A method for controlling the flowering time and the relevant phenotype(leaf, flower, fruit) mediated by the antisense expression vector of apple MdTFL1

Abstract

The present invention relates to a recombinant vector expressing the antisense of MdTFL1 and its uses. Transgenic plants produced by the Agrobacterium-mediated transformation method using the MdTFL1 antisense-expressing recombinant vector containing MdTFL1 in the reverse direction (antisense direction) of the present invention have the MdTFL1 antisense construct gene integrated into the plant's chromosome. Consequently, the phenotypes of the plant's structure, leaves, flowers, and fruits have changed, and the vegetative stage has been reduced, the heading date has been shortened, and seed production has increased. Therefore, the recombinant vector of the present invention can be usefully utilized for controlling the flowering response and shortening the breeding period of perennial crops.

Description

Technology Field

[0001] The present invention MdTFL1 This relates to a recombinant vector expressing antisense and its uses. 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)). In plants, double-stranded RNA molecules can induce sequence-specific silencing. This phenomenon is often referred to as double-stranded RNA ("dsRNA") in plants. This gene-specific silencing is often referred to as RNA interference or RNAi (Fire et al., Nature 391:806–811 (1988)). Other studies have also reported this phenomenon in plants, fungi, and animals (Sharp, Genes and Development 13:139–141 (1999); Matzke et al., Curr. Opin. Genet. Dev, 11:221–227 (2001); Cogoni and Macino, Curr. Opin. Genet.Dev, 10:638–643 (2000); Sharp, Genes and Development 15:485–490 (2001); Waterhouse et al., PNAS USA 95:13959–13964 (1988); Wesley et al., Plant J. 27:581–590 (2001); Grierson, WO 98 / 53083)).

[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... MdTFL1 The purpose is to provide a recombinant vector for inhibiting gene expression.

[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 increasing the number of tobacco seeds.

[0010] In addition, the objective of the present invention is to provide a method for shortening the heading date of rice.

[0011] In addition, the object of the present invention is MdTFL1 The present invention provides a method for producing a transgenic plant in which gene expression is suppressed. means of solving the problem

[0012] To solve the above problem, the present invention MdTFL1 Provides a recombinant vector containing an antisense nucleotide for all or part of a gene.

[0013] In addition, the present invention provides a transformant transformed with the recombinant vector.

[0014] In addition, the present invention relates to a transformed organism that has been transformed into the above-mentioned transformant. MdTFL1 Antisense expression transformation Provides a plant body.

[0015] In addition, the present invention provides a method for increasing the number of tobacco seeds.

[0016] In addition, the present invention provides a method for shortening the heading date of rice.

[0017] In addition, the present invention MdTFL1 A method for producing a transgenic plant in which gene expression is suppressed is provided. Effects of the invention

[0018] The present invention MdTFL1 is included in the reverse direction (antisense direction) MdTFL1 Transgenic plants produced using an antisense-expressing recombinant vector via the Agrobacterium-mediated transformation method MdTFL1 Since the antisense construct gene is integrated into the plant's chromosome, resulting in changes to the plant's structure, leaves, flowers, and fruits, as well as characteristics such as reduced vegetative stages, reduced heading time, and increased seed production, the recombinant vector of the present invention can be usefully utilized for controlling flowering responses and shortening the breeding period of perennial crops. Brief explanation of the drawing

[0019] Figure 1 is a schematic diagram of the T-DNA region of the expression vector and the evaluation results of the transgenic plant: A: Expression band of MdTFL1 mRNA transcript using RT-PCR (MDP0000336547 (SGF29 Tudor-like domain): control); B: Expression vector containing MdTFL1-antisense located between the CaMV 35S promoter and the TNOS terminator for nopaline synthesis: LB: T-DNA right boundary; hptII: Hygromycin phosphotransferase; 35SpolyA: Terminator of the 35S gene; and LB: T-DNA left boundary; C: Target genes through PCR analysis of genomic DNA ( MdTFL1 ) and detection results of the selected marker hptII: PC: Positive control (plasmid DNA of 35S::MdTFL1); NC: Negative control (WT's genomic DNA); M: DNA ladder marker; and 35S::MdTFL1: Each independent transformant line. Figure 2 is a schematic diagram of the T-DNA region of the expression vector. Figure 3 is a figure showing the DNA sequence analysis and protein sequence analysis of the target gene. Figure 4 illustrates the process of producing transgenic tobacco through in vitro tissue culture using the Agrobacterium-mediated transformation method: A: Leaf disc explant from a tobacco seedling used for Agrobacterium-mediated transformation; B: Irregular branches appearing in calluses induced on selective medium MSSE; C: Continuous subculture of irregular branches; D: Propagation of transformed shoots isolated from callus; E: Obtaining seedlings from candidate transgenic plants using a rooting medium; and F to I: Growth process in a culture medium after establishment of a seedling containing roots. Figure 5 illustrates the process of producing transgenic rice through in vitro tissue culture using the Agrobacterium-mediated transformation method: A: Germination of rice seeds; B: Co-culture of embryonic rice callus and transformed Agrobacterium containing an expression vector of the MdTFL1 gene; C: Selection of transgenic rice callus; D: Propagation of transgenic rice callus; and E to F: Culture of transgenic seedlings using a rooting medium. Figure 6 is a figure confirming early flowering of transgenic tobacco expressing MdTFL1 antisense: A: Buds and flowers of 35S::MdTFL1 transgenic tobacco lines of 42 or 46 DAT and WT tobacco; B: Date of flower appearance of transgenic tobacco lines and WT tobacco; and C: Time of first flowering and number of leaves of transgenic tobacco lines and WT tobacco. Figure 7 illustrates bud formation and plant structural changes induced by MdTFL1 antisense expression in tobacco: A: Flowering of branching and inflorescence meristems according to DAT: Yellow arrow: Branch; and Red arrow: Main shoot; B: Total height of the plant and internode distance; and C: Photograph of vertical section between stem tissue / nodes and cell size / number. Figure 8 is a figure analyzing leaf morphology in tobacco by MdTFL1 antisense expression: A: Photo comparing the sizes of the apical, middle, and basal leaves; B: Leaf size and LMA evaluation results; and C: Horizontal section of leaf tissue cell microscope. Figure 9 shows the changes in the flower phenotype in tobacco caused by MdTFL1 antisense expression: A: Total size; B: Anatomical diagram of the flower organs (sepal, petal, and carpel-ovary); C: Size of the flower tubo; and D: Close-up view of the "down-pin" type filament-style position and stigma; and E: Flower shape evaluation result. Figure 10 is a figure confirming changes in seed and fruit production in tobacco due to MdTFL1 antisense expression: A: Tobacco fruit in the early and mature developmental stages; B: Peeled young fruit: Circle marked part: Seed; C: Fruit-bearing branch; D: Seed per fruit; E: Number of fruits per plant; and F: Dry weight of seeds per fruit. Figure 11 shows the heading date in rice due to MdTFL1 antisense expression. Figure 12 is a figure analyzing the semi-dwarf phenotype of transgenic rice expressing MdTFL1 antisense. Figure 13 is a figure analyzing the leaf angle and morphology of transgenic rice expressing MdTFL1 antisense. Figure 14 is a figure confirming the development of a transgenic rice expressing MdTFL1 antisense. Figure 15 is a figure confirming the grain development of transgenic rice expressing MdTFL1 antisense. Figure 16 is a figure confirming the expression pattern of endogenous flowering-related genes in tobacco due to MdTFL1 antisense expression: A: NFL1, NtAP1 and NtSOC1; and B: NtFT. Figure 17 is a figure confirming the expression pattern of endogenous flowering-related genes in tobacco due to MdTFL1 antisense expression: A: CEN; and B: CCD genes including NtCCD8, NtCCD1-3, NtCCD4-1, and NtCCD4-2. Fig. 18 is M This is a diagram confirming the expression patterns of flowering-related genes in rice due to MdTFL1 antisense expression. Specific details for implementing the invention

[0020] 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.

[0021] 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.

[0022] 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.

[0024] In one aspect, the present invention MdTFL1 (GenBank ID: AB052994.1) Containing an antisense nucleotide for all or part of a gene, MdTFL1 This concerns a cassette for inhibiting gene expression.

[0025] In one embodiment, on the cassette MdTFL1 All or part of the gene can be inserted in reverse, and MdTFL1 It is more desirable that the entire gene be inserted in the reverse direction (antisense direction).

[0026] In one embodiment, the cassette is MdTFL1 It may encode antisense RNA, siRNA (small interfering RNA), amiRNA (artificial micro RNA), shRNA (short hairpin RNA), or RNA capable of producing these for all or part of the gene.

[0027] In one embodiment, the antisense nucleotide is MdTFL1 It can be complementary to the gene's transcript.

[0028] Terms of the present invention, " MdTFL1 The "gene" is the gene encoding the TERMINAL FLOWER 1-like protein. MdTFL1 It means, and its nucleotide sequence can be obtained from known databases such as NCBI's GenBank (e.g., GenBank Accession: AB052994.1, etc.). Specifically, the above gene is an 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.

[0029] The term "cassette for inhibiting gene expression" in this invention refers to a target gene MdTFL1 It refers to an expression structure that can inhibit the expression of.

[0030] For the purposes of the present invention, the cassette is a base fragment homologous to the target gene, i.e. MdTFL1 It can have a structure in which all or part of it is introduced in the reverse direction.

[0031] The nucleotide sequences used in the present invention are interpreted to include sequences that exhibit substantial identity with the sequences listed in the sequence list, provided that variations having biologically equivalent activity are taken into account. The term "substantial identity" refers to a sequence that exhibits at least 60% homology, more specifically 70% homology, even more specifically 80% homology, and most specifically 90% homology when the sequence of the present invention is aligned with any other sequence to correspond as much as possible and the aligned sequence is analyzed using an algorithm commonly used in the art.

[0032] In one aspect, the present invention relates to a recombination vector comprising the above cassette.

[0033] In one embodiment, the recombination vector is MdTFL1 It may include antisense nucleotides for all or part of the gene.

[0034] In one embodiment, to the recombination vector MdTFL1 All or part of the gene (GenBank ID: AB052994.1) may be inserted in reverse, and MdTFL1 It is more desirable that the entire gene be inserted in the reverse direction (antisense direction).

[0035] In one embodiment, the recombination vector is MdTFL1 It may encode antisense RNA, siRNA, amiRNA, shRNA, or RNA capable of producing these for all or part of the gene.

[0036] In one embodiment, the recombinant vector may additionally include a transcription regulator, a translation regulator, or a marker capable of confirming gene expression.

[0037] 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.

[0038] 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).

[0039] 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).

[0040] 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.

[0041] 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.

[0042] In one embodiment, the recombinant vector may be pBI121, pROKII, pBI76, pET21, pSK(+), pLSAGPT, pUC, pGEM, pHellsgate8, pPZP, pGA, or pCAMBIA.

[0043] In one embodiment, the recombination vector is MdTFL1 It can be used to inhibit gene expression.

[0044] In one embodiment, the recombinant vector may be a recombinant vector for inducing early flowering, shortening the heading date, inducing semiwarf, shortening the breeding period, controlling plant phenotype, altering plant structure, increasing seeds, or promoting the transition from the vegetative phase to the reproductive phase.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] In one aspect, the present invention relates to a transformant transformed with the recombinant expression vector of the present invention.

[0050] In one embodiment, the transformant may be an Agrobacterium sp. transformant transformed with a recombinant vector, and may be Agrobacterium tumefaciens and Agrobacterium rhizogenes.

[0051] In one embodiment, the transformant comprises the recombinant vector. MdTFL1 It may be Agrobacterium tumefaciens for inhibiting gene expression.

[0052] 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.

[0053] 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)).

[0054] 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.

[0055] 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.

[0056] In one aspect, the present invention relates to a plant body transformed into a transformant comprising a recombinant vector of the present invention.

[0057] In one embodiment, the transformed plant is MdTFL1 By expressing the antisense of the gene MdTFL1 It may be a transgenic plant in which inhibition has been suppressed.

[0058] 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; flowers 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 rice or tobacco.

[0059] In one embodiment, the expression of the NtFT, NFL1, NtAP1, or NtSOC1 genes in the transformed plant may be upregulated.

[0060] In one embodiment, the expression of the NtCCD8, NtCCD1-3, NtCCD4-1, NtCCD4-2, NtCET2, or NtCET4 genes in the transformed plant may be downregulated.

[0061] 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.

[0062] 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.

[0063] In one aspect, the present invention relates to a method for increasing tobacco seeds, comprising the step of introducing the cassette of the present invention into a tobacco.

[0064] In one aspect, the present invention relates to a method for shortening the heading date of rice, comprising the step of introducing the cassette of the present invention into rice.

[0065] In the present invention, the step of introducing the cassette of the present invention into a plant may be the step of introducing a recombinant vector containing said cassette into a plant, and this may be performed by a transformation technique known to those skilled in the art.

[0066] 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.

[0067] 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.

[0069] 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.

[0071] Example 1. MdTFL1 Production of antisense expression vectors

[0072] MdTFL1 (GenBank ID: AB052994.1) To identify the functional characteristics of a gene and the phenotypic changes in plants resulting from its inhibition, MdTFL1A vector expressing the antisense of a gene was constructed. Specifically, total RNA was isolated from various tissues of Fuji apples using the CTAB method, and the expression levels in each tissue were confirmed. The results showed that expression was strong in shoots, flower buds, stems, and roots, but low in leaves, flowers, and fruits (Fig. 1A). Consequently, total RNA from flower buds was used to synthesize cDNA encoding MdTFL1 by RT-PCR using the PrimeScript™ 1st strand cDNA Synthesis Kit (Cat. #6110A, Takara, Kusatsu, Japan). Subsequently, to clone the gene into the expression vector, PCR was performed using primers designed to include KpnI and BamHI restriction enzyme 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 DNA fragments from the target band of the gene were purified using a DNA purification kit (iNtRON MEGAquick-spin™ Plus, Seongnam, Korea). The purified DNA fragments were ligated into the pGEM®-T Easy vector (Cat. # A1360, Promega, Madison, WI, USA) under the control of the cauliflower mosaic virus 35S promoter and the pCAMBIA1300 vector (Cat. # VET1570, Biogene) under the control of the RAmy 3D promoter, respectively, and then transduced into DH5α E. coli cells (Cat. #9057, Takara, Kusatsu, Japan). After analyzing the sequences of the thus prepared plasmid DNA, MdTFL1(GenBank Accession No. AB052994.1) was verified (Macrogen, Seoul, Korea). Subsequently, the above plasmid DNA was treated with BamHI and KpnI restriction enzymes and cloned into the binary vector pCAMBIA1300 (CAMBIA, Canberra, ACT, Australia), which had been treated with the same restriction enzymes. At this time, MdTFL1 Recombinant expression vectors containing the 35S::MdTFL1 antisense construct and the RAmy3D::MdTFL1 antisense construct, respectively, were constructed by ligating the gene into a binary vector in an antisense-directed or reverse (antisense) manner (Figs. 1B and 2).

[0074] Example 2. MdTFL1 Production of Agrobacterium containing an antisense expression vector

[0075] MdTFL1 Agrobacterium was constructed to transform a vector expressing the antisense of into a plant. Specifically, Agrobacterium tumefaciens ( Agrobacterium tumefaciensAfter introducing into EHA105, the transformed EHA105 was 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 with shaking at 200 rpm at 28°C. Plasmid DNA was isolated from the cultured Agrobacterium tumefaciens EHA105, and the presence of the target gene was confirmed. Additionally, alignment of the DNA sequence of the target gene with the protein sequence revealed an error of two nucleotides, but it was confirmed that this did not cause a change in the amino acid sequence (Fig. 3).

[0077] Example 3. MdTFL1 Production of antisense-expressing plants

[0078] 3-1. MdTFL1 Production of transgenic cigarettes expressing antisense genes

[0079] Using the Agrobacterium produced in Example 2 above, tobacco ( N. tabacum The leaf discs of ) were transformed with the 35S::MdTFL1 antisense construct. Specifically, leaf discs from tobacco were 0.5 cm 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::MdTFL1 antisense construct) prepared in Example 2 (Fig. 4A). After Agrobacterium-mediated transformation, the leaf discs were placed upside down on solid MSCO medium containing 4 g / L gelling agent for 3 days under dark conditions (facing 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 washed by gently shaking 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. 4B), after which they were exposed to light and cultured for an additional week. Transformed shoots were excised at 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·m⁻¹. -2 ·s -1 They were cultured and subcultured every 3 weeks (Figs. 4C and D). 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) (Fig. 4E), and after establishing plantlets including roots in vitroThe seedlings were maintained to continue growing in the culture medium (Figs. 4F to 4I). Subsequently, to confirm the insertion of a T-DNA region containing the selected marker hygromycin phosphotransferase (htpII) and the target gene (MdTFL1), leaves were cut from the transformed 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).

[0080] As a result, positive bands of the expected size for both the selection marker (htpII) and the target gene (MdTFL1) were confirmed in leaves isolated from selected seedlings (Fig. 1C), and since the said bands did not appear in WT tobacco plants that had not undergone transformation, the present invention MdTFL1 It was confirmed that the antisense was successfully incorporated into the chromosomes of the transgenic tobacco plants. The 35S::MdTFL1 homozygous transgenic lines selected through this analysis were planted in soil and grown in a greenhouse under long-day conditions of 25°C.

[0082] 3-2. MdTFL1 Production of rice expressing antisense genes

[0083] Rice leaf discs were transformed into a RAmy3D::MdTFL1 antisense construct using the Agrobacterium prepared in Example 2 above. Specifically, mature rice seeds were hulled and surface-sterilized in a clean bench, then inoculated onto a callus induction medium (N6 Cl) at 60 μmol / m² 2Culture was performed for 3 weeks under conditions of s (approx. 2000 lux), a photoperiod of 16 h / 8 h, and 25°C. Subculture was performed after 2 weeks of culture, and calluses from 1 week of subculture were used. After 3 weeks of culture, the endosperm and shoot portions were removed, and the blastoderm-derived calluses were transplanted into new callus induction medium (N6 Cl) and incubated under light at 25°C for 3 days. Subsequently, the blastoderm-derived calluses were co-cultured with the Agrobacterium (including the RAmy3D::MdTFL1 antisense construct) prepared in Example 2 above, and Agrobacterium-mediated transformation was performed as in Example 3-1 above. MdTFL1 Transgenic rice was produced in which an antisense construct gene was integrated into the rice chromosome (Fig. 5).

[0085] Example 4. MdTFL1 Observation of the phenotype of antisense-expressing transgenic tobacco

[0086] 4-1. Early blooming phenotype

[0087] Produced in the above example MdTFL1 As a result of observing the phenotype of transgenic tobacco containing an antisense gene, in vitro An early flowering phenotype was observed in the seedlings (Figs. 4F to 4I). 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. To evaluate the genetic stability of the transgenic plants, the transgenic lines (lines #2 and #6) that showed the earliest flowering were selected, transferred to soil, and germinated into 30 plants / lines. As a result, MdTFL1All 35S::MdTFL1 homozygous T1 plant lines expressing the antisense gene were found to flower significantly earlier than the wild type (WT) (Fig. 6). Floral buds were observed 42 days after transferring to soil, and full bloom occurred at 46 days after transferring, leaving only 6 to 7 leaves (Fig. 6A). In contrast, the WT plants were in a non-flowering vegetative stage, and flowering was delayed by 27 days compared to the aforementioned transgenic plants. The average time to flowering was 48.7 ± 2.3 DAT and 75.8 ± 3.5 DAT for transgenic tobacco and wild-type tobacco, respectively (Fig. 6B). Additionally, the number of leaves required for first flowering was 6.8 ± 0.8 and 13.8 ± 1.0 for transgenic tobacco and wild-type tobacco, respectively (Fig. 6C). In addition, the flowering times of 35S::MdFTL1 transgenic line #2 (#2-T1) and line #6 (#6-T1) were 50.6 ± 0.8 days and 50.7 ± 0.5 days after sowing (DAS), respectively, while the WT was 73.6 ± 4.8 days. Through this, MdTFL1 It was confirmed that the early flowering phenotype of transgenic tobacco containing an antisense gene is conserved in transgenic plants and inherited by the next generation.

[0089] 4-2. Changes in Plant Structure

[0090] Produced in the above example MdTFL1Observation of the phenotype of transgenic tobacco containing the antisense gene revealed that flowering was observed in the (45 DAT) 35S::MdTFL1 transgenic tobacco following the development of the terminal inflorescence meristem, and its main shoot also produced some axillary buds. After the last flower withered (55 DAT), the main shoot did not grow, while the malt rapidly differentiated into lateral shoots. Subsequently, the shoots developed into inflorescence meristems, and both the main shoot and the inflorescence meristems bloomed, whereas in the WT plant, no shoots formed from the main shoot (Fig. 7A). This MdTFL1This indicates that the expression of antisense 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, the internode length of transgenic tobacco was observed to be significantly longer than that of WT tobacco (Fig. 7B), confirming that although WT tobacco has a greater number of nodes than transgenic tobacco, its internode length is shorter. 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 (approximately 1.6 times) in both width and length compared to WT tobacco (Fig. 7C), and the length-to-width (L / W) ratio was 3.2 and 2.2 for transgenic tobacco and WT tobacco, respectively, confirming a significant difference. In addition, the number of cells in WT tobacco was found to be 1.9 times higher than that of transgenic tobacco. Through this, it was confirmed that antisense expression of MdTFL1 not only causes branching buds but also alters plant structure by affecting internode length through cell elongation.

[0092] 4-3. Changes in Leaf Morphology

[0093] Produced in the above example MdTFL1To compare the leaf phenotype of transgenic tobacco containing an antisense 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 leafless tobacco leaves were cut 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.

[0094]

[0095] LM: Mass of perforated leaves (mg); and

[0096] A: πr 2 Area of ​​perforated leaves according to (r: 0.8 cm; π3.14) (cm²) 2 ).

[0097] Observations of changes in leaf morphology in 35S::MdTFL1 antisense transgenic tobacco revealed that the leaf shapes and sizes differed between transgenic tobacco and WT tobacco. Specifically, the leaves of transgenic tobacco were oblanceolate, whereas those of WT tobacco were ovate. In particular, the apical, medial, and basal leaves of transgenic tobacco were observed to be smaller than those of WT tobacco (Fig. 8A). Additionally, the leaf length-to-width (L / W) ratio was found to be larger in transgenic tobacco compared to WT tobacco (2.7 and 2.2, respectively) (Fig. 8B), and LMA was found to be lower in transgenic tobacco compared to WT tobacco (Fig. 8B), indicating that antisense expression of MdTFL1 influenced the development of thin leaves. To identify differences at the cellular level that could affect leaf thickness, the morphology of the leaves was evaluated at the cellular level using an optical microscope, and it was found that the mesophyll tissue layers of the transgenic tobacco leaves were fewer and thinner than those of the WT tobacco leaves (Fig. 8C). In other words, the difference in tobacco leaf thickness was significantly caused by the antisense expression of MdTFL1.

[0099] 4-3. Changes in Flower Morphology

[0100] Produced in the above example MdTFL1Observation of the flower organs of transgenic tobacco containing an antisense gene (35S::MdTFL1-antisense transgenic lines) revealed that the development of flower organs was more distinct in transgenic tobacco 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 tobacco (Figs. 9A and B). Specifically, the sepals of transgenic tobacco were longer and more serrated than those of WT tobacco, while the size of the ovary was found to be the same (Fig. 9B). However, the stigma in transgenic tobacco was found to be smaller than in WT tobacco (Figs. 9C and D). In addition, the filament-style position in the flowers of transgenic tobacco was observed to be "down-pin" (short style-tall filament), whereas in the flowers of WT tobacco, it was observed to be "up-pin" (tall style-short filament). Furthermore, in transgenic tobacco, the stigma (the tip of the style) was located lower than the anther (the tip of the filament), whereas in WT tobacco, the opposite was observed. This suggests that there may be differences in pollination and fertility capabilities. In addition, the shape of the flower organs of the transgenic tobacco was found to be longer and narrower compared to WT (Fig. 9C). When compared using the length-to-width (L / W) ratio of the flower tubo, the average lengths of the flower tubos of the transgenic tobacco and WT were 48.62 ± 1.08 and 40.03 ± 1.41, respectively, and the average widths were 7.08 ± 0.43 and 9.98 ± 0.74, respectively, indicating that the L / W ratio of the transgenic tobacco was 1.72 times greater than that of the WT tobacco (Fig. 9E).In addition, the transgenic tobacco was found to have a higher number of flowers compared to WT tobacco (number of flowers per plant: WT: 13.8 ± 1.5 and transgenic: 22.5 ± 3.6, respectively) (Fig. 9E), which was inferred to affect seed / fruit formation and yield.

[0102] 4-3. Changes in Fruits and Seeds

[0103] Produced in the above example MdTFL1 When the development of the fruit of transgenic tobacco containing an antisense gene was compared with that of WT tobacco, there was no difference in the shape and size of the fruit between the two tobacco lines (Fig. 10A), but the fruit of transgenic tobacco was found to have more seeds than that of WT tobacco (Figs. 10B to D). In addition, the number of fruit of transgenic tobacco was significantly higher than that of WT tobacco (21.7 ± 2.6 and 12.6 ± 1.4, respectively) (Fig. 10E), and the weight of the seeds was also significantly higher in transgenic tobacco (103.6 ± 16.9 mg and 61.0 ± 7.3, respectively) (Fig. 10F).

[0105] Example 5. MdTFL1 Observation of the phenotype of antisense-expressing transgenic rice

[0106] 5-1. Shortening the watering period

[0107] Produced in the above example MdTFL1 When comparing the heading dates of transgenic rice (35S::MdTFL1) containing an antisense gene and WT rice, it was found that the transgenic rice had a heading date up to 20.9 days earlier than WT rice, and WT rice was still in the trophic stage (Fig. 11).

[0109] 5-2. Changes in Rice Length

[0110] Produced in the above example MdTFL1Comparing the lengths of transgenic rice containing an antisense gene and WT rice, it was found that the transgenic rice exhibited a semi-warf phenotype. Specifically, the length of the transgenic rice was on average 20.1 cm shorter than that of WT rice, and notably, the first and second internode lengths were 17.9 ± 4.0 and 11.4 ± 2.1 cm, respectively, which were 4.9 and 4.4 cm shorter than those of WT rice (Fig. 12).

[0112] 5-3. Changes in Leaf Angle and Shape

[0113] Produced in the above example MdTFL1 As a result of comparing the leaf angles and morphology of transgenic rice containing an antisense gene and WT rice, it was found that at 30 DAS (Days after sowing), the development angles of the 3rd and 4th leaves in transgenic rice were 63.5° and 68.0°, respectively, which were significantly higher than those in WT rice (29.3° and 35.4°), and the leaf length of transgenic rice was 9.3 cm shorter than that of WT rice (Fig. 13).

[0115] 5-4. Changes in Seed Development

[0116] Produced in the above example MdTFL1 When comparing the degree of grain development of transgenic rice containing an antisense gene and WT rice, the transgenic rice developed smaller ears than WT rice (Fig. 14), and when comparing grain productivity, the number of grains per ear of transgenic rice and WT rice was 63 ± 5.9 and 45 ± 3.2, respectively (Fig. 15).

[0118] Example 6. MdTFL1 Confirmation of changes in expression of related genes caused by antisense genes

[0119] 6-1. MdTFL1 Antisense expression of... Changes in expression of related genes in tobacco

[0120] Since the transition from the vegetative phase to the reproductive phase regulates floral integrators such as NFL1, AP1, SOC1, FT, and CEN, MdTFL1 The expression levels of genes related to metabolic pathways for flowering, including flowering complexes, were analyzed by qRT-PCR in transgenic tobacco expressing antisense, and for branching-related genes, the expression of CCD genes related to the biosynthesis of strigolactones (SLs), which are phytohormones that regulate branching and affect plant structure, was analyzed.

[0121] As a result, the relative expression levels of NFL1, NtAP1, and NtSOC1 genes were significantly higher in transgenic tobacco compared to WT tobacco (Fig. 16A), NtFT expression was upregulated in transgenic tobacco (Fig. 16B), and CEN gene expression (NtCET2 and NtCET4) was downregulated in transgenic tobacco (Fig. 17A). In addition, CCD genes, including NtCCD8, NtCCD1-3, NtCCD4-1, and NtCCD4-2, were all found to be downregulated in transgenic tobacco (Fig. 17B).

[0123] 6-2. MdTFL1 Changes in flowering gene expression in antisense-expressing rice

[0124] MdTFL1 As a result of analyzing the expression of flowering-related genes in transgenic rice expressing the antisense using qRT-PCR, in the transgenic rice lines (#5-1, #5-2, #5-3, #5-4) MdTFL1 It was confirmed that all expression was induced (Fig. 18).

Claims

Claim 1 delete Claim 2 delete Claim 3 delete Claim 4 delete Claim 5 delete Claim 6 MdTFL1 Gene (Malus domestica) TERMINAL FLOWER 1 A method for shortening the heading date of rice, comprising the step of transforming rice with a recombinant vector containing an antisense nucleotide for all or part of a -like gene. Claim 7 MdTFL1 Gene (Malus domestica) TERMINAL FLOWER 1 A method for inducing semiwarf in rice comprising the step of transforming rice with a recombinant vector containing an antisense nucleotide for all or part of a -like gene. Claim 8 delete Claim 9 delete Claim 10 delete Claim 11 delete Claim 12 delete Claim 13 delete Claim 14 delete Claim 15 delete Claim 16 delete

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