KR20070055193A - Antioxidant gene isolated from melon - Google Patents

Abstract

The present invention relates to an antioxidant gene isolated from melon, and more particularly, to a nucleic acid encoding a polypeptide having an amino acid sequence set forth in SEQ ID NO: 3 and its use. The CmSOD gene of the present invention encodes a superoxide dismutase. Therefore, the CmSOD protein of the present invention and the nucleic acid encoding the same can be very useful for growing plant varieties that overproduce superoxide dismutase.

Antioxidant enzyme, melon, SOD

Description

Antioxidant gene isolated from melon

Figure 1 shows the results of PCR with primers prepared on the basis of well-preserved sequences in the SOD genes of various plants in order to prepare a probe for the screening of the melon SOD gene.

Lane 1: 1 kb ladder marker

Lane 2: PCR product

Figure 2 shows the amino acid sequence (CmSOD, SEQ ID NO: 3), the conventionally known tobacco (NPSOD, GenBank accession no. CAA39444), pepper (CASOD, GenBank accession no. Sweet Potato (IBSOD, GenBank accession no.CAA51654), Chinese Cabbage (BRSOD, GenBank accession no.AAC25568), Baby Pole (ATSOD, GenBank accession no.AAM14107), Corn (ZMSOD, GenBank accession no. The amino acid sequence of the SOD protein of GenBank accession no.AAQ14591), aspen (PTSOD, GenBank accession no.AAD01605), olive tree (OESOD, GenBank accession no.CAD21706), pea (PSSOD, GenBank accession no. This is the result of multiple sequence alignment.

Figure 3 is the result of analyzing the fruit development stage and tissue expression pattern of the CmSOD gene.

0: Immediately after pollination 9: Nine days after pollination

18: Fruit 18 days after pollination 27: Fruit 27 days after pollination

36: 27 days after pollination

L: Leaf MF: Mengung

FF: magnetization S: stem

T: tendril (+): CmSOD gene amplified by PCR

Figure 4 shows the results of Southern blot of CmSOD gene probe for genomic DNA of melon cut by various restriction enzymes.

5 is a schematic diagram of recombinant vectors prepared by inserting the CmSOD gene.

35S: CmSOD (S): Recombinant vector inserting a sense fragment of the CmSOD gene

35S: CmSODRNAi: recombinant vector inserting RNAi fragment of CmSOD gene

6 is a view of a transgenic melon into which a sense fragment of the CmSOD gene is introduced.

Figure 7 is a transgenic melon introduced into the sense fragment of the CmSOD gene It is the result of confirming whether the gene was introduced through PCR.

M: molecular weight marker (1kb ladder marker)

(-): Non-transgenic plant

7-1: Transgenic Plant # 1

(+): Recombinant vector comprising a sense fragment of the CmSOD gene, used for transformation of melon

The present invention relates to an antioxidant gene isolated from melon, and more particularly, to a nucleic acid encoding a polypeptide having an amino acid sequence set forth in SEQ ID NO: 3 and its use.

Most organisms, including plants, are subject to biological stresses such as germs, pests, and viruses, as well as various environmental stresses caused by deterioration of the global environment, and oxygen, which is an essential element for life, is converted to superoxide anion radical and hydrogen peroxide. It is converted into reactive oxygen species such as hydrogen peroxide and hydroxyl radicals. In particular, superoxide anion radical (O ₂ ^-) is an oxygen molecule occurs by reaction with e (2O _{2 ^{+ 2e - → 2 · O 2}} -). These reactive oxygen species are highly reactive and cause severe physiological disorders.

Superoxide dismutase (D superoxide dismutase, hereinafter 'SOD';) is superoxide radical being chemically unavoidably created in the body (O ₂ ^-), an enzyme that converts to H ₂ O _2, cells my defenses Is one of the important enzymes (Fridovich, I. et al., Annu. Rev. Biochem ., 64, 97-112). SOD plays an important role in biological defense mechanisms because it not only removes superoxide anion radicals, but also prevents the formation of hydroxyl radicals. SOD is known to exist in almost all living things. SOD is divided into three types according to the type of metal bonded, that is, Cu, Zn-SOD, Fe-SOD, and Mn-SOD. Cu, Zn-SOD can be found in eukaryotes, Fe-SOD can be found in calm and primitive bacteria, and Mn-SOD can be found in bacteria and eukaryotic mitochondria.

As such, SOD is very important as an environmental resistance factor to remove free radicals in vivo generated by environmental stress. It is suggested that SOD could be used for medicines, foods, and chemicals. In fact, the medicinal use of SOD has been reported to be effective as an anti-inflammatory agent for arthritis, rheumatism, etc., and it has been recognized that it is effective in preventing ischemic heart disease and radiation interference. In addition, a study has been reported on the application of SOD to the skin damaged by UV rays to recover the damaged skin by infiltrating the SOD into the epidermis of the skin (Experimental Dermatology, 6: 116-121, 1997). Development of medicines using SOD as an active ingredient is actively underway in pharmaceutical companies such as the US and Japan, and anti-aging SOD-containing cosmetics are being developed and marketed in Korea.

Therefore, the research to separate and use SOD from the natural world is continuing. To date, SOD genes have been isolated from tobacco, pepper, sweet potato, Chinese cabbage, baby pole, corn, lemon, sashimi, radish, olive tree, and peas. However, no SOD gene has been isolated from melon.

Accordingly, the present inventors have completed the present invention by separating the SOD gene encoding the SOD protein of SEQ ID NO: 3 from the melon while repeatedly studying to separate the SOD gene from the melon.

It is therefore an object of the present invention to provide an SOD gene isolated from melon and its use.

In order to achieve the above object, the present invention provides a polypeptide having an amino acid sequence set forth in SEQ ID NO: 3.

In order to achieve another object of the present invention, the present invention provides a nucleic acid encoding the polypeptide.

In order to achieve another object of the present invention, there is provided a recombinant vector and transforming bacteria containing the nucleic acid.

In addition, to achieve another object of the present invention, the present invention provides a primer for amplifying the nucleic acid.

In order to achieve another object of the present invention, the present invention provides a method for producing a transgenic plant that overproduces a superoxide dismutase comprising introducing the nucleic acid into the plant.

Furthermore, in order to achieve another object of the present invention, the present invention provides a method for searching for antioxidant-related substances of plants by using the polypeptide or its variants, nucleic acids encoding them, fragments or derivatives thereof as probes.

Hereinafter, the present invention will be described in detail.

The present invention is characterized in that it provides for the first time a gene encoding SOD isolated from a melon.

The present invention provides a polypeptide having the amino acid sequence set forth in SEQ ID NO: 3. The polypeptides show very high homology with SODs from a variety of known plants (see FIG. 2). Specifically, the polypeptide according to the present invention shows homology of 89.5% with SOD isolated from sweet potato, 88.2% with SOD isolated from aspen, and 87.5% with SOD isolated from tobacco. In the present invention, the polypeptide of SEQ ID NO: 3 isolated from the melon was named 'CmSOD'.

CmSOD proteins of the invention include naturally occurring or recombinant proteins or proteins having physiological activity substantially equivalent thereto. Proteins having substantially equivalent physiological activity include native / recombinant CmSOD proteins and their functional equivalents and functional derivatives. The term "functional equivalent" refers to an amino acid sequence variant in which some or all of the native protein amino acids are substituted or a part of the amino acids are deleted or added, and have substantially the same physiological activity as the native CmSOD protein. Polypeptides of the invention include polypeptides having at least 90% sequence homology with the CmSOD protein of SEQ ID NO: 3. In addition, the "functional derivative" refers to a protein that has been modified to increase or decrease the physicochemical properties of the protein while maintaining the basic backbone and substantially equivalent physiological activity of the CmSOD protein. For example, this includes structural modifications for altering the stability, shelf life, volatility or solubility of the polypeptide of the present invention.

CmSOD proteins of the present invention can be readily prepared by chemical synthesis known in the art (Creighton, Proteins; Structures and Molecular Principles, WH Freeman and Co., NY, 1983). Representative methods include, but are not limited to, liquid or solid phase synthesis, fragment condensation, F-MOC or T-BOC chemistry (Chemical Approaches to the Synthesis of Peptides and Proteins, Williams et al ., Eds., CRC Press) , Boca Raton Florida, 1997; A Practical Approach, Athert on & Sheppard, Eds., IRL Press, Oxford, England, 1989).

In addition, the CmSOD protein of the present invention can be constructed by genetic engineering methods. First, a DNA sequence encoding the polypeptide of SEQ ID NO: 3 is constructed according to a conventional method. DNA sequences can be constructed by PCR amplification using appropriate primers. Alternatively, DNA sequences may be synthesized by standard methods known in the art, such as using automated DNA synthesizers (such as those sold by Biosearch or Applied Biosystems). The constructed DNA sequence includes a vector comprising one or more expression control sequences (e.g., promoters, enhancers, etc.) that are operatively linked to the DNA sequence to regulate expression of the DNA sequence. The host cell is transformed with the recombinant expression vector formed therefrom. The resulting transformants are cultured under appropriate media and conditions to allow the DNA sequence to be expressed, to recover substantially pure polypeptides encoded by the DNA sequences from the culture. The recovery can be carried out using methods known in the art (eg chromatography). As used herein, "substantially pure polypeptide" means that the polypeptide according to the present invention is substantially free of any other protein derived from a host cell. For genetic engineering methods for the synthesis of CmSOD proteins of the present invention, reference may be made to Maniatis et al ., Molecular Cloning; A laboratory Manual, Cold Spring Harbor laboratory, 1982; Sambrook et al ., Supra; Gene Expression Technology, Method in Enzymology, Genetics and Molecular Biology, Method in Enzymology, Guthrie & Fink (eds.), Academic Press, San Diego, Calif, 1991; And Hitzeman et al ., J. Biol. Chem., 255: 12073-12080, 1990.

The present invention also provides a nucleic acid encoding the CmSOD protein. Preferably, the nucleic acid includes the nucleotide sequence of SEQ ID NO. For example, it may have a nucleotide sequence of SEQ ID NO: 1. More preferably, it may have a nucleotide sequence of SEQ ID NO: 2. Nucleic acids of the invention can include nucleic acids encoding functional equivalents of the CmSOD protein.

Nucleic acids of the invention may be operably linked to expression control sequences. "Operably linked" means that one nucleic acid fragment is combined with another nucleic acid fragment so that its function or expression is affected by the other nucleic acid fragment. Also, "expression control sequence" refers to a DNA sequence that controls the expression of a nucleic acid sequence operably linked in a particular host cell. Such regulatory sequences include promoters for initiating transcription, any operator sequence for regulating transcription, sequences encoding suitable mRNA ribosomal binding sites, and sequences regulating termination of transcription and translation.

Nucleic acids according to the invention can be inserted in the sense direction and / or antisense direction in a suitable expression vector. As used herein, the term "expression vector" refers to a plasmid, virus or other medium known in the art to which a nucleic acid of the present invention can be inserted. Suitable vectors for introducing the nucleic acids of the invention into plant cells include, but are not limited to, Ti-plasmids, root-inducible (Ri) -plasmids and plant viral vectors. Preferably pCAMBIA3300 vector can be used.

Recombinant vectors comprising nucleic acids according to the invention can be introduced into cells using methods known in the art. The cells may be eukaryotic cells such as yeast, plant cells or prokaryotic cells such as E. coli. It may preferably be a bacterium, more preferably E. coli or Agrobacterium microorganisms. By the known methods for introducing the expression vector according to the present invention into a host cell it includes, but are not limited to, Agrobacterium mediated transformation methods (Agrobacterium -mediated transformation), particle (particle gun bombardment method gun bombardment or microparticle bombardment ), Silicon carbide whiskers, sonication, electroporation, and polyglycol-mediated uptake may be used. Preferably, Agrobacterium mediated transformation can be used.

The present invention also provides a primer for amplifying the nucleic acid. Such primers include all primers that can be designed by those skilled in the art to amplify a protein of the invention or a functional equivalent thereof. The primers include all primers capable of amplifying sense, antisense or RNAi fragments of the CmSOD gene according to the present invention. The primer may also comprise a restriction enzyme recognition sequence suitable for easy cloning of the amplified gene within the sequence. The primer for amplifying the sense fragment of the CmSOD gene preferably has a nucleotide sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 12, and SEQ ID NO: 13. Preferably, primer combinations of SEQ ID NOs: 6 and 7 or primer combinations of SEQ ID NOs: 12 and 13 may be used. In addition, the primer for amplifying the antisense fragment of the CmSOD gene preferably has a nucleotide sequence of SEQ ID NO: 10 and SEQ ID NO: 11. It is also a primer for amplifying the RNAi fragment of CmSOD gene containing all the primers for amplifying the sense and antisense fragments of CmSOD gene. Preferably preparing the RNAi fragment of CmSOD gene using the SEQ ID NO: 10 and 11 of the nucleotide sequence primer for the antisense fragment amplification of CmSOD gene having the SEQ ID NO: 12 as the sense fragment amplification primers for the CmSOD gene having the nucleotide sequence of 13 can do.

The CmSOD gene of the present invention can be used to prepare transgenic plants that overproduce SOD by introducing into plants and overexpressing them. "Overexpression" in the above means that the gene is expressed above the level expressed in wild-type plants. Accordingly, the present invention provides a method for producing a transgenic plant that overproduces SOD, comprising introducing a CmSOD gene into the plant. Specifically, the method of the present invention

(a) inserting a nucleic acid encoding a CmSOD protein of the invention into an expression vector; And

(b) introducing the expression vector into the plant.

Preferably, the nucleic acid encoding the CmSOD protein of the present invention in step (a) has a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In addition, the expression vector preferably contains a promoter capable of regulating the expression of the nucleic acid. As the promoter, a promoter (constitutive promoter) which induces the expression of a gene of interest at all times at all times, a promoter which induces tissue-specific expression of a plant, or a promoter which induces development-specific expression of a plant may be used. have. Examples of promoters include CaMV 35S promoters (Odell et al ., Nature, 313: 810-812, 1985), Rsyn7 promoters (US Patent Application No. 08 / 991,601), rice actin promoters (McElroy et al ., Plant Cell 2: 163-171, 1990), ubiquitin promoters (Christensen et al ., Plant Mol. Biol . 12: 619-632, 1989) and ALS promoters (US Patent Application No. 08 / 409,297). In addition, U.S. Patents 5,608,149; The promoters disclosed in 5,608,144 5,604,121 5,569,597 5,466,785, 5,399,680 5,268,463, 5,608,142, and the like can all be used. Preferably a CaMV 35S promoter is used.

Introduction to the plant in step (b) may use a known plant transformation method. For example, as described above, Agrobacterium mediated transformation, particle gun bombardment, silicon carbide whiskers, sonication, electroporation, and precipitation with polyethylene glycol can be used. Preferably, Agrobacterium mediated transformation is used.

The present invention also provides a transgenic plant produced by the above method. Transgenic plants introduced with the CmSOD gene according to the above method overproduce SOD compared to wild-type plants. Therefore, the transgenic plant prepared according to the method of the present invention can suppress free radicals generated excessively by various environmental stresses. In addition, the transgenic plants, fruits and seeds thereof produced according to the present invention may be usefully used as raw materials for pharmaceuticals, foods and cosmetics because they overproduce SOD, and thus have high commercial value. Transgenic plants according to the invention can be asexually propagated by tissue culture. Examples of tissue culture are as follows: Tissues of the transformed plants according to the present invention are cultured in callus induction medium to induce callus, and then cultured in root induction medium to induce rooting. At this time, it is preferable to add a small amount of NAA (naphthalenacetic acid) to the root induction medium. In addition, plant growth promoters such as Gamborg's vitamin solution and IAA (indole-3-acetic acid) may be added. Subsequently, when the root of the root (esophagus) is grown from a plantlet cultured in the root induction medium, the medium is removed and transplanted into a sterile compound soil. At this time, it is preferable to cover the pot with vinyl and seal for about 3 weeks. Then, they are transferred to large pots and re-divided into complete plants.

The plant applicable to the present invention may be a dicotyledonous plant or a monocotyledonous plant. Specifically, food crops such as rice, wheat, barley, corn, soybeans, potatoes, red beans, oats and sorghum; Vegetable crops such as arabidopsis, cabbage, radish, pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, green onion, onion, and carrot; Special crops such as ginseng, tobacco, cotton, sesame, sugar cane, sugar beet, perilla, peanut and rapeseed; Fruit trees such as apple trees, pears, jujube trees, peaches, leeks, grapes, citrus fruits, persimmons, plums, melons, apricots and bananas; Flowers such as roses, gladiolus, gerberas, carnations, chrysanthemums, lilies and tulips; And feed crops such as lygras, redclover, orchardgrass, alphaalpha, tolsquescue and perennial lygras may be used. Preferably melon.

In addition, the present invention provides a method for searching for an antioxidant-related substance of a plant using a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 3, a nucleic acid encoding the same, fragments or derivatives thereof. The antioxidant-related substance may be a gene or a protein, and may also be a compound or extract that is isolated or chemically synthesized from nature. Preferably the method can be carried out using a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 3, a nucleic acid encoding it, a fragment or derivative thereof as a 'probe'. The term 'probe' refers to a medium for finding a desired one. The probe may be labeled with radioisotopes, fluorescent dyes, or colorimetric enzymes and the like to facilitate screening and separation of the locating material. Preferably ³ H, ³² P, ³⁵ S, Fluorescein Isohiocyanate (FITC), Tetrame-thylrhodamine isothiocyanate (TRITC), Biotin, Digoxigennin, Horse-Radish Peroxidase (HRP), Glucose oxidase (HRP) Glucose Oxidase), alkaline phosphatase, and the like. Labeling methods are known in the art. For example, a method of labeling nucleic acids may include uniformly labeling the entire nucleic acid, such as nick translation and random oligonucleotide primers, or as a kind of keying and filling-in. The method of labeling the 5 'end or 3' end can be used. Polypeptides can also be labeled via radioactive oxolation. Radioactive oxo may be directly labeled on tyrosine or histidine of the polypeptide, or may be labeled using Chloramine-T, Iodogen, or lactoperoxidase.

More specifically, the method of the present invention may search for an antioxidant-related protein by analyzing the protein binding aspect with the CmSOD protein, fragment or derivative thereof having the amino acid sequence of SEQ ID NO: 3, and activity of the CmSOD protein according to the present invention. It is possible to search for a substance that inhibits or activates. In addition, the nucleic acid encoding the CmSOD protein or a fragment thereof may be hybridized with cDNA prepared from RNA or mRNA extracted from a plant to search for antioxidant-related genes. In addition, it is possible to directly search for substances that bind the CmSOD gene or its mutant gene or chemicals that inhibit or activate their expression. In addition, the method of the present invention may search for a gene having high sequence homology by comparing the nucleic acid encoding the CmSOD protein with the nucleotide sequence. The method of the present invention is generally used for DNA chips, protein chips, polymerase chain reaction (PCR), Northern blot, Southern blot, Western blot, enzyme immunoreaction (ELISA), 2-D gel analysis, yeast double hybridization reaction ( Screening can be performed in a variety of ways, including yeast two hybrid systems) and in vitro binding assays.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

<Example 1>

From melon CmSOD  Gene Isolation and Sequence Analysis

<1-1> Development of cDNA Library by Melon Fruit Development

Flowering melon ( Cucumis melo cv. Reticulatus F1 Alpha) was artificially inseminated. RNA was extracted from melon fruit at 0 and 9 DAP (day after pollination) and the same amount was mixed. Then, the cDNA library was prepared using the pBK-CMV phagemid vector (Strategene) according to the method suggested by the stratazine cDNA library kit. In addition, in order to produce a library belonging to the latter part of the fruit development step, cDNA library was prepared as described above from melon fruit in 36 and 37 DAP which was the harvest date after artificial insemination. The transformed cells were incubated at 37 ° C. for 45 minutes and then plated in LB medium containing X-gal (80 μg / ml), IPTG (0.55 mM), and kanamycin (50 μg / ml). White colonies containing recombinant DNA were selected and picked into 384 well microtiter plates. The colonies were then transferred to nitrocellulose membranes using an automatic gridding system TAS (BioRobotics, Cambridge, UK).

<1-2> Preparation of the Probe

The primers were prepared by finding well-conserved sites from the base sequences of SOD genes reported in sweet potatoes ( Impomoea batatas ), Populus tremuloides , and tobacco ( Nicotiana plumbaginifolia ). The prepared primer sequences are set forth in SEQ ID NO: 4 and SEQ ID NO: 5. Thereafter, PCR was performed using genomic DNA extracted from the melon leaf as a template. PCR predenatured the template DNA for 4 minutes at 94 ° C., followed by 1 minute at 94 ° C .; 1 minute at 55 ° C .; A total of 30 cycles were repeated for one minute at 72 ° C., and the reaction was finally performed at 72 ° C. for 10 minutes. PCR resulted in an amplified product of about 500 bp size (see Figure 1). After inserting the amplified product into the pGEMT-easy (Promega Co.) vector, the sequencing of the SOD gene was confirmed by sequencing according to methods known in the art. The amplified product was then labeled with [α- ³² P] dCTP and prepared by probe (Rediprime II Rnadom Prime Labeling System, Amersham, # RPN1633).

<1-3> cDNA library screening

To clone the full-length cDNA of the melon SOD gene, the cDNA library prepared from the fruit of the melon in Example <1-1> was screened using the probe prepared in Example <1-2>. The cDNA library was transferred to a nylon membrane, and then hybridized with the probe prepared in Example <1-2> at 65 ° C for 24 hours. Sequential washing for 1 hour in 2 × SSC, 0.1% SDS solution, 1 hour in 1 × SSC, 0.1% SDS, 30 minutes in 0.5 × SSC, 0.1% SDS, and 20 minutes in 0.1 × SSC, 0.1% SDS solution It was. Thereafter, the X-ray film was exposed to light. As a result, clones hybridized with the probe were detected only in 36/37 DAP (results not shown).

<1-4> Electrophoresis and Sequencing

The clones selected in Example <1-3> were inoculated into LB broth and incubated overnight at 37 ° C. Plasmid DNA was isolated from the culture medium, digested with restriction enzymes Eco RI and Xho I, and electrophoresed on a 1% agarose gel. Electrophoresis results confirmed a DNA insert of 733 bp (results not shown). Thereafter, sequencing of the DNA insert was performed. As a result, the clone contained 34 bp 5'UTR, 459 bp coding portion, and 240 bp 3'UTR. The entire nucleotide sequence of the clone was described as SEQ ID NO: 1, and only the sequence of the coding part expressed as a protein among the entire nucleotide sequences was described as SEQ ID NO. The amino acid sequence of the protein encoded from the clone is set forth in SEQ ID NO: 3.

Similarity search () using an amino acid sequence (SEQ ID NO: 3) encoded from the coding part, showed a high similarity to SODs derived from other plants (see Figure 2). In particular, it showed a high homology of 89.5% with SOD (GenBank accession no. CAA51654) isolated from sweet potatoes ( Impomoea batatas ), and Populus tremuloides (GenBank accession no. AAD01605) and tobacco ( Nicotiana plumbaginifolia ; Homologous to SODs isolated from CAA39444), 88.2% and 87.5%, respectively. Thus, the present inventors named the nucleic acid of SEQ ID NO: 2 ' CmSOD gene'.

<Example 2>

CmSOD  Analysis of gene expression patterns by fruit development and tissue

In order to investigate the fruit development and tissue-specific expression patterns of the CmSOD gene isolated in Example 1, the fruit of 0, 9, 18, 27 and 36 DAP by fruit development, and immediately after a few minutes (0 DAP) by tissue ( Total RNA was extracted from leaf, L), male flower (MF), female flower (FF), stem (Stem, S), and tendril (T). Thereafter, the extracted total RNA was used as a template, and a first-strand cDNA was synthesized using Invitrogen's Superscript (# 12371-019). PCR was performed using the synthesized primary-strand cDNA as a template using CmSODF primer (SEQ ID NO: 6) and CmSODR primer (SEQ ID NO: 7). PCR was pre-denatured for 3 minutes at 94 ° C., followed by 1 minute at 94 ° C .; 1 minute at 50 ° C .; A total of 30 cycles were repeated at 1 minute and 30 seconds at 72 ° C., and finally, the reaction was carried out at 72 ° C. for 10 minutes. In this case, PCR was performed using a CmActinF primer (SEQ ID NO: 8) and a CmActinR primer (SEQ ID NO: 9) capable of amplifying the actin gene of melon. Then, the probe and Southern blot prepared in Example <1-2> were performed to confirm whether the band amplified by PCR is the CmSOD gene.

As a result, as shown in Figure 3 , the CmSOD gene was detected at all stages except for 0 DAP when the fruit development was examined, and it was confirmed that the expression is expressed in all other organs except for the maturation and magnetization when examined by tissue .

<Example 3>

CmSOD  Genome structure analysis of genes

Southern blot was performed to analyze the genomic structure of the CmSOD gene. The genomic DNA of the melon was digested with restriction enzymes Hin dIII, Eco RI, Eco RV and Xba I, respectively, and then electrophoresed on 1.0% agarose gel. The DNA on the gel was then transferred to a nylon membrane and hybridized overnight at 65 ° C. with the probe prepared in Example <1-2>. 1 hour in 2x SSC solution containing 0.1% SDS, 1 hour in 1x SSC solution containing 0.1% SDS, 30 minutes in 0.5x SSC solution containing 0.1% SDS, 0.1x containing 0.1% SDS Washed sequentially in SSC solution for 20 minutes. Thereafter, the results were analyzed by exposing it to an X-ray film. As a result, the CmSOD gene was found to show one copy in the melon genome (see FIG. 4 ).

<Example 4>

Construction of recombinant vector for plant transformant development

The inventors prepared a recombinant vector containing a sense fragment or RNAi fragment of the CmSOD gene using the plant transformation pCAMBIA3300 vector (CAMBIA; http://www.cambia.org) as follows.

First, the pCAMBIA3300 vector (CAMBIA; http://www.cambia.org) was digested with Hin dIII and Eco RI, and then the 35S promoter, MCS (multi cloning site) and NOS obtained from the pMBPI vector were digested with the same restriction enzyme. Ligation with fragments.

For the preparation of recombinant vectors comprising sense fragments of the CmSOD gene, the sense fragments of the CmSOD gene were amplified by PCR using CmSODSF primers (SEQ ID NO: 12) and CmSODSR primers (SEQ ID NO: 13). PCR reaction was carried out according to the same method as in Example 1. Amplified PCR products were purified using a kit (Qia quick PCR purification kit, QIAGEN, # 28104). Thereafter, the purified PCR product was digested with Kpn I and Sac I, and then inserted into a pCAMBIA3300 vector containing the 35S promoter and NOS prepared above. The prepared recombinant vector was named '35S: CmSOD (S)' (see FIG. 5 ).

On the other hand, for the production of a recombinant vector containing the RNAi fragment, the pCAMBIA3300 vector containing the 35S promoter and NOS prepared above was cut with Xba I and Bam HI, and then the GUS gene obtained by cutting with the same enzyme was inserted. The antisense fragment of CmSOD gene CmSODASF primer (SEQ ID NO: 10) and CmSODASR primers used (SEQ ID NO: 11) were amplified by PCR, the sense fragment of CmSOD gene CmSODSF primer (SEQ ID NO: 12) and CmSODSR primer (SEQ ID NO: 13) Was amplified by PCR. PCR reaction was carried out according to the same method as in Example 1. Then, the antisense fragment with Xba I and Bam HI, and the sense fragments were each inserted into the front part and the rear part of the GUS gene in the pCAMBIA3300 vector was cut with Kpn I and Sac I (see Fig. 5). The prepared recombinant vector was named '35S: CmSODRNAi'.

Each recombinant vector thus prepared was then introduced into DH10B cells by electroporation. Cells were plated in LB solid medium containing kanamycin (50 mg / L) and cultured overnight at 37 ° C. to select transformants. PCR analysis and DNA restriction enzyme cleavage analysis were performed on the selected colonies to confirm gene insertion (result not shown). Thereafter, each recombinant vector was isolated from the transformants, which were then introduced into Agrobacterium tumefaciens LBA4404, respectively. The transformed Agrobacterium tumefaciens LBA4404 was confirmed by the PCR analysis.

Example 5

Transformation and verification of melon

<5-1> transformation

The transformation of melon was performed as follows. First, the seeds of the melon were germinated in germination medium (GM; MS 4.4 g / L, Sucorse 20 g / L, Agar 8 g / L) for 2 days, and then explants were prepared in cotyledone. The transformed Agrobacterium tumefaciens LBA4404 prepared in Example 4 was co-cultured with explants of melon, respectively. Thereafter, the explants were cultured in pre-culture medium (MPM; LS 4.4g / L, Sucrose 20g / L, BA 0.5uM, Agar 8g / L) for 2 days, and then, selection medium (MSM; LS 4.4g / L, Shoots were induced at Sucrose 20g / L, BA 0.5uM, PPT 5mg / L, Cf 250mg / LAgar 8g / L). After 3-4 weeks, induced shoots were transferred to root induction medium (MRM; LS 2.2g / L, Sucorse 20g / L, Agar 6g / L) and cultured. Both roots were induced in shoots into which the sense fragment or RNAi fragment of the CmSOD gene was inserted, respectively. The root-derived seedlings were purified in-flight and then grown in a greenhouse (see FIG. 6 ).

<5-2> Confirmation of transformation

Genomic DNA was extracted using a kit (DNeasy Plant Mini kit, QIAGEN, # 69104) from the leaves of a seedling plant in which a sense fragment of the CmSOD gene was typically introduced among the transgenic melons prepared in Example <5-1>. . 35S primer / CmSODSR primer combination (SEQ ID NO: 14 / SEQ ID NO: 13) capable of amplifying the 35S promoter and the CmSOD gene using the isolated DNA as a template; And using the CmSODSF primer / NOS primer combination (SEQ ID NO: 12 / SEQ ID NO: 15) that can amplify the CmSOD gene and NOS, PCR was carried out in the same manner as in Example 1 to investigate the transformation. As a result, as shown in Figure 7 , it was confirmed that the sense fragment of the CmSOD gene was correctly inserted into the transgenic melon.

In addition, using a primer combination (SEQ ID NO: 6 and SEQ ID NO: 7) capable of amplifying the CmSOD gene by extracting RNA from the fruit of the transformed plant was confirmed whether the expression of the CmSOD gene in the melon fruit actually.

<Example 6>

Investigation of the Antioxidant Capacity of Transformed Melon Fruits

After harvesting the fruit of each transgenic melon prepared in Example 5, the antioxidant capacity of the CmSOD gene expression according to the present invention was compared and investigated.

<Example 7>

Subsequent Validation of Transformed Melons

Seeds were obtained from each transgenic melon prepared in Example 5. Thereafter, seeds were germinated in a medium containing PPT, and germinated seedlings were grown in soil. Then, the experiments of Examples 4 and 5 were carried out to compare whether the antioxidant ability is seen even in the later generation of the transformed melon.

As described above, in the present invention, the CmSOD gene related to the antioxidant activity of the plant was isolated from the melon. The CmSOD gene of the present invention encodes superoxide dismutase. Therefore, the CmSOD protein of the present invention and the nucleic acid encoding the same can be very useful for growing plant varieties that overproduce superoxide dismutase.

<110> FnP corp., Ltd <120> Antioxidant gene isolated from melon <130> NP05-0111 <160> 15 <170> KopatentIn 1.71 <210> 1 <211> 733 <212> DNA <213> Cucumis melo <400> 1 gaattcggca cgnccttntg agatcacagg aaagatggta aaggctgtag ctgttcttgg 60 aagtagtgag ggtgtgagtg gaactatctt ttttacccaa gaaggagatg gtccaaccac 120 tgttacgggc aatgtttctg gtctcaagcc cgggcctcat ggattccatg ttcacgcctt 180 aggtgacaca accaatggct gcatgtcgac tgggccacat ttcaaccctg ctggaaagca 240 acatggtgct cccgaggacg agaaccgcca tgccggtgat ttagggaaca ttatcgttgg 300 tgaagatggc acggctaact tcaccattac tgacaaccag atttctctct gtggaaagga 360 ctccattatc ggaagggcgg ttgtggttca tggagaccct gatgatcttg gcaagggtgg 420 acatgaactc agcttaagta caggaaatgc tggtgctaga gtagcctgtg gcattattgg 480 tctgcaaggt taagatttgt tgccttcgtt gccgtttcta ttgaagcatt gaacccaaat 540 gctatctagt gcttccaaga atattcctaa actcgcgtga atgaataaaa acgcagcact 600 gtactttttc tttttctttt actttttttg tgacgattag atcttttact tatccatact 660 tgtctgctgt tttattatta tatttctgga aattcggttt cttttttttc ttttcaaaaa 720 aaaaaaaaaa aaa 733 <210> 2 <211> 456 <212> DNA <213> Cucumis melo <400> 2 atggtaaagg ctgtagctgt tcttggaagt agtgagggtg tgagtggaac tatctttttt 60 acccaagaag gagatggtcc aaccactgtt acgggcaatg tttctggtct caagcccggg 120 cctcatggat tccatgttca cgccttaggt gacacaacca atggctgcat gtcgactggg 180 ccacatttca accctgctgg aaagcaacat ggtgctcccg aggacgagaa ccgccatgcc 240 ggtgatttag ggaacattat cgttggtgaa gatggcacgg ctaacttcac cattactgac 300 aaccagattt ctctctgtgg aaaggactcc attatcggaa gggcggttgt ggttcatgga 360 gaccctgatg atcttggcaa gggtggacat gaactcagct taagtacagg aaatgctggt 420 gctagagtag cctgtggcat tattggtctg caaggt 456 <210> 3 <211> 152 <212> PRT <213> Cucumis melo <400> 3 Met Val Lys Ala Val Ala Val Leu Gly Ser Ser Glu Gly Val Ser Gly   1 5 10 15 Thr Ile Phe Phe Thr Gln Glu Gly Asp Gly Pro Thr Thr Val Thr Gly              20 25 30 Asn Val Ser Gly Leu Lys Pro Gly Pro His Gly Phe His Val His Ala          35 40 45 Leu Gly Asp Thr Thr Asn Gly Cys Met Ser Thr Gly Pro His Phe Asn      50 55 60 Pro Ala Gly Lys Gln His Gly Ala Pro Glu Asp Glu Asn Arg His Ala  65 70 75 80 Gly Asp Leu Gly Asn Ile Ile Val Gly Glu Asp Gly Thr Ala Asn Phe                  85 90 95 Thr Ile Thr Asp Asn Gln Ile Ser Leu Cys Gly Lys Asp Ser Ile Ile             100 105 110 Gly Arg Ala Val Val Val His Gly Asp Pro Asp Asp Leu Gly Lys Gly         115 120 125 Gly His Glu Leu Ser Leu Ser Thr Gly Asn Ala Gly Ala Arg Val Ala     130 135 140 Cys Gly Ile Ile Gly Leu Gln Gly 145 150 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> CmSODCF primer <400> 4 gcacggcctt atgagatcac agg 23 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CmSODCR primer <400> 5 gcttcaatag aaacggcaac g 21 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> CmSODF primer <400> 6 cacaggaaag atggtaaagg ctg 23 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CmSODR primer <400> 7 agaaacggca acgaaggcaa c 21 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CmActinF primer <400> 8 gctgtgttcc ccagtattgt tg 22 <210> 9 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CmActinR primer <400> 9 actggcatag agagatagaa cg 22 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> CmSODASF primer <400> 10 gaaggctcta gatcttaacc ttgc 24 <210> 11 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> CmSODASR primer <400> 11 cacaggatcc atggtaaagg ctg 23 <210> 12 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> CmSODSF primer <400> 12 cacaggtacc atggtaaagg ctg 23 <210> 13 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> CmSODSR primer <400> 13 gaaggcaaga gctcttaacc ttgc 24 <210> 14 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> 35S primer <400> 14 tctatctctc tgcagctttc gc 22 <210> 15 <211> 23 <212> DNA <213> Artificial Sequence <220> NOS primer <400> 15 atatgataat catcgcaaga ccg 23

Claims (11)

A polypeptide having an amino acid sequence set forth in SEQ ID NO: 3. A nucleic acid encoding the polypeptide of claim 1. The nucleic acid according to claim 2, which has a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2. A recombinant vector containing the nucleic acid of claim 2. Bacteria transformed with the recombinant vector of claim 4. A primer for amplifying the nucleic acid of claim 2. The primer according to claim 6, wherein the primer is selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13. A method for producing a transgenic plant that overproduces a superoxide dismatuse, comprising introducing the nucleic acid of claim 2 into a plant. A transgenic plant overproducing superoxide dismutase prepared by the method of claim 8. A method of searching for an antioxidant-related active substance of a plant by using a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 2, a nucleic acid encoding the same, a fragment or a derivative thereof as a probe. The method of claim 10, wherein the DNA chip, protein chip, polymerase chain reaction (PCR), Northern blot, Southern blot, Western blot, enzyme immunoassay (ELISA), 2-D gel analysis, yeast two hybridization reaction (yeast two hybrid) system) and an in vitro binding assay.

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