WO2016053022A2 - Method for increasing plant productivity by using ore7 gene, method for reinforcing stress resistance of plants by using ore7 gene and method for delaying plant senescence by using ore7 gene - Google Patents

Abstract

The present invention relates to a method for increasing plant productivity by using an ORE7 gene, a method for reinforcing the stress resistance of plants by using the ORE7 gene and a method for delaying plant senescence by using the ORE7 gene, wherein the ORE7 gene is isolated from Arabidopsis thaliana.

Description

Plant productivity enhancement method, ORE7 stress reinforcement method and plant aging method using ORE7 gene

The present invention relates to a method for increasing plant productivity, a method for enhancing plant stress resistance, and a method for delaying plant aging using the ORE7 gene isolated from Arabidopsis.

Efforts to produce plants with improved growth and stress resistance are important in ensuring the productivity and quality of crops. Therefore, continuous research is being conducted by large agricultural companies such as the US and Europe. Plant aging research is also becoming increasingly important because of its industrial value.

When plants are subjected to various environmental (abiotic) stresses such as high temperature, low temperature, salt, drying, heavy metals, pesticides, as well as biological stresses such as germs, pests, viruses, etc. Reactive oxygen species (ROS) such as superoxide anion radicals (O ₂ ⁻ ), hydrogen peroxide (H ₂ O ₂ ), and hydroxyl radicals that cause physiological disorders, etc. Will change to Excessive generation of these reactive oxygen species in plants causes physiological disturbances in vivo, such as cell membrane degradation, protein degradation, DNA synthesis inhibition, photosynthesis inhibition, and chloroplast destruction, which in turn causes cell death and leaves yellowing (Inse D and Van Montau). M. Curr Opin Biotechnol 1995. 6: 166-172). Since all biological and abiotic stresses occur in the plant ultimately in the form of oxidative stress, oxidative stress resistance is of great importance in the overall plant defense response system, and the production of plants that can cope with these stresses properly is the crop quality. It is also closely related to improvement and productivity. Accordingly, in the development of stress resistant transformants, attempts to express antioxidant genes or to introduce direct stress resistance genes by using promoters such as SWPA2, whose expression is strongly induced under oxidative stress conditions, continue (Kim et al. , Plant Mol Biol. 2003. 51: 831-838).

The suppression of aging of plants is of great industrial importance, not only because of their academic importance, but also because of the possibility of improvement in crop productivity or post-harvest storage efficiency. Plant aging is the final stage of plant development, an age-dependent process of decay at the cellular, tissue, organ, or organismal level, leading to the lethal stage through the growth and development stages. As aging progresses, the plant gradually loses its ability to synthesize and the cellular structures and macromolecules are sequentially degraded, resulting in the loss of homeostasis of cells and eventually death (Buchanan-Wollaston et al., Plant Biotechnology Journal 2003, 1: 3-22; Lim and Nam, Curr. Top. Dev. Biol. 2005, 67: 49-83). The aging of these plants is genetically planned as a series of consecutive biochemical and physiological phenomena, which are very sophisticated and active at the level of cells, tissues, and organs.The effects of internal environmental factors such as plant hormones and external environmental stresses Will receive. Among the plant hormones, cytokinin is a physiologically delayed aging hormone and many aging control techniques have been reported. The Amasino group developed a method for regulating aging-specific cytokinin synthesis by recombining the IPT gene into a senescence-specific SAG12 gene promoter, which showed a 50% increase in productivity in cigarettes that delayed aging. When introduced into the lettuce by the same method it can be seen that the shelf life after harvest significantly increased (McCabe et al., Plant Physiol. 2001, 127 (2): 505-16). In addition, there was a report that the cytokinin level was increased and leaf aging was delayed in tobacco expressing corn homeobox gene (knotted1) in SAG12 gene promoter. Tomatoes have been reported to control the ripening of fruit through ethylene control.Flav-O-Savor, which has been shown to increase the transport and storage of tomatoes by suppressing the expression of polygalacturonase genes related to cell wall degradation, is commercialized. (Giovannoni et al., Plant Cell 1 (1): 53-63, 1989). In addition, reactive oxygen species (ROS) have recently been known to play an important role in plant aging. In particular, catalase isoforms derived from peroxysomes have been proposed by the Zentgraf group with Arabidopsis as a material to control aging of plants with APX1 (Zimmermann P et al., Plant Cell Environ. 2006 Jun; 29 (6): 1049- 60).

On the other hand, molecular control studies related to growth such as plant growth, development, and differentiation are important factors in determining the productivity of useful plants. These growth studies are closely related to aging and stress resistance. Aging may be a limiting factor in crop productivity. Aging can lead to quality loss rates such as yellowing of leaves and loss of nutrients in vegetable crops, etc., and resistance to stress may vary depending on the degree of aging of the plant. Therefore, it is possible to improve agricultural traits such as crop productivity, quality and shelf life through sufficient understanding of plant aging and growth process and experimental demonstration of related gene function.

As part of this effort, the market for genetically modified crops in which genes with agricultural trait enhancement functions have been introduced into plants despite controversy is expanding worldwide. Since the 1980s, transformation technologies have been steadily being developed around the United States, especially in GM crops more than 15 years. Plant transduction of several genes is being attempted through transformation, and this trend is likely to continue as food demand will continue to increase as the world population grows. In Korea, unlike other crops, soybeans have not been able to establish stable and high-efficiency transformation technology, and some laboratories have failed to produce continuous results although there have been some success stories in some laboratories. Although many studies have been conducted to isolate genes and to reveal their functions, it is known that it is difficult to produce transformants that are effective by transforming genes of different species into crops, and soybeans are particularly difficult to produce effective transformants. This is known to be great.

In the present invention, the plant productivity increase effect, stress resistance reinforcement effect, and aging delay effect using the ORE7 gene isolated from Arabidopsis were confirmed.

It is an object of the present invention to provide a method for producing a plant having productivity enhancement characteristics using the ORE7 gene.

Another object of the present invention to provide a method for producing a stress-resistant plant using the ORE7 gene.

Another object of the present invention to provide a method for producing a plant having aging delay properties using the ORE7 gene.

Other and specific objects of the present invention will be presented below.

Applicant for Arabidopsis thaliana ) from ORE7 If a gene (see SEQ ID NO: 1, its protein sequence see SEQ ID NO: 2) is introduced and the gene is introduced into the Arabidopsis, the Arabidopsis exhibits delayed aging characteristics such as prolonged lifespan, delayed yellowing of leaves, and improved photosynthetic efficiency. It has been reported (see Korean Patent Publication No. 2003-0016893).

The inventors have identified the ORE7 gene isolated from the Arabidopsis larvae soybean ( Glycine) as confirmed in the following examples. max L. cv. When introduced to Kwangan, in addition to showing delayed aging characteristics, such as when introduced to Arabidopsis, it also shows productivity enhancement characteristics such as increased individual size and increased yield of seeds, and also shows enhanced resistance to stress such as oxidation. Could.

The present invention is provided based on these experimental results, and in one aspect relates to a method for producing a plant having productivity enhancing properties.

Method of producing a plant having a productivity enhancing feature of the present invention comprises the steps of (a) expressing a gene having a sequence similar to the ORE 7 gene having a nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence of SEQ ID NO: 1 in the plant, and (b ) Screening plants having productivity enhancing properties.

As used herein, "productivity enhancing properties" means that the biomass (size and / or mass) of the whole, stem, root and / or leaves of the plant is increased compared to wild type plants and / or the productivity of the seed of the plant (plant 1). Number and / or mass of seeds per individual) is increased compared to wild type plants.

In addition, in the present specification, "plant" is meant to include mature plants, immature plants (plants), plant seeds, plant cells, plant tissues and the like. When plant cells or plant tissues are used for transformation, the transformed plant cells or plant tissues are described in European Patent EP0116718, European Patent EP0270822, International Patent WO 84/02913, Gould et al. 1991, Plant Physiol 95,426-434, etc., can be used to develop and grow into mature plants.

Also herein, "plant" includes all plants for which productivity gains can give useful results to humans. The meaning of these plants is that crops whose productivity is primarily useful to humans, such as cereals such as rice, wheat, barley, corn, sorghum and oats, soybeans, kidney beans, red beans, mung beans, and lima beans Root and tuber crops such as, peas, potatoes, sweet potatoes, cabbage, cabbage, bok choy, kale, cauliflower, broccoli, young radish, radish, mustard, pepper, spinach, sesame leaf In addition to vegetables, lettuce, garland chrysanthemums, carrots, tomatoes, green onions, sprouts, garlic, carrots, onions, etc., strawberry, watermelon, cucumber, melon, pumpkin, ginseng, tobacco, cotton, sesame, sugar cane , Beets, perilla, rapeseed, apple trees, pears, jujube trees, peaches, lambs, grapes, citrus fruits, persimmons, plums, apricots, bananas, and other bioenergy crops such as switchgrass, silver grass, and reeds. Other lygras, red clover, orchardgrass, alpha alpha, Tall pesque, perennialgrass, rose, gladiolus, gerbera, carnation, chrysanthemum, lily, tulip and the like.

In addition, in the present specification, "gene consisting of a sequence similar to the nucleotide sequence of SEQ ID NO: 1" is a gene encoding the first amino acid of SEQ ID NO: 2 while having a base sequence different from that of the gene of SEQ ID NO: 1 due to codon degeneracy And, as a homologue of genes consisting of the nucleotide sequence of SEQ ID NO: 1 and having a function of increasing the productivity of the plant, due to the evolutionary pathways different according to the type of plant, the nucleotide sequence of SEQ ID NO. It is meant to include all genes made. Herein, the gene consisting of a sequence similar to the nucleotide sequence of SEQ ID NO: 1 is preferably higher in sequence homology with the nucleotide sequence of SEQ ID NO: 1, and most preferably, having 100% sequence homology. On the other hand, in the lower limit of sequence homology, it will be preferable that the gene has a sequence homology of 60% or more with the nucleotide sequence of SEQ ID NO: 1. More specifically, the above sequence homology is 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73 %, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, Higher in order of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% is preferred.

In addition, in the present specification, "expression" means that the gene of the present invention, that is, ORE7 is not expressed in the wild type of the plant to be introduced, but is expressed in the transgenic plant into which the gene is introduced. Such "expression" is directly confirmed by quantifying the expression amount of the gene of SEQ ID NO: 1 or a gene consisting of a sequence similar to the nucleotide sequence of SEQ ID NO: 1 (for example, Real-time PCR method) The protein encoded by the gene can also be quantified and indirectly identified. In addition, depending on the characteristics of the gene can also be confirmed indirectly through the phenotype by the gene.

In the method for producing a plant having a productivity increasing characteristic of the present invention, step (a) may be performed by a genetic engineering method.

Genetic engineering method comprises the steps of: (i) inserting the ORE7 gene of SEQ ID NO: 1 or a gene having a sequence similar to the nucleotide sequence of SEQ ID NO: 1 into an expression vector to be operably linked to a regulatory sequence capable of expressing it, and (ii) transforming the expression vector into a plant.

As used herein, "operably" means that the transcription and / or translation of a gene is linked to be affected. For example, if a promoter influences the transcription of a gene linked to it, the promoter and the gene are operably linked.

In addition, in the present specification, "regulatory sequence" is meant to include all sequences whose presence may affect the transcription and / or translation of a gene linked thereto, and such regulatory sequences include a promoter sequence and a polyadenylation signal. ), Replication start sequence, and the like.

Also in this specification, "promoter" follows the conventional meaning known in the art, specifically located at the top (5 'side) based on the transcription initiation point of a gene, binding to DNA-dependent RNA polymerase By nucleic acid sequences having the function of controlling transcription of one or more genes, including sites, transcriptional initiation sites, transcription factor binding sites, and the like. Such a promoter may be a TATA box upstream of the transcription initiation point (usually at the transcription initiation point (+1) -20 to -30 position), CAAT box (usually approximately -75 position relative to the transcription initiation site if it is of eukaryotes) Present), a 5 'enhancer, a transcription repression factor, and the like.

Available promoters include ORE7 of SEQ ID NO: 1 linked to it. Promoters capable of expressing genes include constitutive promoters (promoters that induce constant expression in all plant tissues), inducible promoters (promoters that induce expression of the gene of interest in response to specific external stimuli, or at specific developmental times or in particular Promoters that specifically induce expression in tissue) can be used. Representative examples of constitutive promoters that can be used include the promoter of the 35S RNA gene of cauliflower mosaic virus (CaMV), and the ubiquitin family of promoters (Christensen et al., 1992, Plant Mol). Biol. 18, 675-689; EP0342926; Cornejo et al., 1993, Plant Mol. Biol. 23, 567-581), rice actin promoter (Zhang et al. 1991, The Plant Cell 3, 1155-1165), etc. Can be mentioned. Examples of inducible promoters that can be used include the yeast metallothionein promoter (Mett et al., Proc. Natl. Acad. Sci., USA, 90: 4567, 1993), which is activated by copper ions, substituted by substituted benzenesulfonamides. In2-1 and In2-2 promoters (Hershey et al., Plant Mol. Biol., 17: 679, 1991), GRE regulatory sequences regulated by glucocorticoids (Schena et al., Proc. Natl. Acad. Sci., USA, 88 : 10421, 1991), ethanol regulating promoters (Caddick et al., Nature Biotech., 16: 177, 1998), light regulating promoters derived from bovine subunits of ribulose bis-phosphate carboxylase (ssRUBISCO) (Coruzzi et al. , EMBO J., 3: 1671, 1984; Broglie et al., Science, 224: 838, 1984), mannino synthase promoter (Velten et al., EMBO J., 3: 2723, 1984), nopalin synthase (NOS) Promoter, octopin synthase (OCS) promoter, heat shock promoter (Gurley et al., Mol. Cell. Biol., 6: 559, 1986; Severin et al., Plant Mol. Biol., 15: 827, 1990) A terlute (glutelin) promoter, a soybean-derived lectin promoter, a cabbage-derived napin promoter, and the like.

The transcription termination sequence is a sequence that acts as a poly (A) addition signal (polyadenylation signal) to enhance the integrity and efficiency of transcription. Examples of transcription termination sequences that can be used include the transcription termination sequence of the nopaline synthase (NOS) gene, the transcription termination sequence of the rice α-amylase RAmy1 A gene, and the transcription termination of the Octopine gene of Agrobacterium tumefaciens. A sequence, a transcription termination sequence of wheat heat shock protein 17, a transcription termination sequence of wheat ubiquitin gene, a transcription termination sequence of rice gluterin gene, a transcription termination sequence of rice lactate dehydrogenase gene, and the like.

The expression vector may include a selection marker gene. "Selection marker gene" as used herein means a gene encoding a trait that enables the selection of plants comprising such marker genes. The marker gene may be an antibiotic resistance gene or may be a herbicide resistance gene. Examples of suitable selection marker genes include genes of adenosine deaminase, genes of dihydrofolate reductase, genes of hygromycin-B-phosphortransferase, genes of thymidine kinase, genes of xanthine-guanine phosphoribosyltransfer Laze gene, phosphinnothricin acetyltransferase gene, etc. are mentioned.

As used herein, the term "transformation" refers to a modification of the genotype of a host plant by the introduction of a hereditary gene, and regardless of the method used for the transformation, the herb gene is a host plant, more precisely a cell of the host plant. Introduced into and integrated into the genome of a cell. Here, the hereditary genes include homologous and heterologous genes, wherein "homologous genes" refer to endogenous genes of a host organism or the same species, and "heterologous genes" are genes that do not exist in the organism to which they are transformed. Say. For example, the Arabidopsis derived gene is a homologous gene for Arabidopsis plants, but a heterologous gene for legume plants.

Meanwhile, a method for transforming a plant with a foreign gene may be a method known in the art, for example, a direct gene transfer method using a gene gun, in in a floral dip planta transformation method, pollen mediated transformation method, protoplast transformation method, virus mediated transformation method, liposome mediated transformation method and the like can be used. In addition, it is also possible to select and use a transformation method suitable for a specific plant, for example, a method for transforming corn is described in US Pat. No. 6,140,553, Fromm et al, 1990, Bio / Technology 8, 833-839, Gordon- Kamm et al, 1990, The Plant Cell 2, 603-618) and the like can be used, methods for transforming rice are described in Shimamoto et al, 1989, Nature 338, 274-276, Datta et al 1990, Bio / Technology 8, 736-740), international patent WO 92/09696, international patent WO 94/00977, international patent WO 95/06722 and the like. Also, in tomato or tobacco transformation (An G. et al., 1986, Plant Physiol. 81: 301-305), Horsch RB et al, 1988, In: Plant Molecular Biology Manual A5, Dordrecht, Netherlands, Kluwer Academic Publishers, pp 1-9, Koornneef M. et al, 1986, In: Nevins DJ. And RA Jones, eds. Tomato Biotechnology, New York, NY, USA, Alan R. Liss, Inc. pp 169 -178) and the like can be used.

Generally used in transforming plants is a method of infecting seedlings, plant seeds and the like with the transformed Agrobacterium.

Such Agrobacterium mediated transformation methods are well known in the art (Chilton et al., 1977, Cell 11: 263: 271; European Patent EP 0116718; US Patent US 4,940,838), and methods suitable for particular plants are also known in the art. Known in See, for example, US Pat. No. 5,159,135 for cotton, US Pat. No. 5,824,877 for soybean, US Pat. No. 5,591,616 for corn, and the like. The Agrobacterium mediated transformation method uses Ti-plasmid, which will contain left and right border sequences that allow the integration of T-DNA into the genome of plant cells.

On the other hand, the selection of the step (b) may be selected by comparing the biomass and / or seed productivity of the plant, or when the selection marker gene is transformed with the transformation at the time of transformation may be selected using the selection marker gene, or These methods can also be mixed and screened.

In another aspect, the present invention relates to a method for producing a plant with enhanced stress resistance.

Method for producing a stress-enhanced plant of the present invention comprises the steps of (a) expressing a gene having a sequence similar to the ORE7 gene having a nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence of SEQ ID NO: 1 in the plant and (b) stress Selecting a plant having enhanced resistance.

As used herein, "stress" means hot stress, cold stress, dry (drought) stress, salt stress and / or oxidative stress.

Step (a) may be performed genetically, as for the genetic engineering method, as described with reference to a method for preparing a plant having productivity enhancing characteristics of the present invention.

The step (b) is selected by comparing the stress resistance of the plant (e.g., the progress of leaf sulfidation, the progression of leaf necrosis, the biomass of the leaves and / or stems, chlorophyll content, photosynthetic efficiency, etc.) When the selection marker gene is transformed together at the time, the selection marker gene may be used for selection, or a combination thereof may be used for selection.

In another aspect, the present invention relates to a method for producing a plant having aging delay properties.

Method of producing a plant having a delayed aging characteristics of the present invention comprises the steps of (a) expressing a gene having a sequence similar to the ORE7 gene having a nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence of SEQ ID NO: 1 in the plant and (b) aging Selecting the plant with delayed properties.

As used herein, the term "aging delay" refers to a property of prolonged plant life as compared to wild-type plants, and specifically, the yellowing and / or necrosis of leaves and / or stems is delayed compared to wild-type plants or the chlorophyll content of plants is wild-type. It is more characteristic than plants or photosynthetic efficiency of plants is higher than wild type plants.

Step (a) may be performed genetically, as for the genetic engineering method, as described with reference to a method for preparing a plant having productivity enhancing characteristics of the present invention.

In step (b), the transformed plant is grown and grown, and the naked eye is selected through the degree of progress of leaf yellowing or the progression of leaf necrosis, or at the time of transformation, the selectable marker gene is transformed together. If selected, the selection may be performed using a selection marker gene, and further, may be selected through a method of quantifying chlorophyll content, photosynthetic efficiency, etc., a method of mixing the above methods, and the like.

In another aspect, the present invention relates to a method for increasing the productivity of a plant.

The method for increasing the productivity of a plant of the present invention is (a) operably linking an ORE7 gene having a nucleotide sequence of SEQ ID NO: 1 or a gene having a sequence similar to the nucleotide sequence of SEQ ID NO: 1 to a regulatory sequence capable of expressing it Inserting into the expression vector preferably and (b) transforming the expression vector into a plant.

In another aspect, the present invention relates to a method for enhancing stress resistance of a plant.

The method of enhancing the stress resistance of a plant of the present invention (a) to enable the ORE7 gene having a nucleotide sequence of SEQ ID NO: 1 or a gene having a sequence similar to the nucleotide sequence of SEQ ID NO: 1 to a regulatory sequence capable of expressing it Inserting into the expression vector so as to be linked, and (b) transforming the expression vector into a plant.

In another aspect, the present invention relates to a method for delaying aging of a plant.

The method for delaying aging of a plant of the present invention is to (a) operably link a gene having a nucleotide sequence of SEQ ID NO. 1 or a gene having a sequence similar to that of SEQ ID NO. 1 to a regulatory sequence capable of expressing it. Inserting into the expression vector and (b) transforming the expression vector into a plant.

Steps (a) and (b) in the above methods are as described with reference to the method for producing a plant having the productivity enhancing properties of the present invention.

In another aspect, the present invention provides a ORE7 gene having a nucleotide sequence of SEQ ID NO: 1 or a gene having a sequence similar to the nucleotide sequence of SEQ ID NO: 1 obtained by the method for producing a plant having a productivity enhancing property of the present invention By expressing it relates to a transgenic plant having productivity enhancing properties.

In a preferred aspect, the plant is a transgenic plant having productivity enhancement properties by introducing and expressing a gene encoding an ORE7 protein consisting of the amino acid sequence of SEQ ID NO: 2, in particular, an ORE7 gene having the nucleotide sequence of SEQ ID NO: 1.

In another aspect, the present invention is a gene having a sequence similar to the ORE7 gene having a nucleotide sequence of SEQ ID NO: 1 or the nucleotide sequence of SEQ ID NO: 1 obtained by the method for producing a plant with enhanced stress resistance of the present invention The present invention relates to a transgenic plant that is enhanced in stress resistance.

In a preferred aspect, the plant is a transgenic plant having enhanced stress resistance by introducing a gene encoding the ORE7 protein consisting of the amino acid sequence of SEQ ID NO: 2, in particular the ORE7 gene having the nucleotide sequence of SEQ ID NO: 1 is introduced to be.

In another aspect, the present invention is a delay in aging of the gene having a nucleotide sequence of SEQ ID NO: 1 or a gene having a sequence similar to the nucleotide sequence of SEQ ID NO: 1 obtained by the method for producing a stress-resistant plant of the present invention The present invention relates to a transgenic plant having characteristics.

In a preferred aspect, the plant is a transgenic plant having enhanced stress resistance by introducing and expressing a gene encoding an ORE7 protein consisting of the amino acid sequence of SEQ ID NO: 2, in particular an ORE7 gene having the nucleotide sequence of SEQ ID NO: 1.

In the present specification, the "transformed plant" refers to a plant cell, a plant tissue, or a plant seed capable of developing and growing as a mature plant, when the gene is introduced and transformed, as well as by mating with the transformed plant. The resulting genome includes modified plants, plant seeds, plant cells.

As described above, according to the present invention, it is possible to provide a method for producing a plant having productivity enhancing characteristics, enhanced stress resistance, or delaying aging using the ORE7 gene.

Figure 1 shows the structure (schematic) of the pB2GW7.0-ORE7 recombinant vector in which the ORE7 gene, which has a function of increasing productivity of plants and has a function of delaying aging and enhancing stress resistance, is introduced in the sense direction.

Figure 2 is a soybean transformation and transformant production process through the pB2GW7.0-ORE7 recombinant vector of FIG. (a) Half seed explants on CCM, 1-after infection, 2-after 5 days inoculation; (b) Shoot induction medium without PPT; (c) Shoot induction medium containing DL-phosphinothricin 10 mg / L for bar selection; (d) Shoot elongation medium with 5 mg / L PPT; (e) Rooting medium; (f) Acclimate putative transgenic plant in the small pot; (g) Grow transgenic plant in the large pot; (h) 5 days after PPT leaf painting T ₀ plants were shown, 1- wild type plant was sensitive, 2- transgenic plant was herbicide resistant.

Figure 3 shows the results of performing genomic PCR to confirm the introduction of the recombinant gene in the leaves of the T ₀ plants of the soybean transformed with the pB2GW7.0-ORE7 recombinant vector of FIG. (a) ORE7 gene; (b) BAR gene; (c) the DNAs between left board and Bar gene; (d) the DNAs between ORE7 gene and right board; PC, binary vector carrying ORE7 and Bar gene as positive control; WT, wild type; # 1 ~ 24, Soybean T ₀ plants transformed with pB2GW7.0-ORE7 recombinant vector.

Figure 4 shows the results of performing RT-PCR to confirm the expression of the recombinant gene in the leaves of the T ₀ plants of the soybean transformed with the pB2GW7.0-ORE7 recombinant vector of FIG. WT, wild type; # 1 ~ 24, soybean T ₀ transformed with pB2GW7.0-ORE7 recombinant vector plant. TUB used as a PCR positive control.

5 is a genomic southern blotting to confirm the number of copies of the recombinant gene in the leaves of the T ₃ line of the soybean transformed with the pB2GW7.0-ORE7 recombinant vector of FIG. It shows the results of the operation. WT, wild type; Soybean T ₃ transformed with # 1, 2, 7, 9, 13, 14, 15, 19 and 24, pB2GW7.0-ORE7 recombinant vector line. DNA molecule size markers are shown at the right of the figure.

6A shows soybean wild type (WT) and vector control (EV) grown to vegetative stage 2, and T ₂ The two-node leaves of transformation lines # 7, # 9, # 14, and # 15 were detached to maintain cancer status and observed the phenotype of the leaves until 14, 18, and 21 days. DAT, days after dark treatment.

Figure 6B shows soybean wild type (WT) and vector control (EV) grown to vegetative stage 2, and T ₂ 2-node leaves of transformation lines # 7, # 9, # 14, and # 15 were detached and maintained in the dark state to investigate the chlorophyll content and photosynthetic efficiency of leaves by Fv / Fm until 14, 18, and 21 days. to be. The SD is shown as error bar. Asterisks indicate significant differences compared to the wild type (P value <0.05).

7A is T ₂ and the growth of beans wild type (WT) to the vegetative stage 2 Transformation line # 7, and # 15 to 50 mM H ₂ O ₂ Figure 2 shows the phenotype of 2-node leaves of V2-stage after 7, 11, and 15 days after stress. DAT, days after H ₂ O ₂ treatment.

Figure 7B shows soybean wild type (WT) and T ₂ grown up to vegetative stage 2. Transformation line # 7, and # 15 to 50 mM H ₂ O ₂ Chlorophyll content of 2-node leaves of V2-stage at 7, 11 and 15 days after stress. The SD is shown as error bar. Asterisks indicate significant differences compared to the wild type (* P value <0.05; ** P value <0.01).

8 shows soybean wild type (WT) and vector control (EV) grown to vegetative stage 2, and T ₂ RT-PCR was used to analyze the expression patterns of aging marker genes in leaves that maintained cancer for 18 days by detaching 2-node leaves of transformation lines # 7, # 9, # 14, and # 15. 18S rRNA was used as a PCR positive control. With GmRbcL GmCab3 is an aging marker gene in soybeans. GmRbcL ; Glycine max chloroplast rbcL gene, GmCab 3 ; Glycine max chlorophyll a / b-binding protein mRNA.

9 shows soybean wild type (WT) and soybean T ₃ transformed with the pB2GW7.0-ORE7 recombinant vector of FIG. 1. Phenotypes of lines # 2, # 7, and # 15 grown for 119 days, 125 days, and 133 days after sowing. DAS, days after sowing.

10 shows soybean wild type (WT) and soybean T ₃ transformed with the pB2GW7.0-ORE7 recombinant vector of FIG. Seed yields and expression rates of the ORE7 gene in lines # 2, # 7, and # 15.

FIG. 11 shows soybean wild type (WT) and soybean T ₃ transformed with the pB2GW7.0-ORE7 recombinant vector of FIG. 1. Figure 21 shows the seed yield and the expression rate of the ORE7 gene. GM ****, soybean T ₃ plant individual transformed with pB2GW7.0-ORE7 recombinant vector. Wild type values are the mean value of 20 individuals and the SD (error bar).

12 shows soybean wild type (WT) and T grown to vegetative stage 2.₃ RT-PCR analysis of the expression patterns of histidine kinase (HK) gene family that functions as cytokinin receptors in cytokinin signaling pathways on 2-node leaves of transformation lines # 2, # 7, and # 15. Shown,TUBWas used as a PCR positive control. Baby poleAHK1 homologue,GmHK7, GmHK9; Baby poleAHK2 homologue,GmKH11; Baby poleAHK3 homologue,GmHK12, GmHK13; Baby poleAHK4 ( CRE1 ) homologue,GmHK14 , GmHK15 , GmHK16, AndGmHK17.

FIG. 13 shows soybean wild type (WT) and T grown to vegetative stage 2.₃ RT-PCR analysis of the expression patterns of genes having the function of histidine phosphotransfer proteins (HP) gene in cytokinin signaling pathways on 2-node leaves of transformation lines # 2, # 7, and # 15 Which representsTUBWas used as a PCR positive control. Baby poleAHP1 homologue,GmHP01 , GmHP03 , GmHP04 , GmHP05; Baby poleAHP2 , 3, 5 homologue,GmHP09 , GmHP10; Baby poleAHP4 homologue,GmHP07 , GmHP08; Baby poleAHP6 homologue,GmPHP01 AndGmPHP03.

14 graphically illustrates the proposed hypothesis for the predominant role of the ORE7 gene in increasing soybean productivity and delaying aging. SAGs: senescence-associated genes of soybean.

Hereinafter will be described with reference to embodiments of the present invention. However, the scope of the present invention is not limited to these examples.

< Example  1> ORE7 Transformation Vector Production and Transformant Production for Soybean

To produce ORE7 soybean transformants, the following procedure was performed.

<Example 1-1> Soybean transformation vector preparation for ORE7

In the Arabidopsis, ORE7 (putative DNA-binding protein ESCAROLA of Arabidopsis thaliana , AT1G20900) gene was transferred to ORE7-F primer (5'-CAC CAT GGA AGG CGG TTA CGA GCA AG-3 ', SEQ ID NO: 3) and ORE7-R primer ( 5'-TTA AAA AGG TGG TCT TGA AGG TG-3 'was isolated and amplified using pENTR / D-TOPO vector (Invitrogen, Carlsbad, Calif., USA) to prepare pENTR-ORE7. . Invitrogen's Gateway® LR Clonase ™ Enzyme mix Kit was used to insert the prepared pENTR-ORE7 into the destination vector pB2GW7.0 (VIB-Ghent University, Ghent, Belgium). The prepared pB2GW7.0-ORE7 vector is Agrobacterium tumefaciens ( Agrobacterium) tumefaciens ) was transformed into EHA105 strain and used as a soybean transformation vector. A schematic diagram of the produced pB2GW7.0-ORE7 vector is shown in FIG. 1. In FIG. 1, BAR refers to a BAR gene (phosphinothricin acetyltransferase gene) that confers resistance to Basta herbicide, RB is a right border, LB is a left border, P35S is a CaMV 35S promoter, and T35S is a CaMV Points to the 35S terminator.

Example 1-2 Soybean Transformation and ORE7 Soybean Transformant Production

Beans ( Glycine max L. cv. Kwangan) Transformation and transformant production process is as follows. First, the scalpel was inserted between the two cotyledons of the soaked seeds, cut vertically up to the hypocotyl and the seedlings were removed. Hypocotyl was cut at about 1 cm below the cotyledon, and one side of the embryonic axis was wound by scalpel 7,8 times. At this time, 5ml of the concentrate is applied to the scalpel and the target site is wounded. Approximately 50 explants were placed in 15 mL co-cultivation / A. tumefaciens , inoculated for 30 minutes after 20 seconds of sonication, vacuum 30 seconds (500 mm.Hg) using a desiccator and diaphragm pump (GAST). . The explant was removed from the tube and placed on sterile filter paper to remove water. Then, one sheet of filter paper was placed and 10 objects were placed (adaxial side down) (Fig. 2a-1). After sealing with Micropore and co-cultured for 5 days at 25 ℃, 18 hours photoperiod (Fig. 2a-2).

After 5 days of co-cultivation, cefotaxime 250 mg / L, vancomycin 50 mg / L and ticarcillin 100 mg / L are used for sterilization. After explants are placed on the filter paper, water is removed and SI (shoot induction) is not selected. 5) One plate per plate on the medium was hypocotyl-fixed to the medium, and the part to be redifferentiated was flattened with the flat side facing up at an angle of about 30 ° (FIG. 2b). Each plate was sealed with a micropore and incubated at 25 ° C for 18 hours at photoperiod. After 2 weeks, explants with shoots were wound on SI-② medium containing antibiotics PPT 10 mg / L. The part is cut off and toothed with adaxial side down (FIG. 2C). Two weeks later, the browned shoot / shoot pad was cut with a scalpel and placed in SEM (shoot elongation medium) medium containing 5 mg / L of antibiotic PPT (FIG. 2D). Every two weeks, the browning area of the shoot was removed by tapping the top of the less pointed scalpel, and the shoot pad was scraped off little by little to allow the medium to be absorbed. After the selection, when the elongated shoot was 4 cm or more, root differentiation was induced in RM medium (FIG. 2E).

After one to two weeks, two or more roots emerged, and the medium was washed with tertiary distilled water and planted in a small pot (6 cm> 6 cm> 5.6 cm) containing 2: 1 mixed top soil and vermiculite. This small pot was placed in a magenta box again and grown at a photoperiod of 25 ° C. and 18 hours (FIG. 2F), and after about 10 days, leaf painting was performed on leaf surface with 100 mg / L DL-PPT (FIG. 2H). Plants confirmed gene introduction by leaf painting are transplanted into big pots (Fig. 2g), and 2, 4 and 10 holes are made in a transparent plastic cover and replaced with a small number every three days. After about 10 days, the plants were treated with Basta (BAYER), induced flower differentiation by single treatment (8 hours light condition, 16 hours dark condition, using paper box), and then transformed to show resistance by Basta treatment. Seeds were transferred to a greenhouse to harvest seeds. The transformation and transformant production process are shown in FIG. 2. We produced the ORE7 soybean transformant through the above transformation process.

Example 1-3 Verification of ORE7 Soybean Transformant Produced

PCR analysis to confirm the introduction of recombinant genes to the soybean transformants produced, RT-PCR analysis to confirm the expression of recombinant genes, and genomic southern blotting to confirm the number of copies of the recombinant genes (Genomic Southern blotting) was performed.

In order to confirm the recombinant gene introduction of the ORE7 soybean transformant, total genomic DNA was isolated from the leaves of the transformants by CTAB (certyltrimethyl-ammonium bromide) method. PCR was performed using the sequences of the introduction gene ORE7 and the selection herbicide resistance gene BAR . The primers used in the case of ORE7 ORE7-GPCR-F (5' -ATG GAA GGC GGT TAC GAG CAA GGC-3 ', SEQ ID NO: 5) and ORE7-GPCR-R (5'- TTA AAA AGG TGG TCT TGA AGG TGT 3 ', SEQ ID NO: 6) and, in the case of the BAR BAR-GPCR-F (5' -CAT GTA ATG CTG CTC AAG GTA CGC-3', SEQ ID NO: 7) and the BAR-GPCR-R (5'- ATG TAA TGC TGC TCA AGG TAC GC-3 ′, SEQ ID NO: 8).

In addition, PCR was performed on the left board (LB) and RB (right board) parts to determine whether the vector was inserted with T-DNA. Primers used were as follows: LB portion was left board-F (5'-TGG CTG GTG GCA GGA TAT ATT GTG-3 ', SEQ ID NO: 9) and BAR-R (5'-AGA CAA GCA CGG TCA ACT TCC) GTA-3 ', SEQ ID NO: 10) and the RB portion is ORE7-RF (5'-GGT CAG GGA CAG TTA GGA GGT AAT-3', SEQ ID NO: 11) and right board-RR (5'-TTA AAC) TGA AGG CGG GAA ACG ACA-3 ', SEQ ID NO: 12).

As a result, it was confirmed that all the ORE7 soybean transformants examined were introduced with the recombinant genes ORE7 and BAR gene and T-DNA of the vector was normally introduced (FIG. 3). This suggests that all of the ORE7 soybean transformants examined had introduced normal vectors.

In order to confirm the expression of recombinant genes for the selected ORE7 soybean transformant, total RNA was extracted from the leaves of the soybean wild type and the ORE7 soybean transformant using RNasey Plant Mini Kit (QIAGEN, Germany). 1 μg of RNA each as a template and 5 minutes at 65 ° C. using Superscript III Reverse Tanscriptase (INVITROGEN, USA); 60 minutes at 50 ° C; And cDNA was synthesized at 70 ° C. for 15 minutes. Then, using the synthesized cDNA as a template, PCR was performed using the primers specific to the following ORE7 and BAR gene, and the TUB gene used as a PCR positive control. PCR denatured template DNA by heating at 94 ° C. for 2 minutes and then 1 minute at 94 ° C .; 1 minute 30 seconds at 55 ° C; And 1 cycle at 72 ℃ one cycle was performed a total of 30 times, followed by a final reaction for 15 minutes at 72 ℃. Then, the PCR product was confirmed by 1% agarose gel electrophoresis, the results are shown in FIG. In the wild type, there was no expression of the recombinant gene ORE7 and the selection marker BAR gene, whereas all the ORE7 soybean transformants examined showed that the normal expression of the recombinant gene ORE7 and the selection marker BAR gene. This fact proves that the transformant is an ORE7 soybean transformant.

Table 1

To confirm the number of copies of a recombinant gene in selected ORE7 soybean transformants, 5 μg genomic DNA isolated from soybean wild type and transformants was cut overnight using restriction enzyme Hind III, and the cut DNA Was fractionated by electrophoresis on 0.8% agarose gel. Fractionated DNA was transferred to a Hybond-N + nylon membrane (Amersham Pharmacia, Piscataway, USA) and then identified through a chemiluminescence system (Roche, Germany) using a digoxigenin (DIG) -labeled DNA probe. DIG-labeled probes consist of ORE7-GS-F primer (5'-ACA GCT TCA ACC GCA GGG CG-3 ', SEQ ID NO: 19) and ORE7-GS-R primer (5'-CGT GGA CGT TTT CCC GGT GCT-3 ', SEQ ID NO: 20) was used to amplify ORE7 and label it with DIG.

Genomic Southern blotting of the ORE7 soybean transformant showed that the # 1, 2, 7, 9, 13, 15, and 19 transformant lines showed two copies, while the # 24 transform line A large number of replicates were shown. On the other hand, in the case of the # 14 transformation line, it appears that one copy number is seen in the southern blot, but it may be considered that several copies overlap in terms of the thickness of the blot (FIG. 5).

< Example  2> ORE7  Characterization of Aging and Stress of Soybean Transformants

Example 2-1 Characterization of Aging of ORE7 Soybean Transformants

In order to analyze the aging characteristics of ORE7 soybean transformants, the 2-node leaves of wild type and transformants grown up to vegetative stage 2 were detached and 3mM MES buffer solution (2- [N-morpholino] -ethanesulfonic acid, After floating to pH 5.8), it remained in the dark state and left for 21 days. Phenotypic observation, chlorophyll content, and photosynthetic efficiency of leaves left for 14, 18, and 21 days after cancer treatment were measured to determine the soybean wild-type and vector control (EV) (pB2GW7.0 vectors without ORE7 gene). Converted soybeans). The above survey was measured for six or more individuals for each variant line.

Phenotypic observations showed that the wild type (WT) and vector control (EV) showed rapid yellowing of leaves after 18 days of cancer treatment, whereas most of the ORE7 transformant lines showed yellowing of leaves after 21 days. In particular, the # 7 transformant line still had green traits after 21 days of cancer treatment (FIG. 6A). These facts suggest that the ORE7 gene provides the trait of delayed aging in soybeans.

Chlorophyll was extracted from each sample leaf using 80% (V / V) acetone to measure chlorophyll content. Chlorophyll content was measured according to the method of Lichtenthaler and Wellburn (Biochemical Society Transduction 603: 591 ~ 592, 1983) using extinction coefficients of 663.2 nm and 664.8 nm. The results are shown in FIG. 6B, and the values presented are the mean ± standard deviation of each of six or more lines per line. In wild-type and vector controls, the chlorophyll content decreased sharply after 18 days of cancer treatment, and the chlorophyll content fell below 20% on the 21st day, whereas the ORE7 soybean transformant showed more than the chlorophyll content on the 18th day of the wild type even on the 21st day. Was keeping it.

Photosynthetic efficiency was measured using Oh et al. (Plant Mol. Biol. 30: 939, 1996). First, the leaves of each day after dark treatment (DAT) were treated with cancer for 15 minutes, and then the fluorescence of chlorophyll was measured using a Plant Efficiency Analyzer (Hansatech). Photosynthetic efficiency was expressed by the photochemical efficiency of PSII (photosystem II) using chlorophyll fluorescence properties, which was the maximum variable fluorescence (Fv) versus the maximum value of fluorescence (Fm). It is expressed as the ratio of (Fv / Fm). Higher values indicate better photosynthetic efficiency.

The results are shown below FIG. 6B and the values presented are the mean value ± standard deviation of each of six or more lines per line. For wild-type and vector controls, the Fv / Fm value decreased to about 60% on day 18 after cancer treatment, and then decreased sharply to about 40% on day 21, while for ORE7 soybean transformants, Fv / Fm was higher than 60% on day 21. It was keeping the value.

From the above results, ORE7 transgenic soybeans have had a life of leaves appeared to have a much longer phenotype compared to the wild type and vector control, the effectiveness of these life extension is ORE7 It is thought to be caused by the delay of biochemical changes due to aging, which is expressed by a decrease in chlorophyll content and a decrease in photosynthetic efficiency by genes.

Example 2-2 Characterization of Stress of ORE7 Soybean Transformants

To investigate the resistance to oxidative stress of the ORE7 soybean transformants, wild-type and transformants were left for 15 days by adding 50 mM H ₂ O ₂ to the 3 mM MES solution. Phenotypic observations and chlorophyll content were examined on 2-node leaves of V2-stage at 7, 11, and 15 days after treatment, and resistance to H ₂ O ₂ stress was analyzed. The above object was made.

In the wild type soybean, the yellowing of leaves appeared clearly from the 7th day of H ₂ O ₂ treatment, and then the sharp yellowing of the leaves occurred, whereas the yellowing of the leaves started from the 11th day of H ₂ O ₂ treatment. Appeared to be. In particular, the # 7 transformation line was observed that the yellowing of the leaves does not occur much even on the 15th day (Fig. 7A). The changes in chlorophyll content showed that wild-type soybeans showed a sharp decrease in chlorophyll content from 7 days of H ₂ O ₂ treatment, and remained at 20%, while ORE7 soybean transformants were nearly 40% at 15 days of H ₂ O ₂ treatment. Was maintained (FIG. 7B). This means that the ORE7 gene provides resistance to oxidative stress in soybeans. In addition, the ORE7 gene was found to provide weak salt resistance in soybeans (data not shown). Therefore, the ORE7 gene is thought to provide many advantages in the development of stress-resistant crops by providing resistance to oxidative and salt stress in plants as well as delaying aging of plants.

Example 2-3 Expression Analysis of Gene Related to Aging in ORE7 Soybean Transformant

To compare the expression patterns of ORE7 soybean aging-related genes with wild species, wild type (WT), vector control (EV), and T ₂ grown up to vegetative stage ₂ Gene expression patterns were confirmed by RT-PCR analysis on leaves that maintained the cancer state for 18 days by detaching 2-node leaves of transformation lines # 7, # 9, # 14, and # 15. 18S rRNA was used as the RT-PCR positive control, and the primers used are shown in Table 2 below.

TABLE 2

GmRbcL ( Glycine max chloroplast rbcL ) and GmCab3 ( Glycine max chlorophyll a / b-binding protein) gene is a gene whose expression is gradually decreased during aging of soybean. The expression of the two genes was higher in the soybean transformation lines # 7, 9, 14, and 15 lines showing the expression of ORE7 compared to wild-type (WT) and vector control (EV) (Fig. 8). This fact suggests that ORE7 soybean transformants delay aging by inhibiting the decrease in expression of the GmRbcL and GmCab3 genes, which are used as markers of aging during aging. Therefore, the ORE7 gene in soybean is thought to induce phenotypic prolongation of leaf life by regulating the expression of aging-related genes at the molecular level and then physiological phenomena such as chlorophyll content and photosynthetic efficiency.

< Example  3> ORE7  Increased productivity of soybean transformants and delayed aging Intertrait  Correlation analysis

Example 3-1 Phenotypic Features of ORE7 Soybean Transformants

To identify the phenotypic characteristics of the ORE7 soybean transformants, wild-type and T ₃ transgenic lines were grown in greenhouses to investigate their phenotypic characteristics, especially aging delay and productivity enhancement. As a result, the # 2 soybean transformation line exhibited strong phenotypic characteristics of prolonged melting until seed harvest, while the # 15 soybean transformation line had both a phenotypic characteristic of delayed aging and productivity-enhancing traits such as increased individual size. It could be confirmed that. On the other hand, the # 7 soybean transformation line was interestingly the same time as the wild type and seed harvest time, there is no phenotypic characteristic of the extension of the melting, but the productivity-increasing traits such as the increase in the individual size was shown to be strong (Figure 9).

Example 3-2 Correlation between Productivity Enhancing Traits and ORE7 Gene Expression Regulation of ORE7 Soybean Transformants

We further examined the seed yield of the soybean transformation lines to further analyze the productivity of the ORE7 soybean transformant. As a result, the # 2 soybean transformation line had a seed yield similar to that of the wild type, but the # 7 and # 15 soybean transformation lines with productivity-increasing traits such as an increase in individual size had a significant seed yield increase compared to the wild type. The # 7 soybean transformation line showed 137% seed yield increase compared to wild type (FIG. 10). As a result of analyzing the increase in seed yield as an indicator of productivity, the increase in the number of pods and the increase in the number of pods in the transformation line led to an increase in seed yield. This fact suggests that although the ORE7 gene has some overlapping functions in providing productivity-improving traits and aging delayed traits such as increased population size and increased seed yield, the productivity-enhanced traits are induced independently of aging delayed expression. Judging.

Quantitative real-time PCR (qRT-PCR) was used to investigate the expression of ORE7 genes in ORE7 soybean transformation lines with differences in phenotypic and productivity gains. qRT-PCR was performed with a CFX-96TM Real-Time system (Bio-Rad) using Ex Taq ^™ Probe. Information on the applied primer / probe sets is shown in Table 3 below.

TABLE 3

As a result, the expression rate of the # 2 soybean transformation line was significantly higher, while the expression rates of the # 7 and # 15 soybean transformation lines were relatively low, especially the expression rate of the # 7 soybean transformation line was significantly lower ( 10). To reconfirm this fact, we seeded T ₃ transformants on the field, harvested seeds at the same time, and then randomly selected transformants to investigate seed yield and ORE7 gene expression rate. The result ORE7 Individuals with high gene expression rates, GM0201, GM2020, GM0203, GM0303, GM0306, and GM1503, had similar seed yields to wild type, while the remaining transgenic individuals with lower ORE7 gene expression rates reaffirmed that seed yields were significantly higher than wild type. Could be (FIG. 11). This fact suggests that, as the team noted earlier in the chromatin engineering of Genomine technology (unpublished), stable expression at low levels of expression of the gene provides increased productivity, and higher expression results in stronger aging delayed traits. Consistent with the team's hypothesis, the increased productivity of the transformants and the provision of the melting prolongation traits were regulated by the regulation of ORE7 gene expression, that is, the higher the gene expression, the longer the aging delayed trait and the gene expression were relatively. It can be concluded that maintaining stabilization at low levels leads to traits of increased productivity.

Example 3-3 Analysis of Molecular Biology of ORE7 Gene in Soybean

Phytohormone cytokinin regulates aging of plants, leading to increased plant productivity (Gan and Amasino, 1995), and also is known to increase plant production by regulating reproductive meristem activity, flower size, and ovule production. Bartrina et al., 2011). We investigated the expression of genes for histidine kinase (HK) and histidine phosphotransfer proteins (HP) that function as cytokinin receptors in the cytokinin signaling pathway to determine whether the ORE7 gene is involved in the cytokinin signaling pathway in soybean.

Beans wild type and histidine kinase gene of soybean analysis in ORE7 beans transformant Arabidopsis AHK1 homologue of GmHK7, GmHK9, Arabidopsis AHK2 homologue of GmKH11, Arabidopsis AHK3 homologue of GmHK12, GmHK13, and Arabidopsis AHK4 (CRE1) homologue GmHK14 , GmHK15 , GmHK16 , and GmHK17 . Expression of the gene was analyzed by RT-PCR, wherein a positive control was used as TUB , and the primers used are shown in Table 4 below.

Table 4

As a result, to the gene expression into hyeonryanggwa proportion of ORE7 gene of the transformant was GmHK13 of the Arabidopsis homologue AHK3 and Arabidopsis homologue of AHK4 GmHK14, GmHK15, GmHK16, and GmHK17 (Fig. 12). This fact suggests that ORE7 gene expression proportionally regulates the expression of GmHK13 , GmHK14 , GmHK15 , GmHK16 , and GmHK17 genes in soybean cytokinin receptor genes. It was hypothesized that # 7 transformants would provide productivity-enhancing traits such as increased seed yield and relatively high expression of transformants, particularly # 2 transformants, with delayed aging traits.

Beans wild and ORE7 histidine phosphotransfer proteins (HP) gene in soybean trait beans analyze the transition body Arabidopsis AHP1 homologue of GmHP01, GmHP03, GmHP04, GmHP05, Arabidopsis AHP2, 3, 5 homologue of GmHP09, GmHP10, Arabidopsis AHP4 homologues GmHP07 , GmHP08 , and Arabidopsis AHP6 homologues GmPHP01 and GmPHP03 . Expression of the gene was analyzed by RT-PCR, wherein a positive control was used as TUB , and the primers used are shown in Table 5 below.

Table 5

The result was the gene expressed from the ORE7 gene hyeonryanggwa proportion of transformants GmHP04 of Arabidopsis AHP1 homologue, interesting AHP2, 3, 5 homologue of GmHP10 the amount of the amount of expression of ORE7 relatively high lower expression than the control On the other hand, when the expression level of ORE7 is relatively low, the expression level was higher than that of the control (FIG. 13). This fact ORE7 gene expression is controlled soybeans histidine phosphotransfer proteins (HP) expression of the gene of the gene family of GmHP04 proportionally, and relatively high expression of the gene ORE7 while acting as a negative regulator for GmHP10 gene ORE7 The relatively low expression of the gene suggests that it acts as a positive regulator for the GmHP10 gene.

Through this analysis, we can hypothesize the following. 1) ORE7 adjusts the GmHK13, GmHK14, GmHK15, GmHK16, and expression of GmHK17 gene of the cytokinin receptor gene proportionally, 2) ORE7 is soybean histidine phosphotransfer proteins (HP) also proportionally controlled GmHP04 gene expression of the genes, 3) the relatively high expression of ORE7 gene, while acting as a negative regulator for GmHP10 gene relatively low expression of ORE7 gene acts as a positive regulator for GmHP10 gene, 4) the expression level increased and the expression of GmHP10 genes GmHP04 gene the amount of reduction, whereas by regulating the expression of senescence-associated genes that provide transfected aging delay, low expression levels and increased expression levels decreased GmHP10 genes GmHP04 gene is increased seed yield of plants, as shown in the preceding information, the biomass increases Provide traits of increased productivity, such as (FIG. 14). This hypothesis could be used as an important indicator for GmHP10 genes to delay plant aging with negative regulator function and to increase plant productivity with positive regulator function.

Claims (15)

1. (a) inserting a gene encoding the amino acid sequence of SEQ ID NO: 2 into an expression vector so as to be operably linked to a regulatory sequence capable of expressing it;

   (b) transforming the expression vector into a plant, and

   (c) a method for producing a plant having a productivity increasing characteristic, comprising selecting a plant having the productivity increasing characteristic.
2. The method of claim 1,

   The gene is a method for producing a plant having productivity enhancement characteristics, characterized in that the ORE7 gene consisting of the nucleotide sequence of SEQ ID NO: 1.
3. The method of claim 1,

   The plant is a method of producing a plant having a productivity increase characteristic, characterized in that the legumes.
4. The plant which has the productivity improvement characteristic obtained by the method of any one of Claims 1-3.
5. (a) inserting a gene encoding the amino acid sequence of SEQ ID NO: 2 into an expression vector so as to be operably linked to a regulatory sequence capable of expressing it;

   (b) transforming the expression vector into a plant, and

   (c) a method for producing stress-enhanced plants, comprising the step of selecting plants with enhanced stress resistance.
6. The method of claim 5,

   The gene is a method for producing stress-enhanced plants, characterized in that the ORE7 gene consisting of the nucleotide sequence of SEQ ID NO: 1.
7. The method of claim 5,

   The plant is a method for producing stress-enhanced plants, characterized in that the legumes.
8. A plant with enhanced stress resistance obtained by the method according to any one of claims 5 to 7.
9. (a) inserting a gene encoding the amino acid sequence of SEQ ID NO: 2 into an expression vector so as to be operably linked to a regulatory sequence capable of expressing it;

   (b) transforming the expression vector into a plant, and

   (c) selecting a plant having a aging retardation property.
10. The method of claim 9,

    The gene is a method for producing a plant having aging delay characteristics, characterized in that the ORE7 gene consisting of the nucleotide sequence of SEQ ID NO: 1.
11. The method of claim 9,

    The plant is a method of producing a plant having a aging delay characteristic, characterized in that the legumes.
12. The plant which has a aging retardation characteristic obtained by the method in any one of Claims 9-11.
13. (a) inserting a gene encoding the amino acid sequence of SEQ ID NO: 2 into an expression vector so as to be operably linked to a regulatory sequence capable of expressing it, and

    (b) a method of increasing the productivity of a plant, comprising the step of transforming the plant with the expression vector.
14. (a) inserting a gene encoding the amino acid sequence of SEQ ID NO: 2 into an expression vector so as to be operably linked to a regulatory sequence capable of expressing it, and

    (b) a method of enhancing stress resistance of a plant, the method comprising transforming the plant with the expression vector.
15. (a) inserting a gene encoding the amino acid sequence of SEQ ID NO: 2 into an expression vector so as to be operably linked to a regulatory sequence capable of expressing it, and

    (b) A method for delaying aging of a plant, comprising the step of transforming the plant with the expression vector.

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