A method for controlling the flowering time and the relevant phenotype(leaf, flower, fruit) mediated by the overexpression vector of apple MdFT1

Abstract

The present invention relates to a recombinant vector that overexpresses MdFT1 and the use thereof for controlling the timing of flowering and phenotype. According to the present invention, in a plant introduced by an Agrobacterium-mediated transformation method using a recombinant vector containing the MdFT1 gene, the overexpression of the MdFT1 gene interferes with the expression level of endogenous flowering regulating genes, thereby promoting early flowering and having a characteristic phenotype of branching growth, leaf shape, flower and fruit development, and in particular, a phenotype in which the production of fruit and seeds increases. Therefore, the recombinant vector of the present invention can be usefully utilized for early fruiting and phenotype control, and for shortening the breeding period of perennial crops.

Description

Technology Field

[0001] The present invention MdFT1 This invention relates to a recombinant vector that overexpresses and its use in controlling the timing of flowering and phenotype. Background Technology

[0002] With the advancement of molecular biology and plant transformation technologies, it has become possible to produce transgenic plants with various desirable traits, such as resistance to pests, fungi, or microbial pathogens, resistance to herbicides, or value-added characteristics. These desirable traits are primarily obtained through the overexpression of transgenes in plants. However, in some cases, it may be desirable to modify plants by altering the expression of specific genes to obtain plants with commercially useful desirable phenotypes or traits. Current methods for altering gene expression typically rely on sense or antisense suppression techniques. Various studies are being conducted in the field of plant biotechnology to successfully genetically engineer plants, including major cultivated varieties. It has been shown that by using genetic engineering technology to inject genes for herbicide resistance, pest and disease resistance, high sugar content, high content of juice nutrients, growth rate control, tolerance to harmful substances, environmental resistance, and beneficial microorganisms, it is possible to contribute to a dramatic increase in productivity through the development of super-high-yielding crops and crops resistant to adverse environments such as low temperature, dryness, and salt damage, as well as pests and diseases. It has also demonstrated the potential to contribute decisively to increased food production by producing crops rich in nutrients or functional components (such as anticancer effects) and crops from which allergy-causing substances have been removed.

[0003] Efforts to improve crop yields through the development of new plant species can be divided into two approaches. One involves reducing crop yield losses by breeding or processing crop varieties with increased tolerance to abiotic stress conditions, such as drought, cold, or salinity, or biotic stress conditions caused by pests or disease-causing pathogens. However, even if these attempts are valuable, they do not provide fundamentally improved crop yields in the absence of stress conditions. The second approach involves breeding or processing new crop varieties with increased basic yield potential. Traditional breeding programs initially achieved improved yields in crop varieties, but yield improvements became less significant thereafter. More recently developed approaches based on molecular biology techniques have sought substantial improvements in crop yields by fundamentally altering the timing, location, or level of expression of plant genes that play a critical role in plant growth and / or development. Although plant genes responsible for plant growth and / or development have been studied over the past 20 years, it remains unclear which of these genes could be clear candidates for improving crop yield due to the complexity of how plant growth regulation is ultimately related to yield characteristics.

[0004] Many complex biochemical pathways in plants have been genetically manipulated by the repression or over-expression of single genes. Enhancement of gene expression in plants has been achieved by introducing red copies of gene coding sequences into plant cells or by incorporating red copies of gene coding sequences into the plant genome. Additionally, in plants, the repression of gene expression, known as gene silencing, occurs at both the transcriptional and post-transcriptional levels, and there are various methods for suppressing the expression of endogenous sequences within host cells. Such methods include antisense inhibition (Smith et al., Nature 334:724–726 (1988)), co-inhibition (Napoli et al., Plant Cell 2:279–289 (1989)), ribozymes (Kohler et al., J.Mol.Biol. 285:1935–1950 (1999)), combination of sense and antisense (Water-House et al., PNAS USA 95:13959–13964 (1998)), promoter silencing (Park et al., Plant J. 9(2):183–194 (1996)), and DNA-binding proteins (Beerli et al., PNAS USA 95:14628–14633 (1997); Liu et al., PNAS USA 94:5525–5530 (1998)).

[0005] However, since such gene regulation in plants occurs through a considerably complex process and it is thought that various genes that have not yet been fully identified are involved, there is a need for a comprehensive analysis of the various phenotypic changes in plants that accompany the regulation of gene expression. The problem to be solved

[0006] The objective of the present invention is to... MdFT1 It is to provide a recombinant vector for gene overexpression.

[0007] In addition, the objective of the present invention is to provide a transformant transformed with the recombinant vector.

[0008] In addition, the objective of the present invention is to provide a plant body transformed into the above-mentioned transformant.

[0009] In addition, the objective of the present invention is to provide a method for promoting seed production of a plant.

[0010] In addition, the objective of the present invention is to provide a method for producing a transgenic plant. means of solving the problem

[0011] To solve the above problem, the present invention MdFT1 We provide a recombinant vector containing the (Malus x domestica flowering locus T) gene.

[0012] In addition, the present invention provides a transformant transformed with the recombinant vector.

[0013] In addition, the present invention provides a plant body transformed into the above-mentioned transformant.

[0014] In addition, the present invention transforms a plant body with the above-mentioned recombinant vector. MdFT1 A method for promoting seed production of a plant, comprising a step of overexpressing a gene, is provided.

[0015] In addition, the present invention MdFT1 A method for producing a transgenic plant that overexpresses is provided. Effects of the invention

[0016] According to the present invention, MdFT1 In plants into which a recombinant vector containing a gene was introduced via the Agrobacterium-mediated transformation method MdFT1 Since the overexpression of the gene interferes with the expression level of the endogenous flowering regulatory gene, thereby promoting early flowering, and possesses a characteristic branching growth, leaf morphology, flower and fruit development phenotype, and particularly increased seed production, the recombinant vector of the present invention can be usefully utilized for early fruiting and phenotype control, and for shortening the breeding period of perennial crops. Brief explanation of the drawing

[0017] Fig. 1 is MdFT1 This is a schematic diagram of the T-DNA region of a vector overexpressing a gene, a diagram showing the results of confirming the presence of a target gene introduced into the vector, and the results of confirming the Agrobecterium into which the vector was introduced: RB: T-DNA right boundary; hptII: Hygromycin phosphotransferase; 35SpolyA: Terminator of the 35S gene; and LB: T-DNA left boundary; M: DNA ladder marker; MdFT1: Target gene; NC: Negative control (genomic DNA of WT); and 35S::MdFT1: Target gene construct. Figure 2 is a figure showing the DNA sequence analysis and protein sequence analysis of the target gene. Figure 3 is a figure illustrating the process of producing transgenic tobacco through in vitro tissue culture using the Agrobacterium-mediated transformation method. Fig. 4 is MdFT1 This is a diagram showing the qPCR analysis of the selection marker (htpII) and target gene (MdFT1) in leaves isolated from genetically modified tobacco seedlings overexpressing the gene: M: DNA ladder marker; PC: Positive control group; NC: Negative control group; WT: Wild tobacco; and 35S::MdFT1: Transformed with a target gene construct MdFT1 Transgenic tobacco that overexpresses genes. Fig. 5 is MdFT1 This is a diagram confirming the flowering of a genetically modified tobacco that overexpresses a gene: 35S::MdFT1: MdFT1 Transgenic tobacco that overexpresses a gene; and WT: Wild tobacco. Fig. 6 is MdFT1This is a diagram confirming early flowering of transgenic tobacco that overexpresses a gene: 35S::MdFT1: MdFT1 Transgenic tobacco that overexpresses genes; NC: Negative control group; and WT: Wild tobacco. Fig. 7 is MdFT1 This is a diagram confirming the first flowering time and the number of leaves of transgenic tobacco overexpressing a gene: 35S::MdFT1: MdFT1 Transgenic tobacco that overexpresses a gene; and WT: Wild tobacco. Fig. 8 is MdFT1 This is a diagram observing the formation of lateral shoots in transgenic tobacco that overexpresses a gene: WT: Wild tobacco; and 35S::MdFT1: MdFT1 Transgenic tobacco that overexpresses genes. Fig. 9 is MdFT1 This is a diagram comparing the length of a genetically modified tobacco that overexpresses a gene with that of a wild-type tobacco: WT: Wild tobacco; and 35S::MdFT1: MdFT1 Transgenic tobacco that overexpresses genes. Fig. 10 is MdFT1 This is a diagram comparing the internode length of genetically modified tobacco with that of wild-type tobacco: Fig. 11 is MdFT1 This figure shows the results of confirming the difference in internode elongation at the cellular level between gene-overexpressing transgenic tobacco and wild-type tobacco using an optical microscope: WT: Wild tobacco; and 35S::MdFT1: MdFT1 Transgenic tobacco that overexpresses genes. Fig. 12 is MdFT1 This is a diagram comparing the leaf phenotype (shape and size) of transgenic tobacco overexpressing the gene with WT: WT: Wild tobacco; and 35S::MdFT1: MdFT1 Transgenic tobacco that overexpresses genes. Fig. 13 is MdFT1 This is a diagram comparing the flower organs of transgenic tobacco overexpressing a gene with WT: WT: Wild tobacco; and 35S::MdFT1: MdFT1 Transgenic tobacco that overexpresses genes. Figures 14 and 15 show the results of comparing the development of fruit (fruit) of transgenic tobacco overexpressing the MdFT1 gene with that of WT tobacco: WT: Wild tobacco; and 35S::MdFT1: MdFT1 Transgenic tobacco that overexpresses genes. Fig. 16 is MdFT1 This figure shows the results of analyzing the expression levels of endogenous flowering genes in gene-overexpressing transgenic tobacco and wild-type tobacco using qRT-PCR: WT: Wild tobacco; and 35S::MdFT1: MdFT1 Transgenic tobacco that overexpresses genes. Fig. 17 is MdFT1 This figure shows the results of qRT-PCR analysis of CCD gene expression in gene-overexpressing transgenic tobacco and wild-type tobacco: WT: Wild tobacco; and 35S::MdFT1: MdFT1 Transgenic tobacco that overexpresses genes. Specific details for implementing the invention

[0018] Hereinafter, the present invention will be described in detail with reference to the attached drawings for embodiments of the present invention. However, the following embodiments are presented as examples of the present invention, and if it is determined that a detailed description of a technology or configuration well known to those skilled in the art may unnecessarily obscure the essence of the present invention, such detailed description may be omitted, and the present invention is not limited thereby. The present invention is capable of various modifications and applications within the scope of the claims set forth below and the equivalents interpreted therefrom.

[0019] Furthermore, the terminology used in this specification is used to appropriately describe preferred embodiments of the present invention, and may vary depending on the intent of the user or operator, or the conventions of the field to which the present invention belongs. Accordingly, the definitions of these terms should be based on the content throughout this specification. Throughout the specification, when a part is described as "comprising" a certain component, unless specifically stated otherwise, this means that it does not exclude other components but may include additional components.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. Any method and material similar or equivalent to those described herein may be used in practice to test the present invention, but preferred materials and methods are described herein. Furthermore, the contents of all publications cited by reference in this specification are incorporated into the present invention.

[0022] In one aspect, the present invention MdFT1 This relates to a recombinant vector containing the (Malus x domestica flowering locus T) gene.

[0023] In one embodiment, the recombinant vector may additionally include a transcription regulator, a translation regulator, or a marker capable of confirming gene expression.

[0024] In one embodiment, the transcription regulator of the recombinant vector may be a Cauliflower Mosaic Virus 35S promoter, an Agrobacterium tumefaciens Ti plasmid nopaline synthase promoter, an octopine synthase promoter, a CASP1 (Casparian line membrane domain protein 1) promoter, or a mannopine synthase promoter, and it is more preferable that it be a Cauliflower Mosaic Virus 35S promoter or an Agrobacterium tumefaciens Ti plasmid nopaline synthase promoter.

[0025] In one embodiment, the marker may be an antibiotic resistance gene, a screening marker gene, a gene encoding beta-glucuronidase (β-glucuronidase encoding gene), chloramphenicol acetyltransferase, luciferase, or a gene encoding fluorescent protein (fluorescent protein encoding gene).

[0026] In one embodiment, the selection marker gene may be selected from the group consisting of neomycin phosphotransferase, hygromycin phosphotransferase, puromycin, histidinol dehydrogenase, guanine phosphotransferase, and zeocin, and it is more preferable that the gene be hygromycin phosphotransferase (htpII).

[0027] In one embodiment, the fluorescent protein may be a green fluorescent protein (GFP), an enhanced green fluorescent protein (EGFP), a yellow fluorescent protein (YFP), a red fluorescent protein (RFP), an orange fluorescent protein (OFP), a cyan fluorescent protein (CFP), a blue fluorescent protein (BFP), a far-red fluorescent protein, or a tetracysteine ​​motif.

[0028] In one embodiment, the recombinant expression vector may include a tag sequence, and the tag may be a His tag, a Myc(c-myc) tag, a FLAG tag, a HA tag, or a T7 tag.

[0029] In one embodiment, the recombinant vector may be pBI121, pROKII, pBI76, pET21, pSK(+), pLSAGPT, pUC, pGEM, pHellsgate8, pPZP, pGA, or pCAMBIA.

[0030] In one embodiment, the recombination vector is MdFT1 It may be for overexpression of the gene (GenBank ID: DQ535887.1).

[0031] In one embodiment, the recombinant vector may be used to induce early flowering or to promote the transition from the vegetative phase to the reproductive phase.

[0032] Terms of the present invention, " MdFT1 The "gene" is of the apple (Malus x domestica). FT (Flowering Locus T) gene MdFT1 It means, and its nucleotide sequence can be obtained from known databases such as NCBI's GenBank (e.g., GenBank Accession: DQ535887.1, etc.). Specifically, the above gene is apple ( Malus domestica More specifically, it may be a gene derived from the flower buds of the Fuji apple variety, but is not limited to this.

[0033] The term "vector" in the present invention may refer to a gene construct comprising an essential regulatory element operably linked to enable the expression of the double-stranded RNA of the target gene, as a means for introducing DNA into a host cell to efficiently suppress the expression of a target gene.

[0034] Specific examples of the above vectors include plasmid vectors, cosmid vectors, bacteriophage vectors, or virus vectors. More specific examples may include Escherichia coli-derived plasmids (pBR322, pBR325, pUC118, pUC119, pET30a, pET30c, and pGEX-GST), Bacillus subtilis-derived plasmids (pUB110 and pTP5), yeast-derived plasmids (YEp13, YEp24, and YCp50), or Ti plasmids. Additionally, animal viruses such as retroviruses, adenoviruses, or vacciniaviruses, insect viruses such as baculoviruses, or plant viruses may be used, and binary vectors such as the pPZP, pGA, and pCAMBIA series may be used, but are not limited thereto as long as the cassette of the present invention can be introduced into a host cell. As a more specific example, the above vector may be pCAMBIA1300 of the pCAMBIA series, but is not limited thereto.

[0035] The term "binary vector" in the present invention refers to a vector designed to function across two vector systems, which is used when the transformation of Agrobacterium by the Ti plasmid is difficult to manipulate due to its size and is divided into two replicants.

[0036] In addition, the vector may be functionally linked to an expression regulatory sequence. As a specific example, the vector may include signal sequences or leader sequences for membrane targeting or secretion in addition to expression regulatory elements such as promoters, operators, start codons, stop codons, polyadenylation signals, and enhancers. As a more specific example, the vector may include a CaMV 35S promoter, but is not limited thereto and may be prepared in various ways depending on the purpose of the invention. Additionally, the vector may include a selectivity marker and may self-replicate or be incorporated into host DNA. The vector of the present invention may be prepared using gene recombination technology well known in the art, and site-specific DNA cleavage and ligation may be performed using enzymes generally known in the art.

[0037] In one aspect, the present invention relates to a transformant transformed with the recombinant expression vector of the present invention.

[0038] In one embodiment, the transformant may be an Agrobacterium sp. transformant transformed with a recombinant vector, and may be Agrobacterium tumefaciens and Agrobacterium rhizogenes.

[0039] In one embodiment, the transformant comprises the recombinant vector. MdFT1 It may be Agrobacterium tumefaciens for inhibiting gene expression.

[0040] In the present invention, Agrobacterium transformed by the recombinant vector genetically transforms a plant by transporting genetic material into the nucleus of a host cell in the form of a type of nucleic acid-protein complex called a T-complex. Once the T-complex enters the nucleus, it reaches the host chromosome and is integrated into the genetic material of that host cell. Agrobacterium genetically transforms the host cell by carrying out various biological processes. In this way, Agrobacterium serves as a special model system for studying the transport of genetic material between bacteria and eukaryotic cells.

[0041] In the present invention, methods for transforming Agrobacterium into a recombinant vector include the CaCl2 method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9:2110-2114(1973)), the Hanahan method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9:2110-2114(1973); and Hanahan, D., J. Mol. Biol., 166:557-580(1983)) and the electroporation method (Dower, WJ et al., Nucleic. Acids Res., 16:6127-6145(1988)).

[0042] The term "Agrobacterium tumefaciens" of the present invention refers to a type of plant crown gall pathogen and, for the purposes of the present invention, may mean a medium for delivering a vector for inhibiting the expression of the target gene of the present invention to a plant.

[0043] In one embodiment, the Agrobacterium tumefaciens may be Agrobacterium tumefaciens LBA4404, GV2260, GV3101, GV3100, GV3850, A136, C58C1, AGL-1, EHA101, or EHA105, but is not limited thereto as long as it can deliver the vector of the present invention to a plant.

[0044] In one aspect, the present invention relates to a plant body transformed into a transformant comprising a recombinant vector of the present invention.

[0045] In one embodiment, the transformed plant is MdFT1 It may be a transgenic plant that overexpresses a gene.

[0046] In one embodiment, the plant body may be any one selected from the group consisting of food crops including rice, wheat, barley, corn, soybeans, potatoes, wheat, red beans, oats, or sorghum; vegetable crops including Arabidopsis thaliana, Chinese cabbage, radish, chili pepper, strawberry, tomato, watermelon, cucumber, cabbage, Korean melon, pumpkin, green onion, onion, or carrot; specialty crops including ginseng, tobacco, cotton, sesame, sugarcane, sugar beet, perilla, peanut, or rapeseed; fruit trees including apple trees, pear trees, jujube trees, peaches, kiwifruit, grapes, citrus fruits, persimmons, plums, apricots, or bananas; floricultural plants including roses, gladiolus, gerberas, carnations, chrysanthemums, lilies, or tulips; and fodder crops including ryegrass, red clover, orchardgrass, alpha-alpha, tall fescue, or perennial ryegrass, and it is more preferable that it be tobacco.

[0047] In one embodiment, in the transformed plant body NtAP1, NtSOC1, NFL1 and NtFTs Gene expression can be upregulated compared to WT plants.

[0048] In one embodiment, the expression of CCD genes in the transformed plant may be downregulated compared to the WT plant, and the CCD genes NtCCD1-3 , NtCCD4-1 and NtCCD4-2 It could be.

[0049] The term "transformation" in this invention refers to the phenomenon of changing genetic traits by introducing genetic material into living cells of a different lineage.

[0050] The term "plant body" in this invention includes not only mature plant bodies but also all plant cells, plant tissues, and plant seeds capable of developing into mature plants.

[0051] In one aspect, the present invention is the present invention MdFT1 The present invention relates to a composition for increasing seed yield comprising, as an active ingredient, a recombinant vector containing the gene (Malus x domestica flowering locus T) (GenBank ID: DQ535887.1) or a transformant containing the same.

[0052] In one aspect, the present invention introduces the recombinant vector of the present invention into a plant body. MdFT1 This relates to a method for enhancing seed production of a plant, comprising a step of overexpressing a gene.

[0053] In the present invention, the step of introducing the recombinant vector of the present invention into a plant may be the step of introducing the recombinant vector including the cassette into a plant, and this may be performed by a transformation technique known to those skilled in the art.

[0054] Specific examples include transformation methods using Agrobacterium, microprojectile bombardment, electroporation, PEG-mediated fusion, microinjection, liposome-mediated method, in planta transformation, vacuum infiltration method, floral meristem dipping method, or Agrobacteria spraying method; more specifically, transformation methods using Agrobacterium may be used, but are not limited thereto.

[0055] In one aspect, the present invention introduces the recombinant vector of the present invention into a plant body. MdFT1 This relates to a method for promoting branching sprouting of plants, comprising the step of overexpressing a gene.

[0056] In one aspect, the present invention relates to a method for producing a transgenic plant, comprising the steps of: producing a recombinant vector of the present invention; transforming the vector into a plant using Agrobacterium; and selecting a transgenic plant.

[0058] The present invention will be explained in more detail through the following examples. However, the following examples are intended only to illustrate the content of the present invention and do not limit the present invention.

[0060] Example 1. MdFT1 Production of overexpression vectors

[0061] MdFT1 To confirm phenotypic changes in plants caused by the overexpression of the (Malus x domestica flowering locus T) gene, MdFT1 An expression vector overexpressing the gene (GenBank ID: DQ535887.1) was constructed. Specifically, total RNA from Fuji apple flower buds was extracted using the CTAB method and RT-PCR with the PrimeScript™ 1st strand cDNA Synthesis Kit (Cat. #6110A, Takara, Kusatsu, Japan). MdFT1cDNA encoding [the gene] was synthesized. Subsequently, to clone the gene into an expression vector, PCR was performed using primers designed to include XbaI and KpnI restriction sites at the 5' ends of the forward and reverse primers, respectively. The PCR products were then analyzed on a 0.8% agarose gel, and the DNA fragment from the target band of the gene was purified using a DNA purification kit (iNtRON MEGAquick-spin™ Plus, Seongnam, Korea). The purified DNA fragment was then subjected to pGEM under the control of the cauliflower mosaic virus 35S promoter. ® After ligating to the -T Easy vector (Cat. # A1360, Promega, Madison, WI, USA) and translating into DH5α E. coli cells (Cat. #9057, Takara, Kusatsu, Japan), the sequence of the thus prepared plasmid DNA was analyzed. MdFT1 The introduction of (GenBank ID: DQ535887.1) was confirmed (Macrogen, Seoul, Korea). Subsequently, the plasmid DNA was treated with XbaI and KpnI restriction enzymes, and then cloned into a binary vector pCAMBIA1300 (CAMBIA, Canberra, ACT, Australia) treated with the same restriction enzymes to construct a recombinant expression vector containing the 35S::MdFT1 construct (Fig. 1).

[0063] Example 2. MdFT1 Production of Agrobacterium containing an overexpression vector

[0064] MdFT1 Agrobacterium was constructed to transform a vector overexpressing into plants. Specifically, Agrobacterium tumefaciens ( Agrobacterium tumefaciens After introduction into EHA105, the cells were spread onto LB agar medium containing 50 μg / mL kanamycin and 100 μg / mL rifampicin and cultured under dark conditions at 28°C for 2 days. Selected transformed Agrobacterium tumefaciens EHA105 colonies were inoculated into 5 mL of LB liquid medium containing antibiotics and cultured overnight at 28°C at 200 rpm. Plasmid DNA was isolated from the cultured Agrobacterium tumefaciens EHA105, and the presence of the target gene was confirmed (Fig. 1). Additionally, the DNA sequence of the target gene and the protein sequence were aligned and confirmed (Fig. 2).

[0066] Example 3. MdFT1 Production of overexpression plants

[0067] The fabricated in Example 2 above MdFT1 Tobacco (using Agrobacterium containing a vector that overexpresses) N. tabacum The leaf discs of ) were transformed with the 35S::MdFT1 construct. Specifically, 0.5 cm leaf discs from tobacco 2The leaf discs were cut to a size and immersed in liquid co-culture medium MSCO (4.3 g / L MS medium containing B5 vitamins, 30 g / L sucrose, 1 mg / L BAP, and 0.1 mg / L NAA) supplemented with 100 μM acetosyringone, along with the Agrobacterium (containing the 35S::MdFT1 construct) prepared in Example 2. After Agrobacterium-mediated transformation, the leaf discs were placed upside down on solid MSCO medium containing 4 g / L gelling agent under dark conditions for 3 days (the axis in contact with the medium). The explants were washed 3 to 4 times with sterile distilled water to remove any remaining Agrobacterium, and then gently washed with sterile distilled water and 500 mg / L cefotaxime for 10 minutes. Subsequently, the specimens were wiped with sterile 3M filter paper and transferred to selective medium MSSE (MSCO + 250 mg / L cefotaxime, 100 mg / L hygromycin B). To induce callus formation, the specimens were cultured under dark conditions at 25°C for 2 to 3 weeks until adventitious shoots appeared (Fig. 3A), after which they were exposed to light and cultured for an additional week. Transformed shoots were excised from the base of the callus, transferred to MSSE, and conditioned under a light / dark photoperiod of 16 h / 8 h with a light intensity of 100 μmol·m². -2 ·s -1 They were cultured and subcultured every 3 weeks (Fig. 3B). To obtain whole plantlets, shoots were transferred to rooting medium MSR (4.3 g / L MS medium containing B5 vitamins, 30 g / L sucrose, 0.1 mg / L IBA, 250 mg / L cefotaxime, 100 mg / L hygromycin B, and 4 g / L gelrite) (Figs. 3C and D), and after establishing plantlets including roots in vitroThe seedlings were continuously maintained to be grown in the culture medium (Figs. 3E to H). Subsequently, the selection marker hygromycin phosphotransferase (htpII) and the target gene ( MdFT1 To confirm the insertion of a T-DNA region containing ), leaves were cut from transgenic seedlings, total RNA was isolated using the RNA Plant Mini Kit (Cat. #74904, Qiagen, Germany), cDNA was synthesized using PrimeScript™, and analyzed by qPCR. The target gene was normalized to the NtActin gene (GenBank Accession No. U60495.1).

[0068] As a result, positive bands of the expected size for both the selection marker (htpII) and the target gene (MdFT1) were confirmed in leaves isolated from selected seedlings, and the said bands did not appear in WT tobacco plants that had not undergone transformation, thus the present invention MdFT1 It was confirmed that it was successfully integrated into the chromosomes of tobacco plants (Fig. 4). In addition, while wild-type tobacco plants with the phenotype grew continuously without flowering (Figs. 3E and F, and Fig. 5), MdFT1 Tobacco plants transformed with [the product] were found to flower (Figs. 3G and H, and Fig. 5). Tobacco plants of the 35S::MdFT1 homozygous transgenic line selected by analysis were planted in soil and grown in a greenhouse under long-day conditions of 25°C.

[0070] Example 4. MdFT1 Observation of the phenotype of overexpressed transgenic tobacco

[0071] 4-1. Confirmation of Early Bloom Phenotype

[0072] Produced in the above example MdFT1 As a result of observing the phenotype of transgenic tobacco that overexpresses the gene, in vitroAn early flowering phenotype was observed in the seedlings, and subsequently, when seedlings including roots were established and maintained in a culture medium for continuous growth instead of being transferred to soil, only the transgenic plants flowered, while the WT plants remained in the vegetative stage (Fig. 5). To evaluate the genetic stability of the transgenic plants, transgenic lines were selected, transferred to soil, and germinated into plants / lines. As a result, MdFT1 All 35S::MdFT1 homozygous T1 plant lines overexpressing the gene flowered up to 23 days earlier than the wild type (WT) (Fig. 6), and the average day of flowering was 50.6 and 73.6 days for transgenic tobacco and wild-type tobacco, respectively (Fig. 6). Furthermore, transgenic tobacco reached full bloom at the stage with 6 to 8 leaves, and some transgenic plants exhibited first flowering immediately after 4 or 5 leaves emerged, whereas WT plants showed a non-flowering vegetative stage (Fig. 7). Through this, MdFT1 It was confirmed that the early flowering phenotype of transgenic tobacco that overexpresses the gene is conserved in the transgenic plants and inherited by the next generation.

[0074] 4-2. Confirmation of changes in plant structure

[0075] Produced in the above example MdFT1 Observation of the structural phenotypes of transgenic tobacco overexpressing the gene revealed that the 35S::MdFT1 transgenic tobacco produced multiple axillary buds on the main shoot, whereas lateral shoots were not formed in the WT plants (Fig. 8). This MdFT1Overexpression of indicates that it causes lateral shoot outgrowth in transgenic tobacco plants. Additionally, while there was no significant difference in overall plant length between transgenic tobacco and WT tobacco (Fig. 9), the internode length of transgenic tobacco was observed to be significantly longer than that of WT tobacco; conversely, it was confirmed that although WT tobacco has a greater number of nodes than transgenic tobacco, its internode length is shorter (Fig. 10). Since this internode elongation in the stem is a result of mitotic activity and cell expansion, the difference in internode elongation at the cellular level between transgenic tobacco and WT tobacco was examined using an optical microscope. The results showed that the cell size of transgenic tobacco was larger than that of WT tobacco in both width and length (Fig. 11). Specifically, the cell length of genetically modified tobacco was found to be 1.5 times longer than that of WT tobacco, and the length-to-width (L / W) ratio was found to be 3.0 and 2.1 for genetically modified and WT tobacco, respectively, confirming a significant difference. In addition, the number of cells in WT tobacco was found to be 1.6 times greater than that of genetically modified tobacco. Through this, MdFT1 It was confirmed that the overexpression of [it] not only causes branching sprouts but also alters plant structure by affecting internode length through cell elongation.

[0077] 4-3. Changes in Leaf Morphology

[0078] Produced in the above example MdFT1To compare the leaf phenotype (shape and size) of transgenic tobacco overexpressing the gene with that of WT, the leaf area of ​​transgenic tobacco and WT tobacco was measured using an LI-3100 area meter (LI-COR Inc., Lincoln, NE, USA), and to measure LMA (leaf mass per area), six leaflets of tobacco without leaf veins were cut per leaf using a cork border with a diameter of 1.6 cm, and the weight of the cut leaves was measured and calculated by applying it to the following mathematical formula 1.

[0079]

[0080] LM: Mass of perforated leaves (mg); and

[0081] A: πr 2 Area of ​​perforated leaves according to (r: 0.8 cm; π3.14) (cm²) 2 ).

[0082] Observations of changes in leaf morphology in 35S::MdFT1 transgenic tobacco revealed differences in leaf shape and size between the transgenic tobacco and WT tobacco. Specifically, the leaves of the transgenic tobacco were oblanceolate, whereas those of WT tobacco were ovate (Fig. 12). Additionally, the leaf length-to-width (L / W) ratio was found to be higher in the transgenic tobacco compared to WT tobacco (2.1 and 1.8, respectively), and the leaf mass per area (LMA) was higher in the transgenic tobacco (13.6 mg / cm²). 2 ) WT tobacco (23.6 mg / cm² 2 It was found to be lower compared to ) (Fig. 12), MdFT1It was found that the overexpression of [it] influenced the development of thin leaves. To confirm differences at the cellular level that could affect leaf thickness, leaf morphology was evaluated at the cellular level using an optical microscope. The results showed that the mesophyll tissue layers in the leaves of transgenic tobacco were fewer than those of WT tobacco, resulting in thinner leaves (Fig. 12). That is, MdFT1 Significant differences in the thickness of tobacco leaves occurred due to the overexpression of

[0084] 4-4. Changes in Flower Shape

[0085] Produced in the above example MdFT1Observation of the flower organs of the gene-overexpressing transgenic tobacco 35S::MdFT1 revealed that the development of flower organs in transgenic tobacco was distinct compared to WT tobacco. While there were no differences in the components of the flower organs (5 sepals, 5 petals, 5 stamens, and 1 pistil), differences in their size and shape were observed between transgenic and WT tobaccos (Fig. 13). Specifically, the length of the flower and flower tubo in transgenic tobacco was longer and the width was smaller compared to WT (Figs. 13A and B). Additionally, the length of the style in transgenic tobacco flowers was shorter than in WT, while the filaments were longer (Figs. 13C and D). Furthermore, the flowers of transgenic tobacco exhibited a "down-pin" filament-style type, whereas the flowers of WT tobacco were observed to have an "up-pin" type (Fig. 13C). In addition, the stigma in the transgenic tobacco flower was found to be smaller compared to WT (Fig. 13D). This suggests that there may be differences in pollination and fertility capabilities. Furthermore, the length of the flower organs in the transgenic tobacco was found to be longer and the width larger compared to WT, and when compared using the length-to-width (L / W) ratio, the L / W ratio of the transgenic tobacco was found to be significantly larger than that of WT tobacco (Fig. 13).

[0087] 4-5. Changes in Fruits and Seeds

[0088] Produced in the above example MdFT1When comparing the development of the fruit of a genetically modified tobacco that overexpresses the gene with that of WT tobacco, there was no significant difference in the shape and size of the fruit (Fig. 14), but the genetically modified tobacco had more flowers and fruits per plant than WT tobacco, and the number of seeds in the fruit of the genetically modified tobacco was significantly increased compared to WT tobacco, and the weight of the seeds was also significantly higher in the genetically modified tobacco (Figs. 14 and 15).

[0090] Example 5. MdFT1 Confirmation of changes in expression of related genes due to overexpression

[0091] 5-1. Changes in Expression of Endogenous Flowering Genes

[0092] As mentioned above MdFT1 Since gene overexpression acted as a floral inducer responsible for the transition from the vegetative phase to the reproductive phase, the expression levels of endogenous floral integrator genes such as NFL1, AP1, SOC1, FT, and CEN were analyzed by qRT-PCR.

[0093] in result, MdFT1 Due to gene overexpression, the expression of flowering inducer genes NtAP1, NtSOC1, NFL1, and NtFTs in transgenic tobacco was upregulated compared to WT tobacco, and the expression of the CEN gene, which is highly expressed in vegetative axillary meristem, was downregulated (Fig. 16).

[0095] 5-2. Changes in the Expression of CCD Genes

[0096] As a result of analyzing the expression of CCD genes involved in the biosynthesis of strigolactones (SLs), which are phytohormones that regulate branching and affect plant structure, using qRT-PCR, MdFT1CCD genes in transgenic tobacco by gene overexpression ( NtCCD1-3 , NtCCD4-1 and NtCCD4-2 It was found that the expression of ) was downregulated (Fig. 17), and through this, it was inferred that the structure of the plant was changed by increasing the length of the nodes through cell elongation and promoting axillary buds and branching sprouts.

Claims

Claim 1 delete Claim 2 delete Claim 3 delete Claim 4 delete Claim 5 MdFT1 Transform tobacco with a recombinant vector containing the (Malus x domestica flowering locus T) gene MdFT1 Overexpressing genes, NtCCD1-3 , NtCCD4-1 and NtCCD4-2 A method for promoting branching buds in tobacco and increasing the internode length of the stem, comprising a step of reducing gene expression. Claim 6 delete Claim 7 delete Claim 8 delete Claim 9 delete Claim 10 delete Claim 11 delete Claim 12 delete Claim 13 delete

Images