KR20030011733A - Transgenic zoysiagrass with reduced shade avoidance - Google Patents

Abstract

PURPOSE: Provided is a transformed Korean-type lawn with shade avoidance reduced which inhibits shade avoidance reaction to grow the transformed agricultural products so that it produces the transformed plants, which are strong, healthy and resistant against the contamination of virus. CONSTITUTION: The transformed Korean-type lawn with shade avoidance reduced as zoysia KCTC-1024BP is produced by the steps of: (i) using a recombinant DNA structure wherein a ubiquitin promoter, a phytochrome A gene and 3' UTR order are configured to transform a zoysia cell; (ii) selecting a vegetable cell transformed in a phytochrome A coating nucleotide order; (iii) regenerating a zoysia vegetable from the vegetable cell transformed in a phytochrome A coating nucleotide order; and (iv) selecting the transformed zoysia class to manifest phytochrome A with order number as 2.

Description

Transgenic zoysiagrass with reduced shade avoidance

The present invention relates to the growth rate and photoreactivity of crop plants through genetic transformation with modified phytochrome A photoreceptors. It relates to the modification of plant structure and photomorphogenesis by molecular biological strategies that design grass-emphasized economically important crop plants, especially transgenic grasses with distinct dwarfism and more branched structures, to respond to environmental aspects. . The present invention is achieved by developing a transformed crop plant through the suppression of negative epithelial response. As a result, the transgenic plants are healthy, robust and more resistant to pathogen infection. Moreover, they can cover the ground and recover from traffic injuries faster than plants, providing plants of high economic value with enhanced adaptive flexibility.

The present invention relates to a transgenic Zoysiagrass consisting of transforming plant cells with a recombinant DNA construct containing a nucleotide sequence that encodes a modified oat phytochrome A in which serine-598 is substituted with alanine to enhance photoreactivity. ( Zoysia japonica Steud.). The resultant transgenic Korean grasses are notable for typical vulva avoidance reactions, such as sparse internodes and leaf stalks, short leaves, dark-green leaf colors, and a dry, sturdy appearance that significantly affect crop yields and environmental adaptations. Decreases.

Joysiagrass, also known as Korean grass, is one of the turfgrass species that is widely cultivated in global warming climates, especially in Far East Asia, including Korea, Japan, and East China. In recent years, growing areas are rapidly spreading in the United States and other countries due to their special characteristics, such as their drought resistance and their ability to quickly recover from traffic disasters. In particular, it grows well in poor soil in virtually all climates. It is therefore widely used for both practical and decorative purposes such as golf courses, athletic fields, boulevards, home gardens and river banks. But his solution has the inherent drawbacks demanded by the consumer. Consumers demand a variety of new things with increased resistance to pathogens, preparations and environmental stresses. Moreover, joysiagrass tends to overgrow during the hot summers and is often labor-required and expensive because it must be moisturized and cut. Overgrown plants are, of course, very sensitive to fungal infections, and large amounts of chemicals, including fungicides, must be applied to control various pathogens, which causes serious environmental pollution. One useful way to solve these problems of joysiagrass cultivation is to genetically design to show reduced negative evasive response. Shading avoidance is an adaptation mechanism of plants to overcome the severe competition of light in nature. Naturally appearing under the canopy of adjacent plants, when the plant grows in the dark or in the shade, stem or hypocotyl expansion causes the expense of leaf and root growth to be drastically stimulated to elongate but with a weak pale appearance. As a result, they are sensitive to pathogen infections and environmental stresses, leading to a significant reduction in harvest production wells. Joysiagrass, genetically designed to have a reduced negative evasive response, will grow slower than the plant, but will grow stronger. With well-established plant molecular biology and plant tissue culture techniques useful to plant biologists, it is possible to introduce virtually any specific target gene into crop plants and to introduce or enhance useful features in predictable ways.

Plant growth and development is regulated through comparable interactions on native developmental signals such as growth hormones and various environmental factors. Light is one of the most important environmental factors, collectively called photomorphogenesis, that affects many aspects of plant growth and development throughout its life, from seed germination, leaf and stem growth, photoperiodic, eroded to flowering. A prominent photomorphologic response important for the survival and reproduction of plants in nature is negative scleroderma. It is a well-known plant development dynamic process that is particularly required in overcoming the intense natural light competition that occurs naturally, such as when plants grow in close proximity and are frequently encountered in naturally occurring, densely grown areas in the forest. Neglective response is an abnormally rapid expansion of stem and leaf stem growth to obtain sufficient light required for photomorphogenesis, but is elongated by inhibition of leaf and root growth and storage organ production, but is shown to cause a fragile and pale appearance. . Although this is an essential mechanism for plant survival, it can be a potential problem in growing crops because crops become thinner, fragile, pale and susceptible to pathogen infection and environmental changes. Various efforts have been made to solve this problem in crop cultivation, and it has recently been found that manipulation of the steep avoidance reaction is a promising way to achieve this.

In a plant, a proximity signal containing negative evasive reactions is radiation reflected from adjacent plants. Photosynthetic pigments absorb most of the visible light (400-700 nm), but light of longer wavelengths (700-800 nm) in the far-red light range is reflected or transmitted, leading to the diffusion of far-red light (FR) under canopy. FR light is recognized by phytochrome photoreceptors. Phytochrome's physiological function works by reversible phototransformation between two distinct spectral forms between red (R) light absorbing Amido's unique photochemically active Pr form and FR light absorbing Pfr form. do. Pfr morphology is important for physiological function in most photomorphologic processes. Thus, there is evidence that phytochrome activity is determined by the R: FR ratio in the light environment. There are at least five or more phytochromes (phyA-phyE) in Arabidopsis. All of these function as well as separate functions. In light green plants, phyA accumulates at high levels and inhibits stem growth in response to λmax light around 710-720 nm. However, in photo-grown plants, phyA is rapidly degraded, and phyB plays a major role in regulating stem growth as a function of the R: FR ratio. PhyB modulates the negative evasion response when the FR content generated under canopy is high. As a result, FR light inhibits stem growth primarily in lush green plants through phyA, but stimulates stem growth in light-grown plants primarily through phyB activity.

In transgenic plants that overexpress phyA, the phyA level is clearly maintained at a specific level sufficient to offset the light-induced degeneration, and apparently exhibits severe dwarfism when grown in a light environment containing a relatively high content of FR wavelengths. Proven. Although designing the fringe avoidance to introduce an external PHYA gene was successful in dicotyledonous plants, transgenic rice plants that overexpress the PHYA gene did not exhibit this dwarf appearance. Two possibilities have been proposed to explain the absence of dwarf appearance in transgenic rice plants. First, phytochromes of monocotyledonous or dicotyledonous plants have distinct functions and specifically function in each plant group. Alternatively, perhaps due to insufficient activity of the promoter used, the expression level of phyA was not sufficient to counteract the light-induced degradation of native phyA. Since all phyAs isolated from monocotyledonous and dicotyledonous plants exhibit high sequence homology and show the same photochemical activity, the latter was presumed to be the cause for the absence of phenotypic changes in transgenic rice plants expressing external phyA. .

An oat phyA mutation was recently obtained in which serine-598 was substituted with alanine and exhibited an overactive photoreaction. Transgenic model plants expressing phyA were more severely dwarfed than expressing wild type phyA when grown in light, and transgenic tuftgrass or rice with modified phyA had wild type phyA regardless of the presence of adjacent plants. It was estimated that this would cause a significantly reduced negative evasive response.

As used herein, the term "genetic transformation" refers to a procedure for introducing a gene (s) or genetic material (s) into a target crop in a predictable manner. Genes or genetic material are stably integrated into the plant genome and transmitted through reproduction as part of the genome of the plant.

The present invention is directed to an oat phytochrome A gene (SEQ ID NO: 1) in which reduced negative epithelial response, such as dwarf appearance and increased branches, is modified to design photoreactivity in various aspects of the most prominent photomorphologic growth and development. Genetic transformation of Joysiagrass. Similar genetic approaches can be applied to genetic transformation of other monocotyledonous plants, such as tuftgrass species or rice, which are closely related.

The oat phytochrome A gene ( PHYA ) used in the present invention is a modified version ( S598A ) of the wild type with serine-598 substituted with alanine. Site-directed mutagenesis altered the codon of serine, AGT, to GCT, the codon of alanine. In total, phytochrome molecules are composed of chromophores covalently bonded at cysteine-321 residues, N-terminal photosensitive domains with phytochromobilin, and some structural motifs required for phytochrome dimerization. And two major structural domains, the C-terminal regulatory domain containing protein kinases. It also contains structural elements that directly interact with various phytochrome interacting factors, such as PIF3, PKS1 and NDPK2. Serine-598 is located in a small hinge region between the two structural domains. In particular, only serine residues are selectively autophosphorylated in Pfr form in vivo. Serine-598 has therefore been proposed to mediate phytochrome function by regulating interdomain signals within phytochrome molecules. Recently, the transformed Arabidopsis plant ( S598A ) with the modified PHYA gene showed an important response of hypersensitivity photoreactivity and negative scleroderma and supported the important role of serine-598 in phytochrome function.

The present invention provides a transgenic joysiagrass that overexpresses the S598A gene under the control of the constitutive ubiquitin promoter (Ubi-P). Transformation lines were separated by herbicide resistance.

Callus from transgenic Joysiagrass was deposited with KCTC-10242BP at the Biotechnology Research Institute Gene Bank of 52, Yuseong-gu, Daejeon, Korea under the Treaty of Budapest on May 10, 2002.

The resulting transgenic joysiagrass shows strong inhibition of the typical negative evasive response. Phenotypic changes observed included dwarf intercutions and scapes, short leaves, more branches and dark-green leaf colors, which are agriculturally useful features on crops. Because transgenic joysiagrass is dwarf but robust (dark-green and robust) it has shown enhanced recovery and pathogen resistance from traffic injuries. Moreover, less often requires mowing or hydration.

These results of transgenic joysiagrass expressing the S598A gene indicate that transgenic crops with reduced negative epithelial response can be produced through similar molecular approaches. They are more robust but can grow at higher densities, an agricultural feature that is particularly useful in rice and other monocots. Moreover, transgenic plants will need less nutrients, moisture and chemicals for growth and pathogen control.

Accordingly, the present invention is directed to : 1) a transformed Zosiagrass ( SoyA japonica Steud.) Or closely related tuftgrass with a modified oat phytochrome A gene (SEQ ID NO: 1) ( S598A ). 2) Provides transgenic joysiagrass (Accession No. KCTC-10242BP) with the S598 gene exhibiting dwarf intercutaneous and pacifiers, short leaves, more branches and dark-green leaf colors.

The technical problem to be achieved in the present invention relates to the growth rate and photoreactivity of crop plants by genetic transformation into modified phytochrome A photoreceptor. It relates to the modification of plant structure and photomorphogenesis by molecular biological strategies that design grass-emphasized economically important crop plants, especially transgenic grasses with distinct dwarfism and more branched structures, to respond to environmental aspects. . The present invention is achieved by developing a transformed crop plant through the suppression of negative epithelial response. As a result, the transgenic plants are healthy, robust and more resistant to pathogen infection. Moreover, they can cover the ground and recover from traffic injuries faster than plants, providing plants of high economic value with enhanced adaptive flexibility.

Figure 1 shows the nucleotide sequence portion of the oat phytochrome A gene (GenBank Accession No. 16110) and the amino acid sequence containing the corresponding serine-598. phyA consists of two functional domains, the N-terminal photosensitive and C-terminal regulatory domains connected by a hinge region. Serine-598 (S) is located within the hinge region and is replaced by alanine (A) by site-directed mutagenesis as shown.

2 shows a recombinant plant expression vector construct with a modified phytochrome A gene ( S598A ). S598A gene transcription is directed by the constitutive ubiquitin promoter (Ubi-P). Plant expression vectors are kanamycin resistance genes (Kan ^R ) for bacterial selection in E. coli and Agrobacterium tumefaciens and herbicide resistance genes (Bar) and hygro for selection in plants. It contains the hygromycin resistance gene (Hgr ^R ). Bar and Hgr ^R expression is indicated by the CaMV 35S promoter (35S-P). S598A gene was ligated using unique BamHI and EcoRI sites. The EcoRI site originally present in the Ubi-P promoter was modified to facilitate subsequent subcloning. RB and LB: right and left edges, respectively. Nos-T and 35S-T: NOS and 35S terminator sequences, respectively.

Figure 3 shows the transgenic Zoiagrass seedlings grown on MS medium containing 5mg / L bialaphos (A). Transformed (S598A) and control (WT) seedlings were grown under the same experimental conditions. Plants were transferred to soil and grown more (B). Note that phenotypic changes such as short leaves (top panel) and more branches (bottom panel) are more apparent.

Figure 4 shows the adult transgenic joysiagrass grown in the soil. Transgenic Joyciagrass plants (S598A) with a modified phytochrome A gene ( S598A ) exhibit dwarf intercutions, leaf scapes, short leaves and more branches compared to control plants. Perhaps due to chloroplast condensation in plant cells, the leaves of transgenic joysiagrass have a dark-green color.

Recently, genetic transformation of a crop plant having a target gene or gene segment has been established as a method for effectively developing a variety of new plant species with new or enhanced useful features. Potential target features suitable for genetic manipulation of crop plants include modified growth rates, adaptive flexibility, enhanced resistance to biological and abiotic stresses, and increased yields. Recent advances in technology and knowledge in plant molecular biology and tissue culture allow the genes of interest to be customarily introduced into any particular plant species. Effective systems for genetic transformation and tissue culture have been developed for joysiagrass, and high-level expression of the introduced genes has been identified in transgenic joysiagrass (Bae et al., 2001).

The detailed description of the invention is disclosed to assist those skilled in the art of practicing the invention. However, modifications and variations of the present invention are possible by those skilled in the art without departing from the direction and scope of the present invention.

The present invention relates to transformed monocotyledonous or dicotyledonous plants, in particular modified oat phytochrome A (SEQ ID NO: Composed of plant cells transformed with a recombinant DNA construct containing a gene sequence encoding 2) and functionally linked to at least one or more expression control sequence elements required for proper expression of the gene in the plant and having a high level And transgenic joysiagrass and related tuftgrass species and rice plants, which confer the ability to maintain the gene expression and inhibit the negative epithelial response.

Justice

"Keep expression";

(1) Intrinsic phytochrome A is light-laible and rapidly decomposes upon light illumination, and additional phytochrome A is used to offset or even suppress the loss by photo-stimulatory decomposition by the acquisition. Meaning that it is continuously supplied from the transgene to a sufficient level;

(2) means that the normal biological activity of phytochromes is modified in such a way that its physiological activity is greatly enhanced compared to that of wild type phytochrome A.

The modified phytochrome A is produced by substituting serine-598 or its functionally equivalent in oat phytochrome or other phytochrome A, respectively, from dicotyledonous or monocotyledonous plants. The modified phytochrome A (SEQ ID NO: 2) may have the same or much greater physiological activity as compared to wild-type phytochrome A in inhibiting negative epithelial responses.

The modified phytochrome A gene (S598A) is applied via conventional genetic transformation procedures using examples within the invention and by other methods well known to those skilled in the art.

"Reduced negative evasive response" refers to inhibition of negative evasive response; At the expense of leaf and root growth regulated by the proximity signal (R: FR ratio) perceived by the phytochrome photoreceptor, it stimulates the swelling of stem (axillary) and pacifier growth, resulting in dwarf of stem (or internode) and pacifier, Branching is increased and leaf color becomes dark-green due to chloroplasts in plant cells. The reduction of the scavenging response is most noticeable when the plant grows in close proximity to adjacent plants.

The transformation of the present invention can be achieved by redistributing more metabolites to the harvestable organs and reducing the negative epithelial response useful for enhancing harvest yields by increasing phytochrome A expression or by modifying the normal biological activity of phytochrome A. When expressed in plants, their biological activity can be greatly enhanced compared to wild type.

For example, serine-598 is located in the hinge region between the N-terminal photosensitive and C-terminal regulatory domains and from recognition in the N-terminal domain to regulation of phytochrome interaction with PIF in the C-terminal domain. It has been suggested to play an important role in signaling between domains (Park et al., 2000). It is preferentially phosphorylated in FR absorbing Pfr phytochromes and is important for phytochrome function. Substitution of serine-598 with alanine was shown to induce a hypersensitive photoresponse to environmental light when expressed in model plant cells, even though it exhibited the same photochemical activity as wild-type phytochrome A.

An "expression control sequence element" induces effective expression of a gene of interest at a high level when properly linked to a gene or gene segment in an expression vector construct and includes, but is not limited to, promoter, enhancer, inhibitor and poly (A) terminator sequences Nucleotide sequence elements that are not. The poly (A) terminator sequence is linked to the 3 'end of the desired gene and controls the addition of poly (A), which is essential for electron termination and mRNA stability and effective translation at the appropriate site.

For example, a promoter that is expressed differently as a gene promoter is linked to the 5 'end of the desired gene and regulates transcription initiation. Promoters useful in the present invention have at least one of the following properties;

(1) constitutive expression of the desired gene or gene segment throughout plant life in all plant organs; or

(2) conditional expression of the desired gene or gene segment only when a particular environmental or developmental signal induces promoter activity; or

(3) organ-specific expression of the desired gene or gene segment in specific plant organs such as flowers, leaves or stems; or

(4) Developmental stage-specific expression of the desired gene or gene segment during specific developmental stages, such as, for example, developmental stage or regenerative growth stage. Two representative promoters used for gene expression in plant cells are the cauliflower mosaic virus 35S (CaMV 35S) promoter and the corn polyubiquitin (Ubi-P) promoter. The latter is particularly useful when expressing a desired gene or gene segment in monocots because it is at least 10 times stronger than the former.

A "plant expression vector" is defined as a recombinant DNA construct consisting of at least one desired gene that is suitably linked to such expression control sequence elements, such as promoter and poly (A) signal sequence, to express the product of the gene at high levels.

The plant expression vector construct (pCUMB-phyA) of the present invention has the following features.

(1) ubiquitin promoter (Ubi-P) and NOS terminator for effective expression of the desired gene; And

(2) herbicide resistance genes (BAR) and hygromycin resistance genes (Hgr ^R ) for selection in higher plant cells; And

(3) kanamycin resistance gene (Kan ^R ) for bacterial selection in E. coli and Agrobacterium tumefaciens; And

(4) A modified oat phytochrome A gene (S598A) in which serine-598 is substituted with alanine and introduced into the pCUMB vector by replacing the GUS-coding sequence with BamHI and EcoRI recognition sites under the control of the Ubi-P promoter.

Desired genes can be prepared synthetically or naturally isolated. It may also consist of a mixture of synthetic and naturally separated DNA components encoding proteins, enzymes, carriers, pumps or phytohormones. In general, synthetic DNA sequences are produced with suitable codons that are preferentially used for gene expression in plant cells.

"Plant transformation" refers to a procedure for introducing a gene of interest or cDNA into a higher plant, preferentially a crop plant, appropriately linked to expression control sequence elements by methods well known to those skilled in the art. Plants may be genetically engineered to exhibit a reduced speech avoidance response by introducing genes or cDNAs in a functionally feasible manner and expressing at an effective level in inducing a reduced speech avoidance response to modify plant structure and growth rate. Can be.

Joysiagrass in the present invention was used as a host plant for the expression of the modified oat phytochrome A gene ( S598A ). Such transgenic joysiagrass exhibits dwarf intercutions and scapes, short leaves and increased branches. It will also show enhanced ability to flexibly adapt to environmental changes and increased resistance to pathogen infection. Moreover, the general maintenance costs required for mowing and hydrating will be greatly reduced in the cultivation of such transgenic joysiagrass.

(Example)

Plant expression vector structure

CDNA encoding oat phytochrome A3 gene (GenBank Accession No. 16110) was reverse-transcribed using PCR primers and PfuTurbo DNA polymerase (Stratagene, La Jolla, CA) with proofreading activity from 3 'to 5' Isolation was performed by running an enzyme-mediated polymerase chain reaction (RT-PCR). The 5 'and 3' PCR primers have BamHI sites and EcoRI recognition sites, respectively, to facilitate subsequent DNA manipulation. PCR products containing genes were double-digested with BamHI and EcoRI and ligated into pGEM3Z (+) cloning vectors double-digested with BamHI and EcoRI in a similar manner to PCR products. The QuickChange ™ Kit (Stratagene) was used for site-directed mutagenesis in the laboratory following the procedure supplied by the manufacturer. The original AGT codon of the oat phytochrome A3 gene was mutated to GCT to replace serine-598 with alanine. Base substitution was demonstrated by direct DNA sequencing. The mutated phytochrome A3 gene ( S598A ) was subsequently subcloned into the BamHI / EcoRI-digested pCUMB vector resulting in a pCUMB-phyA vector. The pCUMB vector is a binary vector containing a resistance gene for kanamycin for bacterial selection in E. coli and Agrobacterium tumefaciens and a resistance gene for hygromycin and herbicides for selection in plant cells. Expression of the S598A gene was under the control of the ubiquitin promoter (Ubi-P). All intermediates and final vector structures were demonstrated by restriction mapping and direct DNA sequencing.

Preparation of Joysiagrass Callus

Mature seeds of Zoysia japonica Steud. Were surface-sterilized with 1 ml of 2% sodium hypochlorite for 15 minutes and then washed three times with sterile secondary-distilled water. Seeds for callus induction were 3% sucrose (w / v), 100 mg / L α-ketoglotamic acid, 4 mg / L thiamine-HCl, 2 mg / L 2,4-D, 0.2 mg / L It was placed on 3MM filter paper-callin MS (Murashige and Skoog, 1962) medium Petri dishes containing BA and 0.2% gelrite (w / v). The medium was titrated to pH 5.8 using HCl before autoclaving at 120 ° C. for 20 minutes at 1.2-1.3 kg / cm 2 pressure. Callus induction was performed in a culture room at 26 ± 1 ° C. for 3 months in the complete dark. Fresh MS containing 1 mg / L of 2,4-D and 0.2 mg / L of BA after subsequent 3-day induction under continuous white light of intensity ³⁰ μmolm ⁻² s ⁻¹ provided by the fluorescent tube Transferred to medium and incubated in the dark for further growth. After 5 weeks of culture in the dark, independent seed-derived callus with green color was transferred and incubated on 3MM filter paper-calin MS medium containing 2,4-D and BA in the dark. Of the four types of callus observed (types I to IV), only type III was further amplified on MS medium containing 4 mg / L of 2,4-D after four weeks of subculturing. It was used as a host plant cell for transformation.

Optimal Conditions for Agrobacterium Infections

Agrobacterium tumefaciens strain EHA105 containing pCUMB-phyA was used for Joysiagrass callus infection. Agrobacterium tumefaciens cells were grown at 28 ° C. overnight with shaking at 160 rpm in 100 ml Erlenmer flasks containing 20 ml LB medium supplemented with 50 mg / L hygromycin and 100 mg / L kanamycin. Cells of 10 ml of culture suspension were collected into 50 ml of polypropylene tubes (Becton Dickinson Labware, USA) by centrifugation at 2500 rpm for 20 minutes and 10 ml of liquid infection medium (calcium-free MS) by gentle vortexing. Salts and vitamins, 30 g / L sucrose, 10 g / L glucose, 100 mg / L betaine, 50 mg / L acetosyringone, 0.01% pluronic F68, 0.01 mg / L BA and 2 g / L gellite, pH 5.2).

Acetosyringone was prepared by dissolving an appropriate amount of powder in dimethyl sulfoxide so that the final concentration was 100 mg / L and stored in the dark at 4 ° C. until use. When necessary, it was added directly to the sterile medium at the appropriate final concentration. Proliferated callus was soaked in acrobacterial cell suspension for 1 minute. Callus after brief dehydration on sterile 3MM filter paper was incubated for 15 days in dark conditions on the same co-culture medium plate as the infection medium except that supplemented with 4 mg / L of 2,4-D. After co-culture, the callus was washed by gentle vortexing in sterile secondary-distilled water supplemented with surfactant (0.02% Pluronic F68) until the wash solution became clear, and finally 1000 mg / L of calbenicillin ( calbenicillin) and sterile secondary-distilled water containing 0.02% surfactant.

Optimized conditions for effective Agrobacterium tumefaciens infection of Zoiagrass Callus were 100-mg / day using Type III as recipient cells, co-cultured for 9 days, excluding 2,4-D and CaCl ₂ from the infection medium. It is to use acetosyringone of L.

The cocultures were then incubated with 3MM filter paper-coated MSCGCB medium (MS salt and vitamin, 30 g / L sucrose, 1 mg / L 2,4-D, 0.01 mg / L BA, 2 g / L gelite and 500 mg / L of carbenicillin, pH 5.8) and incubated in the dark for 2-4 weeks. The callus was then subjected to 3MM filter paper-scaled MSSI medium (MS salt and vitamins, 30 g / L maltose, 1 mg / L BA, 2 g / L gellite and 250 mg / L carbenicillin, for shoot induction). pH 5.8). The induced chute was then subjected to MSRS medium (MS salt and vitamins, 30 g / L sucrose, 1 ml / L BA, 2 g / L gellite, 250 mg / L carbenicillin and 5 mg / L for 4 weeks for rooting and selection). Bialaphos or 10 g / L hygromycin, pH 5.8). Rooting plants were transferred into MSPG medium without MS and non-alafoss (MS salts and vitamins, 30 g / L sucrose, 8 g / L agar, pH 5.8) and further grown. The fully grown plants were then transferred into soil-containing pots and set to 18-hour photoperiod, 30 ° C, 80% relative humidity at 30 μmolm ^-2 s ^-1 irradiation provided by a cool white fluorescent tube. It was grown in a growth chamber controlled by. When the roots were fully developed, the pots were transferred to the green house.

Selection of Transgenic Zosiagrass Plant

5 g / L preparation solution (Meiji Seika, Japan) was sprayed daily for two weeks after the transgenic plants settled in the soil. Two weeks after herbicide administration, the transgenic plants survived the non-alafoss painting and grew adult. However, control plants stopped growing and actually died.

Analysis of the Evasive Response of Transgenic Zosiagrass Plants

Transformation with S598A Pytochrome A Gene Zosiagrass plants were further assessed molecularly to confirm that they were actually transformed with 598A phytochrome A gene, and at the same time they were grown in soil to control the plants under the same growth conditions. Analyzed and compared morphological and photomorphologic features included those related to epidermal avoidance, including plant structure, stem and leaf growth, branching patterns and leaf color.

result

Homozygous lines of seria-598 codons, AGTs substituted with alanine codons, GCTs and transformed with modified oat phytochrome A gene (S598A), indicated by the ubiquitin promoter, were isolated and plant structure, growth rate And morphology was analyzed.

The serine-598 residue is located in the hinge between the two functional domains of the phytochrome molecule and is preferentially phosphorylated in Pfr phytochrome in vivo. Therefore, it has been suggested to play an important role in interdomain signaling by transferring the optical signal recognized by the N-terminal photosensitive domain to the C-terminal regulatory domain. Transfection with a Modified Phytochrome A Gene Arabidopsis plants exhibited a sensitive light response to environmental light and confirmed the importance of the serine-598 residue in phytochrome function. The sequence region of oat phytochrome A containing substitutions with serine-598 and alanine is shown in FIG. 1.

The S598A gene was subcloned into the plant expression vector, pCUMB, and the resulting recombinant vector construct (FIG. 2) was transformed into Joysiagrass using the recently established Agrobacterium-mediated method.

Transformed joysiagrass transformed with S598A gene showed a dwarf appearance overall. More particularly, intercalates and leaf scapes were significantly reduced compared to wild-type plants, and branches also increased at least twice. Moreover the leaves were dark-green in color. This phenotypic change is typical of transgenic model plants with enhanced phytochrome A expression as a result of the drastic reduction in the circumvented response. Although the growth of transgenic joysiagrass plants is greatly inhibited, they are expected to be healthy and robust and more resistant to biological and abiotic stresses, including droughts, traffic injuries and pathogen infections. Moreover, less maintenance costs can be achieved because less mowing and handing is required. Of particular concern is increased resistance to pathogen infections, particularly fungal infections that are prevalent during the hot summer period. As a result, fewer chemical sprays are required.

Thus, as in the case of Joysiagrass used in the present invention, the transformed crop plants with the modified phytochrome A gene are expected to exhibit a reduced negative evacuation response that will be delivered stably and reproducibly to their offspring through reproduction. do.

As described, the present invention can be clearly changed in many ways. However, such changes are not to be regarded as a departure from the scope and scope of the invention, and it is demonstrated to those skilled in the art that all such changes are included within the scope and scope of the claims contained in the invention.

The effect of the present invention relates to the manipulation of crop growth rate and photoreactivity through genetic transformation with modified phytochrome A photoreceptors, which are economically important crop plants, particularly pronounced dwarfism and more branched structures. The present invention relates to a modification of plant structure and photomorphogenesis by a molecular biological strategy for designing a transgenic grass to respond to environmental aspects. As a result, the transgenic plant is healthy, robust and more resistant to pathogen infection.

Claims (5)

Iii ) Zea mays plant ubiquitin promoter; A modified phytochrome A gene of SEQ ID NO: 1 encoding a nucleotide sequence substituted with alanine at serine-598; And a 3 'untranslated sequence consisting of a polyadenylation signal sequence functioning in a plant cell, transforming the Joysiagrass cells with a recombinant DNA construct that is operably linked in the sequence in the 5' to 3 'direction; Ii) selecting plant cells transformed with the phytochrome A coated nucleotide sequence; Iii) regenerating Joysiagrass plants from cells transformed with the phytochrome A coated nucleotide sequence; Iii) selecting a transgenic joysiagrass expressing the phytochrome A of SEQ ID NO: 2; A method for producing genetically transformed Zoysia genus (KCTC-1024BP) having a stepwise negative inhibitory property Genetically transformed Joysia genus produced by the method according to claim 1 Iii ) Zea mays plant ubiquitin promoter; A modified phytochrome A gene of SEQ ID NO: 1 encoding a nucleotide sequence substituted with alanine at serine-598; And transforming the plant cell with a recombinant DNA construct consisting of a 3 'untranslated sequence consisting of a polyadenylation signal sequence functioning within the plant cell, wherein they are operably linked within the sequence in the 5' to 3 'direction; Ii) selecting plant cells transformed with the phytochrome A coated nucleotide sequence; Iii) regenerating Joysiagrass plants from cells transformed with the phytochrome A coated nucleotide sequence; Iii) selecting a transgenic plant expressing the phytochrome A of SEQ ID NO: 2 A method for producing a genetically transformed plant having a negative hedging inhibitory property consisting of steps Genetically transformed plant produced according to the method of claim 3 5. The genetically transformed plant of claim 4, wherein the transgenic plant is a plant belonging to one or more agricultural crops, vegetable crops, horticultural plants, fruit plants and decorative plants.