Lily plant with delayed flower senescence and method for producing the same

Abstract

To provide a technique for preserving the quality of flowers of Liliaceae plants in general that can delay flower senescence.SOLUTION: Provided are double-stranded nucleic acids including factors that suppress LhAPS such as (I): (A) a specific amino acid sequence (LhAPS itself); (B) (B-1) a sequence variant of (A) in which one or more amino acids have been substituted, added, or deleted; (B-2) a sequence variant of (A) having at least about 70% sequence identity therewith; or (B-3) (A) a sequence variant of (A) that hybridizes to (A) under stringent conditions and comprising a sequence having the biological function of (A). The other strand (II) contains the complementary sequence to (I).SELECTED DRAWING: None

Description

Technical Field

[0001] The present disclosure provides a transcription factor family gene (LhAPS) that controls flower senescence and a method of using the same. For example, by a method such as genome editing, RNA interference, or introduction of a mutagen, it is possible to delay flower senescence by partially or completely deleting the expression or function of the LhAPS gene.

Background Art

[0002] In tulips, no effective preservative has been developed, and no gene that controls flower senescence has been identified. In addition, in conventional tulip breeding based on crossing, it takes about three years from crossing to flowering, so it requires a very long time and is inefficient.

[0003] In conventional tulip breeding based on crossing, it takes about three years from crossing to flowering. Therefore, it requires a very long time and is inefficient to repeatedly perform crossing and selection to impart the target traits. In addition, the vase life characteristics of each strain have not been sorted out, and no DNA markers related to vase life have been developed.

Summary of the Invention

Means for Solving the Problems

[0004] There is a need to develop effective quality retention techniques for flowers in general. Therefore, as a result of intensive research, the present disclosure has identified a gene that controls flower senescence in flowers, which is conserved at least in all plants of the Liliaceae family, and has developed a quality retention technique.

[0005] Therefore, the present disclosure provides the following. (Item 1) A nucleic acid molecule comprising a nucleic acid sequence encoding the nucleic acid sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 2. (Item 2) The nucleic acid molecule according to any one of the above items, wherein the nucleic acid molecule consists of the nucleic acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3. (Item 3) A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2. (Item 4) A factor that suppresses the expression of the nucleic acid sequence according to claim 1 or 2 or its sequence variant, and / or the amount or function of the nucleic acid sequence or its sequence variant, or the protein encoded by the nucleic acid sequence or its sequence variant or the polypeptide according to claim 3, wherein the sequence variant is (A-1) A sequence variant having one or more substitutions, additions and / or deletions in the nucleic acid sequence according to claim 1 or 2, (A-2) A sequence variant having at least about 70% sequence identity to the nucleic acid sequence according to claim 1 or 2, (A-3) A sequence variant that hybridizes to the nucleic acid sequence according to claim 1 or 2 under stringent conditions, (A-4) A sequence variant that is an allelic variant according to claim 1 or 2, or (A-5) A sequence variant that is a fragment of (A-1) to (A-4), and the protein encoded by the sequence variant has the biological function of the protein encoded by the nucleic acid sequence according to claim 1 or 2. (Item 5) The factor according to any one of the above items, wherein the factor comprises a protein, polypeptide, oligopeptide, peptide, polynucleotide, oligonucleotide, nucleotide, nucleic acid containing RNA or DNA, polysaccharide, oligosaccharide, lipid, hormone, ligand, signaling substance, synthetic chemical, radiation, or a combination thereof. (Item 6) The factor according to any one of the above items, which is selected from the group consisting of an antisense molecule, an RNAi factor, a factor for genome editing, or a mutagenic factor. (Item 7) A factor that suppresses LhAPS or its homologous gene. (Item 8) The factor according to any of the above items, wherein the factor is a protein, polypeptide, oligopeptide, peptide, polynucleotide, oligonucleotide, nucleotide, nucleic acid including RNA or DNA, polysaccharide, oligosaccharide, lipid, hormone, ligand, signaling substance, synthetic chemical substance, or a complex molecule thereof. (Item 9) The factor according to any of the above items, wherein the factor is in the form of a nucleic acid. (Item 10) The factor according to any of the above items, which is selected from the group consisting of an antisense molecule, an RNAi factor, a factor for genome editing, or a mutagenesis factor. (Item 11) The factor according to any of the above items, wherein the factor is an RNAi factor of LhAPS in the form of a double-stranded nucleic acid. (Item 12) The factor is in a double-stranded form, and one strand is (I) (A) a nucleic acid sequence containing at least about 100 bases of the nucleic acid sequence shown in SEQ ID NO: 1 (LhAPS itself) or the nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 2; or (B) a sequence variant and the other strand is (II) a complementary sequence or an annealing sequence of (I) and the (B) sequence variant includes the (B) sequence variant is (B-1) a sequence variant having one or more substitutions, additions or deletions in the nucleic acid sequence of (A); (B-2) a sequence variant having at least about 70% sequence identity to the nucleic acid sequence of (A); (B-3) a sequence variant that hybridizes under stringent conditions to the nucleic acid sequence of (A); (B-4) a sequence variant that is an allelic variant of (A); or (B-5) a sequence variant that is a fragment of (A) or (B-1) to (B-4), and the protein encoded by the sequence variant has the biological function of the protein encoded by (A). The factor according to any of the above items. (Item 13) The factor according to any one of the above items, wherein (I) does not contain the nucleic acid sequence at positions 28 to 507 of SEQ ID NO: 1. (Item 14) The factor according to any one of the above items, wherein (I) contains the nucleic acid sequence at positions 676 to 986 of SEQ ID NO: 1. (Item 15) The factor according to any one of the above items, which is a vector containing an RNAi factor of LhAPS or its homologous gene. (Item 16) The factor according to any one of the above items, wherein the vector contains a promoter and a terminator. (Item 17) The factor according to any one of the above items, wherein the factor is for genome editing that suppresses the expression and / or function of LhAPS. (Item 18) The factor according to any one of the above items, wherein the factor for genome editing targets the nucleic acid sequence of the following sequence in SEQ ID NO: 1. [Table 3-1] [Table 3-2] [Table 3-3] (Item 19) The factor according to any one of the above items, wherein the factor for genome editing targets the genome editing of the nucleic acid sequence at positions 1 to 507 of SEQ ID NO: 1. (Item 20) A plant cell containing the factor according to any one of the above items or modified by the factor. (Item 21) A plant tissue containing the factor according to any one of the above items or modified by the factor, or a plant tissue containing the cell according to any one of the above items. (Item 22) A plant organ containing the factor according to any one of the above items or modified by the factor, or a plant organ containing the cell according to any one of the above items or the plant tissue according to any one of the above items. (Item 23) A plant body or a part thereof that contains a factor described in any of the above items or is modified by the factor, or a plant body or a part thereof that contains a cell, a plant tissue, or a plant organ described in any of the above items. (Item 24) The plant is a plant of the Liliaceae family, and is a plant body or a part thereof described in any of the above items. (Item 25) The plant is a plant of the Lilium genus, and is a plant body or a part thereof described in any of the above items. (Item 26) The plant body or a part thereof is a bulb, seed, flower, stem, leaf, pollen, scale, corm, cell, tissue, tissue culture, or ovary, or a combination of all or part of them, and is a plant body or a part thereof described in any of the above items. (Item 27) The plant body or a part thereof contains a flower or a part thereof, and is a plant body or a part thereof described in any of the above items. (Item 28) A plant cell in which at least a part of LhAPS or its homologous gene possessed by the plant is suppressed. (Item 29) A plant tissue in which at least a part of LhAPS or its homologous gene possessed by the plant is suppressed, or a plant tissue containing a cell described in any of the above items. (Item 30) A plant organ in which at least a part of LhAPS or its homologous gene possessed by the plant is suppressed, or a plant organ containing a cell described in any of the above items or a plant tissue described in any of the above items. (Item 31) A plant body or a part thereof in which at least a part of LhAPS or its homologous gene possessed by the plant is suppressed, or a plant body or a part thereof containing a cell described in any of the above items, a plant tissue described in any of the above items, or a plant organ described in any of the above items. (Item 32) A cell, tissue, organ, plant body, or a part thereof described in any of the above items, in which the expression and / or function of at least a part of the LhAPS or its homologous gene is suppressed. (Item 33) The inhibition described above is below the criterion of reducing the expression of the vacuolar processing enzyme gene (VPE gene) or the cysteine protease gene (SAG12 gene) by about 50% when measured by the RT-PCR method. The cell, tissue, organ, plant body, or a part thereof according to any one of the above items. (Item 34) The inhibition described above is below the criterion of reducing the expression of the vacuolar processing enzyme gene (VPE gene) by about 50% when measured by the RT-PCR method. The cell, tissue, organ, plant body, or a part thereof according to any one of the above items. (Item 35) The inhibition of LhAPS is achieved by a method selected from the group consisting of a decrease in gene expression level, a decrease in gene copy number, a decrease in gene amplification, a decrease in RNA activity level, a decrease in mRNA abundance, a decrease in mRNA synthesis rate, a decrease in mRNA stability, a decrease in protein activity level, a decrease in protein synthesis, a decrease in protein abundance, a decrease in protein stability, a decrease in protein enzyme activity, or a combination thereof. The cell, tissue, organ, plant body, or a part thereof according to any one of the above items. (Item 36) Whether at least a part of the structural gene of LhAPS or its homologous gene has a mutation as compared with the natural one, and / or whether its gene expression regulator has a mutation as compared with the natural one, and / or whether there is a factor that does not naturally exist and suppresses at least a part of LhAPS or its homologous gene. The cell, tissue, organ, plant body, or a part thereof according to any one of the above items. (Item 37) The mutation or factor is provided by RNA interference or genome editing of the LhAPS gene. The cell, tissue, organ, plant body, or a part thereof according to any one of the above items. (Item 38) A plant cell, plant tissue, plant organ, plant body, or a part thereof in which the gene of LhAPS or its homologous gene is partially or completely mutated or deleted. (Item 39) The plant is a plant of the Liliaceae family, and is the plant cell, plant tissue, plant organ, plant body or a part thereof according to any of the above items. (Item 40) The plant is a plant of the Lilium genus, and is the plant cell, plant tissue, plant organ, plant body or a part thereof according to any of the above items. (Item 41) The plant body or a part thereof is a bulb, seed, flower, stem, leaf, pollen, scale, corm, cell, tissue, tissue culture or ovary, or a combination of all or part of them, and is the plant cell, plant tissue, plant organ, plant body or a part thereof according to any of the above items. (Item 42) The plant body or a part thereof includes a flower or a part thereof, and is the plant cell, plant tissue, plant organ, plant body or a part thereof according to any of the above items. (Item 43) A method for producing a plant body or a part thereof with suppressed flower senescence, which includes providing a state in which at least a part of LhAPS or its homologous gene possessed by the plant body or a part thereof is suppressed in the plant body or a part thereof. (Item 44) The suppression includes suppressing the expression and / or function of at least a part of the LhAPS or its homologous gene as compared with the state before the providing step, and is the method according to any of the above items. (Item 45) The method according to any of the above items further includes measuring or determining by other methods that the expression and / or function of at least a part of the LhAPS or its homologous gene is suppressed as compared with the state before the providing step. (Item 46) The measuring or determining by other methods includes measuring the abundance of mRNA of at least a part of the LhAPS or its homologous gene, the abundance of protein of at least a part of the LhAPS or its homologous gene, the biological function of the protein of at least a part of the LhAPS or its homologous gene, a combination of these, or calculating from the measurement results, and is the method according to any of the above items. (Item 47) The method according to any one of the above items, wherein the suppression of the LhAPS or its homologous gene is achieved by a method selected from the group consisting of a decrease in gene expression level, a decrease in gene copy number, a decrease in gene amplification, a decrease in RNA activity level, a decrease in mRNA abundance, a decrease in mRNA synthesis rate, a decrease in mRNA stability, a decrease in protein activity level, a decrease in protein synthesis, a decrease in protein abundance, a decrease in protein stability, a decrease in protein enzyme activity, or a combination thereof. (Item 48) The method according to any one of the above items, wherein the suppression of the LhAPS or its homologous gene is achieved by performing RNA interference or genome editing of the LhAPS gene. (Item 49) The method according to any one of the above items, wherein the plant is a plant of the Liliaceae family. (Item 50) The method according to any one of the above items, wherein the plant is a plant of the genus Lilium. (Item 51) The method according to any one of the above items, wherein the plant body or a part thereof is a bulb, seed, flower, stem, leaf, pollen, scale, bulblet, cell, tissue, tissue culture, or ovary, or a combination of all or part of them. (Item 52) The method according to any one of the above items, wherein the plant body or a part thereof includes a flower or a part thereof. (Item 53) The method comprises the steps of modifying the LhAPS or its homologous gene in a plant cell to introduce a mutation into a part or all of it or deleting a part or all of it cultivating the plant cell in which the LhAPS or its homologous gene has been modified to obtain a plant body or a part thereof The method according to any one of the above items. (Item 54) The method according to any one of the above items, wherein the modification is performed by antisense, RNA interference, mutagen, or genome editing. (Item 55) A method for extending the vase life of a flower in a plant body or a part thereof, 1) providing a plant body or a part thereof that contains a factor described in any of the above items or is modified by the factor, or a plant body or a part thereof that contains a cell, a plant tissue, or a plant organ described in any of the above items; 2) subjecting the plant body or a part thereof to conditions for flowering; 3) after flowering in the plant body or a part thereof, subjecting it to conditions and a period for maintaining freshness; A method comprising the above steps. (Item 56) The method according to any of the above items, wherein the period is longer than the period when the freshness of the plant body or a part thereof without modification by the factor ends under the conditions for maintaining freshness. (Item 43A) Use of a factor that suppresses LhAPS or its homologous gene in the production of a plant body or a part thereof with suppressed flower senescence. (Item 44A) The use according to any of the above items, wherein the suppression includes suppressing the expression and / or function of at least a part of the LhAPS or its homologous gene as compared to the state before the providing step. (Item 45A) The use according to any of the above items, further comprising measuring or determining by other methods that the expression and / or function of at least a part of the LhAPS or its homologous gene is suppressed as compared to the state before the providing step. (Item 46A) The measuring or determining by other methods includes measuring the abundance of mRNA of at least a part of the LhAPS or its homologous gene, the abundance of protein of at least a part of the LhAPS or its homologous gene, the biological function of at least a part of the LhAPS or its homologous gene, combinations thereof, or calculating from the measurement results. The use according to any of the above items. (Item 47A) The use according to any one of the above items, wherein the suppression of the LhAPS or its homologous gene is achieved by a method selected from the group consisting of a decrease in gene expression level, a decrease in gene copy number, a decrease in gene amplification, a decrease in RNA activity level, a decrease in mRNA abundance, a decrease in mRNA synthesis rate, a decrease in mRNA stability, a decrease in protein activity level, a decrease in protein synthesis, a decrease in protein abundance, a decrease in protein stability, a decrease in protein enzyme activity, or a combination thereof. (Item 48A) The use according to any one of the above items, wherein the suppression of the LhAPS or its homologous gene is achieved by performing RNA interference or genome editing of the LhAPS gene. (Item 49A) The use according to any one of the above items, wherein the plant is a plant of the Liliaceae family. (Item 50A) The use according to any one of the above items, wherein the plant is a plant of the Lilium genus. (Item 51A) The use according to any one of the above items, wherein the plant body or a part thereof is a bulb, seed, flower, stem, leaf, pollen, scale, corm, cell, tissue, tissue culture, or ovary, or a combination of all or part of them. (Item 52A) The use according to any one of the above items, wherein the plant body or a part thereof includes a flower or a part of a flower. (Item 53A) The production method is a step of modifying the LhAPS or its homologous gene in a plant cell to introduce a mutation into a part or the whole thereof or deleting a part or the whole thereof a step of growing a plant cell in which the LhAPS or its homologous gene has been modified to obtain a plant body or a part thereof The use according to any one of the above items, comprising (Item 54A) The use according to any one of the above items, wherein the modification is performed by antisense, RNA interference, mutagen, or genome editing. (Item 55A) Use of a factor that suppresses the LhAPS or its homologous gene in a method for extending the vase life of a flower in a plant body or a part thereof, wherein the method is 1) Providing a plant body or a part thereof that contains a factor described in any of the above items or is modified by the factor, or a plant body or a part thereof that contains a cell, a plant tissue, or a plant organ described in any of the above items; 2) Subjecting the plant body or a part thereof to flowering conditions; 3) After flowering in the plant body or a part thereof, subjecting it to conditions and a period for maintaining freshness; The use comprising the above steps. (Item 56A) The use according to any of the above items, wherein the period is longer than the period when the freshness of the plant body or a part thereof ends without modification by the factor under the conditions for maintaining freshness.

[0006] Therefore, these and other advantages of the present disclosure will be apparent from the following detailed description.

Effect of the Invention

[0007] According to the present disclosure, by suppressing the expression of the transcription factor family gene (LhAPS), it is possible to delay the aging of flowers in general of Liliaceae plants such as lilies and tulips, and extend the ornamental period.

Brief Description of the Drawings

[0008]

Figure 1

Figure 2

[0009] The present disclosure will be described below. Throughout this specification, it should be understood that singular expressions also include the concept of their plural forms unless otherwise specified. Therefore, singular articles (e.g., "a", "an", "the" in English, etc.) should be understood to also include the concept of their plural forms unless otherwise specified. Also, the terms used in this specification should be understood to be used in the meaning commonly used in the art unless otherwise specified. Therefore, unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this disclosure pertains. In case of conflict, this specification (including definitions) shall prevail.

[0010] (Definition) In this specification, "about" means ±10% of the value that follows. It should be understood that this specification does not intend to waive the numerical range in the case where there is no description of "about" even without an explicit description of "about".

[0011] ​As used herein, "LhAPS" refers to a gene having the nucleic acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3, a protein having the amino acid sequence set forth in SEQ ID NO: 2, a derivative thereof having a biological function, or a fragment thereof having a biological function, or a homolog thereof, or a variant encoded by a nucleic acid that hybridizes to the nucleic acid encoding this protein under high stringency conditions or low stringency conditions. Those skilled in the art will understand which of the nucleic acid molecule or the protein, or both, is meant as appropriate in the context. "Variant" or "modified form" also includes, although not intended to be limiting, molecules containing regions substantially homologous to the original protein, such molecules being, in various embodiments, at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% identical over an amino acid sequence of the same size, or when compared to an aligned sequence aligned by a computer homology program known in the art (such as LALIGN (https: / / embnet.vital-it.ch / software / LALIGN_form.html), etc.), or the nucleic acid encoding such a molecule is capable of hybridizing to the sequence encoding the original protein under stringent conditions, moderately stringent conditions, or non-stringent conditions. This is the result of modifying the naturally occurring protein by amino acid substitution, deletion and addition, respectively, and the derivative thereof means a protein that shows that the biological function of the naturally occurring protein may not necessarily be to the same degree. For example, it is also possible to examine the biological function of such a protein by appropriate and available in vitro assays described herein or known in the art.

[0012] In the present disclosure, LhAPS is mainly discussed in lilies, but it is also known that many plants other than lilies, such as tulips (SEQ ID NO: 4), express homologous genes of the LhAPS gene. In particular, the homologous gene of tulips has about 72.1% sequence identity to LhAPS of lilies. Based on such interspecies sequence conservation, it is understood that these plants, especially liliaceae plants, also fall within the scope of the present disclosure.

[0013] As used herein, "having a biological function" refers to a polypeptide, i.e., a fragment or derivative, that has a structural function, a regulatory function, or a biochemical function of a protein, such as a biological activity, according to the aspects related to the polypeptide of the present disclosure, i.e., a fragment or derivative.

[0014] In the context of the present disclosure, a "factor that suppresses (a gene or protein)" directly or indirectly acts on a target gene, its homologous gene, or a protein encoded by these genes, and can temporarily or permanently suppress, reduce, or eliminate the function of the target gene, its homologous gene, or a protein encoded by these genes (for example, a molecule or substance). The factor that suppresses may be an inhibitor (substance), and examples thereof include an antibody or its antigenic fragment, an antisense oligonucleotide, an RNAi factor such as siRNA, a factor for genome editing, a mutagen, a low molecular weight molecule (LMW), a binding peptide, an aptamer, a ribozyme, and a peptidomimetic, etc. For example, it includes a binding protein or binding peptide directed against a target gene, its homologous gene, or a protein encoded by these genes, particularly against the active site of a protein encoded by the target gene or its homologous gene, as well as a nucleic acid directed against a target gene or its homologous gene. The nucleic acid directed against a target gene or its homologous gene refers to, for example, double-stranded or single-stranded DNA or RNA, or its modification or derivative that inhibits the expression or activity of a target gene, its homologous gene, or a protein encoded by these genes, and includes, without limitation, antisense nucleic acid, aptamer, siRNA (small interfering RNA), and ribozyme. The nucleic acid directed against a target gene or its homologous gene also includes, for example, a factor for genome editing that inhibits the expression or activity of the LhAPS gene or its homologous gene. Examples of the factor for genome editing include artificial restriction enzymes (artificial nucleases) and RNA-guided nucleases, etc.As used herein, the "binding protein" or "binding peptide" with respect to the protein encoded by the target gene or its homologous gene refers to a type of protein or peptide that binds to the protein encoded by the target gene or its homologous gene, and includes, but is not limited to, polyclonal antibodies or monoclonal antibodies, antibody fragments, and protein scaffolds directed against the protein encoded by the target gene or its homologous gene.

[0015] As used herein, the "information transmitter" is a substance responsible for information transmission for regulating physiological activities or biological functions in vivo. Representative examples of the "information transmitter" include plant hormones.

[0016] As used herein, the term "antisense molecule" refers to a molecule that can specifically suppress or reduce the expression of a target gene. More specifically, it refers to a molecule that can reduce the protein expression level by specifically decreasing the mRNA amount of a gene having a nucleotide sequence region complementary to a certain nucleotide sequence introduced into a cell. The antisense techniques are roughly classified into a method of directly introducing an RNA molecule complementary to the mRNA derived from a target gene into a cell and a method of introducing a construction vector capable of expressing an RNA complementary to the target gene in the cell. In plants, the latter method is more common. Specifically, in addition to a technique of constructing an expression vector that ligates a single DNA sequence complementary to the nucleotide sequence of a target gene to an appropriate promoter and expresses artificial mRNA under its control and introducing it into a cell, a technique using an expression vector designed to be able to form double-stranded RNA in the cell is used. The basic structure of an antisense molecule is formed by ligating one kind of DNA sequence complementary to a certain target gene under a promoter and further ligating another one of the same in the reverse direction. In the single-stranded mRNA transcribed from this constructed gene, one kind of nucleotide sequence part ligated in the reverse direction is in a complementary relationship, so they pair to form a double-stranded RNA state having a hairpin-like secondary structure, and this causes the degradation of the target gene mRNA according to the RNAi mechanism.

[0017] As used herein, "RNA interference" or "RNAi" is an abbreviation for RNA interference, which is generally known in the art and is a biological process that inhibits or down-regulates gene expression in cells mediated by RNAi factors. For example, it refers to the phenomenon in which homologous mRNA is specifically degraded and the synthesis of gene products is suppressed by introducing a factor that causes RNAi, such as double-stranded RNA (also referred to as dsRNA), into cells, and the technology used therefor. For RNAi, see, for example, Zamore and Haley, 2005, Science, 309, 1519-1524; Vaughn and Martienssen, 2005, Science, 309, 1525-1526; Zamore et al., 2000, Cell, 101, 25-33; Bass, 2001, Nature, 411, 428-429; Elbashiretal., 2001, Nature, 411, 494-498; and Kreutzer et al., International Publication No. 00 / 44895; Zernicka-Goetz et al., International Publication No. 01 / 36646; Fire, International Publication No. 99 / 32619; Plaetinck et al., International Publication No. 00 / 01846; Mello and Fire, International Publication No. 01 / 29058; Deschamps-Depaillette, International Publication No. 99 / 07409 and Li et al., International Publication No. 00 / 44914; Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237; Hutvagner and Zamore, 2002, Science, 297, 2056-60; McManus et al., 2002, RNA, 8, 842-850; Reinhart et al., 2002, gene & Dev., 16, 1616-1626; and Reinhart & Bartel, 2002, Science, 297, 1831.).Also, in this specification, the term RNAi is understood to denote the same as other terms used to describe sequence-specific RNA interference such as post-transcriptional gene silencing, translation inhibition, transcriptional inhibition, epigenetics, etc.

[0018] As used herein, the "RNAi factor" includes nucleic acid molecules or analogs thereof that inhibit gene expression, such as RNA-based molecules. The RNAi factor may be a factor that destroys or cleaves a specific mRNA, or a factor that blocks the processing or translation of RNA such as mRNA. RNAi factors include, but are not limited to, small interfering RNA (siRNA), siRNA prepared by endoribonuclease (esiRNA), short hairpin RNA (shRNA), or microRNA (miRNA), and short interfering nucleic acid (siNA). These factors refer to any nucleic acid molecule that can inhibit or down-regulate gene expression or viral replication by mediating RNA interference "RNAi" or gene silencing in a sequence-specific manner. These terms can also refer to individual nucleic acid molecules, multiple such nucleic acid molecules, or pools of such nucleic acid molecules. These molecules can be double-stranded nucleic acid molecules containing self-complementary sense and antisense regions. Here, the antisense region includes a nucleotide sequence complementary to a nucleotide sequence or a part thereof in the target nucleic acid molecule, and a sense region having a nucleotide sequence corresponding to the target nucleic acid sequence or a part thereof. These molecules can be assembled from two separate oligonucleotides, one strand being the sense strand and the other being the antisense strand. Here, the antisense strand and the sense strand are self-complementary (i.e., the antisense strand and the sense strand form a double-stranded or double-stranded structure, etc., and each strand contains a nucleotide sequence complementary to the nucleotide sequence in the other strand. Here, for example, the double-stranded region can be about 15 to about 30, such as about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs, but can be longer than these. The antisense strand contains a nucleotide sequence complementary to a nucleotide sequence or a part thereof in the target nucleic acid molecule, and the sense strand contains a nucleotide sequence corresponding to the target nucleic acid sequence or a part thereof (e.g., about 15 to about 25 or more nucleotides of the molecule are complementary to the target nucleic acid or a part thereof).Alternatively, these molecules are assembled from a single oligonucleotide, and the self-complementary sense and antisense regions of these molecules are linked by a nucleic acid linker or a non-nucleic acid linker. These molecules can be polynucleotides having a double-stranded, asymmetric double-stranded, hairpin or asymmetric hairpin secondary structure, including self-complementary sense and antisense regions. Here, the antisense region includes a nucleotide sequence complementary to a nucleotide sequence or a part thereof in a separate target nucleic acid molecule, and a sense region having a nucleotide sequence corresponding to the target nucleic acid sequence or a part thereof. These molecules can be circular single-stranded polynucleotides having two or more loop structures and a stem including self-complementary sense and antisense regions. Here, the antisense region includes a nucleotide sequence complementary to a nucleotide sequence or a part thereof in a target nucleic acid molecule, and a sense region having a nucleotide sequence corresponding to the target nucleic acid sequence or a part thereof, and the circular polynucleotide can be processed in vivo or in vitro to generate an active molecule capable of mediating RNAi. These factors can also include single-stranded polynucleotides having a nucleotide sequence complementary to a nucleotide sequence or a part thereof in a target nucleic acid molecule (for example, it is not necessary that a nucleotide sequence corresponding to the target nucleic acid sequence or a part thereof be present within these factors). The single-stranded polynucleotide can further include terminal phosphate groups such as 5'-phosphate (see, for example, Martinez et al., 2002, Cell., 110, 563-574 and Schwarz et al., 2002, Molecular Cell, 10, 537-568), 5',3'-bisphosphate. In certain embodiments, the factors that suppress LhAPS of the present disclosure include separate sense and antisense sequences or regions. Here, the sense region and the antisense region are covalently linked by a nucleotide or non-nucleotide linker molecule known in the art, or are alternately non-covalently linked by ionic interactions, hydrogen bonds, van der Waals interactions, hydrophobic interactions and / or stacking interactions.In certain embodiments, the factor that suppresses LhAPS of the present disclosure comprises a nucleotide sequence complementary to the nucleotide sequence of the target gene. In another embodiment, the factor that suppresses LhAPS of the present disclosure interacts with the nucleotide sequence of the target gene so as to inhibit the expression of the target gene. As used herein, the factor that suppresses LhAPS is not necessarily limited to a molecule containing only RNA, and also includes chemically modified nucleotides and non-nucleotides. In certain embodiments, when the present disclosure is a small interfering nucleic acid molecule, it may lack 2'-hydroxy (2'-OH)-containing nucleotides. In certain embodiments, the present disclosure can be a small interfering nucleic acid that does not require the presence of nucleotides having a 2'-hydroxyl group to mediate RNAi. Thus, when the present disclosure is a small interfering nucleic acid molecule, it may not contain ribonucleotides (e.g., nucleotides having a 2'-OH group). However, if the presence of ribonucleotides in the factor that suppresses LhAPS is not required to maintain RNAi, it may have a linked linker, or other linked or associated groups, moieties or strands, containing one or more nucleotides having a 2'-OH group. In some cases, the factor that suppresses LhAPS of the present disclosure may contain ribonucleotides at about 5, 10, 20, 30, 40 or 50% of the nucleotide positions. As used herein, the factor that suppresses LhAPS may be a nucleic acid molecule capable of mediating sequence-specific RNAi, such as small interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), small interfering oligonucleotide, small interfering nucleic acid, small interfering modified oligonucleotide, chemically modified siRNA, post-transcriptional gene silencing RNA (ptgsRNA).

[0019] As used herein, the "factor for genome editing" refers to a factor that causes genome editing in the introduced cell, tissue, organ or organism. The "factor for genome editing" may be any factor that causes genome editing by a method known in the art, i.e., a method such as CRISPR / Cas9, TALEN, ZFN, meganuclease, etc. Examples of the factor for genome editing include, but are not limited to, artificial restriction enzymes (artificial nucleases), RNA-guided nucleases, guide RNAs (gRNAs), etc.

[0020] As used herein, the "mutation-inducing factor" refers to a factor that promotes the induction of mutations in the target gene. Examples of the "mutation-inducing factor" include chemical substances such as ethyl methanesulfonate (EMS), as well as energy such as light, radiation, heat, electricity, etc.

[0021] As used herein, "homologous gene" refers to a gene with high homology between two or more sequences. The "homology" of a gene refers to the degree of identity between two or more gene sequences relative to each other. Generally, having "homology" means having a high degree of identity or similarity. Therefore, the higher the homology between two genes, the higher the identity or similarity of their sequences. Whether two types of genes have homology can be examined by direct comparison of the sequences or, in the case of nucleic acids, by the hybridization method under stringent conditions. When directly comparing two gene sequences, if the DNA sequences between the gene sequences are typically at least 50% identical, preferably at least 70% identical, more preferably at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identical, those genes have homology. Therefore, as used herein, "homolog" or "homologous gene product" means a protein in another species, preferably a plant, that exhibits the same biological function as the protein component of the complex further described herein. Such homologs may also be referred to as "orthologous gene products". Algorithms for detecting orthologous gene pairs from a particular plant or other species use the entire genomes or partial sequences of these organisms. When analyzing the homology of partial sequences, the LALIGN program (https: / / embnet.vital-it.ch / software / LALIGN_form.html) is typically used in the present disclosure. When analyzing with the LALIGN program, those skilled in the art can compare sequence identity according to the instructions on the website. Details of the analysis method are provided in X. Huang and W. Miller, Adv. Appl. Math. (1991) 12:337-357, etc.Based on the sequence homology of the genes encoding the proteins provided herein to genes of other species, applying conventional techniques to clone each gene, and expressing proteins from such genes, or according to the methods provided herein, or according to other suitable methods well-known in the art, homologs of the proteins described herein can also be isolated by isolating proteins of other species by isolating similar complexes.

[0022] In this specification, numerical values such as "70% or more" for identity, etc. may be, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%, or may be within the range of any two values of those starting values. The above-mentioned "identity" is calculated according to known methods as described above, which is the ratio of the number of identical amino acids in the amino acid sequences between two or more. Specifically, before calculating the ratio, the amino acid sequences of the amino acid sequence groups to be compared are aligned, and gaps are introduced into a part of the amino acid sequence if necessary to maximize the ratio of identical amino acids. Methods for alignment, methods for calculating the ratio, comparison methods, and computer programs related thereto are well-known in the art (for example, BLAST etc. described above). In this specification, "identity" and "similarity" can be represented by values measured by NCBI's BLAST unless otherwise specified. When comparing amino acid sequences by BLAST, the algorithm Blastp can be used with default settings. The measurement results are quantified as Positives or Identities.

[0023] When referring to a gene in this specification, a "vector" means something that can transfer a polynucleotide sequence of interest into a cell of interest. Such vectors include those that can autonomously replicate in host cells such as prokaryotic cells, yeast, animal cells, plant cells, insect cells, animal individuals, and plant individuals, or those that can be integrated into a chromosome and contain a promoter at a position suitable for transcription of the polynucleotides of the present disclosure. In this specification, the vector can be an expression vector, a recombinant vector, or the like.

[0024] In this specification, a "promoter" refers to a region on DNA that determines the start site of gene transcription and directly regulates its frequency, and is a base sequence to which RNA polymerase binds to initiate transcription. The promoter region can usually be estimated by predicting the protein-coding region in the genomic base sequence using DNA analysis software. The predicted promoter region varies for each structural gene, but is usually upstream of the structural gene, but is not limited to this and can also be within or downstream of the structural gene. In the present disclosure, any promoter that can exert the function of a factor causing RNA interference of LhAPS can be used.

[0025] In this specification, a "terminator" refers to a sequence located downstream of the region encoding the protein of a gene and involved in the termination of transcription and the addition of a polyA sequence when DNA is transcribed into mRNA. Examples of terminators include, but are not limited to, the CaMV35S terminator, the terminator of the nopaline synthase gene (Tnos), and the terminator of the tobacco PR1a gene. In the present disclosure, any sequence that exhibits terminator activity in plants can be used.

[0026] As used herein, the "expression" of genes, polynucleotides, polypeptides, proteins, etc. refers to the change of these genes, etc. into another form under certain in vivo effects. Preferably, it refers to the transcription and translation of genes, polynucleotides, etc. into polypeptides, but the production of mRNA by transcription is also a form of expression. For example, the expression level of LhAPS can be determined by any method. Specifically, the expression level of LhAPS can be known by evaluating the amount of LhAPS mRNA, the amount of LhAPS protein, and the biological function of LhAPS protein. The amount of LhAPS mRNA or protein can be determined by the methods detailed elsewhere in this specification or other methods known in the art.

[0027] As used herein, the "suppression of function" of genes, polynucleotides, polypeptides, proteins, etc. refers to the suppression of the biological activity of these genes, etc. The "suppression of function" of LhAPS can be confirmed, for example, by measuring the expression of vacuolar processing enzyme gene (VPE gene) or cysteine protease gene (SAG12 gene) using RT-PCR method. The suppression of the function of LhAPS can be confirmed, for example, when measured by RT-PCR method, the expression of VPE gene or SAG12 gene is reduced by about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, or about 10% compared with their expression in the control, preferably about 50% reduction.

[0028] As used herein, "plant" refers to the general term for organisms belonging to the plant kingdom, characterized by organisms that have chlorophyll, a rigid cell wall, the presence of abundant persistent meristematic tissue, and no ability to move. Typically, "plant" refers to flowering plants that have the formation of cell walls and assimilation by chlorophyll. "Plant" includes both monocotyledonous and dicotyledonous plants. Preferred plants include, for example, plants of the "Liliaceae" family. Representative plants of the "Liliaceae" family include lily, tulip, katakuri, and black lily.

[0029] As used herein, "tissue" of an organism refers to a group of cells that have certain similar functions in that group. Therefore, a tissue can be part of an organ. Within an organ, cells often have the same function, but there may also be a mixture of cells with slightly different functions. Thus, in this specification, a tissue may contain a mixture of various cells as long as they share certain characteristics. "Tissue" includes not only those that make up a differentiated plant body but also plant cell masses such as "callus".

[0030] As used herein, "organ" refers to a structure that has an independent form and is formed by the combination of one or more tissues to perform a specific function. In plants, examples include, but are not limited to, roots, bulbs, stems, trunks, leaves, flowers, seeds, scales, nuts, germ, embryo, fruits, endosperm, and pods.

[0031] As used herein, the term "organism" (or "plant body" in the case of plants) refers to the broadest sense in the art and means an entity (or plant) that exhibits life phenomena. Typically, it has various characteristics such as cell structure, proliferation (self-reproduction), growth, regulation, metabolism, and repair ability, and usually has as its basic attributes growth involving the control of inheritance by nucleic acids and the participation of metabolism controlled by proteins. Organisms include prokaryotes, eukaryotes (such as plants and animals), etc. Preferably, in the present disclosure, the organism can be a plant. In the present specification, preferably, such a plant body can be fertile. More preferably, such a plant body can produce seeds. The gene constructs, agents, compositions, and methods of the present disclosure are intended to function not only in liliaceous plants but also in plants in general.

[0032] As used herein, the term "liliaceous" plants is intended to include the original species and hybrids of plants belonging to the family Liliaceae in biological taxonomy. Examples of liliaceous plants include Lilium, Tulipa, Erythronium, Fritillaria, Amana, Bowiea, Calochortus, Cardiocrinum, Clintonia, Gagea, Lloydia, Medeola, Nomocharis, Notholirion, Prosartes, Streptopus, Tricyrtis, etc.

[0033] As used herein, the term "lily plant" is intended to include the original species and hybrids of plants belonging to the genus Lilium in taxonomy. Therefore, "lily plants" include not only the original species such as Lilium lancifolium, Lilium bulbocodium, Lilium leichtlinii, Lilium japonicum, Lilium auratum, and Lilium sargentiae, but also hybrids such as Oriental Hybrid, Asiatic Hybrid, Longiflorum Hybrid, Martagon Hybrid, and Trumpet Hybrid. In the examples, the aging delay effect has been shown using varieties of the Oriental Hybrid system. However, since the homology of the LhAPS gene is very high among hybrids, it is considered that the aging delay effect will be similarly shown in hybrids of lilies other than the Oriental Hybrid system. Examples of "Oriental Hybrid" include varieties such as Tiara, Casablanca, and Acapulco.

[0034] As used herein, the term "bulb" refers to a storage organ formed by the accumulation of nutrients in specific parts of a plant body, such as roots, stems, and leaves, resulting in deformation and hypertrophy. Representative plants that produce bulbs include lilies, tulips, and hyacinths.

[0035] As used herein, the term "seed" or "species" refers to the reproductive organ of seed plants.

[0036] As used herein, the term "flower" refers to an aggregate of organs for plant reproduction. A "flower" is composed of a combination of a flower axis, perianth, stamen, pistil, etc. In this specification, a "flower" may typically be provided in a state separated from the underground part as a "cut flower", or may be provided in a state where the underground part is retained, such as a "potted flower" or a flower in a flower bed. A "flower" can generally be provided as an object for viewing. A "cut flower" refers to a single flower or a combination of tissues and / or organs containing a flower. For example, a "cut flower" can be a combination of a flower, a stem, and / or leaves. Specifically, a "cut flower" can be a combination of a flower, a stem, and leaves. A "potted flower" refers to a plant cultivated in a flower pot and blooming. A "part of a flower" refers to a part in which at least a part of the elements constituting the above "flower" is missing.

[0037] As used herein, the "perianth" refers to the overall name combining the "corolla" and the "sepal". The "corolla" refers to an organ composed of multiple petals.

[0038] As used herein, the "receptacle" refers to the part of the tip of the stem where the perianth, corolla, petals, stamens, pistils, and sepals are attached.

[0039] As used herein, the "stem" refers to the organ in a plant body that supports flowers and leaves.

[0040] As used herein, the "leaf" refers to the organ in a plant body that performs photosynthesis and respiration.

[0041] As used herein, the "pollen" refers to the cells produced by the anthers present in the stamens of a flower.

[0042] As used herein, the "scale" or "scale leaf" refers to the modified leaf that protects buds and bulbs of a plant body.

[0043] As used herein, the "bulblet" refers to the small bulbs formed around a bulb.

[0044] As used herein, the "spur" refers to the organ in a plant body where the axillary bud stores nutrients and hypertrophies.

[0045] As used herein, the "callus" refers to a mass of undifferentiated plant cells.

[0046] As used herein, "senescence" refers to the phenomenon or process in which, after the mature stage, until an organism dies, the functions of the organism, cells, tissues, or organs, or the functions integrating them, decline. When referring to "senescence of a flower" in the present disclosure, it particularly refers to the phenomenon (or the time course itself) that occurs with the passage of time from after flowering (in some cases, before flowering) until the vase life ends (the ornamental value is lost).

[0047] In this specification, "long-lasting" and "flower senescence" are defined from a horticultural perspective, and are evaluated by the "browning" of the entire flower, or the organs or tissues constituting the flower (e.g., petals or similar organs), and when the b* value increases to a certain extent in the L*a*b* color system using a color difference meter compared to the perianth immediately after flowering (especially when the evaluation is made strict, when the b* value increases by 10 or more in the L*a*b* color system using a color difference meter compared to the perianth immediately after flowering), it is evaluated as "browning", and is regarded as "senescence" in the present disclosure, and the state before being evaluated as "senescence" is expressed as "long-lasting". Also, "suppression of flower senescence" is defined from a horticultural perspective, and in this specification, when the increase in the b* value during flower senescence is significantly suppressed, it is referred to as the suppression of flower senescence. The suppression may be a suppression in the degree of increase, or may be evaluated by extending the time until a certain degree of increase. Flower longevity and senescence can also be evaluated by other evaluation methods (e.g., the state of wilting, the presence or absence of detachment).

[0048] Although not wishing to be bound by theory, senescence is a phenomenon or process in which cells die spontaneously and is considered a type of programmed cell death. When flower cells undergo senescence, autophagy-like phenomena have long been observed in plants such as morning glories.

[0049] "Flower senescence" or its suppression can be evaluated by observing the time from when the entire flower, or the organs or tissues constituting the flower (e.g., perianth, corolla, petals or similar organs) bloom until it undergoes browning (or other equivalent evaluation methods). In this specification, flower senescence is typically evaluated using lily family plants, but the present disclosure is not limited thereto.

[0050] In this specification, the "flowering conditions (or period)" refer to the conditions (or period) under which flowering plants normally grow.

[0051] In this specification, the "long-lasting conditions (or period)" refer to the conditions (or period) under which flowering plants maintain their flowering state.

[0052] As used herein, the terms "protein", "polypeptide", "oligopeptide", and "peptide" are used interchangeably herein and refer to a polymer of amino acids of any length. This polymer may be linear, branched, or cyclic. The amino acids may be natural, non-natural, or modified amino acids. This term may also encompass assemblies of multiple polypeptide chains complexed together. This term also encompasses naturally or artificially modified amino acid polymers. Such modifications include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification (e.g., conjugation with a labeling component). This definition also encompasses, for example, polypeptides containing one or more analogs of amino acids (e.g., including non-natural amino acids), peptide-like compounds (e.g., peptoids), and other modifications known in the art.

[0053] As used herein, "amino acid" may be natural or non-natural, so long as it meets the objectives of the present disclosure.

[0054] As used herein, the terms "polynucleotide", "oligonucleotide", and "nucleic acid" are used interchangeably and refer to polymers of nucleotides of any length. The term also includes "oligonucleotide derivatives" or "polynucleotide derivatives". "Oligonucleotide derivatives" or "polynucleotide derivatives" refer to oligonucleotides or polynucleotides that contain derivatives of nucleotides or in which the linkages between nucleotides are different from the normal ones, and are used interchangeably. Such oligonucleotides specifically include, for example, 2'-O-methyl-ribonucleotides, oligonucleotide derivatives in which the phosphodiester bonds in the oligonucleotide are converted to phosphorothioate bonds, oligonucleotide derivatives in which the phosphodiester bonds in the oligonucleotide are converted to N3'-P5' phosphoramidate bonds, oligonucleotide derivatives in which the ribose and phosphodiester bonds in the oligonucleotide are converted to peptide nucleic acid bonds, oligonucleotide derivatives in which uracil in the oligonucleotide is replaced by C-5 propynyluracil, oligonucleotide derivatives in which uracil in the oligonucleotide is replaced by C-5 thiazoleuracil, oligonucleotide derivatives in which cytosine in the oligonucleotide is replaced by C-5 propynylcytosine, oligonucleotide derivatives in which cytosine in the oligonucleotide is replaced by phenoxazine-modified cytosine, oligonucleotide derivatives in which ribose in DNA is replaced by 2'-O-propylribose, and oligonucleotide derivatives in which ribose in the oligonucleotide is replaced by 2'-methoxyethoxyribose. Unless otherwise indicated, a particular nucleic acid sequence is also intended to include conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the explicitly recited sequence.Specifically, the degenerate codon variants can be achieved by creating an array in which the third position of one or more selected (or all) codons is substituted with a mixed base and / or deoxyinosine residue (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., Mol. Cell Probes 8:91-98 (1994)). As used herein, "nucleic acid" is also used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide. As used herein, "nucleotide" may be either natural or non-natural.

[0055] As used herein, "gene" refers to a factor that defines a genetic trait. It is usually arranged in a certain order on a chromosome. A gene that defines the primary structure of a protein is called a structural gene, and a gene that affects its expression is called a regulatory gene. As used herein, "gene" may refer to "polynucleotide", "oligonucleotide" and "nucleic acid".

[0056] As used herein, "genome" refers to the entirety of the genetic information of an organism. The genome can be encoded in either DNA or RNA. The genome can include non-coding regions together with coding regions that encode proteins.

[0057] Amino acids may be referred to herein by either their generally known three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may likewise be referred to by their generally recognized one-letter codes. In this specification, comparisons of amino acid sequences and base sequence similarities, identities, and homologies are calculated using default parameters with the BLAST sequence analysis tool. The identity search can be performed, for example, using NCBI's BLAST 2.10.1 (released on June 18, 2020). The identity value in this specification usually refers to the value obtained when aligned under default conditions using the above BLAST. However, if a higher value is obtained by changing the parameters, the highest value shall be taken as the identity value. When identity is evaluated in multiple regions, the highest value among them shall be taken as the identity value. Similarity is a numerical value that takes into account similar amino acids in addition to identity.

[0058] As used herein, the term "polynucleotide that hybridizes under stringent conditions" refers to well-known conditions commonly used in the art. Such polynucleotides can be obtained by using, as a probe, a polynucleotide selected from the polynucleotides of the present disclosure, and by methods such as colony hybridization, plaque hybridization, or Southern blot hybridization. Specifically, using a filter immobilized with DNA derived from colonies or plaques, hybridization is performed at 65°C in the presence of 0.7 to 1.0 M NaCl, and then the filter is washed under 65°C conditions using a 0.1 to 2-fold concentration SSC (saline-sodium citrate) solution (the composition of a 1-fold concentration SSC solution is 150 mM sodium chloride and 15 mM sodium citrate), which means a polynucleotide that can be identified. Hybridization can be carried out according to the methods described in experimental manuals such as Molecular Cloning 2nd ed., Current Protocols in Molecular Biology, Supplement 1-38, DNA Cloning 1: Core Techniques, A PRac1tical Approach, Second Edition, Oxford University Press (1995). Here, sequences containing only A sequences or only T sequences are preferably excluded from the sequences that hybridize under stringent conditions. Therefore, the polypeptides used in the present disclosure (for example, the protein encoded by LhAPS) include polypeptides encoded by nucleic acid molecules that hybridize under stringent conditions to the nucleic acid molecules encoding the polypeptides specifically described in the present disclosure.These low stringency conditions include hybridization at 40°C for 18 - 20 hours in a buffer containing 35% formamide, 5x SSC, 50 mM Tris - HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% BSA, 100 μg / ml denatured salmon sperm DNA, and 10% (weight / volume) dextran sulfate, washing at 55°C for 1 - 5 hours in a buffer consisting of 2x SSC, 25 mM Tris - HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS, and washing at 60°C for 1.5 hours in a buffer consisting of 2x SSC, 25 mM Tris - HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS.

[0059] Functional equivalents of LhAPS used in the present disclosure can be found by searching databases, etc. As used herein, "searching" means finding other nucleic acid base sequences having specific functions and / or properties using a certain nucleic acid base sequence, either electronically or by biological or other methods. Examples of electronic searches include, but are not limited to, BLAST (Altschul et al., J. Mol. Biol. 215:403 - 410 (1990)), FASTA (Pearson & Lipman, Proc. Natl. Acad. Sci., USA 85:2444 - 2448 (1988)), Smith and Waterman method (Smith and Waterman, J. Mol. Biol. 147:195 - 197 (1981)), and Needleman and Wunsch method (Needleman and Wunsch, J. Mol. Biol. 48:443 - 453 (1970)). Examples of biological searches include, but are not limited to, stringent hybridization, macroarray or microarray (microarray assay) with genomic DNA attached to a nylon membrane or glass plate, PCR, and in situ hybridization. It is intended that genes used in the present disclosure should include corresponding genes identified by such electronic and biological searches.

[0060] As functional equivalents of the present disclosure, in the amino acid sequence, one or more amino acids can be inserted, substituted, or deleted, or added to one or both ends thereof. As used herein, "insertion, substitution, or deletion of one or more amino acids in the amino acid sequence, or addition to one or both ends thereof" means that modification has been made by a well-known technical method such as site-directed mutagenesis or by natural mutation, and substitution of a plurality of amino acids to the extent that can occur naturally.

[0061] As used herein, an "isolated" biological factor (e.g., a gene (LhAPS), nucleic acid, or protein, etc.) refers to a substance or biological factor from which at least a part of the factors naturally associated with the substance or biological factor has been removed, and is used to distinguish it from natural products. Therefore, usually, the purity of the biological factor in the isolated biological factor is higher (i.e., concentrated) than the state in which the biological factor normally exists.

[0062] As used herein, a "purified" substance or biological factor (e.g., a nucleic acid or protein, etc.) refers to a substance or biological factor from which at least a part of the factors naturally associated with the biological factor has been removed. Therefore, usually, the purity of the biological factor in the purified biological factor is higher (i.e., concentrated) than the state in which the biological factor normally exists. The term "purified" as used herein preferably means that there is at least 75% by weight, more preferably at least 85% by weight, even more preferably at least 95% by weight, and most preferably at least 98% by weight of the same type of biological factor present. The substances used in the present disclosure are preferably "purified" substances.

[0063] As used herein, a "corresponding" amino acid or nucleic acid refers to an amino acid or nucleotide in a polypeptide molecule or polynucleotide molecule that has the same function as, or is predicted to have the same function as, a given amino acid or nucleotide in a reference polypeptide or polynucleotide. For example, in an enzyme molecule, it refers to an amino acid that is present at a similar position in the active site and makes a similar contribution to catalytic activity. For example, in the case of an antisense molecule, it can be a similar portion in the ortholog corresponding to a specific portion of the antisense molecule. A corresponding amino acid can be, for example, a specific amino acid that is cysteinylated, glutathionylated, S-S bonded, oxidized (e.g., oxidation of the methionine side chain), formylated, acetylated, phosphorylated, glycosylated, myristylated, etc. Alternatively, a corresponding amino acid can be an amino acid responsible for dimerization. Such a "corresponding" amino acid or nucleic acid may be a region or domain over a certain range. Therefore, in such a case, it is referred to as a "corresponding" region or domain herein.

[0064] As used herein, a "corresponding" gene (e.g., polynucleotide sequence or molecule) refers to a gene (e.g., polynucleotide sequence or molecule) in a species that has the same function as, or is predicted to have the same function as, a given gene in a reference species. When there are multiple genes having such a function, it refers to those having the same evolutionary origin. Therefore, a gene corresponding to a certain gene can be an ortholog of that gene. Thus, for example, the LhAPS of lily can find a corresponding LhAPS in tulip. Such a corresponding gene can be identified using techniques well known in the art. Therefore, for example, a corresponding gene in a plant (e.g., lily) can be found by searching the sequence database of that plant (e.g., tulip) using the sequence of the reference gene corresponding to the gene (e.g., LhAPS, etc.) as a query sequence, such as SEQ ID NO: 1 or SEQ ID NO: 3.

[0065] As used herein, the term "fragment" refers to a polypeptide or polynucleotide having a sequence length of 1 to n-1 with respect to a full-length polypeptide or polynucleotide (length n). The length of the fragment can be appropriately changed according to the purpose. For example, in the case of a polypeptide, the lower limit of its length can be 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, and more amino acids, and lengths represented by integers not specifically listed here (such as 11, etc.) can also be appropriate as the lower limit. In the case of a polynucleotide, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, and more nucleotides can be mentioned, and lengths represented by integers not specifically listed here (such as 11, etc.) can also be appropriate as the lower limit. It is understood that in this specification, such a fragment falls within the scope of the present disclosure as long as, for example, when the full-length one functions as a marker, the fragment itself also has the function as a marker.

[0066] As used herein, the term "activity" refers to the function of a molecule in the broadest sense. Activity is generally intended to include, but is not limited to, the biological, biochemical, physical, or chemical functions of a molecule. Activity includes, for example, enzyme activity, the ability to interact with other molecules, and the ability to activate, promote, stabilize, inhibit, suppress, or destabilize the functions of other molecules, stability, and the ability to localize at a specific intracellular location. Where applicable, this term also relates to the function of a protein complex in the broadest sense. In the present disclosure, the "activity" of LhAPS is typically transcriptional activity (also referred to herein as "LhAPS transcriptional activity") or gene expression induction activity, and more specifically, it can be senescence-related gene induction activity or programmed cell death-related gene induction activity.

[0067] As used herein, the term "biological function", when referring to a gene or a nucleic acid molecule or polypeptide related thereto, refers to a specific function that the gene, nucleic acid molecule or polypeptide may have in a living body, and examples thereof include, but are not limited to, production of specific antibodies, enzyme activity, imparting resistance, etc. In the present disclosure, for example, functions such as LhAPS and the like promoting or controlling flower senescence can be mentioned, but are not limited thereto. In the present specification, the biological function can be exerted by "biological activity". As used herein, the term "biological activity" refers to the activity that a certain factor (e.g., polynucleotide, protein, etc.) may have in a living body, and includes activities that exhibit various functions (e.g., transcriptional activity or gene expression induction activity), and for example, activities in which another molecule is activated or inactivated by interaction with a certain molecule are also included. When two factors interact, the biological activity includes the binding between the two molecules and the resulting biological changes. For example, when one molecule is precipitated using an antibody and the other molecule also co-precipitates, the two molecules are considered to be bound. Therefore, observing such co-precipitation can be mentioned as one judgment method. As in the present disclosure, in the case of the activity of delaying flower senescence, such an activity is included. For example, when a certain factor is an enzyme, its biological activity includes its enzyme activity. In another example, when a certain factor is a ligand, its biological activity includes the binding of the ligand to the corresponding receptor. Such biological activity can be measured by techniques well-known in the art. Therefore, "activity" refers to indicating or revealing binding (either direct or indirect); affecting a response (i.e., having a measurable effect in response to some exposure or stimulus), and various measurable indicators, for example, the affinity of a compound that directly binds to the polypeptide or polynucleotide of the present disclosure, or for example, the amount of a protein upstream or downstream after some stimulation or event or other similar measures of function can be mentioned.

[0068] As used herein, the “expression” of a gene, polynucleotide, polypeptide, etc. refers to the change of such gene, etc. into another form under certain actions in vivo. Preferably, it refers to the transcription and translation of a gene, polynucleotide, etc. into the form of a polypeptide, but the production of mRNA by transcription can also be an aspect of expression. More preferably, such a polypeptide form can be one that has undergone post-translational processing (a derivative as referred to herein). For example, the expression level of LhAPS can be determined by any method. Specifically, the expression level of LhAPS can be known by evaluating the amount of LhAPS mRNA, the amount of LhAPS protein, and the biological activity of the LhAPS protein. The amounts of LhAPS mRNA and protein can be determined by the methods as described herein.

[0069] As used herein, the “functional equivalent” refers to any entity that has the same target function but a different structure compared to the original entity. Therefore, in the case of “LhAPS or its functional equivalent” or “the group consisting of LhAPS and its functional equivalent”, in addition to LhAPS itself, mutants or variants of LhAPS (e.g., amino acid sequence variants, etc.) that have the effect of delaying flower senescence, and those that can change into LhAPS itself or this mutant or variant of LhAPS at the time of action (e.g., nucleic acids encoding LhAPS itself or mutants or variants of LhAPS, vectors containing such nucleic acids, cells, etc.) are understood to be included. In the present disclosure, it is understood that the functional equivalent of LhAPS can be used in the same manner as LhAPS even if not specifically mentioned.

[0070] The modified amino acid sequence of LhAPS can be one in which, for example, 1 to 30, preferably 1 to 20, more preferably 1 to 9, still more preferably 1 to 5, and particularly preferably 1 to 2 amino acids are inserted, substituted, or deleted, or an addition is made to one or both of its ends. The modified amino acid sequence preferably has an amino acid sequence in which one or more (preferably 1 or several or 1, 2, 3, or 4) conservative substitutions are made in the amino acid sequence of LhAPS. Here, "conservative substitution" means substituting one or more amino acid residues with another chemically similar amino acid residue so as not to substantially modify the function of the protein. For example, it includes cases where a hydrophobic residue is substituted with another hydrophobic residue, or a polar residue is substituted with another polar residue having the same charge. Functionally similar amino acids for which such substitutions can be made are known in the art for each amino acid. Specific examples include, as non-polar (hydrophobic) amino acids, alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, methionine, etc. As polar (neutral) amino acids, glycine, serine, threonine, tyrosine, glutamine, asparagine, cysteine, etc. are included. As positively charged (basic) amino acids, arginine, histidine, lysine, etc. are included. Also, as negatively charged (acidic) amino acids, aspartic acid, glutamic acid, etc. are included.

[0071] As used herein, the terms "agent", "agent", or "factor" (all corresponding to "agent" in English) are used interchangeably in a broad sense and can be any substance or other element (e.g., energy such as light, radiation, heat, electricity, etc.) as long as the intended purpose can be achieved. Such substances include, for example, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (including DNA such as cDNA, genomic DNA, and RNA such as mRNA), polysaccharides, oligosaccharides, lipids, small organic molecules (e.g., hormones, ligands, signaling molecules, small organic molecules, molecules synthesized by combinatorial chemistry, small molecules that can be used as pharmaceuticals (e.g., small molecule ligands, etc.)), and composite molecules thereof, but are not limited thereto. Representative factors specific to a polynucleotide include, but are not limited to, polynucleotides having a certain sequence homology (e.g., 70% or more sequence identity) to the sequence of the polynucleotide and polypeptides such as transcription factors that bind to the promoter region. Representative factors specific to a polypeptide include, but are not limited to, antibodies or derivatives or analogs thereof (e.g., single-chain antibodies) specifically directed to the polypeptide, specific ligands or receptors when the polypeptide is a receptor or ligand, and substrates when the polypeptide is an enzyme.

[0072] (Genetic engineering, gene modification technology) In the present disclosure, genetic engineering and gene modification techniques used to suppress LhAPS or its homologous genes include, for example, genome editing, antisense, RNAi, and mutagenesis.

[0073] As used herein, "genome editing" refers to a technique for introducing mutations so as to specifically modify (e.g., suppress, decrease, enhance, or increase) the expression of a target gene. More specifically, it is a technique that uses site-specific nucleases to cleave at a target position in the genomic sequence of a target gene, thereby generating single-stranded or double-stranded breaks at specific positions within the genome. The breaks in the genome thus generated are usually repaired by processes such as homologous recombination (HDR) and non-homologous end joining (NHEJ). Random deletion or insertion of several bases in the target gene causes gene mutations such as nonsense mutations, missense mutations, and / or frameshifts, resulting in the failure to produce a normal (biologically functional) protein. Examples of genome editing techniques include techniques such as CRISPR / Cas9, TALEN, ZFN, and meganuclease. General techniques of genome editing are outlined, for example, in Cox et al., Nat. Med. 21: 121-131 (2015); Zhang et al., Genome Biol. 19: 210 (2018). Introducing mutations into a target gene using genome editing is excellent in that it can specifically introduce mutations into a specific gene and more efficiently create varieties having desired properties than conventional techniques such as mutagenesis and repeated crossing. In addition, in the system in Japan at the time of filing of the present application, plant cells, plant tissues, plant organs, plants, or parts thereof produced by the "genome editing" technique without residual foreign genes are also advantageous in that they can be used without undergoing safety review under the Cartagena Act.

[0074] When introducing mutations into genes by genome editing, methods using complexes such as CRISPR / Cas9, where the part involved in binding to the target DNA is RNA and the part involved in DNA cleavage is protein, or TALEN, ZFN, etc., where both the part involved in binding to the target DNA and the part involved in DNA cleavage are proteins, can be mentioned. For example, in the method using CRISPR / Cas9, guide RNA, Cas9, etc. are introduced into target cells, and in the method using TALEN or ZFN, a fusion protein in which a DNA binding domain and a nuclease are fused is introduced into target cells, whereby mutations can be introduced into the genome. Examples of methods for introducing into target cells include the Agrobacterium method, the RNA virus vector method, the plasma treatment method, the particle gun (bombardment) method, the PEG method, the electroporation method, etc.

[0075] As a suitable method for introducing mutations into the genome by genome editing, it may be a method of generating mutations when the cleavage site is naturally repaired after cleaving the target base sequence, or a DNA fragment having one or several base mutations in a sequence homologous to the target base sequence is introduced into the cell, and after cleaving the target base sequence, one or several base mutations are inserted when the cleavage site is repaired using the introduced DNA fragment as a template. Note that the number of bases is usually 2 to 6 bases.

[0076] The CRISPR / Cas9 system consists of three factors: CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA), and Cas9 protein. The Cas9 protein derived from Streptococcus pyogenes recognizes NGG (where N is either G, A, T, or C), which are three bases downstream of the target genomic sequence, as the PAM sequence (Proto-spacer Adjacent Motif), and cleaves the three bases upstream of it. A common method in this technical field is to introduce a DNA double-strand break at a targeted site on genomic DNA by expressing guide RNA (gRNA), which is formed by ligating tracrRNA to the 3' end of crRNA complementary to the target gene sequence, and Cas9. In this specification, the guide RNA is designed such that the expression of the LhAPS gene is suppressed or reduced, or the LhAPS gene is knocked out. That is, it is designed to express a protein with a partial deletion, substitution, or addition of an amino acid sequence with respect to the nucleic acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3.

[0077] The ZFN (Zinc Finger Nuclease) system is a system that utilizes an artificial restriction enzyme consisting of a zinc finger domain (ZF) that recognizes and binds to target DNA and a FokI domain that cleaves DNA. In this system, the zinc finger domain recognizes a specific gene sequence on the genome, and the FokI domain cleaves the DNA in this region, resulting in a DNA double-strand break.

[0078] The TALEN (Transcription Activator-Like Effector Nucleases) system is a system that uses an artificial nuclease having the transcription factor TAL effector (TALE) of bacteria belonging to the genus Xanthomonas as a DNA binding domain. The mechanism of action of genome editing by TALEN is similar to that of ZFN. It recognizes and binds to the target DNA, and the FokI domain cleaves the DNA in this region, resulting in a double-strand break of the DNA. The DNA binding domain of TALE has a repeat structure with 34 amino acids as one unit (module), and it is known that one module recognizes one base. The 12th and 13th amino acid residues in the module are called Repeat variable di-residue (RVD), and the RVD contributes to the binding specificity and stabilization of the base.

[0079] As methods for introducing mutations into target genes by genome editing, for example, there are a transformation method using Agrobacterium, and a method of directly introducing a genome editing factor into protoplasts. The transformation method using Agrobacterium is a method of introducing a genome editing factor into plant cells by infecting plant cells with Agrobacterium bacteria containing a vector expressing the genome editing factor. That is, plant cells or plant tissues isolated from a target plant (for example, a liliaceae plant) are incubated with Agrobacterium bacteria containing a vector expressing the genome editing factor to introduce the genome editing factor into the plant cells. Then, by performing cell culture or tissue culture under appropriate culture conditions, genome-edited callus is generated. Then, by inducing differentiation of the genome-edited callus, a plant in which the genome of the whole plant body is edited can be obtained by regenerating the genome-edited plant body. The method of directly introducing a genome editing factor into protoplasts is a method of directly introducing a genome editing factor into plant cells without using Agrobacterium and a vector. That is, protoplasts are prepared from plant cells derived from a target plant (for example, a liliaceae plant), and the genome editing factor is directly introduced into the protoplasts by the PEG method, the particle gun method, or the like. Then, by incubating the protoplasts under appropriate culture conditions, a plant in which the genome is edited can be obtained.

[0080] As a method for introducing mutations into the genome by mutants, for example, from natural mutants, mutants induced by radiation such as UV irradiation and gamma rays, and mutants induced by chemicals such as ethyl methanesulfonate (EMS), those having mutations in the gene are selected by the PCR method, the TILLING (Targeting Induced Local Lesions in Genomes) method, the HRM (High Resolution Melting) method, etc., and a method of obtaining those in which mutations have occurred in the gene can be mentioned.

[0081] Antisense activity is usually achieved by a nucleic acid sequence having a length of at least 8 consecutive nucleotides that is complementary to the nucleic acid sequence of the target gene. Such nucleic acid sequences are preferably at least 9 consecutive nucleotides in length, more preferably 10 consecutive nucleotides in length, even more preferably 11 consecutive nucleotides in length, 12 consecutive nucleotides in length, 13 consecutive nucleotides in length, 14 consecutive nucleotides in length, 15 consecutive nucleotides in length, 20 consecutive nucleotides in length, 25 consecutive nucleotides in length, 30 consecutive nucleotides in length, 40 consecutive nucleotides in length, 50 consecutive nucleotides in length, and can be nucleic acid sequences. Such nucleic acid sequences include nucleic acid sequences that are at least 70% homologous, more preferably at least 80% homologous, even more preferably 90% homologous, 95% homologous to the above-described sequences. Such antisense activity is preferably complementary to the sequence at the 5' end of the nucleic acid sequence of the target gene. Such antisense nucleic acid sequences also include those having one or several or one or more nucleotide substitutions, additions and / or deletions with respect to the above-described sequences. Therefore, as used herein, "antisense activity" includes, but is not limited to, a decrease in the expression level of a gene.

[0082] General antisense technology is described in textbooks (Murray, JA et al., eds., Antisense RNA and DNA, Wiley-Liss Inc, 1992). Subsequently, a phenomenon called RNA interference (RNAi) was revealed in further research, leading to the development of antisense technology. RNAi is a phenomenon in which when short double-stranded RNAs (about 20 bases in length) with sequences homologous to the target gene are introduced into cells, the mRNA of the target gene homologous to the RNA sequence is specifically degraded and the expression level decreases. This phenomenon, initially discovered in Caenorhabditis elegans, has been found to be a universal phenomenon in organisms including plants. It has been elucidated that the molecular-level mechanism by which the expression of the target gene is suppressed by antisense technology undergoes the same process as this RNAi. Conventionally, an expression vector was constructed by ligating a single DNA sequence complementary to the nucleotide sequence of the target gene to an appropriate promoter and expressing artificial mRNA under its control, and then introducing it into cells. In recent findings, expression vectors designed to be able to form double-stranded RNA in cells are used. The basic structure is created by ligating one DNA sequence complementary to a certain target gene under a promoter, and then ligating another one in the reverse direction. In the single-stranded mRNA transcribed from this constructed gene, since a portion of the nucleotide sequence ligated in the reverse direction is in a complementary relationship, they pair to form a double-stranded RNA state with a hairpin-like secondary structure, which causes the degradation of the target gene's mRNA according to the mechanism of RNAi. An example of its use in Arabidopsis thaliana has been reported in plants (Smith, NA et al., Nature 407, 319-320, 2000). Also, a review of RNAi in general has been compiled (Morita and Yoshida, Protein, Nucleic Acid, Enzyme 47, 1939-1945, 2002). The content described in these documents is incorporated herein by reference in its entirety.

[0083] As used herein, "RNA interference" or "RNAi" is an abbreviation for RNA interference, which is generally known in the art and is a biological process that inhibits or downregulates gene expression in cells mediated by factors that cause RNAi. For example, it refers to the phenomenon in which homologous mRNA is specifically degraded and the synthesis of gene products is suppressed by introducing a factor that causes RNAi, such as double-stranded RNA (also referred to as dsRNA), into cells, as well as the techniques used therefor. As used herein, "RNAi" may also be used synonymously with, in some cases, "factor that causes RNAi", "factor that induces RNAi", "RNAi factor", and the like.Regarding RNAi, see, for example, Zamore and Haley, 2005, Science, 309, 1519-1524; Vaughn and Martienssen, 2005, Science, 309, 1525-1526; Zamore et al., 2000, Cell, 101, 25-33; Bass, 2001, Nature, 411, 428-429; Elbashiretal., 2001, Nature, 411, 494-498; and Kreutzer et al., WO 00 / 44895; Zernicka-Goetz et al., WO 01 / 36646; Fire, WO 99 / 32619; Plaetinck et al., WO 00 / 01846; Mello and Fire, WO 01 / 29058; Deschamps-Depaillette, WO 99 / 07409 and Li et al., WO 00 / 44914; Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237; Hutvagner and Zamore, 2002, Science, 297, 2056-60; McManus et al., 2002, RNA, 8, 842-850; Reinhart et al., 2002, gene & Dev., 16, 1616-1626; and Reinhart & Bartel, 2002, Science, 297, 1831. Also, in this specification, the term "RNAi" is understood to be synonymous with other terms used to describe sequence-specific RNA interference such as post-transcriptional gene silencing, translation inhibition, transcriptional inhibition, epigenetics, etc. In this specification, an "RNAi agent" may be anything as long as it causes "RNAi".

[0084] In this specification, factors that cause RNAi include, for example, RNA or a modified form thereof that contains a double-stranded portion at least 10 nucleotides in length, including a sequence having at least about 70% homology to a part of the nucleic acid sequence of the target gene or a sequence that hybridizes under stringent conditions, but is not limited thereto. Here, this factor preferably includes a 3' overhang, and more preferably, the 3' overhang is DNA having a length of 2 nucleotides or more (for example, it can be DNA having a length of 2 to 4 nucleotides).

[0085] Alternatively, RNAi factors used in the present disclosure include, for example, but are not limited to, pairs of short inverted complementary sequences (for example, 15 bp or more, for example, 24 bp, etc.).

[0086] Although not bound by theory, one of the mechanisms by which RNAi is thought to work is that when a molecule that causes RNAi, such as dsRNA, is introduced into a cell, in the case of relatively long RNA (for example, 40 base pairs or more), an RNaseIII-like nuclease called Dicer, which has a helicase domain, cuts the molecule from the 3'-end by approximately 20 base pairs at a time in the presence of ATP, generating short double-stranded RNA (also called siRNA). In this specification, "siRNA" is an abbreviation for short interfering RNA, whether artificially chemically synthesized, biochemically synthesized, synthesized in vivo, or short double-stranded RNA of 10 base pairs or more formed by degradation of double-stranded RNA of about 40 bases or more in vivo. Usually, it has a 5'-phosphate, 3'-OH structure, and the 3'-end protrudes by about 2 bases. A protein specific to this siRNA binds to form RISC (RNA-induced-silencing-complex). This complex recognizes and binds to mRNA having the same sequence as the siRNA, and cleaves the mRNA at the central part of the siRNA by RNaseIII-like enzyme activity. Regarding the relationship between the sequence of the siRNA and the sequence of the mRNA to be cleaved as a target, it is preferably 100% identical. However, for base mutations at positions outside the center of the siRNA, the cleavage activity by RNAi does not completely disappear, but partial activity remains. On the other hand, base mutations in the central part of the siRNA have a great impact, and the cleavage activity of mRNA by RNAi is extremely reduced. Utilizing such properties, for mRNA with mutations, siRNA with the mutation arranged in the center can be synthesized and introduced into cells to specifically degrade only the mRNA containing the mutation. Therefore, in the present disclosure, siRNA itself can be used as a factor that causes RNAi, and a factor that generates siRNA (for example, typically dsRNA of 40 base pairs or more) can be used as such a factor.

[0087] Also, while not wishing to be bound by theory, it is contemplated that, apart from the above pathway, the antisense strand of siRNA binds to mRNA and acts as a primer for RNA-dependent RNA polymerase (RdRP), resulting in the synthesis of dsRNA, which again serves as a substrate for Dicer, generating new siRNAs and amplifying the effect. Thus, in the present disclosure, siRNA itself and factors that give rise to siRNA are also useful. Indeed, in insects, for example, 35 molecules of dsRNA can almost completely degrade the mRNA in cells with 1,000 or more copies, from which it is understood that siRNA itself and factors that give rise to siRNA are useful.

[0088] In the present disclosure, double-stranded RNAs having a length of about 20 bases or less (for example, typically about 21 to 23 bases in length), which are referred to as siRNAs, can be used. Such siRNAs can be used for the treatment, prevention, prognosis, etc. of diseases, since gene expression is suppressed by expressing them in cells, suppressing the expression of the pathogenic gene targeted by the siRNA. The siRNAs used in the present disclosure may take any form as long as they can cause RNAi.

[0089] In another embodiment, the factor that causes RNAi of the present disclosure can be a short hairpin RNA (shRNA) having a protrusion at the 3' end. As used herein, "shRNA" refers to a molecule of about 20 base pairs or more that forms a double-stranded structure within the molecule and has a hairpin-like structure by containing a partially palindromic base sequence in single-stranded RNA. Such shRNAs are artificially chemically synthesized. Alternatively, such shRNAs can be generated by synthesizing RNA in vitro using T7 RNA polymerase with DNA having a hairpin structure in which the DNA sequences of the sense strand and the antisense strand are linked in the reverse direction. Without wishing to be bound by theory, after being introduced into cells, such shRNAs are degraded intracellularly to a length of about 20 bases (representatively, for example, 21 bases, 22 bases, 23 bases), cause RNAi in the same manner as siRNAs, and it should be understood that they have the treatment effect of the present disclosure. It should be understood that such effects are exerted in a wide range of organisms such as insects, plants, and animals (including mammals). Thus, since shRNAs cause RNAi in the same manner as siRNAs, they can be used as an active ingredient of the present disclosure. ShRNAs can also preferably have a 3' protruding end. The length of the double-stranded portion is not particularly limited, but can preferably be about 10 nucleotides or more in length, more preferably about 20 nucleotides or more in length. Here, the 3' protruding end can preferably be DNA, more preferably DNA having a length of at least 2 nucleotides or more, and even more preferably DNA having a length of 2 to 4 nucleotides. The factor that causes RNAi used in the present disclosure can be either artificially synthesized (e.g., chemically or biochemically) or naturally occurring, and there is no essential difference in the effect of the present disclosure between the two. In the case of chemically synthesized ones, purification is preferably performed by liquid chromatography or the like.

[0090] The factors that cause RNAi used in the present disclosure can also be synthesized in vitro. In this synthesis system, antisense and sense RNAs are synthesized from template DNA using T7 RNA polymerase and a T7 promoter. After annealing these in vitro and introducing them into cells, RNAi is caused through the mechanism as described above, and the effects of the present disclosure are achieved. Here, such RNAs can be introduced into cells by any appropriate method, for example, the calcium phosphate method. Factors that cause RNAi of the present disclosure also include single strands that can hybridize with mRNA, or factors such as all similar nucleic acid analogs thereof. Such factors are also useful in the present disclosure.

[0091] In this specification, examples of vectors include those that can replicate and be isolated and purified in common bacteria used in genetic experiments (a representative example being Escherichia coli strains derived from Escherichia coli K12 strain). This is necessary for constructing the target gene to be introduced into plants. Specifically, for example, there are commercially available constructed plasmids such as E. coli's pBR322 plasmid, pUC18, pUC19, pBluescript, and pGEM-T. When directly introducing a gene fragment into a plant cell for transformation using methods such as the electroporation method, polyethylene glycol method, or particle gun method, the construction of the gene to be introduced can be carried out using such commonly available commercial plasmids. Further, as a special example of a vector, when transforming a plant cell using the gene introduction method via Agrobacterium, a plasmid called a "binary vector" having a nucleotide sequence corresponding to the replication origins of both E. coli and Agrobacterium, and the border sequences (Left border and Right Border) derived from T-DNA indicating the border regions that can be introduced into plants is required. Examples include pBI101 (commercially available from Clontech), pBIN (Bevan, N., Nucleic Acid Research 12, 8711-8721, 1984), pBINPlus (van Engelen, FA et al., Tranegenic Research 4, 288-290, 1995), pTN or pTH (Fukuoka H et.al., Plant Cell Reports 19, 2000), pPZP (Hajdukiewicz P et al., Plant Molecular Biology 25, 989-994, 1994), etc., but are not limited thereto. In addition, as a vector that can be used in plants, a tobacco mosaic virus vector is also exemplified. However, since this type of vector does not introduce the target gene into the plant chromosome, its use is limited when it is not necessary to propagate the gene-introduced plant via seeds, but it can be used in the present disclosure.

[0092] In this specification, the technique for introducing a nucleic acid molecule into a cell can be any technique, for example, transformation, transduction, transfection, etc. Such techniques for introducing nucleic acid molecules are well-known and commonly used in the art. For example, Ausubel F. A. et al. (eds.) (1988), Current Protocols in Molecular Biology, Wiley, New York, NY; Sambrook J. et al. (1987) Molecular Cloning: A Laboratory Manual, 2nd Ed. and its 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, Separate Volume of Experimental Medicine "Gene Transfer & Expression Analysis Experimental Methods", Yodosha, 1997, etc. The introduction of genes can be confirmed using the methods described in this specification such as Northern blot and Western blot analysis or other well-known and commonly used techniques.

[0093] Also, as the method for introducing a vector, any method as described above for introducing DNA into a cell can be used, for example, transfection, transduction, transformation, etc. (for example, calcium phosphate method, liposome method, DEAE dextran method, electroporation method, method using a particle gun (gene gun), etc.), lipofection method, spheroplast method [Proc. Natl. Acad. Sci. USA, 84, 1929 (1978)], lithium acetate method [J. Bacteriol., 153, 163 (1983)], the method described in Proc. Natl. Acad. Sci. USA, 75, 1929 (1978).

[0094] As used herein, the "gene transfection reagent" refers to a reagent used to promote the transfection efficiency in a gene transfection method. Such gene transfection reagents include, for example, but are not limited to, cationic polymers, cationic lipids, polyamine-based reagents, polyimine-based reagents, calcium phosphate, etc. Specific examples of reagents used in transfection include commercially available reagents from various sources, such as Effectene Transfection Reagent (cat.no.301425, Qiagen, CA), TransFastTM Transfection Reagent (E2431, Promega, WI), TfxTM-20 Reagent (E2391, Promega, WI), SuperFect Transfection Reagent (301305, Qiagen, CA), PolyFect Transfection Reagent (301105, Qiagen, CA), LipofectAMINE 2000 Reagent (11668-019, Invitrogen corporation, CA), JetPEI(×4)conc. (101-30, Polyplus-transfection, France), and ExGen 500 (R0511, Fermentas Inc., MD), etc., but are not limited thereto.

[0095] When the present disclosure is used in plants, methods well-known to those skilled in the art, such as the method via Agrobacterium and the method of direct introduction into cells, can be used for introducing a plant expression vector into plant cells. As the method via Agrobacterium, for example, the method of Nagel et al. (Nagel et al. (1990), Microbiol. Lett., 67, 325) can be used. This method first transforms Agrobacterium by electroporation with an expression vector suitable for plants, for example, and then introduces the transformed Agrobacterium into plant cells by the method described by Gelvin et al. (edited by Gelvin et al. (1994), Plant Molecular Biology Manual (Kluwer Academic Press Publishers)). As the method of directly introducing a plant expression vector into cells, the electroporation method (see Shimamoto et al. (1989), Nature, 338:274-276; and Rhodes et al. (1989), Science, 240:204-207), the particle gun method (see Christou et al. (1991), Bio / Technology 9:957-962), and the polyethylene glycol (PEG) method (see Datta et al. (1990), Bio / Technology 8:736-740) can be mentioned. These methods are well-known in the art, and a method suitable for the plant to be transformed can be appropriately selected by those skilled in the art.

[0096] Cells into which a plant expression vector has been introduced are first selected by drug resistance such as hygromycin resistance and kanamycin resistance. Then, they can be redifferentiated into plant tissues, plant organs, and / or plants by methods well-known in the art. Furthermore, seeds can be obtained from the plants. The expression of the introduced gene can be detected by Northern blotting or PCR. If necessary, the expression of the protein as the gene product can be confirmed, for example, by Western blotting.

[0097] Although the present disclosure has been shown to be particularly useful in plants, it can also be used in other organisms. The molecular biology techniques used in the present disclosure are well-known and commonly used in the art, for example, as described in Ausubel F.A. et al. (eds.) (1988), Current Protocols in Molecular Biology, Wiley, New York, NY; Sambrook J et al. (1987) Molecular Cloning: A Laboratory Manual, 2nd Ed. and its 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, and "Gene Transfer & Expression Analysis Experimental Methods" in Experimental Medicine Separate Volume, Yodosha, 1997, etc.

[0098] In methods of transformation, physical methods include the polyethylene glycol method (PEG method), electroporation method, microinjection method, and particle gun method. These methods are highly useful in that they can be applied to both monocotyledonous and dicotyledonous plants. However, in the polyethylene glycol method and the electroporation method, since the cell wall becomes an obstacle, protoplasts must be used, and there is a problem in that the frequency of integration of the introduced gene into the chromosomal DNA of plant cells is low. In addition, in the microinjection method using callus or tissue without using protoplasts, there are many difficulties regarding the thickness of the needle and the fixation of the tissue. In the particle gun method using tissue, there are also problems such as mutations appearing in a chimeric form. In addition, in these physical methods, generally, the introduced foreign gene is likely to be integrated as a multi-copy gene in an incomplete state into the nuclear genome. It is known that when a foreign gene is introduced in multiple copies, the gene is likely to be inactivated.

[0099] For the method of introducing an isolated gene using an organism, there are the Agrobacterium method, the viral vector method, and the method using pollen as a vector, etc. Since these methods introduce genes using plant callus, tissues or plants without using protoplasts, they have the advantages that the culture period is not long and they are less likely to be affected by obstacles such as somaclonal variation. Among these, the method using pollen as a vector has few experimental examples yet, and there are many unknown parts as a plant transformation method. The viral vector method has the advantage that the gene to be introduced spreads throughout the whole plant infected with the virus, but there are problems in that there is no guarantee that it will be transmitted to the next generation as it is only amplified and expressed in each cell, and that long DNA fragments cannot be introduced. The Agrobacterium method has many advantages, such as the ability to introduce DNA of about 20 kbp or more into the chromosome without major rearrangement, the low copy number of the introduced gene, and high reproducibility. Since Agrobacterium is outside the host range for monocotyledonous plants such as Gramineae plants, the introduction of foreign genes into Gramineae plants has conventionally been carried out by physical methods as described above. However, in recent years, in plants with established culture systems such as rice among monocotyledonous plants, the Agrobacterium method has come to be applied, and rather, the Agrobacterium method is now preferably used.

[0100] In the introduction of foreign genes by the Agrobacterium method, when a low-molecular phenolic compound such as acetosyringone synthesized by the plant acts on the Vir region of the Ti plasmid, the T-DNA region is excised from the Ti plasmid and integrated into the nuclear chromosomal DNA of plant cells through several processes. In dicotyledonous plants, since the plant itself has a synthesis mechanism for such phenolic compounds, foreign genes can be easily introduced by the leaf disk method, etc., and the reproducibility is also high. In contrast, in monocotyledonous plants, since the plant itself does not synthesize such phenolic compounds, it has been difficult to produce transgenic plants by Agrobacterium. However, by adding acetosyringone at the time of Agrobacterium infection, it is now possible to introduce foreign genes into monocotyledonous plants.

[0101] In the present disclosure, in the transformant, the nucleic acid molecule of interest (transgene) may or may not be introduced into the chromosome. Preferably, the nucleic acid molecule of interest (transgene) is introduced into the chromosome, and more preferably, it is introduced into both of two chromosomes.

[0102] (Plant) Preferred plants are not limited to ornamental plants for viewing flowers, but also include any plant for which it is useful to prevent flower senescence, such as crops, trees, lawns, weeds, etc. Unless otherwise indicated, plants include any of plant bodies, plant organs, plant tissues, plant cells, and seeds. Examples of plant organs include roots, bulbs, leaves, stems, flowers (or parts of organs or tissues constituting the flower, such as petals or similar organs (e.g., perianth)), pollen, bracts, nuts, and fruits. Also, the plant may be provided as a combination of plant tissues or plant organs, and in this case, for example, it may be provided as a cut flower or potted flower (a combination of flower, stem, leaf and / or root). Examples of plant cells include callus and suspension culture cells.

[0103] As methods for genome editing in plant cells, any of them can be used as described in detail elsewhere in this specification. For example, the methods described in Yan, R.; Wang, Z.; Ren, Y.; Li, H.; Liu, N.; Sun, H. Establishment of Efficient Genetic Transformation Systems and Application of CRISPR / Cas9 Genome Editing Technology in Lilium pumilum DC. Fisch. and Lilium longiflorum White Heaven. Int. J. Mol. Sci. 2019, 20, 2920., and the methods described in JP 2020-130054 A, etc. are exemplified.

[0104] As a method for introducing a recombinant vector into a plant cell, any method for introducing DNA into a plant cell can be used as long as it is described in detail elsewhere in this specification. For example, Agrobacterium (Japanese Patent Laid-Open No. 59-140885, Japanese Patent Laid-Open No. 60-70080, WO94 / 00977), electroporation method (Japanese Patent Laid-Open No. 60-251887), method using a particle gun (gene gun) (Japanese Patent No. 2606856, Japanese Patent No. 2517813), etc. are exemplified.

[0105] In this specification, plant cultivation can be carried out by any method known in the art. Methods for cultivating plants are described in "Mastering Lilies" (edited by Hideo Imai, Noh Bunka Kyokai, 2006), etc.

[0106] For the culture, dedifferentiation, differentiation, and regeneration of plant cells, plant tissues, and plants, techniques and media known in the art are used. Such media include, for example, Murashige-Skoog (MS) medium, Gamborg B5 (B) medium, White medium, Nitsch&Nitsch (Nitsch) medium, etc., but are not limited thereto. These media are usually used with an appropriate amount of plant growth regulators (plant hormones) added.

[0107] To create a plant cell, plant tissue, plant organ, or plant or a part thereof in which the gene of LhAPS or its homologous gene is partially or completely mutated or deleted, the whole plant or a part thereof may be mutated by exposing it to a conventional mutagen. Alternatively, a part of the plant may be dedifferentiated to form callus or cells, and the callus or cells may be modified by genetic engineering or the like to obtain callus or cells containing the factor of the present disclosure or in which the factor of the present disclosure is modified.

[0108] As used herein, in the case of plants, "redifferentiation" means the phenomenon in which cells in an undifferentiated state differentiate into more differentiated entities such as functional cells, tissues, or entire individuals. For example, by redifferentiating callus, tissue pieces such as cells (leaves, roots, etc.) can be formed, and by growing these tissue pieces, organs or plants can be formed.

[0109] Methods for redifferentiating transformants into plants are well known in the art. Such methods include, but are not limited to, those described in Rogers et al., Methods in Enzymology 118:627-640 (1986); Tabata et al., Plant Cell Physiol., 28:73-82 (1987); Shaw, Plant Molecular Biology: A practical approach. IRL press (1988); Shimamoto et al., Nature 338:274 (1989); Maliga et al., Methods in Plant Molecular Biology: A laboratory course. Cold Spring Harbor Laboratory Press (1995). Therefore, those skilled in the art can appropriately use the above well-known methods according to the transgenic plants aimed at, and redifferentiate them. The transgenic plants thus obtained have the target gene introduced therein, and the introduction of such a gene can be confirmed using the methods described herein such as Northern blot and Western blot analysis or other well-known conventional techniques.

[0110] (Preferred Embodiment) The preferred embodiments of the present disclosure will be described below. It is understood that the embodiments provided below are for a better understanding of the present disclosure, and the scope of the present invention should not be limited to the following description. Therefore, it is obvious that those skilled in the art can make appropriate modifications within the scope of the present invention in consideration of the descriptions in this specification.

[0111] <LhAPS gene and protein> In a representative aspect of the present disclosure, there are provided the LhAPS gene and the LhAPS protein encoded by this gene, which have been newly discovered by the present disclosure. LhAPS is one of the transcription factor family genes whose expression level increases during flower senescence. However, as a result of creating a transformant (LNR4) in which the expression of LhAPS was suppressed, it was observed in tulips (Oriental hybrid line), which are plants of the Liliaceae family, that the period until the flowers turn brown was extended (see Examples). These results indicate that LhAPS is a transcription factor family gene that controls (promotes) flower senescence, and that flower senescence can be delayed by suppressing the expression of LhAPS. In the Oriental hybrid line, the sequence homology of the LhAPS gene is very high, at 99% or more, between the Tiara variety used in the examples and other varieties (the Casablanca variety and the Acapulco variety). Therefore, it is understood that the suppression of senescence by the suppressor of LhAPS is not specific to the variety. In addition, it is understood that in other Liliaceae plants as well, the ornamental period can be extended by suppressing the expression of LhAPS orthologs by genome editing, genetic recombination, or suppressors of LhAPS such as inhibitors. In particular, the homologous gene of the LhAPS gene possessed by tulips (genus Tulipa of the Liliaceae family) (having the nucleotide sequence described in SEQ ID NO: 4) has about 72.1% sequence identity with the nucleic acid sequence encoding tulip LhAPS (SEQ ID NO: 1). Therefore, it is considered that if it is a sequence variant having at least about 70% sequence identity, flower senescence can be delayed by suppressing its expression. The LhAPS protein commonly has a region predicted to bind to DNA conserved at least in Liliaceae plants.In particular, although not wishing to be bound by theory, in liliaceae plants, programmed cell death occurs during flower senescence, and the expression of a common gene group is induced (see, for example, van Doorn WG, Woltering EJ. Physiology and molecular biology of petal senescence. Journal of Experinental Botany 59:453-80, 2008), and it is understood that a similar regulatory mechanism exists. It is understood that the inhibition of the activity of the LhAPS ortholog also has the same effect in plants other than lilies. It is understood that the factors of the present disclosure also have the same effect in plants other than the examples. The factors of the present disclosure may be any factor that can temporarily or permanently suppress, reduce or eliminate the function of LhAPS, including proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (including DNA such as cDNA and genomic DNA, and RNA such as mRNA), factors for genome editing, polysaccharides, oligosaccharides, lipids, small organic molecules (such as hormones, ligands, signaling substances, small organic molecules, molecules synthesized by combinatorial chemistry, small molecules that can be used as pharmaceuticals (such as small molecule ligands, etc.)), and complex molecules thereof, but are not limited thereto. In some cases, they may also be other elements other than substances (such as mutagenic factors such as energy such as light, radiation, heat, and electricity).

[0112] The present disclosure is effective in the case of Liliaceae plants (for example, plants of the genus Lilium or Tulipa) where the aging effect progresses due to browning of the flower within a few days after flowering. Aging in Liliaceae plants (for example, plants of the genus Lilium or Tulipa) is different from the wilting and in-rolling of flowers commonly seen in other plants, so the aging suppression effect of the technology of the present disclosure is significantly useful. For example, also, the flowers of lilies and tulips are composed of three inner perianths and three outer perianths, while the flowers of typical plants other than Liliaceae plants do not have the same composition, for example, are composed of petals or are composed of a corolla in which the petals are fused together, and it is particularly noteworthy that the structure of three inner perianths and three outer perianths can also suppress aging. Furthermore, the effect in monocotyledonous plants is also considered to be particularly noteworthy.

[0113] A representative nucleic acid sequence of the LhAPS gene includes the nucleic acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 or a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 2. Therefore, in one aspect, the present disclosure provides a nucleic acid molecule comprising the nucleic acid sequence shown in SEQ ID NO: 1 or a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 2. In a more specific aspect, the nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NO: 1. In a further specific aspect, the nucleic acid molecule consists of the nucleic acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3.

[0114] In one aspect, the present disclosure provides a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2. In a more specific aspect, the amino acid sequence of the polypeptide comprises the sequence shown in SEQ ID NO: 2. In a further specific aspect, the amino acid sequence of the polypeptide consists of the sequence shown in SEQ ID NO: 2.

[0115] It is understood that the present disclosure is effective in suppressing flower senescence by suppressing the LhAPS homologous gene not only in plants of the genus Lily in the family Liliaceae, but also in any plant having the LhAPS homologous gene, particularly in all plants of the family Liliaceae. Without wishing to be bound by theory, this is because the functions of the LhAPS homologous gene are considered to be substantially similar, at least in all plants of the family Liliaceae, and although the LhAPS gene was found in the present disclosure, it has been found to be commonly found in all plants of the family Liliaceae in the analysis in the present disclosure.

[0116] <Suppressor of LhAPS> In one aspect, the present disclosure provides a factor that suppresses the function of the nucleic acid sequence or amino acid sequence described in <LhAPS gene and protein>, or a modified sequence thereof. The modified sequence of the nucleic acid sequence is: (A-1) a sequence variant having one or more substitutions, additions and / or deletions in the nucleic acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, or the nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 2; (A-2) a sequence variant having at least about 70% sequence identity to the nucleic acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, or the nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 2; (A-3) a sequence variant that hybridizes under stringent conditions to the nucleic acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, or the nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 2; (A-4) a sequence variant that is an allelic variant of the nucleic acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, or the nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 2; or (A-5) a sequence variant that is a fragment of (A-1) to (A-4), and the protein encoded by the sequence variant has the biological function of the protein encoded by the nucleic acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, or the nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 2. The modified sequence of the amino acid sequence includes: (a) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 or a fragment thereof; (b) a polypeptide in which one or more amino acids are mutated in the amino acid sequence shown in SEQ ID NO: 2, and the mutation is selected from the group consisting of substitution, addition and / or deletion, and has the same type of biological activity as the biological activity of the polypeptide in (a); (c) a polypeptide encoded by a splice variant or allelic variant of the nucleotide sequence shown in SEQ ID NO: 1; (d) a polypeptide encoded by a homologous gene of the gene encoding the amino acid sequence shown in SEQ ID NO: 2; or (e) a polypeptide having an amino acid sequence with at least about 70% identity to any one of the polypeptides in (a) to (d) and having the same type of biological activity as the biological activity of the polypeptide in (a).The factor may be a protein, polypeptide, oligopeptide, peptide, polynucleotide, oligonucleotide, nucleotide, nucleic acid including RNA or DNA, polysaccharide, oligosaccharide, lipid, hormone, ligand, signaling substance, synthetic chemical, radiation, a combination thereof, or a complex factor. In a more specific aspect, the factor is selected from the group consisting of an antisense molecule, an RNAi factor, a factor for genome editing, and a mutagenic factor.

[0117] In one aspect, the present disclosure provides a factor that suppresses LhAPS. Suppression of LhAPS can be achieved, for example, by a method selected from the group consisting of a decrease in gene expression level, a decrease in gene copy number, a decrease in gene amplification, a decrease in RNA activity level, a decrease in mRNA abundance, a decrease in mRNA synthesis rate, a decrease in mRNA stability, a decrease in protein activity level, a decrease in protein synthesis, a decrease in protein abundance, a decrease in protein stability, a decrease in protein enzyme activity, or a combination thereof. The factor that suppresses LhAPS can be any substance as long as it partially or completely suppresses the biological function of the LhAPS gene or protein in a plant. The factor may be a protein, polypeptide, oligopeptide, peptide, polynucleotide, oligonucleotide, nucleotide, nucleic acid including RNA or DNA, polysaccharide, oligosaccharide, lipid, hormone, ligand, signaling substance, synthetic chemical, radiation, a combination thereof, or a complex factor. In a more specific aspect, the factor is selected from the group consisting of an antisense molecule, an RNAi factor, a factor for genome editing, and a mutagenic factor. In this specification, it has been demonstrated that an LhAPS expression suppression line was created by RNAi factor and genome editing, and the aging of flowers was delayed (see Examples).

[0118] In one embodiment, the factor is in the form of nucleic acid. Examples of inhibitors in the form of nucleic acid include aptamers, ribozymes, antisense oligonucleotides, RNAi factors such as siRNA, and the like. In other embodiments, factors in the form of protein can also be used. Examples of such factors in the form of protein include factors for genome editing such as artificial nucleases, antibodies or antigenic fragments, binding peptides, peptidomimetics, and the like. Alternatively, the factor of the present disclosure may be other molecules such as small molecules.

[0119] In one embodiment, the factor is a factor that causes RNA interference of LhAPS in the form of double-stranded nucleic acid.

[0120] In a specific embodiment, the factor of the present disclosure is in double-stranded form, and one strand thereof contains (I) (A) a nucleic acid sequence shown in SEQ ID NO: 1 (LhAPS itself) or at least about 20 bases, about 50 bases, about 100 bases, about 150 bases, about 200 bases of the sequence encoding SEQ ID NO: 2 (or minimally including any number between these numbers); or (B) a sequence variant, and the other strand contains (II) a complementary sequence or an annealing sequence of (I), and the (B) sequence variant is (B-1) a sequence variant having one or several substitutions, additions or deletions in (A); (B-2) a sequence variant having at least about 70%, preferably at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to the sequence of (A); or (B-3) a sequence variant that hybridizes under stringent conditions to the sequence of (A); (B-4) a sequence variant that is an allelic variant of (A); or (B-5) a sequence variant that is a fragment of (A) or (B-1) to (B-4), and the protein encoded by the sequence variant has the biological function of the protein encoded by (A).

[0121] In a further specific embodiment, the factor of the present disclosure is in a double-stranded form, and one of its strands (I) does not contain the 480-base sequence at positions 30-507 of SEQ ID NO: 1. Without wishing to be bound by theory, the base sequence at positions 30-507 of SEQ ID NO: 1 corresponds to a DNA binding site, and targeting this site may also suppress the expression of genes other than the LhAPS gene. Therefore, it is desirable to target regions other than this site. In a more specific embodiment, one strand (I) contains the nucleic acid sequence at positions 726-1036 of SEQ ID NO: 1. Specific embodiments are not limited to this. For example, it is understood that any range may be used as long as the length is 98-853 bases (Wesley et al., Plant Journal 27:581-590). Also, appropriate RNAi factors can be designed with reference to Wesley et al., Plant Journal 27:581-590, etc.

[0122] In plant RNAi, it is generally considered that mRNA degradation can be induced using the sequence of any region of the transcript (mRNA) of the target gene. From the principle of RNAi, it is obvious to those skilled in the art that mRNA degradation will occur as long as the transcribed sequence of the target gene is included (see, for example, Wesley et al. Plant Journal 27:581-590). Although any region can be selected, preferably, when targeting the sequence of a region with very high conservation (a sequence common among multiple genes), there is a possibility of degrading the mRNA of genes having the same sequence in addition to the target gene. Therefore, it is advantageous to exclude the conserved region from the target sequence. Also in the RNAi construct of LhAPS of the present disclosure, those performed in the examples have excluded regions with relatively high conservation just in case. It is known that RNAi can be induced by targeting the 5'UTR and 3'UTR (untranslated regions) instead of the coding region (see, for example, Wesley et al. Plant Journal 27:581-590). In plants, siRNA can also be used, but preferably, it is understood that longer RNAi factors can also be used (in plants, longer dsRNA is expressed, but ultimately it is processed into siRNA of about 20 bases to induce silencing).

[0123] In one aspect, the present disclosure provides a vector comprising a nucleic acid that causes RNA interference of LhAPS. The nucleic acid that causes RNA interference of LhAPS included herein is in any form exemplified by the factors that suppress LhAPS of the present disclosure, and it is understood that any nucleic acid can be used as long as it is a nucleic acid that causes RNA interference of LhAPS.

[0124] In another embodiment, the vector of the present disclosure comprises a promoter and / or a terminator.

[0125] In one aspect, the present disclosure provides a factor for genome editing that causes the function and / or suppression of the function of LhAPS. The factor for genome editing may be any factor used in techniques such as CRISPR / Cas9, TALEN, ZFN, meganuclease, etc.

[0126] When CRISPR / Cas9 is selected as the genome editing technique, a guide RNA having a sequence of 23 bases including NGG (where N is any base of ATGC) called a PAM sequence at the 3' end is used. The sequence of the guide RNA can be designed by a program such as CRISPR-P. The sequence of the guide RNA used for genome editing is preferably one that targets genome editing in the nucleic acid sequence of positions 1 to 507 of SEQ ID NO: 1, and examples of the sequence of the guide RNA include the sequences shown in the following table.

Table 3-1

Table 3-2

Table 3-3

[0127] In one aspect, the present disclosure provides a plant cell containing a factor that suppresses the LhAPS of the present disclosure or modified by the factor. In another aspect, a plant tissue containing the factor or modified by the factor or a plant tissue containing the plant cell is provided. In yet another aspect, a plant organ containing the factor or modified by the factor or a plant organ containing the plant cell or the plant tissue is provided. In yet another aspect, a plant body or a part thereof containing the factor or modified by the factor, or a plant body or a part thereof containing the plant cell, the plant tissue, or the plant organ is provided.

[0128] In a preferred embodiment, the plant cell of the present disclosure is a plant cell of the Liliaceae family. Such plant cells include, for example, the genus Lilium, the genus Tulipa, the genus Erythronium, the genus Fritillaria, the genus Amana, the genus Bowiea, the genus Calochortus, the genus Cardiocrinum, the genus Clintonia, the genus Gagea, the genus Lloydia, the genus Medeola, the genus Nomocharis, the genus Notholirion, the genus Prosartes, the genus Streptopus, the genus Tricyrtis, etc., and examples include, but are not limited to, lily, tulip, erythronium, and black lily. In a more preferred embodiment, the plant cell of the present disclosure is a lily cell.

[0129] In one embodiment, a plant body or a part thereof containing a factor that suppresses LhAPS includes a bulb, a seed, a flower, a stem, a leaf, pollen, a scale, an ovule, a cell, a tissue, a tissue culture, or an embryo sac, or a combination of all or part of them. In a specific embodiment, for example, it can be provided in the form of the whole flower, or an organ constituting the flower such as a petal, a corolla, and a perianth, or a combination of organs including the flower (for example, a cut flower or a potted flower, etc.).

[0130] (Method for producing a plant in which LhAPS or its homologous gene is suppressed) In another aspect, the present disclosure provides a plant cell in which LhAPS is suppressed more than in its natural state. The "natural state" means a state in which the expression level of LhAPS is equivalent to that of the housekeeping gene Elongation factor 1 alpha (EF1alpha) at the peak of expression. Here, since the expression level of LhAPS is low on the 0th day after flowering but reaches a peak on about the 4th to 5th day after flowering, the expression level on about the 4th to 5th day after flowering is used. Such cells, in which LhAPS is suppressed more than in its natural state, can be achieved by introducing a factor that suppresses LhAPS of the present disclosure into plant cells. When obtaining such plant cells using a factor that suppresses LhAPS, conventionally, transduction is performed using a vector containing a drug resistance gene (for example, a hygromycin resistance gene), and plant cells into which a mutation has been inserted can be screened by screening using the drug resistance marker. Alternatively, plants in which LhAPS is suppressed may be selected by PCR or the like for plants or cells in which LhAPS has mutated from a population obtained by subjecting a plant body or plant cells to mutagenesis treatment with a mutagen or the like. In this case, for example, in the case of lily, those having a lower level of expression or activity than the natural LhAPS disclosed in the present disclosure can be selected. The same can be done for other plants. Alternatively, in addition to the method of introducing a suppressing factor and screening, methods such as zinc finger nuclease or gene targeting may be used to modify the sequence of endogenous LhAPS and suppress its expression. Regarding the application of zinc finger nuclease in plants in the practice of the present disclosure, reference can be made to Keishi Osakabe, Yuriko Osakabe and Seiichi Toki, Site-directed mutagenesis in Arabidopsis using custom-designed zinc-finger nucleases. Proc. Nat. Acad. Sci. USA (2010) June 29, 107(26): 12034-12039.Regarding gene targeting, reference can be made to Molecular breeding of a novel herbicide-tolerant rice by gene targeting. Endo, Masaki; Osakabe, Keishi; Ono, Kazuko; Handa, Hirokazu; Shimizu, Tsutomu; Toki, Seiichi. The Plant Journal vol.52 issue 1 October 2007. p.157-166; Rie Terada, Hiroko Urawa, Yoshishige Inagaki, Kazuo Tsugane, and Shigeru Iida: Efficient gene targeting by homologous recombination in rice. Nature Biotechnology 20(10)1030-1034 (2002).

[0131] That the plant cells, tissues, organs or plants obtained by screening have LhAPS suppressed compared to the natural state can be confirmed, for example, by a method selected from the group consisting of a decrease in gene expression level, a decrease in gene copy number, a decrease in gene amplification, a decrease in RNA activity level, a decrease in mRNA abundance, a decrease in mRNA synthesis rate, a decrease in mRNA stability, a decrease in protein activity level, a decrease in protein synthesis, a decrease in protein abundance, a decrease in protein stability, a decrease in protein enzyme activity, or a combination thereof. A decrease in gene expression level can typically be confirmed by techniques such as real-time PCR (RT-PCR). A decrease in protein expression level can typically be confirmed by techniques such as Western Blotting. In addition, in the case of LhAPS, a decrease in protein enzyme activity can be measured by the expression level of downstream genes or reporter assays and the like.

[0132] Screening may be performed in any state of cells, tissues, organs, or plants. Preferably, screening for drug resistance is performed on callus. The presence or absence of gene introduction and the introduction of mutations by genome editing are analyzed by PCR or sequencing using DNA extracted from adventitious buds (shoots) induced from callus. The decrease in LhAPS gene expression and protein amount is analyzed in the perianth.

[0133] Plant cells having the traits of interest of the present disclosure can be redifferentiated into tissues, organs, or plants by methods known in the art.

[0134] Once a plant having the traits of interest of the present disclosure is obtained as a transformant, the transformant may be used as a pollen parent or a seed parent, and a new variety having the traits of interest of the present disclosure may be produced by a conventional crossing method.

[0135] In a preferred embodiment, the plant cells of the present disclosure are plant cells of the Liliaceae family. Examples of such plant cells include, but are not limited to, genus Lilium, genus Tulipa, genus Erythronium, genus Fritillaria, genus Amana, genus Bowiea, genus Calochortus, genus Cardiocrinum, genus Clintonia, genus Gagea, genus Lloydia, genus Medeola, genus Nomocharis, genus Notholirion, genus Prosartes, genus Streptopus, genus Tricyrtis, etc., and examples include, but are not limited to, lily, tulip, erythronium, and fritillary. In a more preferred embodiment, the plant cells of the present disclosure are cells of lily.

[0136] In one embodiment, the suppression of flower senescence in the present disclosure only requires that the senescence of some or all of the components constituting the flower be suppressed. The components constituting the flower include the following: petals, corolla, perianth, stamens, pistils, sepals, receptacle, floral axis. The suppression of flower senescence can be the suppression of senescence of at least one, at least two, at least three, at least four or more of the components constituting the flower. In a preferred embodiment, it may be advantageous if the senescence of any one of the components constituting the flower, for example, petals, corolla, perianth, etc., is suppressed. In a preferred embodiment, the suppression of flower senescence only requires that the suppression of senescence of any one of petals, corolla, perianth, etc. be achieved, or the suppression of senescence of the whole flower may also be achieved. Regarding the suppression of flower senescence, for stamens, pistils, etc., the suppression of senescence does not necessarily have to be achieved. In various embodiments, the suppression of flower senescence typically refers to the "petals" senescing, the corolla senescing, the perianth senescing, the perianth in a state of being attached to the receptacle senescing, the part of the whole flower excluding stamens and pistils or the whole flower (i.e., including all the components constituting the flower such as perianth, floral axis corresponding to the stem, stamens, pistils, etc.) senescing.

[0137] (Method for extending the lifespan of flowers) In another aspect, the present disclosure provides a method for extending the vase life of a flower in a plant body or a part thereof. The method for extending the vase life of a flower includes: 1) providing a cell (e.g., a plant cell), a plant tissue, or a plant body or a part thereof that contains or is modified by the factor described in <Suppressor of LhAPS>; 2) subjecting the cell, the plant tissue, or the plant body or a part thereof to flowering conditions; and 3) after flowering in the cell, the plant tissue, or the plant body or a part thereof, subjecting it to vase life conditions and a period. The period can be longer than the period when the vase life of the plant body or a part thereof without modification by the factor ends under the vase life conditions. For example, when a variety of the Oriental Hybrid strain of lily, "Tiara", is used, the period when the vase life ends without modification by the factor is about 5 days. In this case, the period when the vase life of the plant body or a part thereof modified by the factor ends can be 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, etc. In a preferred embodiment, the plant body or a part thereof can be the whole flower, or an organ constituting the flower such as petals, corolla, and perianth, or a combination of organs containing the flower (e.g., cut flowers or potted flowers, etc.).

[0138] (Screening) In another aspect, the present disclosure provides a method for screening an agent that delays flower senescence using the nucleic acid molecule (LhAPS or a variant thereof) of the present disclosure or the protein encoded thereby. As the screening method, methods known in the art can be applied. This method includes (i) a step of contacting LhAPS or a substance, cell, plant, etc. containing the same with a test substance; and (ii) a step of detecting the expression of LhAPS in the substance, cell, plant, etc. after contacting with the test substance. In step (i), cells, etc. used are those of Liliaceae plants. The agent that delays flower senescence can be selected from substances or factors that suppress the expression or activity of LhAPS in the test substance. Such substances or factors can be further introduced into a plant or plant cell to confirm whether they can delay flower senescence.

[0139] (General technology) The molecular biological, biochemical, and microbiological techniques used in this specification are well-known and commonly used in the art. For example, Sambrook J. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, F.M. (1987). Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, F.M. (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Innis, M.A. (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, F.M. (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, F.M. (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Innis, M.A. et al. (1995). PCR Strategies, Academic Press; Ausubel, F.M. (1999).These methods are described in Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; Sninsky, JJ et al. (1999); PCR Applications: Protocols for Functional Genomics, Academic Press; Special Edition of Experimental Medicine: "Gene Introduction & Expression Analysis Experimental Methods" Yodosha, 1997; "PCR Experimental Protocols for Plants" Shujunsha (1997); and Experimental Protocols for Model Plants Shujunsha (1996), the relevant parts of which (possibly in their entirety) are incorporated herein by reference.

[0140] DNA synthesis techniques and nucleic acid chemistry for producing artificially synthesized genes are described, for example, in Gait, MJ (1985). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Gait, MJ (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F. (1991). Oligonucleotides and Analogues: A Practical Approach, IRL Press; Adams, RL et al. (1992). The Biochemistry of the Nucleic Acids, Chapman & Hall; Shabarova, Z. et al. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, GM et al. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, GT (1996). Bioconjugate Techniques, Academic Press, and the like, the relevant portions of which are incorporated herein by reference.

[0141] References such as scientific literature, patents, and patent applications cited in this specification are incorporated herein by reference in their entirety to the same extent as if each were specifically recited.

[0142] As described above, the present disclosure has been described by showing preferred embodiments for ease of understanding. Hereinafter, the present disclosure will be described based on examples. However, the above description and the following examples are provided for illustrative purposes only and not for the purpose of limiting the present invention. Therefore, the scope of the present invention is not limited to the embodiments or examples specifically described herein, but is limited only by the claims.

Example

[0143] If necessary, the handling of plants and the like used in the following examples was carried out in accordance with the standards established by the Japanese government, or the National Agriculture and Food Research Organization, or the Niigata Agricultural Research Institute. In addition, although the reagents specifically used were the products described in the examples, equivalent products of other manufacturers (SIGMA-ALDRICH, Wako Pure Chemical Industries, Nacalai Tesque, Kanto Chemical, etc.) can also be substituted.

[0144] (Plant materials and growing conditions) In the following examples, the plant used was "Tiara", a variety of the Oriental Hybrid strain of lily. "Tiara" was obtained from domestic bulb sales companies such as Yamatoki Noen Co., Ltd. or Nakamura Noen Co., Ltd. The bulbs were planted in horticultural soil (adjusted peat moss, BVB) and cultivated in a greenhouse. The vase life of the flowers was evaluated by cutting the flowers immediately after blooming and placing them in distilled water in a room (temperature 20°C, PPFD 10 μmol·m -2 ·s -1 , 12-hour day length). The end of the vase life was defined as the time when at least half of the perianths had turned brown, or when the number of perianths showing browning was at least half or more.

[0145] (Example 1: Isolation of LhAPS) As a result of intensive research on the lily genome, the present inventors newly discovered a gene that may be related to flower senescence. This gene was isolated, 3'-RACE and 5'-RACE were performed based on the isolated partial sequence, and the nucleotide sequence including the full length of the ORF was determined. The RNeasy plant mini kit (Qiagen) was used for RNA extraction, and the PrimeScript RT Master Mix (Takara bio) was used for cDNA synthesis. PrimeSTAR HS DNA polymerase (Takara bio) was used for PCR. A cDNA clone containing the full length ORF was obtained by RT-PCR using a primer set for full length cloning (LhAPS-Full-F20: 5'-TCCTCCTCCTCCGACCAC-3' (SEQ ID NO: 6), LhAPS-Full-R1191: 5'-CAATCCATACAATCTACAAACTTTTCC-3' (SEQ ID NO: 7)). The obtained cDNA clone was cloned into the pGEM-T easy vector (Promega), and the nucleotide sequence was determined and named LhAPS.

[0146] (Example 2: Production of LhAPS expression-suppressed transformants) The transformant with suppressed LhAPS expression was created using the RNAi method. A 311-base pair LhAPS cDNA fragment (726 - 1036 bases) was cloned into pDONR201 (Invitrogen), and then inserted into the plant transformation vector pH7GWIWG(II) (Karimi M, et al. 2002. GATEWAY vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci 7: 193 - 195.) with the hygromycin resistance gene (HPT) using LR ClonaseII Enzyme mix (Invitrogen). The resulting transformation vector pH7-APS contains the LhAPS cDNA fragment in the antisense and sense directions downstream of the cauliflower mosaic virus 35S promoter, and a 35S terminator downstream of that. pH7-APS was introduced into cultured cells derived from the filaments of the lily cultivar "Tiara" using Agrobacterium strain EHA101. Specifically, the filaments of the lily cultivar "Tiara" were cultured in a callus induction medium (MS medium excluding -1 gelangum, 2 mg / L -1 picloram, and adding MgSO 4 ·7H 2 O) to induce callus. The callus was subcultured to an adventitious bud regeneration medium that induces both growth and redifferentiation (the above composition with the picloram addition concentration changed to 0.1 mg / L -1 and further adding 1 mg / L -1 thidiazuron) and cultured for 14 days. After inoculating the callus with Agrobacterium strain EHA101 carrying the transformation vector pH7-APS, it was selected in an adventitious bud regeneration medium with 15 mg / L -1 hygromycin added. The regenerated adventitious buds were transferred to a bulb enlargement medium (4 g / L -1The bulbs were then transplanted onto a hormone-free MS medium supplemented with gellan gum to encourage bulb formation, and after approximately 12 months, they were transplanted into horticultural soil in a special screened greenhouse for acclimation. The acclimated bulbs were then grown and allowed to flower. Four types of LhAPS expression suppression lines (LNR4) were obtained (LNR4-2, LNR4-10, and LNR4-15). The shelf life of the flowering lilies was evaluated using the method described above. The presence or absence of the introduced gene was confirmed by PCR amplification of the HPT gene.

[0147] (Results and Discussion) Table 1 shows the flower shelf life of the wild type and the LhAPS expression suppression line (LNR4). In Table 1, the wild type flower shelf life was 5 days, while the three strains of the LhAPS expression suppression line had a shelf life of 8 to 9 days. Figure 1 shows photographs of the wild type and the LhAPS expression suppression line from flowering to senescence. In Figure 1, in the wild type, three or more perianths were partially browned on the fifth day after flowering, whereas in the LhAPS expression suppression line, no browning of the perianth was observed even on the eighth day after flowering, and three or more perianths were partially browned on the ninth day. This result shows that suppression of LhAPS delays the senescence of the perianth compared to the wild type, and extends the flower shelf life (ornamental period) by about 1.5 to 2 times. [Table 1]

[0148] Example 3: Analysis of gene expression by quantitative real-time RT-PCR RNA was extracted from the inner perianth of flowers on the 4th day after flowering of wild-type and LhAPS expression-suppressed lines using the RNeasy plant mini kit (Qiagen). PrimeScript RT Master Mix (Takara bio) was used for cDNA synthesis, and SYBR Premix EX TaqII (Takara bio) was used for PCR. PCR was performed using a Thermal Cycler Real Time System (Model TP600, Takara bio). The primers for amplifying LhAPS used in PCR were LhAPS-QF-679 (5’-TCACACTTTGGGCTTCACTG-3’ (SEQ ID NO: 8)) and LhAPS-QR-777 (5’-CCACTACCCTAAAACCACACA-3’ (SEQ ID NO: 9)). The expression level was normalized using LhEF1alpha, which is an internal control, and calculated as the relative expression level. The primers for amplifying LhEF1alpha were LhEF1a-QF2-729 (5’-TGAGGTGTTCTTCTATTCATGTCC-3’ (SEQ ID NO: 10)) and LhEF1a-QR2-867 (5’-CCAAATAGAGCAGCAAAGCA-3’ (SEQ ID NO: 11)).

[0149] (Results and Discussion) The relative expression levels of the LhAPS gene in the perianth on the 4th day after flowering of wild-type and LhAPS expression-suppressed lines are shown in Table 2 and Figure 2 below. It is shown that the gene expression of LhAPS in all LhAPS expression-suppressed lines (LNR4-2, LNR4-10, and LNR4-15) was significantly reduced compared to the wild-type. From this result, it was shown that the LhAPS expression-suppressed lines created in this example had more than 10-fold suppression of LhAPS expression compared to the wild-type. [Table 2]

[0150] (Example 4: Creation of transformants with mutations introduced into LhAPS by genome editing) Using a genome editing method utilizing CRISPR / Cas9, a mutant was introduced into LhAPS to create a transformant with suppressed or absent LhAPS expression. A gene expression cassette expressing a guide RNA and a Cas9 gene was inserted into the plant transformation vector pIG121-Hm (Ohta et al., Plant Cell Physiol. 31: 805-813 (1990)) having a hygromycin resistance gene (HPT). The obtained transformation vector pIG-APS contains a Cas9 gene downstream of the cauliflower mosaic virus 35S promoter, a gene expression cassette containing an actin terminator downstream thereof, and a guide RNA sequence described in Table 3 downstream of the rice U6 promoter, and downstream thereof, a gene expression cassette containing a guide RNA scaffold sequence and a poly T sequence. pIG-APS is introduced into cultured cells derived from the filaments of the lily cultivar "Tiara" using the Agrobacterium EHA101 strain. Specifically, the filaments of the lily cultivar "Tiara" are cultured in a callus induction medium (MS medium excluding 4 g L -1 gelangum, 2 mg L -1 picloram was added, and MgSO 4 ·7H 2 O) to induce callus. The callus is subcultured in an adventitious bud regeneration medium that simultaneously induces growth and redifferentiation (the picloram addition concentration was changed to 0.1 mg L -1 from the above composition, and 1 mg L -1 thidiazuron was added) and cultured for 14 days. After inoculating the callus with the Agrobacterium EHA101 strain having the transformation vector pIG-APS, selection is carried out in an adventitious bud regeneration medium supplemented with 15 mg L -1 hygromycin. The regenerated adventitious buds are transplanted to a bulb enlargement medium (MS medium supplemented with 4 g L -1 gelangum and excluding hormones) every 3 weeks to promote bulb formation, and after about 12 months, they are transplanted to horticultural soil in a specific net room for acclimatization. The acclimatized bulbs are grown and flowered. The presence or absence of the transgene is confirmed by PCR amplification of the HPT gene using DNA extracted from adventitious buds. The presence or absence of the introduced mutation is confirmed by PCR amplification of the region containing the target sequence using DNA extracted from adventitious buds and sequencing.

[0151] Alternatively, introduction of mutations by genome editing can also be achieved by directly injecting a complex of guide RNA and Cas9 protein into plant cells by particle bombardment without integrating the Cas9 gene and the guide RNA expression cassette into the plant genome. For example, the method described in Zhen Liang, Kunling Chen, Yi Zhang, Jinxing Liu1, Kangquan Yin, Jin-Long Qiu & Caixia Gao. Genome editing of bread wheat using biolistic delivery of CRISPR / Cas9 in vitro transcripts or ribonucleoprotein. Nature protocol 13:413-430 is exemplified. Specifically, a complex of guide RNA and Cas9 protein is introduced into the shoot apical meristem of lily by particle bombardment. The shoot apical meristem after the introduction treatment by particle bombardment is cultured by a method known in the art to regenerate adventitious buds. DNA is extracted from the regenerated adventitious buds, and the region containing the target sequence is sequenced to confirm the presence or absence of mutations. The adventitious buds in which the introduction of mutations is confirmed are transplanted to a bulb enlargement medium (MS medium supplemented with 4 g L -1 agar gum and without hormones) every three weeks to promote bulb formation, and transplanted to horticultural soil in a specific net room after about 12 months for acclimatization. The acclimatized bulbs are grown to flower.

[0152] (Example 5: Verification of LhAPS Expression in the Tepals of Different Lily Varieties) In this example, varieties of lily other than "Tiara" are used to verify the expression of LhAPS in the perianth. Based on the LhAPS gene sequence of "Tiara", primers for LhAPS amplification are designed, and RT-PCR is performed using RNA extracted from the perianth of the target lily to isolate LhAPS of the target lily. After determining the nucleotide sequence, the expression of LhAPS is analyzed by real-time PCR. Specifically, RNA is extracted from the perianth using the RNeasy plant mini kit (Qiagen), and cDNA is synthesized using the PrimeScript RT Master Mix (Takara bio). PrimeSTAR HS DNA polymerase (Takara bio) is used for PCR. The obtained cDNA clone is cloned into the pGEM-T easy vector (Promega), and the nucleotide sequence is determined. For expression analysis, RNA is extracted from the inner perianth of the flower on the 4th day after flowering using the RNeasy plant mini kit (Qiagen), and cDNA is synthesized using the PrimeScript RT Master Mix (Takara bio). Then, PCR is performed using SYBR Premix EX TaqII (Takara bio). A Thermal Cycler Real Time System (Model TP600, Takara bio) is used for PCR. The expression level of LhAPS is normalized using LhEF1alpha, which is an internal control, and calculated as the relative expression level.

[0153] (Example 6: Screening of inhibitors) Isolate the protein that interacts with the LhAPS protein and develop a system that can monitor the interaction with the LhAPS protein in vitro (96-well plate). Specifically, a reporter such as a fluorescent protein is used to measure the degree of inhibition of the interaction by the compound. In the implementation, the search methods for molecular target therapeutic drugs in cancer research can be referred to. Using the developed monitoring system, chemical screening is performed using a compound library to find a drug that inhibits the activity of the LhAPS protein.

[0154] As described above, the present disclosure has been illustrated using preferred embodiments of the present disclosure. However, it is understood that the scope of the present invention should be construed only by the claims. It is understood that patents, patent applications, and documents cited herein should be incorporated by reference into this specification as if the content thereof were specifically set forth herein.

Industrial Applicability

[0155] The present disclosure may be used in the field of flower gardening. Cut flowers with extended vase life (e.g., lilies) are commercially valuable. In other Liliaceae plants such as tulips, suppression of the expression of LhAPS orthologs can also delay flower senescence.

Sequence Listing Free-Text

[0156] SEQ ID NO: 1: A nucleic acid sequence corresponding to the translation region of LhAPS. SEQ ID NO: 2: The amino acid sequence of the LhAPS protein. SEQ ID NO: 3: The full-length nucleic acid sequence of LhAPS including the untranslated region. SEQ ID NO: 4: A partial-length nucleic acid sequence of the homologous gene of LhAPS in tulip. SEQ ID NO: 5: A partial-length amino acid sequence of the homologous gene of LhAPS in tulip. SEQ ID NO: 6: The sequence of the primer used for cloning the LhAPS gene. SEQ ID NO: 7: The sequence of the primer used for cloning the LhAPS gene. SEQ ID NO: 8: The sequence of the primer used for RT-PCR of the LhAPS gene. SEQ ID NO: 9: The sequence of the primer used for RT-PCR of the LhAPS gene. SEQ ID NO: 10: The sequence of the primer used for RT-PCR of the LhEF1alpha gene. SEQ ID NO: 11: The sequence of the primer used for RT-PCR of the LhEF1alpha gene. Array numbers 12 to 100: Sequences of guide RNAs used for genome editing.

Claims

1. A plant cell in which the expression of LhAPS encoded by the nucleic acid sequence shown in SEQ ID NO: 1 or represented by a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 2 is suppressed using RNA interference or genome editing, or the expression of a polypeptide having the amino acid sequence shown in SEQ ID NO: 2 is suppressed using RNA interference or genome editing, wherein the plant cell is a plant cell of the genus Allium.

2. A plant tissue in which the expression of LhAPS encoded by the nucleic acid sequence shown in SEQ ID NO: 1 or represented by a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 2 is suppressed using RNA interference or genome editing, or the expression of a polypeptide having the amino acid sequence shown in SEQ ID NO: 2 is suppressed using RNA interference or genome editing, or a plant tissue containing the cell according to claim 1, wherein the plant tissue is a plant tissue of the genus Allium.

3. A plant organ in which the expression of LhAPS encoded by the nucleic acid sequence shown in SEQ ID NO: 1 or represented by a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 2 is suppressed using RNA interference or genome editing, or the expression of a polypeptide having the amino acid sequence shown in SEQ ID NO: 2 is suppressed using RNA interference or genome editing, or a plant organ containing the cell according to claim 1 or the plant tissue according to claim 2, wherein the plant organ is a plant organ of the genus Allium.

4. A plant in which the expression of LhAPS having the nucleic acid sequence shown in SEQ ID NO: 1 or having a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 2 is suppressed using RNA interference or genome editing, or the expression of a polypeptide having the amino acid sequence shown in SEQ ID NO: 2 is suppressed using RNA interference or genome editing, or a plant containing the cell according to claim 1, the plant tissue according to claim 2, or the plant organ according to claim 3, wherein the plant is a plant of the genus Allium.

5. The plant according to claim 4, wherein the plant is Allium and the expression of the LhAPS is suppressed by 10 times or more compared to the wild type.

6. The plant according to claim 4 or 5, wherein the plant is a bulb, seed, flower, stem, leaf, pollen, scale, corm, cell, tissue, tissue culture, or ovary, or a combination of all or part thereof.

7. The plant according to any one of claims 4 to 6, wherein the plant body includes flowers.

8. A method for extending the longevity of flowers in a plant body, comprising: 1) providing a plant body, a cell according to claim 1, a plant tissue according to claim 2, a plant organ according to claim 3, or a plant body according to any one of claims 4 to 7, wherein the expression of LhAPS encoded by the nucleic acid sequence shown in SEQ ID NO: 1 or having a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 2 is suppressed, or the expression of a polypeptide having the amino acid sequence shown in SEQ ID NO: 2 is suppressed using RNA interference or genome editing, and the plant body is a plant body of the genus Lily; 2) subjecting the plant body to flowering conditions; 3) subjecting the plant body to longevity conditions and a period after flowering.

9. The method according to claim 8, wherein the period is longer than the period when the longevity of the plant body without modification by the RNA interference or genome editing ends under the longevity conditions.

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