2, sweet potato ( Ipomoea batatas IbMT2 ( Ipomoea ) Related to Heavy Metal Resistance from L. cv Yulmi) batatas metallothionein 2) provides a protein.

The range of IbMT2 proteins according to the present invention includes proteins having the amino acid sequence represented by SEQ ID NO: 2 isolated from sweet potatoes 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. "Substantially homogeneous physiological activity" means activity that enhances heavy metal resistance of a plant.

The invention also includes fragments, derivatives and analogues of the IbMT2 protein. As used herein, the terms “fragment”, “derivative” and “analogue” refer to a polypeptide that possesses substantially the same biological function or activity as the IbMT2 polypeptide of the invention. Fragments, derivatives, and analogs of the present invention comprise (i) polypeptides substituted with one or more conservative or nonconservative amino acid residues (preferably conservative amino acid residues), wherein the substituted amino acid residues are encoded by a genetic code. Or (ii) a polypeptide having substituent (s) at one or more amino acid residues, or (iii) another compound (a compound capable of extending the half-life of a polypeptide, such as polyethylene glycol) A polypeptide derived from a bound mature polypeptide, or (iv) an additional amino acid sequence (eg, a leader sequence, a secretion sequence, a sequence used to purify the polypeptide, a proteinogen sequence or a fusion protein) and It may be a polypeptide derived from said polypeptide bound. Such fragments, derivatives and analogs as defined herein are well known to those skilled in the art.

The present invention also provides a gene encoding the IbMT2 protein. The gene of the present invention may be DNA or RNA encoding the IbMT2 protein. DNA includes cDNA, genomic DNA or artificial synthetic DNA. DNA can be single stranded or double stranded. DNA may be a coding strand or a non-coding strand.

 Preferably, the gene of the present invention may include the nucleotide sequence shown in SEQ ID NO: 1.

Polynucleotides encoding a mature polypeptide represented by SEQ ID NO: 2 include a coding sequence encoding only a mature polypeptide; Sequences encoding mature polypeptides and various additional coding sequences; Mature polypeptides (and any additional coding sequences) and sequences encoding noncoding sequences.

The term "polynucleotide encoding a polypeptide" refers to a polynucleotide encoding a polypeptide, or a polynucleotide further comprising additional coding and / or noncoding sequences.

The invention also relates to variants of said polynucleotides encoding polypeptides comprising the same amino acid sequence as described herein, or fragments, analogs, and derivatives thereof. Polynucleotide variants may be naturally occurring allelic variants or non-naturally occurring variants. Such nucleotide variants include substitutional variants, deletional variants, and insertional variants. As is known in the art, allelic variants are alternatives to polynucleotides, which may include one or more substituted, deleted or inserted nucleotides, which do not result in a substantial functional change in the polypeptide encoded by the variant. Do not.

In addition, the present invention provides a poly-hybridized hybridization with a sequence having at least 50%, preferably at least 70%, more preferably at least 80% identity with a nucleotide sequence of SEQ ID NO. It relates to nucleotides. The present invention particularly relates to polynucleotides that hybridize to the polynucleotides described herein under stringent conditions. In the present invention, "stringent conditions" include (1) hybridization and washing under 0.2 × SSC, 0.1% SDS, lower ionic strength such as 60 ° C. and higher temperature; Or (2) hybridization in the presence of denaturing agents such as 50% (v / v) formamide, 0.1% bovine serum / 0.1% Ficoll and 42 ° C .; Or (3) hybridization that occurs between only two sequences having at least 80%, preferably at least 90%, more preferably at least 95% identity. In addition, the biological function and activity of the polypeptide encoded by the hybridizable polynucleotide are identical to the biological function and activity of the mature polypeptide represented by SEQ ID NO: 2.

In accordance with an embodiment of the invention, it should be understood that the IbMT2 gene is preferably derived from sweet potatoes. However, embodiments of the invention have high homology (eg, 60% or more, ie, 70%, 80%, 85%, 90%, 95%, even 98% sequence identity) with the sweet potato IbMT2 gene and differ from others. It also includes other genes derived from plants. Sequencing methods and means for determining sequence identity or homology (eg BLAST) are well known in the art.

The present invention also provides a recombinant vector comprising the IbMT2 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, 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. 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.

In the present invention, the polynucleotide sequence encoding the IbMT2 protein can be inserted into a recombinant expression vector. The term "recombinant expression vector" means a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus, or other vector. In principle, any plasmid and vector can be used as long as it can replicate and stabilize in the host. An important feature of the expression vector is that it has an origin of replication, a promoter, a marker gene and a translation control element.

Expression vectors comprising an IbMT2 protein-encoding DNA sequence and appropriate transcriptional / translational control signals can be constructed by methods well known to those skilled in the art. Such methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence can be effectively linked to a suitable promoter in the expression vector to drive mRNA synthesis. Expression vectors may also include ribosomal binding sites and transcription terminators as translation initiation sites.

Preferred examples of recombinant vectors of the invention are Ti-plasmid vectors capable of transferring part of themselves, the so-called T-region, to plant cells when present in a suitable host such as Agrobacterium tumerfaciens. 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. Preferably, the recombinant vector may be, but is not limited to, a pKBS1-1 vector, a pBI101 vector, and a pCAMBIA vector.

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, and chloramphenicol. Resistance genes include, but are not limited to.

In the recombinant vector of the present invention, the promoter may be, but is not limited to, CaMV 35S, actin, ubiquitin, pEMU, MAS or histone promoter. The term "promoter " refers to the region of DNA upstream from the structural gene and refers to a DNA molecule to which an RNA polymerase binds to initiate transcription. A "plant promoter" is a promoter capable of initiating transcription in plant cells. A "constitutive promoter" is a promoter that is active under most environmental conditions and developmental conditions or cell differentiation. Constructive promoters may be preferred in the present invention because the choice of transformants can be made by various tissues at various stages. Thus, the constitutive promoter does not limit the selection possibilities.

In the recombinant vectors of the present invention, conventional terminators can be used, for example nopalin synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, Agrobacterium tumefaciens (Agrobacterium tumefaciens) Terminator of the octopine gene, but is not limited thereto. Regarding the need for terminators, it is generally known that such regions increase the certainty and efficiency of transcription in plant cells. Therefore, the use of a terminator is highly desirable in the context of the present invention.

The present invention also provides a host cell transformed with the recombinant vector of the present invention. The host cell capable of continuously cloning and expressing the vector of the present invention in a prokaryotic cell while being stable can be used in any host cell known in the art, for example, E. coli JM109, E. coli BL21, E. coli RR1. , Bacillus genus strains, such as E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus thuringiensis, and Salmonella typhimurium, Serratia marcensons, and various Pseudomonas Enterobacteria such as species and strains. The host cell is preferably E. 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 lines (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cell lines), and plant cells. The host cell is preferably a plant cell.

The method of carrying the vector of the present invention into a host cell is performed by using the CaCl ₂ method or one method (Hanahan, D., J. Mol. Biol., 166: 557-580 (1983)) when the host cell is a prokaryotic cell. And the electroporation method. When the host cell is a eukaryotic cell, the vector is injected into the host cell by microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, and gene bombardment .

Transformation of a plant means any method of transferring DNA to a plant. Such transformation methods do not necessarily have a period of regeneration and / or tissue culture. 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.

The present invention also provides a method for enhancing heavy metal resistance of a plant comprising the step of transforming said recombinant vector into plant cells. Preferably, the heavy metal may be calcium, copper, manganese, magnesium or lead, but is not limited thereto.

In addition, the present invention provides a transgenic plant with enhanced heavy metal resistance produced by the above method and its seed. Preferably, the plant may be a monocotyledonous plant or a dicotyledonous plant, but is not limited thereto.

The monocotyledonous plants include Alismataceae, Hydrocharitaceae, Juncaginaceae, Schuchzeriaceae, Pomomotontonaceae, Najadaceae, Zosteraceae, Liliaceae, Haemodoraceae, Agavaceae, Amaryllidaceae, Dioscoreaceae, Pontederiaceae, Iridaceae, Burmanniaceae, Juncaceae, Commelinaceae , Eriocaulaceae, Panaxaceae (Gramineae, Gramineae, Poaceae), Araceae, Lemnaceae, Sparganiaceae, Typhaceae, Cycloaceae, Cyperaceae, Musaceae ), Ginger (Zingiberaceae), Cannaceae (Cannaceae) or Orchidaceae (Orchidaceae), but is not limited thereto.

The dicotyledonous plants are Asteraceae (Dolaceae, Diapensiaceae), Asteraceae (Clethraceae), Pyrolaceae, Ericaceae, Myrsinaceae, Primaceae (Primulaceae), Plumbaginaceae, Persimmonaceae (Ebenaceae) , Styracaceae, Stink bug, Symplocaceae, Ash (Oleaceae), Loganiaceae, Gentianaceae, Menyanthaceae, Oleaceae, Apocynaceae , Asclepiadaceae, Rubiaceae, Polemoniaceae, Convolvulaceae, Boraginaceae, Verbenaceae, Labiatae, Solanaceae, Scrophulariaceae , Bignoniaceae, Acanthaceae, Sesame (Pedaliaceae), Fructose (Orobanchaceae). Gesneriaceae, Lentibulariaceae, Phrymaceae, Plantaginaceae, Caprifoliaceae, (Perox Adoxaceae), Valerianaceae, Dipsacaceae, Campanaceae ( Campanulaceae, Compositae, Myricaceae, Sapaceae, Juglandaceae, Salicaceae, Birchaceae, Beechaceae, Fagaceae, Elmaceae, Moraceae , Urticaceae, Santalaceae, Mistletoe, Lothanthaceae, Polygonaceae, Landaceae, Phytolaccaceae, Nyctaginaceae, Pomegranate, Azizaceae (Portulacaceae), Caryophyllaceae, Chinopodiaceae, Amaranthaceae, Cactaceae, Magnoliaceae, Illiciaceae, Lauraceae, Cassia family, Cecidiphyllaceae, Ranunculus eae), Berberidaceae, Lardizabalaceae, Bird breeze (Mentaceae, Menispermaceae), Nymphaeaceae, Ceratophyllaceae, Cabombaceae, Saururaceae , Piperaceae, Chloranthaceae, Aristolochiaceae, Actinidiaceae, Camellia, Theaceae, Guttaiferae, Droseraceae, Papaveraceae ), Capparidaceae, Cruciferaceae (Mustaceae, Cruciferae), Planeaceae (Plataceae, Platanaceae), Verruaceae, Hamamelidaceae, Pheasant (Snaphaceae, Crassulaceae), Panaxaceae (Saxifragaceae) Eucommiaceae, Pittosporaceae, Rosaceae, Leguminosae, Oxalidaceae, Geraniaceae, Tropaeolaceae, Zygophyllaceae, Linaceae Euphorbiaceae), star moss (Callitrichaceae), Rutaceae, Simaroubaceae, Meliaceae, Polygalaceae, Anacardiaceae, Mapleaceae, Aceraceae, Sapindaceae, Mapleaceae Hippocastanaceae, Sabiaceae, Balsam, Balsaminaceae, Aquifoliaceae, Nova, Celastraceae, Staphyleaceae, Buxaceae, Empetraceae, Rhamnaceae, Vitaceae, Elaeocarpaceae, Tiliaceae, Malvaceae, Sterculiaceae, Adenaceae, Thymelaeaceae, Eraeagnaceae (Flacourtiaceae), Violaceae, Passifloraceae, Tamaricanaceae, Elatinaceae, Begoniaceae, Cucurbitaceae, Buddha (Lythraceae), Pomegranate Punica ceae), Onnagraceae, Haloragaceae, Bataceae (Alangiaceae), Dogwood (Hornaceae, Cornaceae), Arboraceae (Agaraceae) or Umbelliferae (Apiaceae) It may be, but is not limited thereto.

In addition, the present invention comprises the steps of transforming plant cells with the recombinant vector; And

It provides a method for producing a heavy metal resistant transformed plant comprising the step of regenerating a plant from the transformed plant cells.

The method of the present invention comprises transforming plant cells with a recombinant vector according to the present invention, which transformation can be mediated by, for example, Agrobacterium tumefiaciens. The method also includes the step of regenerating the transgenic plant from said transformed plant cell. The method for regenerating the transformed plant from the transformed plant cell may use any method known in the art.

Transformed plant cells should be re-differentiated into whole plants. Techniques for the regeneration of mature plants from callus or protoplast cultures are well known in the art for numerous different species (Handbook of Plant Cell Culture, Vol. 1-5, 1983-1989 Momillan, N.Y.).

The present invention also provides a composition for enhancing heavy metal resistance of plants, comprising the IbMT2 gene of the present invention. In the composition of the present invention, the IbMT2 gene may be composed of the nucleotide sequence of SEQ ID NO: 1. The composition of the present invention contains an IbMT2 gene derived from sweet potato as an active ingredient, and can improve heavy metal resistance of the plant by transforming the gene IbMT2 into the plant. The plant is as described above.

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 IbMT  Gene Cloning , Sequencing and softness analysis

Total RNA was isolated from the leaves of Yumi plant, a variety of sweet potato (Ipomoea batatas (L.) Lam) using QIAGEN's RNeasy mini kit. One strand cDNA was synthesized using Invitrogen's SuperScript III First-Strand Synthesis System for RT-PCR. To isolate metallothionein genes, blasted with metallothionein genes from the website of http://plantta.tigr.org/ (TIGR Plant Transcript Assemblies) (Brassica campestris ssp.pekinensis) Two metallothionein-like genes and one gene induced by aging were found. Considering these as potential type 1, type 2 and type 3 metallothionein, primers were prepared for cloning three genes from Ipomoea batatas L. cv Yulmi.

The base sequences of the primers for cloning the type 1 metallothionein gene are forward primers (5'-ATGTCTTCCGGTTGCAAG-3 '; SEQ ID NO: 3) and reverse primers (5'-ACAGTTGCAAGGGTC-3'; SEQ ID NO: 4). The base sequences of the primers for cloning the type 2 metallothionein gene are forward primers (5'-ATGTCTTGCTGCGGAGGA-3 '; SEQ ID NO: 5) and reverse primers (5'-ACATTTGCAAGGTCG-3'; SEQ ID NO: 6). The base sequences of the primers for cloning the type 3 metallothionein gene are forward primers (5'-ATGTCGGACAAGTGCGGA-3 '; SEQ ID NO: 7) and reverse primers (5'-GTGGCCACAGGTGCG-3'; SEQ ID NO: 8). PCR was performed using Clonetech's advantage2 polymerase, and the PCR product of the expected size was cloned using a pGEMTeasy cloning vector (promega), followed by sequencing to confirm the entire nucleotide sequence.

The cDNA gene was named IbMT1, IbMT2 and IbMT3 cDNA according to the type 1, type 2 and type 3 classification of the metallothionein genes, respectively. The total length of the IbMT1 cDNA gene is 201 bp and encodes 67 amino acids. The total length of the IbMT2 cDNA gene is 249 bp and encodes 83 amino acids. The total length of the IbMT3 cDNA gene is 195 bp and encodes 65 amino acids (FIG. 1).

As a result of blasting the gene from the database, IbMT1 gene was identified as sweet potato ( Ipomoea). batatas L. cv. Kokei and I. batatas L. cv. Tainong57) showed 99% homology with the metallothionein-like type 1 genes (GenBank: AF116845.1 and GenBank: AB193156.1). IbMT2 gene is a sweet potato ( I. batatas L. cv. Tainong57) showed 96% homology with the metallothionein-like type 2 gene (GenBank: AF177760.1). IbMT3 gene is a sweet potato ( I. batatas L. cv. Tainong57) showed 100% homology with the metallothionein-like protein gene (GenBank: AF242374.1).

As a result of blasting the gene of the present invention in a database with genes other than sweet potatoes, IbMT1 was identified as Mimulus guttatus ) showed the highest homology with the putative metallothionein gene, as well as Panax ginseng. ginseng ), Arabidopsis thaliana ), Populus trichocarpa and Betula platyphylla homology with the putative metallothionein genes. IbMT2 gene is known as Camellia sinensis ) showed the highest homology with the putative metallothionein gene of the genus , but also Oenanthe javanica and Nicotiana plumbaginifolia ) and potatoes ( Solanum homologous to gene of tuberosum ). IbMT3 gene is cotton ( Gossypium In addition it showed estimated metallothionein gene with the highest homology hirsutum), peanuts (Arachis hypogaea ), wild rice ( Porteresia coarctata ), barley ( Hordeum vulgare ) and bamboo ( Phyllostachys) edulis ) showed homology with the putative metallothionein gene (FIG. 2 and 3).

Example  2: sweet potato IbMT  Analysis of gene expression by tissue

It was confirmed that the IbMT gene of the present invention is expressed in the off-white sweet potato Yulmi, the purple sweet potato, Shinjami, and the yellow sweet potato, Shinhwangmi varieties (data not shown). Among the sweet potato varieties, RT-PCR was performed to analyze tissue expression patterns of IbMT gene in Yumi sweet potato.

Leaves, stems, roots and storage roots were isolated from sweet potato plants to separate total RNA using QIAGEN's RNeasy mini kit, and a single-strand cDNA was synthesized using Invitrogen's SuperScript III First-Strand Synthesis System for RT-PCR. As primers, primers used for cloning the IbMT1, IbMT2 and IbMT3 genes were used in Example 1.

As a result, the IbMT gene was strongly expressed in the leaves and the roots of the IbMT1 gene, but weakly expressed in the stems and the roots. IbMT2 was expressed in the stem and weakly expressed in the roots, but not in the leaves and roots. IbMT3 gene was strongly expressed in the leaves and stems corresponding to the ground portion, but weakly expressed in the root and storage roots (FIG. 4A). In addition, as a result of examining the expression of three IbMT genes in Yuli callus, only IbMT1 gene was specifically expressed in callus, and other IbMT2 and IbMT3 genes were not expressed (FIG. 4B). Therefore, IbMT genes are found throughout the plant, but each gene is expressed in a different pattern.

Example  3: by various external stress treatment IbMT  Gene expression analysis

Experiments were carried out on yulmi varieties of sweet potato callus in the growth phase 7 days after passage. Plant hormones involved in dry stress and plant defense mechanisms (apsis acid, etepon, methyl jasmonate and salicylic acid), heavy metals (iron, copper, zinc and manganese), and abiotic stresses MV, sodium chloride, hydrogen peroxide and low temperature ( cold) was treated separately. Callus was treated to a depth about 1/5 submerged in each solution, and the callus was sampled after 3 and 18 hours. In order to perform RT-PCR, RNA was isolated by the method described in Examples 1 and 2, and cDNA was synthesized, respectively. RT-PCR was performed with the IbMT gene specific primers used in the above examples.

As a result, IbMT gene showed the following expression. When treated with plant hormones, IbMT1 gene was strongly induced 3 hours after treatment with abscic acid, etepon and methyl jasmonate, but salicylic acid showed a slight decrease in expression. After 18 hours, the expression was increased by salicylic acid, and the increase by methyl jasmonate was sustained, whereas the increase by Apsis acid and etepon, which was strongly induced after 3 hours of treatment, decreased to the control level. Could be (FIG. 5A).

When heavy metals were treated, the IbMT1 gene increased strongly after 3 hours of iron and manganese treatment, but after 18 hours of treatment, it decreased to the level before treatment, and in the case of copper, expression was increased after 18 hours of treatment. No increase in expression was seen by zinc treatment (FIG. 5B).

After 3 hours of nonbiological stress treatment such as MV, sodium chloride, hydrogen peroxide and low temperature, the expression of IbMT1 gene was slightly increased only by low temperature, but decreased by MV, sodium chloride and hydrogen peroxide. However, after 18 hours of treatment, it was confirmed that the IbMT1 gene was strongly induced by sodium chloride and low temperature (FIG. 5C).

Example  4: IbMT  Determination of Heavy Metal Resistance by Gene Expression of Escherichia Coli

In order to confirm whether the IbMT gene of the present invention actually shows resistance to heavy metals, the IbMT gene was cloned into an E. coli expression vector. For this purpose, primers were prepared by binding the BamHI restriction enzyme sequence at the 5 'end and the SalI restriction enzyme sequence at the 3' end. The base sequences of the primers for cloning the type 1 metallothionein gene were forward primers (5'-GCggatcc ATGTCTTCCGGTTGCAAG-3 '; SEQ ID NO: 9) and reverse primers (5'-CTgtcgac ACAGTTGCAAGGGTC-3'; SEQ ID NO: 10). to be. The base sequences of the primers for cloning the type 2 metallothionein gene are forward primers (5'-GCggatccATGTCTTGCTGCGGAGGA-3 '; SEQ ID NO: 11) and reverse primers (5'-CTgtcgac ACATTTGCAAGGTCG-3'; SEQ ID NO: 12). . The base sequences of the primers for cloning the type 3 metallothionein gene are forward primers (5'-GCggatcc ATGTCGGACAAGTGCGGA-3 '; SEQ ID NO: 13) and reverse primers (5'-CTgtcgac GTGGCCACAGGTGCG-3'; SEQ ID NO: 14) to be.

PCR was performed using Clonetech's advantage2 polymerase, and PCR products of expected size were digested with BamHI and SalI restriction enzymes and cloned into pET28a expression vector (novagen), an E. coli expression vector. Through sequencing, the entire nucleotide sequence was confirmed. The identified expression vector was transformed into E. coli expression strain BL21 (DE3) cells to induce the expression of IbMT protein using IPTG. Escherichia coli was grown at 37 ° C. until the OD value of absorbance 600 nm became 0.4 to 0.6, and then IPTG was added to a final concentration of 1 mM to continue growing at 25 ° C. to induce the production of IbMT recombinant protein. E. coli was harvested from the culture medium overnight, and then the cells were disrupted by ultrasound to extract soluble and insoluble proteins. SDS-PAGE analysis confirmed the production of IbMT protein.

In the case of IbMT1 protein, expression was not confirmed in the crude extract, and purification was performed in the Ni-column with his-tag at the N-terminus of the vector (FIG. 6A). In the case of IbMT2 protein, expression was confirmed in the crude extract of insoluble protein (FIG. 6B), and in the case of IbMT3 protein, expression was observed in the crude extract of soluble protein (FIG. 6C).

The growth of E. coli containing the IbMT gene in E. coli medium containing heavy metals such as calcium, copper, cadmium, iron, manganese, magnesium, lead and zinc was compared with E. coli transformed with an empty vector. The IbMT1 and IbMT2 genes showed some resistance to heavy metals such as calcium, copper, manganese, magnesium, and lead, but did not grow at all in the medium containing cadmium and zinc, and the empty vector in the medium containing iron. It showed the same growth as the expressing Escherichia coli. The IbMT3 gene showed the same heavy metal resistance as IbMT1 and IbMT2, but E. coli showed much better growth. Therefore, the IbMT genes of the present invention were directly confirmed to have resistance to heavy metals such as calcium, copper, manganese, magnesium and lead (Table 1).

Heavy Metal Resistance of IbMTs Expressed in Escherichia Coli calcium Copper cadmium iron manganese magnesium lead zinc IbMT1 + + NG - + + + NG IbMT2 + + NG - + + + NG IbMT3 +++ +++ NG - +++ +++ +++ NG

+: Grows well,-: same as empty vector, NG: no growth.

<110> Korea Research Institute of Bioscience and Biotechnology <120> IbMT2 gene from Ipomoea batatas and uses <130> PN10044 <160> 14 <170> KopatentIn 1.71 <210> 1 <211> 249 <212> DNA <213> Ipomoea batatas <400> 1 atgtcttgct gcggaggaaa ctgtggctgc ggctctgcct gcaagtgcgg cagcggctgt 60 ggaggatgtg gcatgttccc tgatgtggag aatgtcaaga ctgtcaccct tattcagggt 120 gttgcaccag tgaacaacaa cacttttgag ggagctgaga tgggcgcaga gggaggagat 180 gggtgcaact gtggatcagg cagctgcagc tgtggaccag cctgtgcctg cgacccttgc 240 aaatgttga 249 <210> 2 <211> 82 <212> PRT <213> Ipomoea batatas <400> 2 Met Ser Cys Cys Gly Gly Asn Cys Gly Cys Gly Ser Ala Cys Lys Cys   1 5 10 15 Gly Ser Gly Cys Gly Gly Cys Gly Met Phe Pro Asp Val Glu Asn Val              20 25 30 Lys Thr Val Thr Leu Ile Gln Gly Val Ala Pro Val Asn Asn Asn Thr          35 40 45 Phe Glu Gly Ala Glu Met Gly Ala Glu Gly Gly Asp Gly Cys Asn Cys      50 55 60 Gly Ser Gly Ser Cys Ser Cys Gly Pro Ala Cys Ala Cys Asp Leu Ala  65 70 75 80 Asn val         <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 3 atgtcttccg gttgcaag 18 <210> 4 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer <400> 4 acagttgcaa gggtc 15 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 5 atgtcttgct gcggagga 18 <210> 6 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer <400> 6 acatttgcaa ggtcg 15 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 7 atgtcggaca agtgcgga 18 <210> 8 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer <400> 8 gtggccacag gtgcg 15 <210> 9 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 9 gcggatccat gtcttccggt tgcaag 26 <210> 10 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer <400> 10 ctgtcgacac agttgcaagg gtc 23 <210> 11 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 11 gcggatccat gtcttgctgc ggagga 26 <210> 12 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer <400> 12 ctgtcgacac atttgcaagg tcg 23 <210> 13 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 13 gcggatccat gtcggacaag tgcgga 26 <210> 14 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer <400> 14 ctgtcgacgt ggccacaggt gcg 23

Claims (11)

Ipomoea , consisting of the amino acid sequence of SEQ ID NO: 2 batatas IbMT2 ( Ipomoea ) Related to Heavy Metal Resistance from L. cv Yulmi) batatas metallothionein 2) protein. Gene encoding the 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. A method of enhancing heavy metal resistance of a plant, comprising the step of transforming the recombinant vector of claim 4 into a plant cell, wherein said plant is overexpressed. 7. The method of claim 6, wherein the heavy metal is calcium, copper, manganese, magnesium or lead. Transgenic plant with enhanced heavy metal resistance produced by the method of claim 6. Seeds of plants according to claim 8. Transforming a plant cell with the recombinant vector of claim 4; And
Method for producing a heavy metal resistant transformed plant comprising the step of regenerating the plant from the transformed plant cells. A composition for enhancing heavy metal resistance of a plant, comprising the gene of claim 2.

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