KR20170014024A - Method for enhancing drought resistance of rose - Google Patents

Abstract

The present invention relates to a method for improving the drought resistance of roses. More specifically, the present invention relates to a method for improving the dry resistance of roses by introducing a superoxide dismutase 2 (SOD2) gene derived from Escherichia coli into roses .
According to the present invention, it is possible to effectively improve the weather resistance of roses. The improved roses according to the present invention can survive for a longer period of time even when there is no smooth water supply, which is expected to greatly contribute to the reduction of damages due to drought or malfunction of the irrigation system.

Description

[0001] The present invention relates to a method for enhancing the resistance to roses,

The present invention relates to a method for improving the drought resistance of roses. More specifically, the present invention relates to a method for improving the dry resistance of roses by introducing a superoxide dismutase 2 (SOD2) gene derived from Escherichia coli into roses .

The new varieties of major flower crops, including roses, are being developed primarily through breed or interspecific hybridization techniques. Cross-breeding techniques such as interspecific or interspecific hybridization are the main means of developing new varieties, but it is not possible to develop varieties with characteristics not possessed by genetic resources, and even if the characteristics of genetic resources are possessed, It is impossible to develop varieties with such characteristics. Transgenic techniques are a way to overcome the limitations of this hybrid breeding.

In the case of roses, one of the three cut flowers in the world, there have been reports of numerous regeneration studies carried out proactively in order to obtain transgenic plants. However, studies on transformation have been conducted by Firoozabady et al. (1994) Since its first report, until recently, only a few studies have been reported by three or four research teams worldwide. However, as reported at the end of 2006 by Suntory in Japan and obtained commercialized blue rose transgenic plants at the end of 2009, the transgenic technology that has been used for major breeding techniques only in rice and soybean crops until recently, It has been suggested that it is not easy to acquire a transgenic plant of rose until now. The majority of foreign reports that succeeded in acquiring the rose transformants described above, including the report of Tanaka (2009) by Suntory Co., have used callus or somatic embryo callus derived from leaves (including petiole) and stem fragments The gene was introduced through Agrobacterium or a gene gun, but the efficiency was low and only a few cultivars were studied. In Korea, the Korean Institute of Bioscience and Biotechnology (KRIBB) has attempted to develop callus induction and transformation technology for rosette somatic embryogenesis for 4 years from 1996 to 2000, and Kim et al. (2004) 2002) reported that a somatic embryo-derived callus derived from 'Tineke' was used as an explant to obtain a GFP transgenic transformant. However, in Korea, except for this research team, It has not been developed.

On the other hand, the active oxygen generated through the activity of various oxidase and peroxidase enzymes can damage the intracellular macromolecules of the rose and kill the cells in response to the internal generation signal or external environmental stimulus. These environmental stresses can easily damage the roses, which can cause a lot of damage to the roses. Therefore, it is very urgent to research and develop the rosin transgenic plants which can enhance the stress resistance to reduce the damage.

Accordingly, the inventors of the present invention have conducted various studies on a method for enhancing various stress resistance of roses. As a result, it has been found that the introduction of superoxide dismutase 2 (SOD2) gene derived from Escherichia coli, drought resistance, and the present invention has been completed.

Korean Patent No. 10-1077209 Korean Patent No. 10-1239643

SUMMARY OF THE INVENTION Accordingly, it is a primary object of the present invention to provide a method for effectively improving the resistance to dryness of roses.

According to one aspect of the present invention, the present invention provides a method for expressing superoxide dismutase 2 (SOD2) derived from Escherichia coli, which is represented by the amino acid sequence of SEQ ID NO: 1, The present invention provides a method for improving the drought resistance of roses.

Superoxide dismutase 2 (SOD2) belongs to the iron / manganese superoxide dismutase family and is known to function as an antioxidant defense mechanism. SOD2 derived from Escherichia coli , which is used in the present invention, can be represented by the amino acid sequence of SEQ ID NO: 1 as MnSOD.

The sequence of SEQ ID NO: 3 is known as the SOD2 gene sequence derived from E. coli. Here, a promoter and the like are included, and a site encoding the amino acid of SOD2 is a 141 to 761 base portion (SEQ ID NO: 2).

In the present invention, the improvement or improvement of the resistance to dryness means that the rose can survive longer in a dry environment than a control, that is, a rose without the SOD2 gene .

In the method of the present invention, the gene to be introduced into and expressed in the cell is preferably represented by the nucleotide sequence of SEQ ID NO: 2. However, the present invention is not limited thereto, and it is considered that a sequence in which a codon is changed so as to encode the amino acid sequence of SEQ ID NO: 1 is also possible.

In the method of the present invention, it is preferable that the gene is regulated in transcription by a 35S promoter derived from a mosaic virus (P35S). However, the present invention is not limited to this, and other promoters capable of operating in roses may be applicable.

In the method of the present invention, it is preferable to use an agrobacterium-mediated transformation method as a method for introducing a gene into a cell.

In the method of the present invention, it is preferable that the transformation is carried out using the recombinant vector containing the gene and represented by Fig.

In the method of the present invention, the cell is preferably a somatic embryo or embryogenic callus derived from roots of roses.

According to the present invention, it is possible to effectively improve the weather resistance of roses. The improved roses according to the present invention can survive for a longer period of time even if there is no smooth water supply, which is expected to greatly contribute to the reduction of damage to the farm due to subsequent drought or malfunction of the watering device.

1 is a schematic diagram showing the SOD2 gene insertion pPZP200-SOD2-Bar vector.
Fig. 2 shows the appearance of roots-derived somatic embryo callus in the cabin of rose.
FIG. 3 shows the process from the introduction of the SOD2 gene to the step of obtaining a transformant.
FIG. 4 shows the result of PCR analysis that confirmed the introduction of the SOD2 gene.
FIG. 5 is a southern blot analysis result in which the copy number of SOD2 gene introduced was confirmed.
FIG. 6 shows a result of real-time PCR analysis of the SOD2 gene expression of the transcript.
FIG. 7 is a western blot analysis result showing the protein expression of the SOD2 gene.
FIG. 8 is a graph showing the relative moisture content (%) of the non-transformant (control) (NT) and the SOD2 gene transfected transformant 8 strain on the seventh day of no-treatment.
FIG. 9 is a graph showing relative ionic linkage (%) at 7 days between the non-transformant (control) (NT) and the SOD2 gene transfected transformant 8 lines.
Fig. 10 shows the growth state on the second day after resumption of irrigation (control group) and SOD2 transfected transformant for 7 days.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.

Example 1. Preparation of SOD2 gene insertion recombinant vector and transformation of Agrobacterium

A pBI-SOD recombinant vector cloned into the pBI121 vector was used so that the SOD2 gene of SEQ ID NO: 2 was transcribed by P35S (35S promoter). The P35S-SOD2 region (about 1.5 kb) of the recombinant vector pBI-SOD was introduced into the expression vector pPZP200-Bar for plant transformation, which was not problematic for commercialization, using the HindIII and SacI restriction enzyme recognition sites. As a result, pPZP200 -SOD2-Bar recombinant vector (SEQ ID NO: 4) (see Fig. 1).

This pPZP200-SOD2-Bar recombinant vector was transformed into Agrobacterium strain LBA4404, and the transformed strain was mixed with glycerol 1: 1 and stored in a -70 ° C freezer.

Example 2. Preparation of somatic embryo callus

According to the method of Korean Patent No. 10-1077209, somatic embryogenic calli of roses were prepared.

KR056002 roses selected from the cross between Tineke and mirinae gold varieties were used. The somatic embryogenic calli derived from root roots of this rose were collected to a size of 2 cm in diameter, and 2,4-D 3 mg ℓ ^-1 , L-proline 300 mg ℓ ^-1 , sucrose 30 g ℓ ^-1 , phytagel 4 g ℓ ^-1 , and the like. The somatic embryo (including embryogenic callus) (hereinafter referred to as "embryogenic calli"), which is round, smooth and white in appearance, was propagated in subculture on SH medium (Schenk & Hildebrandt medium) (See Fig. 2).

Example 3: SOD2 gene introduction and regeneration Plant acquisition

end. Agrobacterium culture

1 ml of Agrobacterium tumefaciens LBA4404 stock solution, which is stored at -70 ° C in a freezer, was added to LB liquid medium supplemented with 50 μg / l of spectinomycin and incubated at 170 to 200 rpm To an OD _{600 of} 1.6 to 1.9.

I. Gene transfer through Agrobacterium infection and culture

Agrobacterium culture was centrifuged at 6,000 rpm for 7 minutes to obtain cells, and the obtained cells were suspended in a liquid culture medium for somatic embryo callus proliferation (1/2 volume of the culture medium) in which acetosyringone was added at a concentration of 50 μM 2.5 ml of the prepared KR056002 somatic embryo (including embryogenic callus) was infected for 30 minutes and then placed on a sterilized filter paper to remove the bacterial suspension from the specimen. Then, acetosyringone was added at a concentration of 50 μM and somatic cells The cells were transferred to embryogenesis callus-growing solid medium (pH 5.2) and cultured for 3 days at a temperature of 25 ± 2 ° C.

All. Plant regeneration and proliferation in selected medium

SOD2 gene insertion Somatic embryos (including embryogenic callus) co-cultured with LBA4404 for 3 days were washed with a medium for somatic embryogenesis callus growth containing 250 mg · l ^-1 cefotaxim, and then washed with sterile filter paper Respectively. From the germ-infected somatic embryos (including embryogenic callus) from which the washings had been removed, the selection media were subcultured at 3 to 6 week intervals until the 41 KR056002 regenerated plants were obtained (see Tables 1 and 3) ).

Planting of regeneration plant in selection medium after introducing gene for environmental improvement SOD2 kind
(system) Transformant
Number of acquisition systems Up to transfection after gene transfer
Subculture course KR056002 32 (2 times) → germination induction selection medium (1 time) → shoot induction selection medium (8 times) → shoot elongation selection medium (2 times) → shoot initiation selection medium (3 times) → shoot elongation selection medium 2 times) → Daesin colostrum selection medium (2 times) → Shinshohei selection medium (1 time) → Shinsho kidney selection medium (1 time) → Daesinchok induction selection medium (6 times)

Example 4. Confirmation of gene transfer, transcript and protein expression of regenerated plant

After the introduction of the SOD2 gene by the PCR analysis, it was confirmed whether or not the gene of the regenerated plant was introduced in the selection medium (see FIG. 4), and Southern blot analysis confirmed that the SOD2 gene was introduced into 1 to 3 copies 5).

In addition, it was confirmed by real-time PCR analysis that the transgene of the transgene was normally expressed (see Fig. 6), and the protein of the transgene was normally expressed by Western blot analysis (Fig. 7 Reference).

Example 5:

58 seedlings of 8 SOD2 transformants with 3 weeks of rooting and 44 ~ 54 days of extracorporeal purification were thoroughly watered together with non - transformants and then treated at 25 ℃ for 7 days. linkage (cell membrane integrity status index), and recovery of growth after rehydration, the relative moisture content of the transformants before and after treatment was higher than that of the non - transformants by 0.1 ~ 11% But the transgenic plants were found to restore the growth of most individuals. In addition, relative ion linkage, which indicates the state of cell membrane integrity under stress conditions, was found to be 5 to 74% lower than that of untransformed transformants, thus demonstrating excellent ability to withstand daily and severe stress conditions 8, 9 and 10).

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Method for enhancing drought resistance of rose <130> PA-D15103 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 206 <212> PRT <213> Escherichia coli <400> 1 Met Ser Tyr Thr Leu Pro Ser Leu Pro Tyr Ala Tyr Asp Ala Leu Glu   1 5 10 15 Pro His Phe Asp Lys Gln Thr Met Glu Ile His His Thr Lys His His              20 25 30 Gln Thr Tyr Val Asn Aslan Ala Asn Ala Ala Leu Glu Ser Leu Pro Glu          35 40 45 Phe Ala Asn Leu Pro Val Glu Glu Leu Ile Thr Lys Leu Asp Gln Leu      50 55 60 Pro Ala Asp Lys Lys Thr Val Leu Arg Asn Asn Ala Gly Gly His Ala  65 70 75 80 Asn His Ser Leu Phe Trp Lys Gly Leu Lys Lys Gly Thr Thr Leu Gln                  85 90 95 Gly Asp Leu Lys Ala Ala Ile Glu Arg Asp Phe Gly Ser Val Asp Asn             100 105 110 Phe Lys Ala Glu Phe Gly Lys Ala Ala Ala Ser Arg Phe Gly Ser Gly         115 120 125 Trp Ala Trp Leu Val Leu Lys Gly Asp Lys Leu Ala Val Val Ser Thr     130 135 140 Ala Asn Gln Asp Ser Pro Leu Met Gly Glu Ala Ile Ser Gly Ala Ser 145 150 155 160 Gly Phe Pro Ile Met Gly Leu Asp Val Trp Glu His Ala Tyr Tyr Leu                 165 170 175 Lys Phe Gln Asn Arg Arg Pro Asp Tyr Ile Lys Glu Phe Trp Asn Val             180 185 190 Val Asn Trp Asp Glu Ala Ala Ala Arg Phe Ala Ala Lys Lys         195 200 205 <210> 2 <211> 621 <212> DNA <213> Escherichia coli <400> 2 atgagctata ccctgccatc cctgccgtat gcttacgatg ccctggaacc gcacttcgat 60 aagcagacca tggaaatcca ccacaccaaa caccatcaga cctacgtaaa caacgccaac 120 gcggcgctgg aaagcctgcc agaatttgcc aacctgccgg ttgaagagct gattaccaaa 180 ctggaccagc tgccagcaga caagaaaacc gtactgcgca acaacgctgg cggtcacgct 240 aaccacagcc tgttctggaa aggtctgaaa aaaggcacca ccctgcaggg tgacctgaaa 300 gcggctatcg aacgtgactt cggctccgtt gataacttca aagcagaatt tgaaaaagcg 360 gcagcttccc gctttggttc cggctgggca tggctggtgc tgaaaggcga taaactggcg 420 gtggtttcta ctgctaacca ggattctccg ctgatgggtg aagctatttc tggcgcttcc 480 ggcttcccga ttatgggcct ggatgtgtgg gaacatgctt actacctgaa attccagaac 540 cgccgtccgg actacattaa agagttctgg aacgtggtga actgggacga agcagcggca 600 cgttttgcgg cgaaaaaata a 621 <210> 3 <211> 1053 <212> DNA <213> Escherichia coli <400> 3 tcgggcattt tcctgcaaaa ccataccctt acgaaaagta cggcattgat aatcattttc 60 aatatcattt aattaactat aatgaaccaa ctgcttacgc ggcattaaca atcggccgcc 120 cgacaatact ggagatgaat atgagctata ccctgccatc cctgccgtat gcttacgatg 180 ccctggaacc gcacttcgat aagcagacca tggaaatcca ccacaccaaa caccatcaga 240 cctacgtaaa caacgccaac gcggcgctgg aaagcctgcc agaatttgcc aacctgccgg 300 ttgaagagct gattaccaaa ctggaccagc tgccagcaga caagaaaacc gtactgcgca 360 acaacgctgg cggtcacgct aaccacagcc tgttctggaa aggtctgaaa aaaggcacca 420 ccctgcaggg tgacctgaaa gcggctatcg aacgtgactt cggctccgtt gataacttca 480 aagcagaatt tgaaaaagcg gcagcttccc gctttggttc cggctgggca tggctggtgc 540 tgaaaggcga taaactggcg gtggtttcta ctgctaacca ggattctccg ctgatgggtg 600 aagctatttc tggcgcttcc ggcttcccga ttatgggcct ggatgtgtgg gaacatgctt 660 actacctgaa attccagaac cgccgtccgg actacattaa agagttctgg aacgtggtga 720 actgggacga agcagcggca cgttttgcgg cgaaaaaata atcatttgcc gcctgctgca 780 atgaggcgta taggccgcat atcagcttaa aaaatgaacc atcgccaacg gcggtggttt 840 ttttgtgatc aatttcaaaa taaaaacaat gatccgaata aaaataaaac agcgtttcaa 900 ttgatgtggt tttgacactt ttatgattaa atgaatgtct atcttcgttt ccatcaacac 960 tgatgctcca ttgaggaatt acgcatcagc ccttaaaaat atgccgacag gtgatggaaa 1020 tgcagataaa acgctcgatt gagaaaatcc cgg 1053 <210> 4 <211> 10154 <212> DNA <213> Artificial Sequence <220> <223> pPZP200-SOD2-Bar <400> 4 gtttacccgc caatatatcc tgtcaaacac tgatagttta aactgaaggc gggaaacgac 60 aatctgatcc aagctcaagc taagcttgca tgcctgcagg tccccagatt agccttttca 120 atttcagaaa gaatgctaac ccacagatgg ttagagaggc ttacgcagca ggtctcatca 180 agacgatcta cccgagcaat aatctccagg aaatcaaata ccttcccaag aaggttaaag 240 atgcagtcaa aagattcagg actaactgca tcaagaacac agagaaagat atatttctca 300 agatcagaag tactattcca gtatggacga ttcaaggctt gcttcacaaa ccaaggcaag 360 taatagagat tggagtctct aaaaaggtag ttcccactga atcaaaggcc atggagtcaa 420 agattcaaat agaggaccta acagaactcg ccgtaaagac tggcgaacag ttcatacaga 480 gtctcttacg actcaatgac aagaagaaaa tcttcgtcaa catggtggag cacgacacac 540 ttgtctactc caaaaatatc aaagatacaga tctcagaaga ccaaagggca attgagactt 600 ttcaacaaag ggtaatatcc ggaaacctcc tcggattcca ttgcccagct atctgtcact 660 ttattgtgaa gatagtggaa aaggaaggtg gctcctacaa atgccatcat tgcgataaag 720 gaaaggccat cgttgaagat gcctctgccg acagtggtcc caaagatgga cccccaccca 780 cgaggagcat cgtggaaaaa gaagacgttc caaccacgtc ttcaaagcaa gtggattgat 840 gtgatatctc cactgacgta agggatgacg cacaatccca ctatccttcg caagaccctt 900 cctctatata aggaagttca tttcatttgg agagaacacg ggggactcta gaggatcccc 960 gggatactgg agatgaatat gagctatacc ctgccatccc tgccgtatgc ttacgatgcc 1020 ctggaaccgc acttcgataa gcagaccatg gaaatccacc acaccaaaca ccatcagacc 1080 tacgtaaaca acgccaacgc ggcgctggaa agcctgccag aatttgccaa cctgccggtt 1140 gaagagctga tcaccaaact ggaccagctg ccagcagaca agaaaaccgt actgcgcaac 1200 aacgctggcg gtcacgctaa ccacagcctg ttctggaaag gtctgaaaaa aggcaccacc 1260 ctgcagggtg acctgaaagc ggctatcgaa cgtgacttcg gctccgttga taacttcaaa 1320 gcagaatttg aaaaagcggc agcttcccgc tttggttccg gctgggcatg gctggtgctg 1380 aaaggcgata aactggcggt ggtttctact gctaaccagg attctccgct gatgggtgaa 1440 gctatttctg gcgcttccgg cttcccgatt atgggcctgg atgtgtggga acatgcttac 1500 tacctgaaat tccagaccg ccgtccggac tacattaaag agttctggaa cgtggtgaac 1560 tgggacgaag cagcggcacg ttttgcggcg aaaaaataat catttgccgc ctgctggagc 1620 tccggccatg ctagagtccg caaaaatcac cagtctctct ctacaaatct atctctctct 1680 atttttctcc agaataatgt gtgagtagtt cccagataag ggaattaggg ttcttatagg 1740 gtttcgctc tgtgttgagc atataagaaa cccttagtat gtatttgtat ttgtaaaata 1800 cttctatcaa taaaatttct aattcctaaa accaaaatcc agtgacctct gcaggtcccc 1860 agattagcct tttcaatttc agaaagaatg ctaacccaca gatggttaga gaggcttacg 1920 cagcaggtct catcaagacg atctacccga gcaataatct ccaggaaatc aaataccttc 1980 ccaagaaggt taaagatgca gtcaaaagat tcaggactaa ctgcatcaag aacacagaga 2040 aagatatatt tctcaagatc agaagtacta ttccagtatg gacgattcaa ggcttgcttc 2100 acaaaccaag gcaagtaata gagattggag tctctaaaaa ggtagttccc actgaatcaa 2160 aggccatgga gtcaaagatt caaatagagg acctaacaga actcgccgta aagactggcg 2220 aacagttcat acagagtctc ttacgactca atgacaagaa gaaaatcttc gtcaacatgg 2280 tggagcacga cacacttgtc tactccaaaa atatcaaaga tacagtctca gaagaccaaa 2340 gggcaattga gacttttcaa caaagggtaa tatccggaaa cctcctcgga ttccattgcc 2400 cagctatctg tcactttatt gtgaagatag tggaaaagga aggtggctcc tacaaatgcc 2460 atcattgcga taaaggaaag gccatcgttg aagatgcctc tgccgacagt ggtcccaaag 2520 atggaccccc acccacgagg agcatcgtgg aaaaagaaga cgttccaacc acgtcttcaa 2580 agcaagtgga ttgatgtgat atctccactg acgtaaggga tgacgcacaa tcccactatc 2640 cttcgcaaga cccttcctct atataaggaa gttcatttca tttggagaga acacggggga 2700 ctctaggggg atctaccatg agcccagaac gacgcccggc cgacatccgc cgtgccaccg 2760 aggcggacat gccggcggtc tgcaccatcg tcaaccacta catcgagaca agcacggtca 2820 acttccgtac cgagccgcag gaaccgcagg agtggacgga cgacctcgtc cgtctgcggg 2880 agcgctatcc ctggctcgtc gccgaggtgg acggcgaggt cgccggcatc gcctacgcgg 2940 gcccctggaa ggcacgcaac gcctacgact ggacggccga gtcgaccgtg tacgtctccc 3000 cccgccacca gcggacggga ctgggctcca cgctctacac ccacctgctg aagtccctgg 3060 aggcacaggg cttcaagagc gtggtcgctg tcatcgggct gcccaacgac ccgagcgtgc 3120 gcatgcacga ggcgctcgga tatgcccccc gcggcatgct gcgggcggcc ggcttcaagc 3180 acgggaactg gcatgacgtg ggtttctggc agctggactt cagcctgccg gtaccgcccc 3240 gtccggtcct gcccgtcacc gagatctgat gaccccgaat ttccccgatc gttcaaacat 3300 ttggcaataa agtttcttaa gattgaatcc tgttgccggt cttgcgatga ttatcatata 3360 atttctgttg aattacgtta agcatgtaat aattaacatg taatgcatga cgttatttat 3420 gagatgggtt tttatgatta gagtcccgca attatacatt taatacgcga tagaaaacaa 3480 aatatagcgc gcaaactagg ataaattatc gcgcgcggtg tcatctatgt tactagatcg 3540 ggaattcaat tcggcgttaa ttcagtacat taaaaacgtc cgcaatgtgt tattaagttg 3600 tctaagcgtc aatttgttta caccacaata tatcctgcca ccagccagcc aacagctccc 3660 cgaccggcag ctcggcacaa aatcaccact cgatacaggc agcccatcag tccgggacgg 3720 cgtcagcggg agagccgttg taaggcggca gactttgctc atgttaccga tgctattcgg 3780 aagaacggca actaagctgc cgggtttgaa acacggatga tctcgcggag ggtagcatgt 3840 tgattgtaac gggacagag cgttgctgcc tgtgatcaat tcgggcacga acccagtgga 3900 cataagcctg ttcggttcgt aagctgtaat gcaagtagcg tatgcgctca cgcaactggt 3960 ccagaacctt gccgaacgc agcggtggta acggcgcagt ggcggttttc atggcttgtt 4020 atgactgttt ttttggggta cagtctatgc ctcgggcatc caagcagcaa gcgcgttacg 4080 ccgtgggtcg atgtttgatg ttatggagca gcaacgatgt tacgcagcag ggcagtcgcc 4140 ctaaaacaaa gttaaacatc atgggggaag cggtgatcgc cgaagtatcg actcaactat 4200 cagaggtagt tggcgtcatc gagcgccatc tcgaaccgac gttgctggcc gtacatttgt 4260 acggctccgc agtggatggc ggcctgaagc cacacagtga tattgatttg ctggttacgg 4320 tgaccgtaag gcttgatgaa acaacgcggc gagctttgat caacgacctt ttggaaactt 4380 cggcttcccc tggagagagc gagattctcc gcgctgtaga agtcaccatt gttgtgcacg 4440 acgacatcat tccgtggcgt tatccagcta agcgcgaact gcaatttgga gaatggcagc 4500 gcaatgacat tcttgcaggt atcttcgagc cagccacgat cgacattgat ctggctatct 4560 tgctgacaaa agcaagagaa catagcgttg ccttggtagg tccagcggcg gaggaactct 4620 ttgatccggt tcctgaacag gatctatttg aggcgctaaa tgaaacctta acgctatgga 4680 actcgccgcc cgactgggct ggcgatgagc gaaatgtagt gcttacgttg tcccgcattt 4740 ggtacagcgc agtaaccggc aaaatcgcgc cgaaggatgt cgctgccgac tgggcaatgg 4800 agcgcctgcc ggcccagtat cagcccgtca tacttgaagc tagacaggct tatcttggac 4860 aagaagaaga tcgcttggcc tcgcgcgcag atcagttgga agaatttgtc cactacgtga 4920 aaggcgagat caccaaggta gtcggcaaat aatgtctagc tagaaattcg ttcaagccga 4980 cgccgcttcg ccggcgttaa ctcaagcgat tagatgcact aagcacataa ttgctcacag 5040 ccaaactatc aggtcaagtc tgcttttatt atttttaagc gtgcataata agccctacac 5100 aaattgggag atatatcatg catgaccaaa atcccttaac gtgagttttc gttccactga 5160 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta 5220 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa 5280 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact 5340 gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca 5400 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt 5460 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg 5520 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag 5580 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta 5640 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat 5700 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg 5760 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc 5820 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac 5880 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc 5940 gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg 6000 tgcggtattt cacaccgcat atggtgcact ctcagtacaa tctgctctga tgccgcatag 6060 ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc 6120 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac 6180 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac 6240 gcgcgaggca gggtgccttg atgtgggcgc cggcggtcga gtggcgacgg cgcggcttgt 6300 ccgcgccctg gtagattgcc tggccgtagg ccagccattt ttgagcggcc agcggccgcg 6360 ataggccgac gcgaagcggc ggggcgtagg gagcgcagcg accgaagggt aggcgctttt 6420 tgcagctctt cggctgtgcg ctggccagac agttatgcac aggccaggcg ggttttaaga 6480 gttttaataa gttttaaaga gttttaggcg gaaaaatcgc cttttttctc ttttatatca 6540 gtcacttaca tgtgtgaccg gttcccaatg tacggctttg ggttcccaat gtacgggttc 6600 cggttcccaa tgtacggctt tgggttccca atgtacgtgc tatccacagg aaagagacct 6660 tttcgacctt tttcccctgc tagggcaatt tgccctagca tctgctccgt acattaggaa 6720 ccggcggatg cttcgccctc gatcaggttg cggtagcgca tgactaggat cgggccagcc 6780 tgccccgcct cctccttcaa atcgtactcc ggcaggtcat ttgacccgat cagcttgcgc 6840 acggtgaaac agaacttctt gaactctccg gcgctgccac tgcgttcgta gatcgtcttg 6900 aacaaccatc tggcttctgc cttgcctgcg gcgcggcgtg ccaggcggta gagaaaacgg 6960 ccgatgccgg gatcgatcaa aaagtaatcg gggtgaaccg tcagcacgtc cgggttcttg 7020 ccttctgtga tctcgcggta catccaatca gctagctcga tctcgatgta ctccggccgc 7080 ccggtttcgc tctttacgat cttgtagcgg ctaatcaagg cttcaccctc ggataccgtc 7140 accaggcggc cgttcttggc cttcttcgta cgctgcatgg caacgtgcgt ggtgtttaac 7200 cgaatgcagg tttctaccag gtcgtctttc tgctttccgc catcggctcg ccggcagaac 7260 ttgagtacgt ccgcaacgtg tggacggaac acgcggccgg gcttgtctcc cttcccttcc 7320 cggtatcggt tcatggattc ggttagatgg gaaaccgcca tcagtaccag gtcgtaatcc 7380 cacacactgg ccatgccggc cggccctgcg gaaacctcta cgtgcccgtc tggaagctcg 7440 tagcggatca cctcgccagc tcgtcggtca cgcttcgaca gacggaaaac ggccacgtcc 7500 atgatgctgc gactatcgcg ggtgcccacg tcatagagca tcggaacgaa aaaatctggt 7560 tgctcgtcgc ccttgggcgg cttcctaatc gacggcgcac cggctgccgg cggttgccgg 7620 gattctttgc ggattcgatc agcggccgct tgccacgatt caccggggcg tgcttctgcc 7680 tcgatgcgtt gccgctgggc ggcctgcgcg gccttcaact tctccaccag gtcatcaccc 7740 agcgccgcgc cgatttgtac cgggccggat ggtttgcgac cgtcacgccg attcctcggg 7800 cttgggggtt ccagtgccat tgcagggccg gcagacaacc cagccgctta cgcctggcca 7860 accgcccgtt cctccacaca tggggcattc cacggcgtcg gtgcctggtt gttcttgatt 7920 ttccatgccg cctcctttag ccgctaaaat tcatctactc atttattcat ttgctcattt 7980 actctggtag ctgcgcgatg tattcagata gcagctcggt aatggtcttg ccttggcgta 8040 ccgcgtacat cttcagcttg gtgtgatcct ccgccggcaa ctgaaagttg acccgcttca 8100 tggctggcgt gtctgccagg ctggccaacg ttgcagcctt gctgctgcgt gcgctcggac 8160 ggccggcact tagcgtgttt gtgcttttgc tcattttctc tttacctcat taactcaaat 8220 gagttttgat ttaatttcag cggccagcgc ctggacctcg cgggcagcgt cgccctcggg 8280 ttctgattca agaacggttg tgccggcggc ggcagtgcct gggtagctca cgcgctgcgt 8340 gatacgggac tcaagaatgg gcagctcgta cccggccagc gcctcggcaa cctcaccgcc 8400 gatgcgcgtg cctttgatcg cccgcgacac gacaaaggcc gcttgtagcc ttccatccgt 8460 gacctcaatg cgctgcttaa ccagctccac caggtcggcg gtggcccata tgtcgtaagg 8520 gcttggctgc accggaatca gcacgaagtc ggctgccttg atcgcggaca cagccaagtc 8580 cgccgcctgg ggcgctccgt cgatcactac gaagtcgcgc cggccgatgg ccttcacgtc 8640 gcggtcaatc gtcgggcggt cgatgccgac aacggttagc ggttgatctt cccgcacggc 8700 cgcccaatcg cgggcactgc cctggggatc ggaatcgact aacagaacat cggccccggc 8760 gagttgcagg gcgcgggcta gatgggttgc gatggtcgtc ttgcctgacc cgcctttctg 8820 gttaagtaca gcgataacct tcatgcgttc cccttgcgta tttgtttatt tactcatcgc 8880 atcatatacg cagcgaccgc atgacgcaag ctgttttact caaatacaca tcaccttttt 8940 agacggcggc gctcggtttc ttcagcggcc aagctggccg gccaggccgc cagcttggca 9000 tcagacaaac cggccaggat ttcatgcagc cgcacggttg agacgtgcgc gggcggctcg 9060 aacacgtacc cggccgcgat catctccgcc tcgatctctt cggtaatgaa aaacggttcg 9120 tcctggccgt cctggtgcgg tttcatgctt gttcctcttg gcgttcattc tcggcggccg 9180 ccagggcgtc ggcctcggtc aatgcgtcct cacggaaggc accgcgccgc ctggcctcgg 9240 tgggcgtcac ttcctcgctg cgctcaagtg cgcggtacag ggtcgagcga tgcacgccaa 9300 gcagtgcagc cgcctctttc acggtgcggc cttcctggtc gatcagctcg cgggcgtgcg 9360 cgatctgtgc cggggtgagg gtagggcggg ggccaaactt cacgcctcgg gccttggcgg 9420 cctcgcgccc gctccgggtg cggtcgatga ttagggaacg ctcgaactcg gcaatgccgg 9480 cgaacacggt caacaccatg cggccggccg gcgtggtggt gtcggcccac ggctctgcca 9540 ggctacgcag gcccgcgccg gcctcctgga tgcgctcggc aatgtccagt aggtcgcggg 9600 tgctgcgggc caggcggtct agcctggtca ctgtcacaac gtcgccaggg cgtaggtggt 9660 caagcatcct ggccagctcc gggcggtcgc gcctggtgcc ggtgatcttc tcggaaaaca 9720 gcttggtgca gccggccgcg tgcagttcgg cccgttggtt ggtcaagtcc tggtcgtcgg 9780 tgctgacgcg ggcatagccc agcaggccag cggcggcgct cttgttcatg gcgtaatgtc 9840 tccggttcta gtcgcaagta ttctacttta tgcgactaaa acacgcgaca agaaaacgcc 9900 aggaaaaggg cagggcggca gcctgtcgcg taacttagga cttgtgcgac atgtcgtttt 9960 cgaagacgg ctgcactgaa cgtcagaagc cgactgcact atagcagcgg aggggttgga 10020 tcaaagtact ttaaagtact ttgatcccga ggggaaccct gtggttggca tgcacataca 10080 aatggacgaa cggataaacc ttttcacgcc cttttaaata tccgattatt ctaataaacg 10140 ctcttttctc ttag 10154

Claims (6)

(2), which expresses the superoxide dismutase 2 (SOD2) derived from Escherichia coli, which is represented by the amino acid sequence of SEQ ID NO: 1, into a cell and expressing the same. How to improve.
The method according to claim 1,
Wherein the gene is represented by the nucleotide sequence of SEQ ID NO: 2.
The method according to claim 1,
Wherein the expression causes the transcription of the gene to be regulated by a mosaic virus-derived 35S promoter.
The method according to claim 1,
Characterized in that an agrobacterium-mediated transformation method is used as a method for introducing the gene into a cell.
5. The method of claim 4,
Wherein the transformation is carried out using the recombinant vector containing the gene and represented by Fig. 1.
5. The method of claim 4,
Wherein said cell is a somatic embryo or embryogenic callus derived from roots of roses.

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