KR20080067435A - The production of fruit tree for transforming tree form by using mads-box gene - Google Patents

Abstract

A method for preparing a tree-form transformed woody plant is provided to transform the tree-form of a fruit tree without controlling growth through branch allurement by introducing an MdMADS2 gene isolated from apple into the tree. A method for preparing a tree-form transformed woody plant such as a flowering plant comprises the steps of: (a) preparing an expression vector including an MdMADS2(U78948) gene; (b) introducing the expression vector into a woody plant cell using Agrobacterium, a gene gun method, electroporation, microinjection or a direct DNA absorption method to transform the woody plant cell; and (c) tissue-culturing the transformed woody plant cell to re-differentiate it. A transgenic fruit tree is characterized in that a tree-form is transformed by transducing the MdMADS2 gene thereinto. Further, the tree-form transform is one of drooped condition, rolling condition or branch-twisted condition.

Description

Production of fruit tree for transforming tree form by using MADS-Box gene}

1 is MdMDS2 Here is a comparison of phenotypes of transgenic Fuji varieties with the genes introduced:

    a: wild type; And

b: MdMADS2 Genetically Modified Fuji Varieties.

2 is MdMADS2 Here is a comparison of the phenotypes of the transgenic gala varieties with the genes introduced:

    a: wild type; And

b: MdMADS2 Genetically Transgenic Gala Varieties.

3 is MdMADS2 Here is a comparison of the phenotypes of the transgenic Macintosh strains with the genes introduced:

    a: wild type; And

b: MdMADS2 Genetically modified Macintosh varieties.

4 is MdMADS2 PCR confirmed the presence of NPTII gene in transgenic apples into which the gene was introduced:

a: MdMADS2 Transgenic Fuji variety into which the gene is introduced;

b: MdMADS2 Transgenic gala varieties into which genes have been introduced; And

c: MdMADS2 Genetically modified transgenic macaque varieties.

5 is MdMADS2 Southern blot results using NPTII gene probe in transgenic apples with the genes introduced:

a: MdMADS2 Transgenic Fuji variety into which the gene is introduced;

b: MdMADS2 Transgenic gala varieties into which genes have been introduced;

c: MdMADS2 Transgenic Macintosh varieties with genes introduced;

H: Hind III restriction enzyme treatment group; And

X: Xba I restriction enzyme treatment.

6 is apple and MdMADS2 of wild type Fuji varieties A comparison of the transgenic Fuji varieties of apples:

    a: wild-type Fuji varieties;

    b: cut section of wild type Fuji varieties; And

c: Cut section of the transgenic Fuji variety into which the MdMADS2 gene was introduced.

The present invention relates to a method for producing an orchard crop for tree deformation, and more particularly, to MdMADS2 isolated from apples. The present invention relates to a method for producing a fruit crop in which a transgenic gene is used to generate a male strain.

In general, fruit crops require three to five years of juvenile, and are young crops, and they have to be grown in the same region for many years. In addition, the crown area is relatively large, and the breeding period is very long, and the effort and cost are high. The competitiveness of the fruit industry should be based on such factors as good varieties, production techniques and growing conditions. In many cases, the characteristics of the developed varieties are excellent, but due to the cultivation environment, the characteristics of the fruit are insufficient. To compensate for this, a lot of labor has been put into production such as high-density cultivation, stabilization of water washing, redness and redness work, bagging, and reflection film installation to improve quality such as high coloring, fruit and high sugar content. In particular, the fruit trees such as apples and pears are perennials, growing not only as giant trees with the passage of time, but also because they grow to a size that does not reach the height of humans because of their growth characteristics. It will be able to perform a variety of tasks such as enemy, work, bagging and harvesting. In order to overcome this problem, branch attracting function with a function of suppressing the branch from growing to the sky is utilized, and the conventional new branch attracting method is to insert a toothpick between the stem and the branch when the branch is young for attracting a new branch. Open or hang the ends of the concrete manned weight, or wire attracted and branched attracted rings, etc. have been developed and used. However, the stabilization of water washing through eggplant induction takes more than seven years and requires a lot of labor, and due to such labor-intensive cultivation conditions, it is difficult to secure international competitiveness of fruit crops.

In Latin America, an advanced country of apple cultivation, the stabilization of apple trees with water-shrinkage stabilizes watersheds, leading to breakthrough efforts to reduce production, increase unit productivity, and lower production costs. Sewage-type water washing of apple trees not only has the advantages of securing solar light and improving breathability, but also has a great effect on reducing labor labor during cultivation, such as spraying of pesticides, pruning, red / red, bagging, and harvesting. There is an advantage. However, such sewage stabilization of the sewage type has been made through branch incentives, and it takes more than seven years.

MADS-box genes represent a family of genes consisting of 30 or more amino acid sequences encoding transcription factors with conserved regions called MADS-boxes. Many of these genes are known to be involved in different organ differentiation of plants through regulation at the level of transcription. Arabidopsis Among the MADS- box genes classified in thaliana ), AP1 ( APETALA1 ) and AP 2 ( APETALA 2) were identified as genes of group A. AP3 ( APETALA ) and PI ( PISTIL L) were group B genes, AG ( AGAMOUS). Genes have been classified as Group C genes (Gunter Theissen et) al ., Plant Molecular Biology , 2000, 42: 115-149. FBP7 ( floral binding protein 7 ) and FBP11 ( floral binding protein11 ) has been newly classified as a group D gene because it was found to be a gene that affects the development of the embryo by cosuppression in transformed petunias (GC Angenent et. al ., Plant Cell , 1995, 7: 1569-1582). However, it has not been reported that any MADS-box gene affects the change of tree crops.

The MdMADS2 gene was first reported by Sung et al. In 1999 as a MADS- box gene isolated from apples, and has been found to be expressed in all flower organs except for stamens and atria. In addition, MdMADS2 gene isolated from apples was transformed into tobacco and analyzed for its function, and early flowering and dwarfing of tobacco were observed. Therefore, the MdMADS2 gene has been reported to play an important role in early flowering of flowers (Sung et. al ., Plant Physiology , 1999, 120: 969-978).

Genetic techniques have been actively conducted in crop improvement studies, but no studies have been reported to induce the change of the fruit tree crop by the introduction of foreign genes. However, it can be said that the industrial utilization is large because it can bring about the effect of shortening the time for stabilizing washing, reducing the labor force and improving economic efficiency through the change of the fruit tree by introducing foreign genes.

Thus, the present inventors have isolated MdMADS2 from apple As a result of the introduction of the gene, the present invention was completed by confirming that the water crop was changed in the fruit crops without performing the flush stabilization work.

An object of the present invention is a method of producing an orchard crop that can cause a change in the shape of the tree without causing water stabilization through eggplant induction, an expression vector including the gene causing the perturbation, a plant cell transduced with the expression vector and the expression vector It is to provide a transgenic plant that has been transduced.

In order to achieve the above object, the present invention provides a method for producing a tree plant modified.

In addition, the present invention MdMADS2 Provided are fruit crop transgenic plants that are transduced with the gene to produce a maleic change.

Hereinafter, the present invention will be described in detail.

The inventors have found that MdMADS2 has a significant effect on the early flowering of flowers. Gene was isolated from young flower bud of apple Fuji cultivar MdMADS2 MdMADS2 by inserting the gene into the plant transformation vector Gene expression vectors were prepared and named “pMdMADS2.” The pMdMADS2 was introduced into Agrobacterium tumefaciens LBA4404 to produce transgenic apple varieties. The work duration is more than 7 years and a lot of labor is required .. As a result of comparing the produced apple varieties with wild apple varieties, the MdMADS2 of the present invention was used. Genetically transduced apple varieties (Fuji, Gala, and Macintosh) produced water deflection without special flush stabilization. In addition to the sewage phenomenon, a rolling phenomenon and branch twisting phenomenon were also observed (see FIGS. 1 to 3). This phenomenon facilitates wheat cultivation and branching of fruit trees, which can reduce the labor input during cultivation, such as pesticide spraying, cell work, red / red fruiting, bagging, and harvesting. Can be competitive from the breed. In addition, the MdMADS2 gene was isolated from the apple Fuji varieties, but it was confirmed that a change in the tree type was common in the gala variety and the Macintosh variety of the apple varieties. Through this MdMADS2 of the present invention It can be seen that the method of producing fruit trees using genes can induce tree strains in various fruit trees.

The inventors of the present invention in the transgenic apple varieties MdMADS2 The genes were inserted normally (see Figure 4), at least 1 copy (copy), more than 3 copies confirmed that the gene was introduced (see Figure 5). However, regardless of the number of copies it can be seen that only one copy of the male deformation to be achieved in the present invention effectively occurs (see Figs. 1 to 3).

We use non-transformed apple varieties and MdMADS2 Fruits were compared between transgenic apple varieties. As a result, it was observed that there was no change in the properties of the fruits (see Table 1 and Fig. 6).

Therefore, the method of manufacturing the modified tree plant according to the present invention tolerates wheat cultivation and eggplant induction of fruit trees to reduce the labor input during cultivation, such as pesticide spraying and pruning, red / red, bagging, harvesting, water washing By reducing the work time required for stabilization work, it can be usefully used for growing competitive woody plants. It is also possible to produce a uniquely shaped ornamental tree without going through the branches and pruning.

The present invention provides a method for producing a tree modified tree plant.

The present inventors provide the following preparation method: 1) preparing an expression vector comprising the MdMADS2 (U78948) gene; 2) transforming plant cells by introducing the expression vector of step 1) into tree plant cells and co-culturing the tree plant cells; And 3) regenerating by tissue culture of the transformed woody plant cells of step 2).

In the above production method, the male deformation means sewage phenomenon, rolling field or branch twist of a tree plant.

In the above production method, the woody plant is preferably a flowering plant. It is preferable that the flowering plant is a rosaceae, and the rosaceae is preferably a Japonica subfamily, Rosaceae subfamily, Andoaceae or Pear subfamily. The maple tree subfamily is preferably, but not limited to, a maple tree, a tail-poplar tree or a noodle tree. The rosaceae is preferably, but not limited to, chick flower, yellow plum, raspberry, bearberry, white nectar, persimmon, rose or brier. The adoaceae is preferably cherry, cherry, peach, plum, plum or apricot, but is not limited thereto. The pear subfamily is preferably an apple tree, a pear tree or a Chinese quince tree, but is not limited thereto. The varieties of the apple tree are preferably a Fuji variety, a gala variety, a Macintosh variety, a Tsugaru variety, a Hongro variety, a Red Daricious variety, a Golden Daricious variety or a mutant variety, but are not limited thereto. Apple varieties known to those skilled in the art may be used in the production method of the present invention.

In the production method, the sequence of the MdMADS2 gene of step 1) is preferably SEQ ID NO: 7, but is not limited thereto, and the MdMADS2 Any gene having a sequence homology of 85% or more can be used in the present invention. The pGA1530 vector, which is a plant transformation vector used as the expression vector, is not limited thereto, and any plant transformation vector known to those skilled in the art may be used in the production method of the present invention.

In the above production method, the introduction method of step 2) is a method using Agrobacterium, gene gun method, electroporation, microinjection, direct DNA absorption method (Gasser CS and Fraley) RT, Science , 1989, 244: 1293-1299), but is not limited thereto. Any gene transduction method of plant cells known to those skilled in the art may be used in the present invention. The Agrobacterium ( Agrobacterium) is preferably Agrobacterium tumefaciens ( Agrobacterium tumefaciens ), but is not limited thereto.

In addition, the present invention MdMADS2 Provided are fruit crop transgenic plants that are transduced with the gene to produce a maleic change.

Regeneration through tissue culture carried out to obtain the transgenic plant of the present invention is preferably carried out through a general transgenic plant production process.

The transgenic plants are preferably apples, pears or peaches, more preferably apples, and the varieties of apples are Fuji varieties, gala varieties, Macintosh varieties, Tsugaru varieties, Hongro varieties and Red Derris varieties. It is preferable that the varietal varieties or varieties of varieties, but not limited to, apple varieties known to those skilled in the art may be used in the production method of the present invention.

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> MdMADS2 Gene Cloning and  Transformation Vector Construction for Fruit Tree Change

Apple ( Malus domestica Borkh cv. CDNA library (SUNG. SK et . al , Plant Physiol. , 1999, 120: 969-978) in vivo removal ( in A cDNA pool was prepared by separating cDNAs according to an in vivo excision method (stratagene, USA, Catalog # 200450). MdMADS2 from GenBank (NIH, USA), Gene Sequencing Database After searching for the gene (U78948), a forward primer (SEQ ID NO: 1: 5'-AAGCTTATGGGGAGGGGAAG-3 ') and a 3' terminal ORF (open reading frame) containing a nucleotide sequence of Hind III restriction enzyme in the 5 'terminal ORF of the gene ), A reverse primer (SEQ ID NO: 2: 5'-TCTAGACTATTCATTAAGGTGGCG-3 ') containing a nucleotide sequence of Xba I restriction enzyme was prepared. PCR was performed using the prepared primer pairs as a template of the cDNA pool of apple buds. The composition of the PCR reaction was 100 ng cDNA template, 1 μM primer, 1 × Ex Taq buffer, 0.2 mM dNTP, 2.5 units of polymerase ( TaKaRa). Ex Taq : TaKaRa, Japan) was used. Reaction conditions were thermally denatured at 94 ° C. for 5 minutes using a Perkin Elemer 9600, and the thermal denaturation, primer binding and extension extensions were 94 ° C./30 sec., 58 ° C., respectively. 35 cycles were carried out at 1 ° C./1 min and 72 ° C./1 min, and then the entire amplification process was terminated by end elongation at 72 ° C./5 min. The amplified PCR product was cloned into pGEM-T Easy vector (Promega, USA), followed by T7 primer (SEQ ID NO: 3: 5'-TAATACGACTCACTATAGGG-3 ') and SP6 primer (SEQ ID NO: 4: 5'- TATTTAGGTGACACT ATAG-3'). The nucleotide sequence of the amplification product was examined to confirm that the ORF of the MdMADS2 gene was inserted into the vector.

MdMADS2 produced above The vector containing the gene was cleaved with Hind III and Xba I and then cloned forward to the pGA1530 vector, which is a plant transformation vector digested with the same enzyme. Plants transformed pGA1530 vector for switching has a kanamycin resistance has got a Ney emitter 35S promoter and T7 terminator has a NPT Ⅱ (neomycin phosphotransferase) gene as a selection gene (Plant molecular Biology Manual , 1988, A3: 1-19). The recombinant plasmid in which MdMADS2 thus prepared was inserted in the forward direction was named 'pMdMADS2'. And Agrobacterium tumefaciens LBA4404 (Hoekema, A. et) al ., 1983, Nature , 303, 179-181) and then strains introduced with the recombinant plasmid pMdMADS2 in kanamycin medium were selected.

< Example  2> MdMADS2 Genes introduced Transformation  Apple productions

To prepare transgenic apples into which the pMdMADS2 gene was introduced: 1) apple in-flight passage; 2) harvesting inoculation sections; 3) Agrobacterium (Agrobacterium) inoculation and co-culture; And 4) the experiment was carried out in a sequential step such as rooting medium selection.

Cultivation of apple shoots contained 1 mg / L BA, 0.3 mg / L IBA, 0.5 mg / L GA, 30 g / L sucrose and 8 g / L plant agar in MS mineral salts and vitamin medium. After the addition was adjusted to pH 5.8, a medium sterilized at 121 ° C and 1.2 atm for 15 minutes in a sterilizer was used. After 4 weeks, shoots grown from growth medium were healed with rooting medium (1/2 MS medium, IBA 0.3 mg / L, sucrose 15 g / L, plant agar 8 g / L). The leaves were used as inoculation sections. Agrobacterium tumefaciens LBA4404 prepared according to Example 1 (Hoekema, A. et al ., 1983, Nature , 303, 179-181) were incubated for 20 hours at 220 rpm, 28 ° C in liquid YEB medium containing 10 mg / L of kanamycin and 3 mg / L of tetracycline. The concentration at the time of culture was OD ₆₀₀ value 0.7. Agrobacterium tumefaciens LBA4404 Suspension culture medium (OD ₆₀₀ value 1.3) was inoculated by shaking culture at 150 rpm, 22 ° C for 4 hours. The inoculated leaves were absorbed with sterile dried sterilized paper and then co-cultured with conventional coculture medium [MS inorganic salts, LS vitamins, IBA 0.1 mg / L, TDZ 5 mg / L, sucrose 30 g / L, dicin agar ( daishin agar) 8 g / L] was cultured for 3 days in medium containing 200 μM of acetylgon. After 3 days, leaf sections were placed on redistribution selection medium containing 50 mg / L of kanamycin and 350 mg / L of cefotaxime, followed by 4 weeks of cancer culture and then 2 weeks of light culture followed by 2 weeks of light culture. Treated. The basic composition of the selection medium was MS inorganic salt, LS vitamin, IBA 0.1 mg / L, TDZ 5 mg / L, sucrose 30 g / L, agar 8 g / L. Individuals re-differentiated from the selection medium were transplanted into growth medium without kanamycin, and the grown shoots were healed in rooting medium added with 30 mg / L of kanamycin for reselection. Successful rootings were removed from the culture vessels, the medium was removed with sterile water, implanted in a pot of sterile sand, and then saturated to maintain the saturation of the artificial soil (vermiculite: perlite: horticultural soils = 1: 1). 1: 1), which were transferred to the quarantine greenhouse and then transplanted to the package.

< Example  3> MdMADS2  Phenotypic Analysis of Apples with Genes

MdMADS2 By comparing the phenotype of the transgenic apple with the phenotype of the nontransgenic apple, MdMADS2 The function of the gene was analyzed.

As a result, MdMADS2 The apples to which the genes were introduced showed a common phenotype in which the fruit tree had sewage, rolling, and twisting of branches regardless of the variety (Fuji, Gala, Macintosh).

MdMADS2 In the case of the Fuji varieties in which the gene was introduced, the non-transformant had a branch extending upward, but the transgenic Fuji showed a phenotype of sewage, rolling, and twisting of the branches (FIG. 1). Also MdMADS2 In the case of the gala varieties in which the genes were introduced, the non-transformant had a stiff up branch, but in the case of the transgenic gala varieties, the sewage phenomenon, the rolling phenomenon, and the twisting of the branches occurred (Figure 2). MdMADS2 also works for Macintosh Transformed varieties showed a common phenotype of sewage, rolling, and twisting of branches (Figure 3).

These results showed that MdMADS2 cloned from apple Fuji varieties The genes were found to induce a change in tree type regardless of the apple variety.

The present inventors have MdMADS2 through the presence of the NPTII gene targeting MdMADS2 transgenic apple varieties It was confirmed that the gene was introduced. The NPTII gene forward primer (SEQ ID NO: 5'-GAGGCTATTCGGCTATGACTG-3 ') and the NPT II gene reverse primer (SEQ ID NO: 6: 5'-ATCGGGAGCGGCGATACCGTA-3') were used to confirm the introduction of MdMADS2 by PCR. . The PCR reaction composition used was 200 ng of genomic DNA extracted from MdMADS2 transgenic apple leaves using a kit (DNeasy Plant Mini kit, # 69104 QIAGEN, USA), using a primer of 1 μM, 1 X Ex Taq buffer. , 0.2 mM dNTP, 2.5 units of polymerase ( TaKaRa Ex Taq : TaKaRa, Japan) was used. Reaction conditions were thermally denatured at 94 ° C. for 5 minutes using a Perkin Elemer 9600, and the thermal denaturation, primer binding and extension extensions were 94 ° C./30 seconds, 57, respectively. 35 cycles were carried out at 1 ° C./1 min and 72 ° C./1 min, and then the entire amplification process was terminated by end elongation at 72 ° C./10 min.

As a result, it was confirmed that the MdMADS2 gene was normally introduced in all of the Fuji varieties, the gala varieties, and the Macintosh strains of apples (FIG. 4).

< Example  4> MdMADS2  Southern of transgenic apple Blot  Gene copy number analysis through

The present inventors analyzed the copy number of the transgene through Southern blot of the apple varieties (Fuji, Gala, McIntosh Weiss) transformed with the MdMADS2 gene. Specifically MdMADS2 Genomic DNA was extracted from a transgenic apple leaf using a kit (DNeasy Plant Mini kit, # 69104 QIAGEN,). 20 μg of the isolated genomic DNA was digested with Hin dIII and Xba I restriction enzymes, and then electrophoresed on 1.0% agarose gel. The electrophoresis gel was reacted with 0.25 N HCl for 10 minutes to depurination and then washed twice with distilled water. Thereafter, the mixture was reacted in a denature solution (0.5 M NaOH, 1.5 M NaCl) for 30 minutes, and neutralized by reacting in a neutralization solution (1.5 M NaCl, 0.5 M Tris pH7.5) for 30 minutes. The neutralized agarose gel was transferred to a positively charged nylon membrane (Roche, Germany) for 16 hours and the DNA-transferred nylon membrane was fixed with a UV cross-linker. NPTII probes labeled with digoxigenin-dUTP using SEQ ID NOs: 5 and 6 were prepared using PCR DIG probe synthesis kit (Roche, Germany) to prepare DNA fragments for use as probes. Produced. After that, the nylon-transferred nylon membrane was placed in a buffer (DIG Easy Hyb buffer, Roche, Germany) and subjected to prehybridization for 30 minutes, and the NPTII probe labeled with dioxygenin was subjected to a water bath at 100 ° C. After denaturation at, hybridization was performed at 42 ° C. for at least 16 hours. After completion of the hybridization, the nylon membrane was reacted with a primary washing solution (0.1% SDS, 2 × SSC) for 30 minutes at room temperature, and a secondary washing solution (0.1% SDS) at 68 ° C. , 0.5 × SSC) for 30 minutes. The washed nylon membrane was then subjected to antibody hybridization using a kit (DIG Wash and Block Buffer kit; Roche, Germany) and a dioxygenin antibody (anti-digoxigenin-AP; Roche, Germany), followed by a washing reaction. . In addition, it was reacted with a color kit (chemiluminescene substrate CDP-Star, Roche, Germany) and was then exposed to X-ray film.

As a result, the transgenic Fuji varieties into which MdMADS2 was introduced were present in many copies of 3 or more copies (FIG. 5A), and in the case of the transgenic gala variety, 1 to 2 copies (FIG. 5B). In addition, it was confirmed that one copy of the MdMADS2 gene was introduced in the case of the transformed Macintosh. In contrast, NPTII genes were not detected in non-transformants with no phenotype. With these results, regardless of the number of copies of the introduced gene MdMADS2 MdMADS2 When the genes were introduced, it was found that the transformations represented by the sewage, rolling, and torsional distortions were made in the transformants.

< Example  5> MdMADS2 Comparison of Fruit Characteristics of Transgenic Apples

MdMADS2 In order to compare the fruit characteristics of transgenic apples with non-transformants, 20 fruits were harvested during the ripening season (Fuji, Gala, and Macintosh). The items included were weight, skin color, sugar content, acidity and seed count.

As a result, as shown in Table 1, MdMADS2 Transformed Fuji, Gala, and Macintosh litchi varieties all exhibited similar fruit characteristics as non-transformants. These results indicate that MdMADS2 The transgenic apples did not differ from the non-transformants in the nature of the fruit, and only a change in the water, such as sewage, rolling, and torsion was found (Fig. 6).

In the method of manufacturing the fruit crops of the present invention, by controlling the expression of the MdMADS2 gene, it is possible to control the water change of the fruit tree without performing the flush stabilization work. In addition, since the fruit of the fruit-changed fruit is not different from the fruit of the wild fruit, the manufacturing method of the present invention is useful in developing competitive varieties of fruit crops by reducing the working time and the labor force in the cultivation of fruit crops, which are administered to flush stabilization work. It can be used and is useful for producing a unique shaped ornamental tree.

<110> Dongbu Hannong Chemicals Co., Ltd. <120> The production of fruit for transforming tree form by using          MADS-Box gene <130> 6p-09-51 <160> 7 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HindIII sense primer <400> 1 aagcttatgg ggaggggaag 20 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> XbaI antisense primer <400> 2 tctagactat tcattaaggt ggcg 24 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> T7 primer <400> 3 taatacgact cactataggg 20 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> SP6 primer <400> 4 tatttaggtg acactatag 19 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> NPTII gene sense primer <400> 5 gaggctattc ggctatgact g 21 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> NPTII antisense primer <400> 6 atcgggagcg gcgataccgt a 21 <210> 7 <211> 1230 <212> DNA <213> Artificial Sequence <220> <223> MdMADS2 gene sequence <400> 7 gtcactcata tatagacaga gaaggagaga gaccagaccc ctaccttaga gagagagaga 60 gagcagaagc catctgtgtg tcaactggtt ctttctctcc catttttctt ggtttcttgg 120 tgggatttct ggtttctcta aactaagaga tcagttcagc aggaacaacc gtatatatat 180 tactaggatt attaattatt tatttataat aataaataat tgttagagag attatgggga 240 ggggaagggt gcagctgaag agaattgaga acaagatcaa caggcaggtg accttctcaa 300 agagaaggtc ggggctgatg aagaaagctc atgagatttc tgtgctttgt gatgctgagg 360 ttgctttgat tatcttctcc accaagggca agctctttga gtactccaat gattcctgca 420 tggaaaggat cctggaaagg tacgaaagat actcatatac agagaggcag cttcttgcaa 480 atgataatga atccactgga agctggactc tggaacatgc aaagctcaag gctagggtgg 540 aggttttaca aagaaatcaa agacactata tgggagaaga tctccaatcc ttaagtctca 600 aagagcttca aaatttagag caacagcttg attctgcact gaagcacata aggtcaagaa 660 agaaccaagt tatgtacgaa tcgatttctg agctccagaa gaaggataag gcattgcagg 720 agcaaaacaa cttgctggca aagaaggtga aggagaagga gaacgcagta gctcaacagg 780 cccaattgga gcatgtgcag gagcagaggt tgaactcctc ttcctccctt cttccacggg 840 cattgcagtc cttgaatttc ggcagcgggt ccaattacca ggccattaga tcaagtgaag 900 gaataccagg agataatcag cagtatggag atgaaacccc aactccacat agacctaaca 960 tgctgctgcc cgcttggatc gtccgccacc ttaatgaata gaagaattca tcaccatgaa 1020 ataatattat ctccaaaggg aataatatta tgtgacatca attaatcaag taaacatatt 1080 atacttatat atatatatat atatatatct atatatctat accaagaaat taattggtaa 1140 tgtgcatgat gaaaacccta gactcgatct atctctaaat gtgtatcgta tgttggaatt 1200 cattaatata tcttaagggc ttgtttggta 1230  

Claims (15)

1) preparing an expression vector comprising the MdMADS2 (U78948) gene; 2) transforming the woody plant cells by introducing the expression vector of step 1) into woody plant cells; And 3) A method for producing a modified tree plant, characterized in that it comprises the step of tissue culture of the transformed wood plant cells of step 2). The method according to claim 1, wherein the introduction method of step 2) is a method using Agrobacterium, a gene gun method, an electroporation method, a microinjection method, or a direct DNA absorption method. Way. The method according to claim 1, wherein the male deformation is sewage, rolling, or branch twist. The method according to claim 1, wherein the tree plant is a flowering plant. The method according to claim 4, wherein the flowering plant is rosaceae. 6. The method according to claim 5, wherein the rosaceae is Zapaceae, Rosaceae, Rhododendron or Pear subfamily. 7. The method according to claim 6, wherein the meadowsweet subfamily is meadowsweet, assorted poplar or noodle. 7. The method according to claim 6, wherein the rosaceae is a chick flower, a yellow plum, a raspberry, a bear, a white-eyed tree, a flower, a rose or a brier. The method according to claim 6, wherein the adoaceae is cherry, cherry, peach, plum, plum or apricot. The method according to claim 6, wherein the pear tree subfamily is an apple tree, a pear tree or a Chinese quince tree. The method of claim 10, wherein the apple is a Fuji variety, a gala variety, a Macintosh variety, a Tsugaru variety, a Hongro variety, a Red Daricious variety, a Golden Daricious variety or a mutant variety. The method of claim 1, wherein the sequence of MdMADS2 (U78948) of step 1) is SEQ ID NO: 7. An orchard crop transformed plant in which the MdMADS2 gene is transduced to cause a maleoid change. The transgenic plant of claim 13, wherein the fruit crop is an apple, pear or peach. 15. The transgenic plant of claim 14, wherein the apple is a Fuji variety, a gala variety, a Macintosh variety, a Tsugaru variety, a erythroma variety, a Red Daricious variety, a Golden Daricious variety, or a mutant variety.

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