JP2023038009A - Lily flower senescence delay method - Google Patents

Abstract

To develop effective quality maintenance techniques for flowers in general, including Liliaceae plants.SOLUTION: Provided are double stranded nucleic acids comprising factors that suppress LhHFS, for example, (I) (A) the sequence shown in SEQ ID NO: 1 (LhHFS itself) or the sequence encoding SEQ ID NO: 2; or (B) (B-1) a sequence variant of (A) having one or more substitutions, additions, or deletions, (B-2) a sequence variant of (A) having at least about 70% sequence identity, or (B-3) a sequence variant of (A) that hybridizes to the sequence of (A) under stringent conditions and that contains a sequence having the biological function of (A). The other strand (II) contains the complementary strand to (I).SELECTED DRAWING: None

Description

The present disclosure provides transcription factor family genes (LhHFS) that regulate floral senescence and methods of use thereof. For example, flower senescence can be delayed by partially or completely deleting the expression or function of the LhHFS gene by means of genome editing, RNA interference, introduction of a mutagen, or the like.

In lilies, no effective shelf life extenders have been developed, and no genes controlling flower senescence have been identified. In addition, conventional lily breeding based on crossbreeding takes about three years from crossbreeding to flowering, which is extremely time-consuming and inefficient.

In conventional lily breeding based on crossing, it takes about three years from crossing to flowering, so it takes a very long time to confer target traits by repeating crossing and selection, which is inefficient. In addition, the shelf life of each line has not been organized, and DNA markers related to shelf life have not been developed.

For flowers in general, the development of effective quality maintenance technology is required. Therefore, as a result of intensive research, the present disclosure has identified a gene that is conserved at least in all Liliaceae plants and that controls flower senescence, and has achieved development of quality preservation technology.

（項目２１） 前記ゲノム編集用の因子は配列番号１のうち塩基１～４８３位の核酸配列のゲノム編集を標的にするものである、上記項目のいずれかに記載の因子。
（項目２２） 上記項目のいずれかに記載の因子を含むかまたは該因子によって改変された植物細胞。
（項目２３） 上記項目のいずれかに記載の因子を含むかまたは該因子によって改変された植物組織または上記項目のいずれかに記載の細胞を含む植物組織。
（項目２４） 上記項目のいずれかに記載の因子を含むかまたは該因子によって改変された植物器官または上記項目のいずれかに記載の細胞もしくは上記項目のいずれかに記載の植物組織を含む植物器官。
（項目２５） 上記項目のいずれかに記載の因子を含むかまたは該因子によって改変された植物体またはその一部、あるいは上記項目のいずれかに記載の細胞、上記項目のいずれかに記載の植物組織もしくは上記項目のいずれかに記載の植物器官を含む植物体またはその一部。
（項目２６） 前記植物は、ユリ科の植物である、上記項目のいずれかに記載の植物体またはその一部。
（項目２７） 前記植物は、ユリ属の植物である、上記項目のいずれかに記載の植物体またはその一部。
（項目２８） 前記植物体またはその一部が、球根、種子、花、茎、葉、花粉、りん片、木子、細胞、組織、組織培養物もしくはむかご、またはそれらの全部もしくは一部の組合せである、上記項目のいずれかに記載の植物体またはその一部。
（項目２９） 前記植物体またはその一部が花またはその一部を含む、上記項目のいずれかに記載の植物体またはその一部。
（項目３０） その植物が有するＬｈＨＦＳまたはその相同遺伝子の少なくとも一部が抑制された植物細胞。
（項目３１） その植物が有するＬｈＨＦＳまたはその相同遺伝子の少なくとも一部が抑制された植物組織、あるいは上記項目のいずれかに記載の細胞を含む植物組織。
（項目３２） その植物が有するＬｈＨＦＳまたはその相同遺伝子の少なくとも一部が抑制された植物器官、あるいは上記項目のいずれかに記載の細胞もしくは上記項目のいずれかに記載の植物組織を含む植物器官。
（項目３３） その植物が有するＬｈＨＦＳまたはその相同遺伝子の少なくとも一部が抑制された植物体またはその一部、あるいは上記項目のいずれかに記載の細胞、上記項目のいずれかに記載の植物組織もしくは上記項目のいずれかに記載の植物器官を含む植物体またはその一部。
（項目３４） 前記ＬｈＨＦＳまたはその相同遺伝子の少なくとも一部の発現および／または機能が抑制されている、上記項目のいずれかに記載の細胞、組織、器官あるいは植物体またはその一部。
（項目３５） 前記抑制は、ＲＴ－ＰＣＲ法で測定した場合に、ｖａｃｕｏｌａｒ ｐｒｏｃｅｓｓｉｎｇ ｅｎｚｙｍｅ遺伝子（ＶＰＥ遺伝子）、またはｃｙｓｔｅｉｎｅ ｐｒｏｔｅａｓｅ遺伝子（ＳＡＧ１２遺伝子）の発現を約５０％低減させるという基準を下回るものである、上記項目のいずれかに記載の細胞、組織、器官あるいは植物体またはその一部。
（項目３６） 前記抑制は、ＲＴ－ＰＣＲ法で測定した場合に、ｖａｃｕｏｌａｒ ｐｒｏｃｅｓｓｉｎｇ ｅｎｚｙｍｅ遺伝子（ＶＰＥ遺伝子）の発現を約５０％低減させるという基準を下回るものである、上記項目のいずれかに記載の細胞、組織、器官あるいは植物体またはその一部。
（項目３７） 前記ＬｈＨＦＳの抑制が、遺伝子発現レベルの低下、遺伝子コピー数の低下、遺伝子増幅の低下、ＲＮＡ活性レベルの低下、ｍＲＮＡ存在量の低下、ｍＲＮＡ合成速度の低下、ｍＲＮＡ安定性の低下、タンパク質活性レベルの低下、タンパク質合成の低下、タンパク質存在量の低下、タンパク質安定性の低下、タンパク質酵素活性の低下、またはこれらの組合せからなる群より選択される方法によって達成される、上記項目のいずれかに記載の細胞、組織、器官あるいは植物体またはその一部。
（項目３８） 前記ＬｈＨＦＳまたはその相同遺伝子の少なくとも一部の構造遺伝子が天然のものと比較して変異を有するか、および／またはその遺伝子発現調節因子が天然のものと比較して変異を有するか、および／またはＬｈＨＦＳまたはその相同遺伝子の少なくとも一部を抑制する天然には存在しない因子を有する、上記項目のいずれかに記載の細胞、組織、器官あるいは植物体またはその一部。
（項目３９） 前記変異または因子は、ＬｈＨＦＳ遺伝子のＲＮＡ干渉またはゲノム編集によって提供されるものである、上記項目のいずれかに記載の細胞、組織、器官あるいは植物体またはその一部。
（項目４０） ＬｈＨＦＳまたはその相同遺伝子の遺伝子が一部または全部が変異または欠損した植物細胞、植物組織、植物器官あるいは植物体またはその一部。
（項目４１） 前記植物は、ユリ科の植物である、上記項目のいずれかに記載の植物細胞、植物組織、植物器官あるいは植物体またはその一部。
（項目４２） 前記植物は、ユリ属の植物である、上記項目のいずれかに記載の植物細胞、植物組織、植物器官あるいは植物体またはその一部。
（項目４３） 前記植物体またはその一部が、球根、種子、花、茎、葉、花粉、りん片、木子、細胞、組織、組織培養物もしくはむかご、またはそれらの全部もしくは一部の組合せである、上記項目のいずれかに記載の植物細胞、植物組織、植物器官あるいは植物体またはその一部。
（項目４４） 前記植物体またはその一部が花またはその一部を含む、上記項目のいずれかに記載の植物細胞、植物組織、植物器官あるいは植物体またはその一部。
（項目４５） 花の老化が抑制された植物体またはその一部の生産方法であって、該植物体またはその一部において、該植物体またはその一部が有するＬｈＨＦＳまたはその相同遺伝子の少なくとも一部が抑制された状態を提供することを含む、方法。
（項目４６） 前記抑制は、前記提供工程前の状態よりも前記ＬｈＨＦＳまたはその相同遺伝子の少なくとも一部の発現および／または機能が抑制されることを含む、上記項目のいずれかに記載の方法。
（項目４７） 前記提供工程前の状態よりも前記ＬｈＨＦＳまたはその相同遺伝子の少なくとも一部の発現および／または機能が抑制されていることを測定または他の方法で決定することをさらに含む、上記項目のいずれかに記載の方法。
（項目４８） 前記測定または他の方法で決定することは、前記ＬｈＨＦＳまたはその相同遺伝子の少なくとも一部のｍＲＮＡの存在量、前記ＬｈＨＦＳまたはその相同遺伝子の少なくとも一部のタンパク質の存在量、前記ＬｈＨＦＳまたはその相同遺伝子の少なくとも一部のタンパク質の生物学的機能、これらの組合せを測定し、あるいは測定結果から計算することを含む、上記項目のいずれかに記載の方法。
（項目４９） 前記ＬｈＨＦＳまたはその相同遺伝子の抑制が、遺伝子発現レベルの低下、遺伝子コピー数の低下、遺伝子増幅の低下、ＲＮＡ活性レベルの低下、ｍＲＮＡ存在量の低下、ｍＲＮＡ合成速度の低下、ｍＲＮＡ安定性の低下、タンパク質活性レベルの低下、タンパク質合成の低下、タンパク質存在量の低下、タンパク質安定性の低下、タンパク質酵素活性の低下、またはこれらの組合せからなる群より選択される方法によって達成される、上記項目のいずれかに記載の方法。
（項目５０） 前記ＬｈＨＦＳまたはその相同遺伝子の抑制が、ＬｈＨＦＳ遺伝子のＲＮＡ干渉またはゲノム編集を実行することによって達成される、上記項目のいずれかに記載の方法。
（項目５１） 前記植物は、ユリ科の植物である、上記項目のいずれかに記載の方法。
（項目５２） 前記植物は、ユリ属の植物である、上記項目のいずれかに記載の方法。
（項目５３） 前記植物体またはその一部が、球根、種子、花、茎、葉、花粉、りん片、木子、細胞、組織、組織培養物もしくはむかご、またはそれらの全部もしくは一部の組合せである、上記項目のいずれかに記載の方法。
（項目５４） 前記植物体またはその一部が花またはその一部を含む、上記項目のいずれかに記載の方法。
（項目５５） 前記方法は、
植物細胞におけるＬｈＨＦＳまたはその相同遺伝子を改変して、その一部または全体に変異を導入するかあるいはその一部または全体を欠損させる工程
ＬｈＨＦＳまたはその相同遺伝子が改変された植物細胞を育てて植物体またはその一部を得る工程
を含む、上記項目のいずれかに記載の方法。
（項目５６） 前記改変が、アンチセンス、ＲＮＡ干渉、突然変異誘発因子、ゲノム編集によって行われる、上記項目のいずれかに記載の方法。
（項目５７） 植物体またはその一部における花の日持ちを延長する方法であって、
１）上記項目のいずれかに記載の因子を含むかまたは該因子によって改変された植物体またはその一部、あるいは上記項目のいずれかに記載の細胞、上記項目のいずれかに記載の植物組織もしくは上記項目のいずれかに記載の植物器官を含む植物体またはその一部を提供する工程と、
２）該植物体またはその一部を、開花する条件に供する工程と、
３）該植物体またはその一部において開花後、日持ちする条件および期間に供する工程と
を包含する方法。
（項目５８） 前記期間は、前記日持ちする条件下で、前記植物体またはその一部の、前記因子による改変のない場合の日持ちが終了する期間よりも長い期間である、上記項目のいずれかに記載の方法。
（項目４５Ａ） ＬｈＨＦＳまたはその相同遺伝子を抑制する因子の、花の老化が抑制された植物体またはその一部の生産方法における使用。
（項目４６Ａ） 前記抑制は、前記提供工程前の状態よりも前記ＬｈＨＦＳまたはその相同遺伝子の少なくとも一部の発現および／または機能が抑制されることを含む、上記項目のいずれかに記載の使用。
（項目４７Ａ） 前記提供工程前の状態よりも前記ＬｈＨＦＳまたはその相同遺伝子の少なくとも一部の発現および／または機能が抑制されていることを測定または他の方法で決定することをさらに含む、上記項目のいずれかに記載の使用。
（項目４８Ａ） 前記測定または他の方法で決定することは、前記ＬｈＨＦＳまたはその相同遺伝子の少なくとも一部のｍＲＮＡの存在量、前記ＬｈＨＦＳまたはその相同遺伝子の少なくとも一部のタンパク質の存在量、前記ＬｈＨＦＳまたはその相同遺伝子の少なくとも一部のタンパク質の生物学的機能、これらの組合せを測定し、あるいは測定結果から計算することを含む、上記項目のいずれかに記載の使用。
（項目４９Ａ） 前記ＬｈＨＦＳまたはその相同遺伝子の抑制が、遺伝子発現レベルの低下、遺伝子コピー数の低下、遺伝子増幅の低下、ＲＮＡ活性レベルの低下、ｍＲＮＡ存在量の低下、ｍＲＮＡ合成速度の低下、ｍＲＮＡ安定性の低下、タンパク質活性レベルの低下、タンパク質合成の低下、タンパク質存在量の低下、タンパク質安定性の低下、タンパク質酵素活性の低下、またはこれらの組合せからなる群より選択される方法によって達成される、上記項目のいずれかに記載の使用。
（項目５０Ａ） 前記ＬｈＨＦＳまたはその相同遺伝子の抑制が、ＬｈＨＦＳ遺伝子のＲＮＡ干渉またはゲノム編集を実行することによって達成される、上記項目のいずれかに記載の方法。
（項目５１Ａ） 前記植物は、ユリ科の植物である、上記項目のいずれかに記載の使用。
（項目５２Ａ） 前記植物は、ユリ属の植物である、上記項目のいずれかに記載の使用。
（項目５３Ａ） 前記植物体またはその一部が、球根、種子、花、茎、葉、花粉、りん片、木子、細胞、組織、組織培養物もしくはむかご、またはそれらの全部もしくは一部の組合せである、上記項目のいずれかに記載の使用。
（項目５４Ａ） 前記植物体またはその一部が花またはその一部を含む、上記項目のいずれかに記載の使用。
（項目５５Ａ） 前記生産方法は、
植物細胞におけるＬｈＨＦＳまたはその相同遺伝子を改変して、その一部または全体に変異を導入するかあるいはその一部または全体を欠損させる工程
ＬｈＨＦＳまたはその相同遺伝子が改変された植物細胞を育てて植物体またはその一部を得る工程
を含む、上記項目のいずれかに記載の使用。
（項目５６Ａ） 前記改変が、アンチセンス、ＲＮＡ干渉、突然変異誘発因子、ゲノム編集によって行われる、上記項目のいずれかに記載の使用。
（項目５７Ａ） ＬｈＨＦＳまたはその相同遺伝子を抑制する因子の、植物体またはその一部における花の日持ちを延長する方法における使用であって、前記方法は、
１）上記項目のいずれかに記載の因子を含むかまたは該因子によって改変された植物体またはその一部、あるいは上記項目のいずれかに記載の細胞、上記項目のいずれかに記載の植物組織もしくは上記項目のいずれかに記載の植物器官を含む植物体またはその一部を提供する工程と、
２）該植物体またはその一部を、開花する条件に供する工程と、
３）該植物体またはその一部において開花後、日持ちする条件および期間に供する工程と
を包含する使用。
（項目５８Ａ） 前記期間は、前記日持ちする条件下で、前記植物体またはその一部の、前記因子による改変のない場合の日持ちが終了する期間よりも長い期間である、上記項目のいずれかに記載の使用。 Accordingly, the present disclosure provides:
(Item 1) (A) 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; or (B) a nucleic acid molecule comprising a sequence variant,
The (B) sequence variant is
(B-1) a sequence variant having one or more substitutions, additions and/or deletions in the nucleic acid sequence of (A);
(B-2) a sequence variant having at least about 90% 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),
A nucleic acid molecule, wherein the protein encoded by the sequence variant has the biological function of the protein encoded by (A).
(Item 2) The nucleic acid molecule according to any one of the above items, wherein the nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NO:1.
(Item 3) 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 4) (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 mutations include substitutions, additions and/or deletions, and the polypeptide of (a) a polypeptide having a biological activity analogous to that of
(c) a polypeptide encoded by a splice 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) having at least about 90 identity to the polypeptide of any one of (a)-(d) % and has a biological activity similar to that of the polypeptide of (a).
(Item 5) An agent that suppresses the expression of the nucleic acid sequence according to any of the above items and/or the amount or function of the nucleic acid sequence or the protein encoded by the nucleic acid sequence or the polypeptide according to any of the above items.
(Item 6) The factors include proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids including RNA or DNA, polysaccharides, oligosaccharides, lipids, hormones, ligands, signaling substances, synthetic The agent of any of the preceding items that is chemical, radiation, or a combination thereof.
(Item 7) The factor according to any of the above items, which is selected from the group consisting of an antisense molecule, an RNAi factor, a genome-editing factor, or a mutagen.
(Item 8) A factor that represses LhHFS or its homologous gene.
(Item 9) The homologous gene is
(i)
A sequence variant of 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, wherein the sequence variant comprises
(B-1) a sequence variant having one or more substitutions, additions and/or deletions in the 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;
(B-2) a sequence variant having at least about 70% sequence identity with the 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;
(B-3) a sequence variant that hybridizes under stringent conditions to 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;
(B-4) a sequence variant that is an allelic variant of the nucleic acid sequence shown in SEQ ID NO: 1 or the nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 2, or (B-5) 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, or a sequence variant that is a fragment of (B-1) to (B-4),
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 the amino acid sequence shown in SEQ ID NO: 2, or (ii)
(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 mutations include substitutions, additions and/or deletions, and the polypeptide of (a) a polypeptide having a biological activity analogous to that of
(c) a polypeptide encoded by a splice 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) having at least about 70 identity to the polypeptide of any one of (a)-(d) % and has a biological activity similar to that of the polypeptide of (a). factor.
(Item 10) The factors include proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids including RNA or DNA, polysaccharides, oligosaccharides, lipids, hormones, ligands, signaling substances, synthetic An agent according to any of the preceding items, which is a chemical substance, or a compound molecule thereof.
(Item 11) The factor according to any of the above items, wherein the factor is in nucleic acid form.
(Item 12) The factor according to any of the above items, which is selected from the group consisting of an antisense molecule, an RNAi factor, a genome-editing factor, or a mutator.
(Item 13) The factor according to any of the above items, wherein the factor is an LhHFS RNAi factor in the form of a double-stranded nucleic acid.
(Item 14) The factor is in double-stranded form, one strand of which is (I)
(A) a nucleic acid sequence comprising at least about 100 bases of the 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; or (B) a sequence variant, the other strand comprising:
(II) comprising complementary or annealing sequences of (I);
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),
The factor according to any of the above items, wherein the protein encoded by the sequence variant has the biological function of the protein encoded by (A).
(Item 15) The factor according to any of the above items, wherein (I) does not contain the nucleic acid sequence of bases 22-483 of SEQ ID NO:1.
(Item 16) The factor according to any of the above items, wherein (I) comprises a nucleic acid sequence of bases 574 to 864 in SEQ ID NO:1.
(Item 17) The factor according to any of the above items, which is a vector containing an RNAi factor of LhHFS or its homologous gene.
(Item 18) The element according to any of the above items, wherein the vector comprises a promoter and a terminator.
(Item 19) The factor according to any one of the above items, wherein the factor is a genome-editing factor that suppresses the expression and/or function of LhHFS.
(Item 20) The factor according to any one of the above items, wherein the factor for genome editing targets the following nucleic acid sequence of SEQ ID NO:1.

(Item 21) The factor according to any of the above items, wherein the factor for genome editing targets genome editing of the nucleic acid sequence of bases 1 to 483 in SEQ ID NO:1.
(Item 22) A plant cell containing or modified by the factor according to any of the above items.
(Item 23) A plant tissue containing or modified by the factor according to any of the above items or a plant tissue containing the cell according to any of the above items.
(Item 24) A plant organ containing or modified by the factor according to any of the above items, or a plant organ containing the cell according to any of the above items or the plant tissue according to any of the above items. .
(Item 25) A plant body or part thereof containing or modified by the factor according to any of the above items, or the cell according to any of the above items, or the plant according to any of the above items. A plant or part thereof comprising a tissue or plant organ according to any of the above items.
(Item 26) The plant or part thereof according to any one of the above items, wherein the plant belongs to the Liliaceae family.
(Item 27) The plant or part thereof according to any one of the above items, wherein the plant belongs to the genus Lilium.
(Item 28) The plant body or part thereof is a bulb, seed, flower, stem, leaf, pollen, scale, tree, cell, tissue, tissue culture or germ, or a combination of all or part thereof. A plant or part thereof according to any of the above items.
(Item 29) The plant or part thereof according to any of the above items, wherein the plant or part thereof comprises a flower or part thereof.
(Item 30) A plant cell in which at least part of LhHFS or its homologous gene in the plant is suppressed.
(Item 31) A plant tissue in which at least a portion of LhHFS or a homologous gene thereof possessed by the plant is suppressed, or a plant tissue containing the cell according to any one of the above items.
(Item 32) A plant organ in which at least part of LhHFS or a homologous gene thereof possessed by the plant is suppressed, or a plant organ comprising the cell according to any of the above items or the plant tissue according to any of the above items.
(Item 33) A plant in which at least a portion of LhHFS or a homologous gene thereof possessed by the plant is suppressed, or a portion thereof, or the cell according to any one of the above items, the plant tissue according to any one of the above items, or A plant body or part thereof comprising the plant organ according to any of the above items.
(Item 34) The cell, tissue, organ or plant body or part thereof according to any one of the above items, wherein the expression and/or function of at least part of the LhHFS or its homologous gene is suppressed.
(Item 35) The suppression is below the standard of reducing the expression of the vascular 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 or plant body or part thereof according to any of the above items.
(Item 36) Any one of the above items, wherein the suppression is below the standard of reducing the expression of the vascular processing enzyme gene (VPE gene) by about 50% when measured by RT-PCR method. A cell, tissue, organ or plant or part thereof.
(Item 37) Suppression of LhHFS reduces gene expression level, gene copy number, gene amplification, RNA activity level, mRNA abundance, mRNA synthesis rate, and mRNA stability. , decreased protein activity levels, decreased protein synthesis, decreased protein abundance, decreased protein stability, decreased protein enzymatic activity, or combinations thereof. Any cell, tissue, organ or plant or part thereof.
(Item 38) Whether the structural gene of at least a part of the LhHFS or its homologous gene has a mutation compared to a natural one, and/or whether the gene expression regulator thereof has a mutation compared to a natural one , and/or a cell, tissue, organ or plant or part thereof, having a non-naturally occurring factor that represses at least part of LhHFS or a homologous gene thereof.
(Item 39) The cell, tissue, organ or plant body or part thereof according to any one of the above items, wherein the mutation or factor is provided by RNA interference or genome editing of the LhHFS gene.
(Item 40) A plant cell, plant tissue, plant organ, plant body, or a part thereof in which a part or all of the gene of LhHFS or its homologous gene is mutated or deleted.
(Item 41) A plant cell, plant tissue, plant organ, plant body, or a part thereof according to any one of the above items, wherein the plant belongs to the Liliaceae family.
(Item 42) The plant cell, plant tissue, plant organ, plant body, or part thereof according to any one of the above items, wherein the plant belongs to the genus Lilium.
(Item 43) The plant body or part thereof is a bulb, seed, flower, stem, leaf, pollen, scale, tree, cell, tissue, tissue culture or germ, or a combination of all or part thereof. A plant cell, plant tissue, plant organ or plant body or part thereof according to any of the above items.
(Item 44) The plant cell, plant tissue, plant organ or plant body or part thereof according to any of the above items, wherein the plant body or part thereof comprises a flower or a part thereof.
(Item 45) A method for producing a plant or part thereof in which flower senescence is suppressed, wherein at least one of LhHFS or a homologous gene thereof possessed by the plant or part thereof A method comprising providing a state in which a portion is suppressed.
(Item 46) The method according to any of the above items, wherein the suppression includes suppressing the expression and/or function of at least a part of the LhHFS or its homologous gene as compared to the state before the providing step.
(Item 47) The above item, further comprising measuring or otherwise determining that the expression and/or function of at least a portion of the LhHFS or its homologous gene is suppressed compared to the state before the providing step. The method according to any one of
(Item 48) The measurement or other determination includes the abundance of mRNA of at least a portion of said LhHFS or a homologous gene thereof, the abundance of protein of at least a portion of said LhHFS or a homologous gene thereof, and the abundance of said LhHFS A method according to any of the preceding items, comprising measuring or calculating from the results of measurements the biological function of at least part of the protein, or a homologous gene thereof, or a combination thereof.
(Item 49) Suppression of the LhHFS or its homologous gene reduces gene expression level, gene copy number, gene amplification, RNA activity level, mRNA abundance, mRNA synthesis rate, mRNA reduced stability, reduced protein activity levels, reduced protein synthesis, reduced protein abundance, reduced protein stability, reduced protein enzymatic activity, or combinations thereof. A method according to any of the preceding items.
50. The method of any of the preceding items, wherein the suppression of LhHFS or its homologous gene is achieved by performing RNA interference or genome editing of the LhHFS gene.
(Item 51) The method according to any one of the above items, wherein the plant belongs to the Liliaceae family.
(Item 52) The method according to any one of the above items, wherein the plant belongs to the genus Lilium.
(Item 53) The plant body or part thereof is a bulb, seed, flower, stem, leaf, pollen, scale, tree, cell, tissue, tissue culture or germ, or a combination of all or part thereof. A method according to any of the preceding items.
(Item 54) A method according to any of the preceding items, wherein the plant or part thereof comprises a flower or part thereof.
(Item 55) The method includes:
A step of modifying LhHFS or a homologous gene thereof in a plant cell to introduce a mutation in part or the whole thereof, or to delete a part or the whole thereof A plant body by growing plant cells in which LhHFS or a homologous gene thereof is modified or part thereof.
(Item 56) The method according to any one of the above items, wherein the modification is performed by antisense, RNA interference, a mutagen, or genome editing.
(Item 57) A method for extending the vase life of flowers in a plant or part thereof, comprising:
1) A plant body or part thereof containing or modified by any of the above factors, or a cell of any of the above items, a plant tissue of any of the above items, or a step of providing a plant body or a part thereof comprising the plant organ according to any of the above items;
2) subjecting the plant or part thereof to flowering conditions;
3) subjecting the plant body or part thereof to a condition and a period of keeping for a long time after flowering;
(Item 58) Any one of the above items, wherein the period is longer than the period in which the plant body or part of the plant body or part thereof lasts under the long-lasting condition without being modified by the factor. described method.
(Item 45A) Use of a factor that suppresses LhHFS or a homologous gene thereof in a method for producing a plant body or part thereof in which flower senescence is suppressed.
(Item 46A) 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 LhHFS or its homologous gene relative to the state before the providing step.
(Item 47A) The above item, further comprising measuring or otherwise determining that the expression and/or function of at least a part of the LhHFS or its homologous gene is suppressed compared to the state before the providing step. any use described in
(Item 48A) Said measuring or otherwise determining may include the abundance of mRNA of at least a portion of said LhHFS or its homologous gene, the abundance of protein of said LhHFS or at least a portion of its homologous gene, said LhHFS Use according to any of the preceding items, including measuring or calculating from the results of measurements the biological function of a protein of at least part of it, or a homologous gene thereof, or a combination thereof.
(Item 49A) Suppression of the LhHFS or its homologous gene reduces gene expression level, gene copy number, gene amplification, RNA activity level, mRNA abundance, mRNA synthesis rate, mRNA reduced stability, reduced protein activity levels, reduced protein synthesis, reduced protein abundance, reduced protein stability, reduced protein enzymatic activity, or combinations thereof. , use according to any of the above items.
50A. The method of any of the preceding items, wherein the suppression of LhHFS or its homologous gene is achieved by performing RNA interference or genome editing of the LhHFS gene.
(Item 51A) Use according to any one of the above items, wherein the plant belongs to the Liliaceae family.
(Item 52A) Use according to any one of the above items, wherein the plant belongs to the genus Lilium.
(Item 53A) The plant body or part thereof is a bulb, seed, flower, stem, leaf, pollen, scale, tree, cell, tissue, tissue culture or germ, or a combination of all or part thereof. A use according to any of the above items.
(Item 54A) Use according to any of the preceding items, wherein the plant or part thereof comprises a flower or part thereof.
(Item 55A) The production method includes:
A step of modifying LhHFS or a homologous gene thereof in a plant cell to introduce a mutation in part or the whole thereof, or to delete a part or the whole thereof A plant body by growing plant cells in which LhHFS or a homologous gene thereof is modified or part thereof.
(Item 56A) The use according to any of the above items, wherein the modification is performed by antisense, RNA interference, mutagenesis, or genome editing.
(Item 57A) Use of a factor that represses LhHFS or a homologous gene thereof in a method for prolonging flower vase life in a plant or part thereof, the method comprising:
1) A plant body or part thereof containing or modified by any of the above factors, or a cell of any of the above items, a plant tissue of any of the above items, or a step of providing a plant body or a part thereof comprising the plant organ according to any of the above items;
2) subjecting the plant or part thereof to flowering conditions;
3) subjecting the plant body or part thereof to a condition and a period of keeping for a long time after flowering;
(Item 58A) Any one of the above items, wherein the period is longer than the period during which the plant body or part of the plant body or part thereof lasts under the long-lasting condition without being modified by the factor. Use as indicated.

Accordingly, these and other advantages of the present disclosure will be apparent upon reading the detailed description below.

According to the present disclosure, by suppressing the expression of a transcription factor family gene (LhHFS), it is possible to delay flower senescence and extend the ornamental period of general Liliaceae plants such as lilies and tulips. .

FIG. 1 shows photographs of the state of flowers after flowering of the wild-type (Cont) and LhHFS expression-suppressed line (LNR18-2). The number of days after flowering is shown at the top of the photo. The top row of photographs shows wild-type lilies after 0-7 days, and the bottom row of photographs shows LhHFS-suppressed lines of lilies after 0-9 days. The wild type exhibited browning of the perianth 5 days after flowering, and the LhHFS expression-suppressed line exhibited browning of the perianth 8 days after flowering. The arrows indicate that the wild-type strain finished keeping its shelf life 4 days after flowering, and the LhHFS expression-suppressed line finished its shelf life 7 days after flowering. FIG. 2 shows the relative expression level of the LhHFS gene in the perianth of the wild-type (Cont) and LhHFS expression-suppressed line (LNR18) on day 4 after flowering. The vertical axis of the graph indicates the relative expression level of the LhHFS gene, and the axis of the graph corresponds to the wild type (Cont) and LhHFS expression-suppressed lines (LNR18-2 and LNR18-15) from the left.

The present disclosure is described below. It should be understood that throughout this specification, expressions in the singular also include the concept of the plural unless specifically stated otherwise. Thus, articles in the singular (eg, “a,” “an,” “the,” etc. in the English language) should be understood to include their plural concepts as well, unless specifically stated otherwise. Also, it should be understood that the terms used in this specification have the meanings commonly used in the art unless otherwise specified. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict, the present specification (including definitions) will control.

(definition)
As used herein, "about" means ±10% of the value that follows. It should be understood that, in this specification, even if there is no explicit description of "about," it is not intended to waive any numerical range in the absence of a description of "about."

As used herein, "LhHFS" refers to the gene having the nucleic acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3 and the protein having the amino acid sequence set forth in SEQ ID NO: 2, found in this disclosure, biological A functional derivative thereof, or a biologically functional fragment thereof, or a homologue thereof, or a mutation encoded in a nucleic acid that hybridizes under high or low stringency conditions to a nucleic acid encoding this protein. point to the body Those skilled in the art will understand that either nucleic acid molecules or proteins, or both, are meant as appropriate, depending on the context. A "mutant" or "variant" also includes, but is not intended to be limited to, molecules containing regions of substantial homology to the original protein, and such molecules may be used in various embodiments. , aligned over amino acid sequences of the same size, or by performing an alignment by a computer homology program known in the art (eg, LALIGN (https://embnet.vital-it.ch/software/LALIGN_form.html), etc.) A nucleic acid that is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% identical when compared to a sequence of a sequence that is or encodes such a molecule is It can hybridize to the original protein-encoding sequence under stringent, moderately stringent, or non-stringent conditions. It is the product of modifying naturally occurring proteins by amino acid substitutions, deletions and additions, respectively, the derivatives of which still exhibit the biological function of the naturally occurring protein, although not necessarily to the same degree. , means protein. The biological function of such proteins can be determined, for example, by suitable and available in vitro assays described herein or known in the art.

In the present disclosure, lilies are mainly discussed as LhHFS, but it is also known that many plants other than lilies, such as tulip (SEQ ID NO: 4), express homologous genes of the LhHFS gene. In particular, the homologous gene of tulip has about 71% sequence identity to the LhHFS of lily, and based on such interspecies sequence conservation, these plants, particularly Liliaceae, are also subject to the present disclosure. is understood to fall within the range of

As used herein, "having a biological function" refers herein to the structural function of the protein, such as biological activity, according to the aspect to which the polypeptides, ie fragments or derivatives, of the present disclosure relate. Refers to a polypeptide, ie fragment or derivative, having a , regulatory, or biochemical function.

In the context of the present disclosure, a "repressor (of a gene or protein)" means a gene of interest or its homologous gene, or a protein encoded by these genes, which acts directly or indirectly to suppress the gene of interest or homologous genes thereof, or factors (eg, molecules or substances) that can temporarily or permanently suppress, reduce, or eliminate the function of proteins encoded by these genes. The inhibitory factor may be an inhibitor (substance), examples of which include antibodies or antigenic fragments thereof, antisense oligonucleotides, RNAi factors such as siRNA, genome editing factors, mutagens, Examples include low molecular weight molecules (LMWs), binding peptides, aptamers, ribozymes and peptidomimetics, which are directed against genes of interest or their homologous genes, or proteins encoded by these genes. Also included are binding proteins or binding peptides specifically directed against the active site of a protein encoded by the gene of interest or its homologous gene, as well as nucleic acids directed against the gene of interest or its homologous gene. Nucleic acids directed against genes of interest or their homologous genes can be, for example, double- or single-stranded DNA or RNA, or their Refers to modifications or derivatives and includes, without limitation, antisense nucleic acids, aptamers, siRNA (small interfering RNA) and ribozymes. The nucleic acid for the gene of interest or its homologous gene includes, for example, a genome editing factor that inhibits the expression or activity of the LhHFS gene or its homologous gene. nucleases) and RNA-guided nucleases. As used herein, the term "binding protein" or "binding peptide" for a protein encoded by a gene of interest or a homologous gene thereof refers to a type of protein that binds to a protein encoded by a gene of interest or a homologous gene thereof. or peptides, and include, but are not limited to, polyclonal or monoclonal antibodies, antibody fragments and protein scaffolds directed against proteins encoded by the gene of interest or its homologous genes.

As used herein, the term "signaling substance" refers to a substance responsible for transmitting information for regulating physiological activity or biological function in vivo. Plant hormones are typical examples of “signaling substances”.

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, a molecule capable of decreasing the protein expression level by specifically decreasing the amount of mRNA of a gene having a nucleotide sequence region complementary to that sequence, depending on a certain nucleotide sequence introduced into the cell. Say. Antisense techniques include direct introduction into cells of RNA molecules complementary to mRNA produced from target genes, and introduction of constructive vectors capable of expressing RNA complementary to target genes into cells. The latter method is more common in plants. Specifically, one DNA sequence that is complementary to the nucleotide sequence of the target gene is ligated to an appropriate promoter to construct an expression vector that expresses the artificial mRNA under its control, and introduced into the cell. In addition to the technique of using an expression vector designed to construct a double-stranded RNA in a cell, a technique of using an expression vector is used. The basic structure of an antisense molecule is made by linking one type of DNA sequence complementary to a certain target gene under a promoter, and then linking the same DNA sequence to another in the opposite direction. In the single-stranded mRNA transcribed from this construction gene, one type of nucleotide sequence portion connected in the opposite direction is in a complementary relationship, so that it pairs to form a double-stranded RNA state with a hairpin-like secondary structure. , which causes mRNA degradation of the target gene according to the mechanism of RNAi.

As used herein, "RNA interference" or "RNAi" is an abbreviation for RNA interference, which is commonly known in the art and is a biological method that inhibits or downregulates gene expression in cells mediated by RNAi agents. It's a process. For example, by introducing an RNAi-causing factor such as double-stranded RNA (also referred to as dsRNA) into a cell, the homologous mRNA is specifically degraded and the synthesis of the gene product is suppressed, and the technology used therefor Say. For RNAi, see, for example, Zamore and Haley, 2005, Science, 309, 1519-1524; Vaughn and Martinssen, 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; Publication No. 00/01846; Mello and Fire, WO 01/29058; Deschamps-Depaillette, WO 99/07409 and Li et al., WO 00/44914; 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, as used herein, the term RNAi is intended to be synonymous with other terms used to describe sequence-specific RNA interference, such as post-transcriptional gene silencing, translational inhibition, transcriptional inhibition, and epigenetics. understood.

As used herein, "RNAi agents" include nucleic acid molecules or analogs thereof, such as RNA-based molecules, that inhibit gene expression. An RNAi agent may be an agent that destroys or cleaves a specific mRNA, or an agent that blocks the processing or translation of RNA such as mRNA. RNAi agents include silencing RNAs (siRNAs), endoribonuclease-prepared siRNAs (esiRNAs), short hairpin RNAs (shRNAs), or microRNAs (miRNAs), and short interfering nucleic acids (siNAs); It is not limited to these. These agents refer to any nucleic acid molecule capable of inhibiting or down-regulating gene expression or viral replication by mediating RNA interference "RNAi" or gene silencing in a sequence-specific manner. These terms can also refer to an individual nucleic acid molecule, a plurality of such nucleic acid molecules, or a pool of such nucleic acid molecules. These molecules can be double-stranded nucleic acid molecules comprising self-complementary sense and antisense regions. Here, an antisense region includes a nucleotide sequence that is complementary to a nucleotide sequence or portion thereof in a target nucleic acid molecule, and a sense region that has a nucleotide sequence or portion thereof corresponding to the target nucleic acid sequence. These molecules can be assembled from two separate oligonucleotides, one strand being the sense strand and the other the antisense strand. Here, the antisense and sense strands are self-complementary (i.e., the antisense and sense strands form a duplex or double-stranded structure, such that each strand follows a nucleotide sequence in the other strand). including nucleotide sequences that are complementary, where, for example, the double-stranded region is from 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.An antisense strand comprises a nucleotide sequence that is complementary to a nucleotide sequence or a portion thereof in a target nucleic acid molecule, The sense strand comprises a nucleotide sequence corresponding to a target nucleic acid sequence or portion thereof (eg, about 15 to about 25 or more nucleotides of the molecule are complementary to the target nucleic acid or portion thereof); , these molecules are assembled from a single oligonucleotide, and the self-complementary sense and antisense regions of these molecules are joined by a nucleic acid or non-nucleic acid linker.These molecules are self-complementary can be a polynucleotide having a double-stranded, asymmetric double-stranded, hairpin or asymmetric hairpin secondary structure comprising a positive sense region and an antisense region, wherein the antisense region is in a separate target nucleic acid molecule A nucleotide sequence that is complementary to a nucleotide sequence or a portion thereof and a sense region having a nucleotide sequence that corresponds to a target nucleic acid sequence or a portion thereof These molecules have two or more loop structures and are self-complementary. can be a circular single-stranded polynucleotide having a stem that includes a sense region and an antisense region, where the antisense region is complementary to a nucleotide sequence or portion thereof in the target nucleic acid molecule. A circular polynucleotide comprising a nucleotide sequence and a sense region having a nucleotide sequence corresponding to a target nucleic acid sequence or a portion thereof, is processed in vivo or in vitro to produce an active molecule capable of mediating RNAi. These agents can also include a single-stranded polynucleotide having a nucleotide sequence that is complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof (e.g., these agents can be a target nucleic acid sequence or a portion thereof) It is not necessary that the nucleotide sequence corresponding to the moiety be present within these elements.) Single-stranded polynucleotides may be attached at the 5' phosphate (e.g., Martin ez et al. , 2002, Cell. , 110, 563-574 and Schwarz et al. , 2002, Molecular Cell, 10, 537-568), a terminal phosphate group such as a 5',3'-diphosphate. In certain embodiments, the LhHFS-suppressing agents of this disclosure comprise separate sense and antisense sequences or regions. Here, the sense and antisense regions are covalently linked by a nucleotide or non-nucleotide linker molecule known in the art, or by ionic interactions, hydrogen bonding, van der Waals interactions, hydrophobic interactions and/or They are alternately non-covalently bound by stacking interactions. In certain embodiments, the agents that suppress LhHFS of the disclosure comprise a nucleotide sequence that is complementary to a nucleotide sequence of a target gene. In another embodiment, the agents that suppress LhHFS of the present disclosure interact with the nucleotide sequence of the target gene to inhibit expression of the target gene. As used herein, agents that suppress LhHFS are not necessarily limited to molecules comprising only RNA, but also include chemically modified nucleotides and non-nucleotides. In certain embodiments, when the disclosure is a small interfering nucleic acid molecule, it may lack 2'hydroxy (2'-OH) containing nucleotides. In certain embodiments, the disclosure can be small interfering nucleic acids that do not require the presence of nucleotides with 2' hydroxyl groups to mediate RNAi. Thus, when the disclosure is a short interfering nucleic acid molecule, it may not contain ribonucleotides (eg, nucleotides with 2'-OH groups). However, if the presence of ribonucleotides within the factor that suppresses LhHFS is not necessary to maintain RNAi, then an attached linker, or other linkage or It may have associated groups, moieties or chains. In some cases, the LhHFS-suppressing agents of the present disclosure may comprise ribonucleotides at about 5, 10, 20, 30, 40 or 50% of the nucleotide positions. As used herein, agents that suppress LhHFS are nucleic acid molecules that can mediate sequence-specific RNAi, such as small interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA. (shRNA), small interfering oligonucleotides, small interfering nucleic acids, small interfering modified oligonucleotides, chemically modified siRNA, post-transcriptional gene silencing RNA (ptgsRNA).

As used herein, the term "genome-editing factor" refers to a factor that causes genome editing in a cell, tissue, organ, or organism into which it is introduced. A "genome-editing agent" may be any agent that results in genome editing by techniques known in the art, ie, CRISPR/Cas9, TALENs, ZFNs, meganucleases, and the like. Agents for genome editing include, but are not limited to, artificial restriction enzymes (artificial nucleases) and RNA-guided nucleases, guide RNAs (gRNAs), and the like.

As used herein, the term "mutator" refers to a factor that promotes mutation induction in a target gene. "Mutagenic factors" include chemical substances such as ethylmethanesulfonic acid (EMS), as well as energy such as light, radiation, heat and electricity.

As used herein, "homologous gene" refers to a gene with high homology between two or more sequences. "Homology" of a gene refers to the degree of identity between two or more gene sequences, and 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 determined by direct sequence comparison or, in the case of nucleic acids, hybridization under stringent conditions. When two gene sequences are directly compared, the DNA sequences are typically at least 50% identical, preferably at least 70% identical, more preferably at least 80%, 90% identical between the gene sequences. The genes are homologous if they are 95%, 96%, 97%, 98% or 99% identical. Thus, "homologue" or "homologous gene product" as used herein refers to a protein in another species, preferably a plant, that exerts the same biological function as the protein constituents of the complexes further described herein. means. Such homologues may also be referred to as "orthologous gene products." Algorithms for detecting orthologous gene pairs from certain plants or other species use whole genomes or partial sequences of these organisms. When analyzing partial sequence homology, the LALIGN program (https://embnet.vital-it.ch/software/LALIGN_form.html) is typically used in this disclosure. When analyzed by the LALIGN program, one skilled in the art can follow the instructions on the website to compare sequence identities. For details of the analysis method, refer to X.P. Huang and W. Miller, Adv. Appl. Math. (1991) 12:337-357. Based on the sequence homology of the genes encoding the proteins provided herein to the genes of other species, conventional techniques are applied to clone the respective genes and to express the proteins from such genes. or by isolating analogous complexes according to the methods provided herein or according to other suitable methods known in the art. It is also possible to isolate homologues of the proteins described herein.

In the present specification, "70% or more", which is a numerical value such as identity, is, 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%, and may be within the range of any two of these starting numerical values. The above-mentioned "identity" is calculated according to the known method as described above, based on the ratio of the number of homologous amino acids in two or more amino acid sequences. Specifically, prior to calculating percentages, the amino acid sequences of the amino acid sequences being compared are aligned and gaps are introduced into portions of the amino acid sequences where necessary to maximize the percentage of identical amino acids. do. Alignment methods, ratio calculation methods, comparison methods, and computer programs related thereto are conventionally well known in the art (eg, BLAST, etc., as described above). As used herein, “identity” and “similarity” can be expressed as values measured by NCBI BLAST unless otherwise specified. Blastp can be used with default settings as an algorithm when comparing amino acid sequences with BLAST. The measurement results are quantified as Positives or Identities.

When referring to genes herein, "vector" refers to something capable of transferring a polynucleotide sequence of interest into a cell of interest. Such vectors are capable of autonomous replication or integration into chromosomes in host cells such as prokaryotic cells, yeast, animal cells, plant cells, insect cells, animal and plant cells, Those containing a promoter at a position suitable for transcription of the polynucleotide of the present disclosure are exemplified. A vector herein can be an expression vector, a recombinant vector, and the like.

As used herein, the term "promoter" refers to a region on DNA that determines the transcription initiation site of a gene and directly regulates its frequency, and is a nucleotide sequence that binds RNA polymerase and initiates transcription. The promoter region can be estimated by predicting the protein-coding region in the genomic base sequence using DNA analysis software. The putative promoter region varies from structural gene to structural gene, but is usually upstream of the structural gene, but is not limited thereto, and can also be present within or downstream of the structural gene. Any promoter that allows the function of the LhHFS RNA interference factor can be used in the present disclosure.

As used herein, the term "terminator" refers to a sequence that is located downstream of the protein-encoding region of a gene and involved in the termination of transcription and the addition of a poly-A sequence when DNA is transcribed into mRNA. Terminators include, but are not limited to, CaMV35S terminator, nopaline synthase gene terminator (Tnos), tobacco PR1a gene terminator. Any sequence that exhibits terminator activity in plants can be used in the present disclosure.

As used herein, "expression" of genes, polynucleotides, polypeptides, proteins and the like means that the genes and the like undergo certain actions in vivo and become different forms. Preferably, it means that genes, polynucleotides, etc. are transcribed and translated into a form of a polypeptide, but being transcribed to produce mRNA is also an aspect of expression. For example, the expression level of LhHFS can be determined by any method. Specifically, the expression level of LhHFS can be determined by evaluating the amount of LhHFS mRNA, the amount of LhHFS protein, and the biological function of LhHFS protein. The amount of LhHFS mRNA and protein can be determined by methods detailed elsewhere herein or by other methods known in the art.

As used herein, "suppression of function" of genes, polynucleotides, polypeptides, proteins, etc. means that the biological activity of the gene, etc. is suppressed. "Suppression of function" of LhHFS can be confirmed by measuring the expression of the vascular processing enzyme gene (VPE gene) or the cysteine protease gene (SAG12 gene) using, for example, the RT-PCR method. Suppression of the function of LhHFS means that, for example, the expression of the VPE gene or the SAG12 gene is about 90%, about 80%, about It can be confirmed by a reduction of 70%, about 60%, about 50%, about 40%, about 30%, about 20%, or about 10%, preferably about 50%.

As used herein, "plant" is a collective term for organisms belonging to the kingdom Plantae, characterized by the presence of chlorophyll, a rigid cell wall, abundant persistent embryonic tissue, and an inability to move. Typically, plants refer to flowering plants that have cell wall formation and anabolism through chlorophyll. "Plant" includes both monocotyledonous and dicotyledonous plants. Preferable plants include, for example, plants of the "liliaceae" family. Plants of the "liliaceae" typically include lily, tulip, dogtooth violet, and black lily.

As used herein, the term "tissue" of an organism refers to a population of cells that have a uniform, similar effect in the population. A tissue may thus be part of an organ. Organs often have cells with the same function, but there are cases where cells with slightly different functions coexist. may be mixed. "Tissue" includes not only those constituting differentiated plants but also plant cell masses such as "callus".

As used herein, the term "organ" refers to a structure that has an independent form and is formed by combining one or more types of tissues to perform a specific function. Plants include, but are not limited to, roots, bulbs, stems, stems, leaves, flowers, seeds, scales, xylems, germs, embryos, fruits, endosperms, and germs.

As used herein, the term "organism" (or "plant body" in the case of plants) is used in the broadest sense in the art, and refers to an entity (or plant) that engages in life phenomena, typically a cell structure , proliferation (self-reproduction), growth, regulation, substance metabolism, repair ability, etc., and usually have genetics governed by nucleic acids and proliferation involving metabolism governed by proteins as basic attributes. Organisms include prokaryotes, eukaryotes (plants, animals, etc.) and the like. Preferably, in the present disclosure the organism may be a plant. Here, preferably such plants may be fertile. More preferably, such plants are capable of producing seeds. It is contemplated that the genetic constructs, agents, compositions and methods of this disclosure will function in plants in general, not just Liliaceae plants.

As used herein, the term “liliaceae” plant is intended to include original species and hybrids of plants belonging to the taxonomic Liliaceae family. Examples of plants of the Liliaceae family include Lilium, Tulipa, Erythronium, Fritillaria, Amana, Bowie, and Calochortus. , Cardiocrinum, Clintonia, Gagea, Lloydia, Medeola, Nomocharis, Notholirion, Prosartes, Takeshimaran The genus (Streptopus), the genus Tricyrtis, etc. are mentioned.

As used herein, the term “lilium” plant is intended to include progenitors and hybrids of plants belonging to the taxonomic family Liliaceae, genus Lilium. Therefore, plants of the "lily genus" include progenitors such as longiflorum, lily lily, lily Asiaticus, tiger lily, day lily, and lily lily, as well as oriental hybrids, asiatic hybrids, longiflorum hybrids, martagon hybrids, and Hybrids such as trumpet hybrids are also included. In the examples, the senescence retarding effect is shown using the Oriental hybrid variety, but since the homology of the LhHFS gene is very high between the hybrids, in lily hybrids other than the Oriental hybrid, It is thought that the anti-aging effect is also shown in the same way. Examples of "oriental hybrids" include cultivars such as Tiara, Casablanca, and Acapulco.

As used herein, the term “bulb” refers to a storage organ formed by accumulating nutrients in specific parts of a plant, such as roots, stems, and leaves, resulting in deformation and enlargement. Plants that produce bulbs typically include lilies, tulips, hyacinths, and the like.

As used herein, "seed" or "seed" refers to the reproductive organs of a seed plant.

As used herein, the term "flower" refers to an assembly of plant reproductive organs. "Flower" refers to a combination of flower axis, perianth, stamens, pistils, etc. As used herein, "flowers" may be provided in a state where they are separated from the underground, typically as "cut flowers", or "flowers in pots", or flowers whose underground parts are retained, such as flowers in flowerbeds. may be provided as-is. A "flower" can be generally provided as an object of appreciation. "Cut flower" refers to a flower alone or a combination of tissues and/or organs comprising a flower. For example, "cut flowers" can be a combination of flowers, stems and/or leaves and the like. Specifically, "cut flowers" can be a combination of flowers, stems, leaves, and the like. "Pot flower" refers to a flowering plant that is cultivated in a flower pot. A "part of a flower" refers to the absence of at least a part of the elements constituting the above-mentioned "flower".

The term "perianth" as used herein refers to the entire name combining "corolla" and "calyx". A "corolla" refers to an organ composed of multiple petals.

As used herein, the term "perianth" refers to a portion of the stem tip where perianth, corolla, petal, stamen, pistil, and sepal are joined together.

As used herein, the term "stem" refers to an organ that supports flowers and leaves in a plant.

As used herein, the term “leaf” refers to an organ that performs photosynthesis and respiration in a plant body.

As used herein, "pollen" refers to cells produced by anthers present in the stamens of flowers.

As used herein, the term “melanoma” or “melanoma leaf” refers to metamorphic leaves that protect buds, bulbs, and the like of plants.

As used herein, the term "tree" refers to small bulblets formed around bulbs.

As used herein, the term “mukago” refers to an organ in a plant that is enlarged by a side bud storing nutrients.

As used herein, the term “callus” refers to an undifferentiated plant cell mass.

As used herein, the term "senescence" refers to a phenomenon or process in which the function of an organism, cell, tissue or organ, or the function that integrates them, declines, which occurs from the maturity stage until the organism dies. say. When referring to "flower senescence" in the present disclosure, a phenomenon that occurs with the passage of time (or the passage of time itself) from after flowering (in some cases, before flowering) to the end of shelf life (losing ornamental value) ).

As used herein, “shelf life” and “flower senescence” are defined from a horticultural point of view, and “browning” of the entire flower or organs or tissues (e.g., petals or similar organs) that constitute the flower. ”, and in the L*a*b* color system using a color difference meter, if the b* value increases to a certain extent compared to the flower cover immediately after flowering (especially when the evaluation is strict, the color In the L * a * b * color system using a color difference meter, when the b * value increases by 10 or more compared to the perianth immediately after flowering), it is evaluated as "browning", and the " The state before being evaluated as "aging" is expressed as "longevity". In addition, the term "suppression of flower senescence" is defined from a horticultural point of view. called suppression. Suppression may be suppression in the degree of increase and may be assessed by prolongation of time to a certain degree of increase. Longevity and aging can also be evaluated by other evaluation methods (eg, withering state, presence or absence of detachment).

While not wishing to be bound by theory, senescence is the phenomenon or process by which cells spontaneously die and is considered a type of programmed cell death. During the senescence of flower cells, autophagy-like phenomena have long been observed in plants such as morning glory.

"Flower senescence" or its suppression is defined as the period from the time the whole flower, or the organs or tissues that make up the flower (e.g., perianth, corolla, petals, or similar organs) blooms until it undergoes browning (or can be assessed by observing the time of other equivalent assessment methods). Although flower senescence is typically evaluated herein using Liliaceae plants, the present disclosure is not limited thereto.

As used herein, "blooming conditions (or period)" refers to conditions (or period) under which a flowering plant normally grows.

As used herein, the term "long-life condition (or period)" refers to the condition (or period) under which a flowering plant maintains its flowered state.

As used herein, "protein", "polypeptide", "oligopeptide" and "peptide" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear, branched, or cyclic. Amino acids may be naturally occurring or non-naturally occurring, and may be modified amino acids. The term can also include multiple polypeptide chains assembled into a complex. The term also includes natural or artificially modified amino acid polymers. Such modifications include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification (eg, conjugation with a labeling component). This definition also includes, for example, polypeptides containing one or more analogs of amino acids (including, for example, unnatural amino acids), peptide-like compounds (eg, peptoids), and other modifications known in the art. be done.

As used herein, "amino acid" may be either naturally occurring or non-naturally occurring, as long as the purposes of the present disclosure are met.

As used herein, "polynucleotide," "oligonucleotide," and "nucleic acid" are used interchangeably herein to refer to polymers of nucleotides of any length. The term also includes "oligonucleotide derivatives" or "polynucleotide derivatives." "Oligonucleotide derivative" or "polynucleotide derivative", used interchangeably, refer to oligonucleotides or polynucleotides that contain derivatives of nucleotides or that have unusual linkages between nucleotides. Specific examples of such oligonucleotides include, for example, 2′-O-methyl-ribonucleotides, oligonucleotide derivatives in which phosphodiester bonds in oligonucleotides are converted to phosphorothioate bonds, and phosphodiester bonds in oligonucleotides. is converted to an N3′-P5′ phosphoramidate bond, an oligonucleotide derivative in which ribose and phosphodiester bonds in the oligonucleotide are converted to peptide nucleic acid bonds, uracil in the oligonucleotide is converted to C- 5-propynyluracil-substituted oligonucleotide derivative, oligonucleotide derivative wherein uracil in oligonucleotide is substituted with C-5 thiazole uracil, oligonucleotide derivative wherein cytosine in oligonucleotide is substituted with C-5 propynylcytosine, oligo Oligonucleotide derivatives in which cytosine in nucleotides is replaced with phenoxazine-modified cytosine, oligonucleotide derivatives in which ribose in DNA is replaced with 2′-O-propyl ribose, and two riboses in oligonucleotide Examples include oligonucleotide derivatives substituted with '-methoxyethoxyribose. Unless otherwise indicated, a particular nucleic acid sequence also includes conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences thereof, as well as sequences explicitly indicated. is intended to include Specifically, degenerate codon substitutions create sequences in which the third position of one or more selected (or all) codons is replaced with mixed bases and/or deoxyinosine residues. (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)). "Nucleic acid" is also used interchangeably herein with gene, cDNA, mRNA, oligonucleotide, and polynucleotide. As used herein, "nucleotides" may be natural or non-natural.

As used herein, the term “gene” refers to factors that define genetic traits. They are usually arranged in a certain order on the chromosome. A gene that defines the primary structure of a protein is called a structural gene, and a gene that controls its expression is called a regulatory gene. As used herein, "gene" may refer to "polynucleotide," "oligonucleotide," and "nucleic acid."

As used herein, the term "genome" refers to the entire genetic information of an organism. A genome can be encoded in either DNA or RNA. A genome can include non-coding regions as well as coding regions that encode proteins.

Amino acids may be referred to herein by either their commonly known three letter symbols or by the one letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may likewise be referred to by their commonly recognized one-letter code. As used herein, comparisons of similarity, identity and homology of amino acid and nucleotide sequences are calculated using the sequence analysis tool BLAST with default parameters. The identity search can be performed using, for example, NCBI BLAST 2.10.1 (published June 18, 2020). The value of identity in the present specification usually refers to the value when aligned under the default conditions using the above BLAST. However, if a higher value is obtained by changing the parameters, the highest value is taken as the value of identity. When identity is evaluated in multiple regions, the highest value among them is taken as the identity value. Similarity is a number that takes into account similar amino acids in addition to identity.

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 colony hybridization, plaque hybridization, Southern blot hybridization, or the like using a polynucleotide selected from among the polynucleotides of the present disclosure as a probe. Specifically, using a filter on which colony- or plaque-derived DNA is immobilized, hybridization is performed at 65° C. in the presence of 0.7-1.0 M NaCl, followed by 0.1- to 2-fold concentration. SSC (saline-sodium citrate) solution (composition of 1-fold SSC solution is 150 mM sodium chloride, 15 mM sodium citrate), means a polynucleotide that can be identified by washing the filter under conditions of 65 ° C. do. Hybridization is described in Molecular Cloning 2nd ed. , Current Protocols in Molecular Biology, Supplements 1-38, DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University Press (1995). Here, sequences that hybridize under stringent conditions preferably exclude sequences containing only A sequences or only T sequences. Thus, polypeptides (e.g., LhHFS-encoded proteins) used in this disclosure include nucleic acids that hybridize under stringent conditions to nucleic acid molecules encoding the polypeptides specifically described in this disclosure. Polypeptides encoded by the molecules are also included. These low stringency conditions are 35% formamide, 5×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) Hybridization at 40° C. for 18-20 hours in buffer containing dextran sulfate, in buffer consisting of 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. , 1-5 hours at 55° C., and 1.5 hours at 60° C. in a buffer consisting of 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. include.

Functional equivalents of LhHFS used in this disclosure can be found by searching databases and the like. As used herein, "searching" means using one nucleobase sequence electronically or biologically or otherwise to find other nucleobase sequences with specific functions and/or properties. say. For electronic searching, BLAST (Altschul et al., J. Mol. Biol. 215:403-410 (1990)), FASTA (Pearson & Lipman, Proc. Natl. Acad. Sci., USA 85:2444- 2448 (1988)), the Smith and Waterman method (Smith and Waterman, J. Mol. Biol. 147:195-197 (1981)), and the Needleman and Wunsch method (Needleman and Wunsch, J. Mol. Biol. 48:443). -453 (1970)), but are not limited thereto. Biological searches include stringent hybridization, macroarray in which genomic DNA is attached to a nylon membrane or the like or microarray attached to a glass plate (microarray assay), PCR and in situ hybridization, and the like. is not limited to As used herein, genes used in this disclosure are intended to include corresponding genes identified by such electronic and biological searches.

As functional equivalents of the present disclosure, one or more amino acid insertions, substitutions or deletions in the amino acid sequence, or additions to one or both termini thereof can be used. As used herein, "insertion, substitution or deletion of one or more amino acids in an amino acid sequence, or addition to one or both ends thereof" refers to well-known techniques such as site-directed mutagenesis. It means that it has been modified by a method or by natural mutation, such as substitution of a plurality of amino acids to the extent that it can occur naturally.

As used herein, an "isolated" biological agent (e.g., gene (LhHFS), nucleic acid or protein, etc.) means that at least a portion of the agent or biological agent that is naturally associated with the agent or biological agent has been removed. It is used to distinguish from natural products. Thus, the purity of the biological agent in an isolated biological agent is usually higher (ie, concentrated) than the state in which the biological agent is normally present.

As used herein, a "purified" substance or biological agent (eg, nucleic acid or protein) refers to one from which at least a portion of the factors naturally associated with the biological agent have been removed. Thus, the purity of the biological agent in a purified biological agent is generally higher (ie, more concentrated) than the state in which the biological agent is normally present. As used herein, the term "purified" preferably refers to at least 75%, more preferably at least 85%, even more preferably at least 95%, and most preferably at least 98% by weight of It means that the same type of biological agent is present. Materials used in this disclosure are preferably "purified" materials.

As used herein, a "corresponding" amino acid or nucleic acid has or has the same effect in a given polypeptide or polynucleotide molecule as a given amino acid or nucleotide in a polypeptide or polynucleotide to which it is compared. Amino acids or nucleotides that are predicted to be similar, eg, in an enzyme molecule, to amino acids that are in similar positions in the active site and contribute similarly to catalytic activity. For example, an antisense molecule can be the same portion in an ortholog that corresponds to a particular portion of the antisense molecule. The corresponding amino acid can be, for example, cysteinylated, glutathionylated, S—S bond formation, oxidized (eg of the methionine side chain), formylated, acetylated, phosphorylated, glycosylated, myristylated, etc. can be an amino acid of Alternatively, the corresponding amino acid can be the amino acid responsible for dimerization. Such "corresponding" amino acids or nucleic acids may be regions or domains spanning a range. Accordingly, such cases are referred to herein as "corresponding" regions or domains.

As used herein, a "corresponding" gene (e.g., polynucleotide sequence or molecule) has or is predicted to have the same effect in a species as a given gene in a species to which it is compared. A gene (eg, a polynucleotide sequence or molecule) and, if multiple genes having such an effect, are of the same evolutionary origin. Thus, a gene corresponding to a gene can be an ortholog of that gene. Thus, LhHFS in lilies can find corresponding LhHFS in tulips, for example. Such corresponding genes can be identified using techniques well known in the art. Therefore, for example, a corresponding gene in a certain plant (for example, lily) can be obtained by using a sequence such as SEQ ID NO: 1 or SEQ ID NO: 3 as a query sequence for a reference gene for the corresponding gene (for example, LhHFS). It can be found by searching a plant (eg, tulip) sequence database.

As used herein, "fragment" refers to a polypeptide or polynucleotide having a sequence length from 1 to n-1 relative to a full-length polypeptide or polynucleotide (length of n). The length of the fragment can be changed as appropriate according to its purpose. For example, the lower limit of the length of the polypeptide is 15, 20, 25, 30, 40, 50 and more amino acids are included, and lengths represented by integers not specifically recited herein (eg, 11, etc.) are also suitable as lower limits. obtain. Also, in the case of polynucleotides, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides are included, specifically recited herein. A length expressed as an integer that does not contain a number (eg, 11, etc.) may also be suitable as a lower bound. As used herein, such fragments are understood to fall within the scope of the present disclosure as long as the fragment itself also functions as a marker, eg, if the full-length version functions as a marker.

As used herein, "activity" refers to the function of a molecule in its broadest sense. Activity generally includes, but is not limited to, any biological, biochemical, physical or chemical function of the molecule. Activities include, for example, activating, promoting, stabilizing, inhibiting, repressing, or destabilizing enzymatic activity, ability to interact with other molecules, and functions of other molecules. stability, ability to localize to specific subcellular locations. Where applicable, the term also relates to the function of protein complexes in the broadest sense. In the present disclosure, the "activity" of LhHFS is typically transcription activity (herein also referred to as "LhHFS transcription activity") or gene expression induction activity, more specifically aging-related gene induction activity or It may be programmed cell death-associated gene inducing activity.

As used herein, the term "biological function" refers to a specific function that the gene, nucleic acid molecule or polypeptide can have in vivo when referring to a gene or a nucleic acid molecule or polypeptide related thereto. Examples include, but are not limited to, generation of specific antibodies, enzymatic activity, imparting resistance, and the like. In the present disclosure, for example, the function of LhHFS and the like to promote or control flower senescence can be exemplified, but is not limited thereto. As used herein, a biological function can be exerted by "biological activity." As used herein, the term "biological activity" refers to an activity that a certain factor (e.g., polynucleotide, protein, etc.) can have in vivo, and includes various functions (e.g., transcriptional activity or gene expression induction). activity), and includes, for example, activities in which interaction with one molecule activates or inactivates another molecule. When two agents interact, their biological activity is determined by the binding between the two molecules and the resulting biological changes, e.g., when one molecule is precipitated with an antibody, the other Two molecules are considered bound when the molecules also co-precipitate. Therefore, observing such coprecipitation is one method of determination. As in the present disclosure, reference to activities that delay floral senescence encompasses such activities. For example, if an agent is an enzyme, its biological activity includes its enzymatic activity. Another example includes binding to the receptor to which the ligand corresponds when the agent is a ligand. Such biological activity can be measured by techniques well known in the art. Thus, "activity" indicates or reveals binding (either directly or indirectly); affecting a response (i.e., having a measurable effect in response to some exposure or stimulus); Refers to various measurable indicators, such as the affinity of a compound that binds directly to a polypeptide or polynucleotide of the present disclosure, or, for example, the amount of upstream or downstream protein after some stimulus or event or other Measures of similar function are included.

As used herein, "expression" of a gene, polynucleotide, polypeptide, etc. means that the gene or the like undergoes a certain action in vivo and changes into another form. Preferably, genes, polynucleotides, etc. are transcribed and translated into a form of polypeptide, but transcription to produce mRNA can also be an aspect of expression. More preferably, such polypeptide forms may be post-translationally processed (derivatives as referred to herein). For example, the expression level of LhHFS can be determined by any method. Specifically, the expression level of LhHFS can be determined by evaluating the amount of LhHFS mRNA, the amount of LhHFS protein, and the biological activity of LhHFS protein. The amount of LhHFS mRNA and protein can be determined by methods such as those described herein.

As used herein, the term "functional equivalent" refers to anything that has the same intended function but differs in structure from the original entity of interest. Therefore, when referring to "LhHFS or its functional equivalent" or "group consisting of LhHFS and its functional equivalents", in addition to LhHFS itself, variants or variants of LhHFS (e.g., amino acid sequence variants, etc.) which has the effect of delaying flower senescence, and which can be changed into LhHFS itself or a variant or variant of this LhHFS at the time of action (e.g., LhHFS itself or a variant of LhHFS or nucleic acids encoding the variants, and vectors, cells, etc. containing the nucleic acids). It is understood that functional equivalents of LhHFS can be used similarly to LhHFS in this disclosure, even if not specifically mentioned.

The modified amino acid sequence of LhHFS is, for example, 1 to 30, preferably 1 to 20, more preferably 1 to 9, still more preferably 1 to 5, particularly preferably 1 to 2 amino acid insertions, substitutions, Alternatively, deletions or additions to one or both termini thereof may be made. The modified amino acid sequence is preferably an amino acid sequence having one or more (preferably one or several or one, two, three, or four) conservative substitutions in the amino acid sequence of LhHFS. may As used herein, "conservative substitution" means replacement of one or more amino acid residues with another, chemically similar amino acid residue that does not substantially alter the function of the protein. For example, replacing one hydrophobic residue with another hydrophobic residue, replacing one polar residue with another polar residue having the same charge, and the like. Functionally similar amino acids with which such substitutions can be made are known in the art for each amino acid. Specific examples of nonpolar (hydrophobic) amino acids include alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, methionine, and the like. Polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, cysteine, and the like. Examples of positively charged (basic) amino acids include arginine, histidine, and lysine. In addition, negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

As used herein, "agent", "agent" or "factor" (both equivalent to the English equivalent of agent) are used broadly and interchangeably to describe any agent capable of achieving its intended purpose. It may be matter or other elements (eg, energy such as light, radiation, heat, electricity, etc.). Such substances include, for example, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (including DNA such as cDNA and genomic DNA, RNA such as mRNA), poly Saccharides, oligosaccharides, lipids, organic small molecules (e.g., hormones, ligands, signaling substances, organic small molecules, molecules synthesized by combinatorial chemistry, small molecules that can be used as pharmaceuticals (e.g., small molecule ligands, etc.), etc.) , including but not limited to these complex molecules. Polynucleotide-specific factors typically include polynucleotides that are complementary to the sequence of the polynucleotide with a certain degree of sequence homology (for example, sequence identity of 70% or more); Examples include, but are not limited to, polypeptides such as transcription factors that bind to promoter regions. Agents specific to a polypeptide typically include an antibody specifically directed against the polypeptide, a derivative thereof, or an analogue thereof (e.g., single-chain antibody); or specific ligands or receptors where the polypeptide is an enzyme, substrates thereof where the polypeptide is an enzyme, and the like.

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

As used herein, “genome editing” refers to a technique for introducing mutations so as to specifically modify (for example, suppress, decrease, improve or increase) the expression of a target gene. More specifically, techniques that use site-specific nucleases to cleave target genes at target locations in the genomic sequence, thereby generating single- or double-stranded breaks at specific locations within the genome. is. The breaks in the genome thus generated are usually repaired by processes such as homologous recombination (HDR) and non-homologous end repair (NHEJ). Genetic mutations such as nonsense mutations, missense mutations, and/or frameshifts are caused by random deletion or insertion of a few bases in the target gene, resulting in the production of normal (biologically functional) proteins. Gone. Techniques for genome editing include techniques such as CRISPR/Cas9, TALEN, ZFN, and meganuclease. General techniques for genome editing are described, for example, in Cox et al. , Nat. Med. 21: 121-131 (2015); Zhang et al. , Genome Biol. 19: 210 (2018). The introduction of mutations into target genes using genome editing is more efficient than conventional methods such as mutagenesis or repeated crossbreeding, by specifically introducing mutations into specific genes and producing cultivars with the desired properties. It is excellent in that it can be produced efficiently. In addition, under the Japanese system at the time of the filing of this application, plant cells, plant tissues, plant organs, or plant bodies or parts thereof produced by "genome editing" technology without remaining foreign genes are prohibited under the Cartagena Act. It is also advantageous in that it can be used without undergoing a safety review.

When a mutation is introduced into a gene by genome editing, the part involved in binding to the target DNA is RNA, and the part involved in DNA cleavage is a complex such as CRISPR / Cas9, or the part involved in binding to the target DNA. and a method using TALENs, ZFNs, etc., in which both of the portions involved in DNA cleavage are proteins. For example, in the method using CRISPR / Cas9, guide RNA, Cas9, etc. are introduced into the target cell, and in the method using TALEN, ZFN, a fusion protein in which the DNA binding domain and the nuclease are fused is introduced into the target cell. , mutations can be introduced into the genome. Methods for introduction into target cells include the Agrobacterium method, the RNA virus vector method, the plasma treatment method, the particle gun (bomberment) method, the PEG method, the electroporation method, and the like.

A suitable method for introducing mutations into the genome by genome editing may be a method of generating mutations when the cleavage site is naturally repaired after cleaving the target nucleotide sequence, or a method of generating mutations when the cleavage site is naturally repaired after cleaving the target nucleotide sequence. A DNA fragment having one or several nucleotide mutations in the sequence is introduced into cells, and after cleaving the target nucleotide sequence, one or several nucleotide mutations are inserted when the cleavage site is repaired using the introduced DNA fragment as a template. It may be a method of Incidentally, the number of bases is usually 2 to 6 bases.

The CRISPR/Cas9 system consists of three factors: CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA), and Cas9 protein. The Cas9 protein from the Streptococcus pyogenes strain replaces the three bases NGG (where N is either G, A, T or C) downstream of the target genomic sequence with the PAM sequence (Proto-spacer Adjacent Motif ) and cleaves its 3 salt phase upstream. A common approach in the art is to express Cas9 with a guide RNA (gRNA) linked to the tracrRNA at the 3′ end of the crRNA complementary to the target gene sequence, to target It introduces a DNA double-strand break at the site. As used herein, the guide RNA is designed such that the expression of the LhHFS gene is suppressed or reduced, or the LhHFS gene is knocked out. That is, the nucleic acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 is designed to express a protein in which the amino acid sequence is partially deleted, substituted, or added.

A 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 at this region, resulting in a double-strand break in the DNA.

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

Techniques for introducing mutations into target genes by genome editing include, for example, transformation using Agrobacterium, and methods for directly introducing genes for genome editing into protoplasts. Transformation using Agrobacterium is a technique for introducing genome-editing factors into plant cells by infecting plant cells with Agrobacterium bacteria containing vectors that express genome-editing factors. . That is, a plant cell or plant tissue isolated from a plant of interest (for example, a plant of the family Liliaceae) is incubated with an Agrobacterium bacterium containing a vector that expresses a factor for genome editing, and a gene editing factor for genome editing is added to the plant cell. Introduce a factor. Genome-edited callus is then generated by performing cell or tissue culture under appropriate culture conditions. Thereafter, the genome-edited callus is differentiated to regenerate the genome-edited plant body, thereby obtaining a plant body in which the genome of the entire plant body is edited. 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 plant of interest (eg, Liliaceae plant), and a factor for genome editing is introduced directly into the protoplasts by the PEG method, particle gun method, or the like. After that, by incubating the protoplasts under appropriate culture conditions, a genome-edited plant can be obtained.

Methods for introducing mutations into the genome by means of mutants include, for example, natural mutants, UV irradiation, irradiation such as gamma rays, and mutants induced by chemical substances such as ethylmethanesulfonic acid (EMS). , by selecting those with mutations in the gene by the PCR method, TILLING (Targeting Induced Local Lesion in Genomes) method, HRM (High Resolution Melting) method, etc., to obtain those with mutations in the gene method of doing so.

Antisense activity is usually achieved with a nucleic acid sequence of at least 8 contiguous nucleotides in length complementary to the nucleic acid sequence of the gene of interest. Such nucleic acid sequences are preferably at least 9 contiguous nucleotides long, more preferably 10 contiguous nucleotides long, even more preferably 11 contiguous nucleotides long, 12 contiguous nucleotides long, 13 contiguous nucleotides long. contiguous nucleotides in length, 14 contiguous nucleotides in length, 15 contiguous nucleotides in length, 20 contiguous nucleotides in length, 25 contiguous nucleotides in length, 30 contiguous nucleotides in length, 40 contiguous nucleotides in length It can be a nucleic acid sequence, 50 contiguous nucleotides long, nucleotides long. 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 sequences described above. Such antisense activity is preferably complementary to sequences at the 5' end of the nucleic acid sequence of the gene of interest. Such antisense nucleic acid sequences also include those having one or several or more than one nucleotide substitutions, additions and/or deletions relative to the sequences described above. Therefore, as used herein, "antisense activity" includes, but is not limited to, reduction in gene expression level.

Antisense techniques in general are described in textbooks (Murray, JAH eds., Antisense RNA and DNA, Wiley-Liss Inc, 1992). Furthermore, subsequent studies revealed a phenomenon called RNA interference (RNAi), which led to the development of antisense technology. In RNAi, when a short double-stranded RNA (about 20 bases) having a sequence homologous to a target gene is introduced into cells, mRNA of the target gene homologous to the RNA sequence is specifically degraded. This is a phenomenon in which the expression level decreases. This phenomenon, which was first discovered in nematodes, has been found to be a universal phenomenon in living organisms, including plants. It was clarified that they undergo a similar process. Conventionally, one DNA sequence that is complementary to the nucleotide sequence of the target gene is ligated to an appropriate promoter to construct an expression vector that expresses the artificial mRNA under its control and introduce it into cells. was done. Recent findings use expression vectors designed to construct double-stranded RNA in cells. The basic structure is made by linking one type of DNA sequence complementary to a certain target gene under a promoter, and then linking the same thing to another one in the opposite direction. In the single-stranded mRNA transcribed from this construction gene, one type of nucleotide sequence portion connected in the opposite direction is in a complementary relationship, so that it pairs to form a double-stranded RNA state with a hairpin-like secondary structure. , which causes mRNA degradation of the target gene according to the RNAi mechanism. In plants, it has been reported to be used in Arabidopsis thaliana (Smith, NA et al., Nature 407, 319-320, 2000). RNAi in general is summarized in a review article (Morita and Yoshida, Protein, Nucleic Acid, Enzyme 47, 1939-1945, 2002). The contents described in these documents are incorporated herein by reference in their entireties.

As used herein, "RNA interference" or "RNAi" is an abbreviation for RNA interference, commonly known in the art, that inhibits or downregulates gene expression in cells mediated by factors that cause RNAi. It is an academic process. For example, by introducing an RNAi-causing factor such as double-stranded RNA (also referred to as dsRNA) into a cell, the homologous mRNA is specifically degraded and the synthesis of the gene product is suppressed, and the technology used therefor Say. In the present specification, "RNAi" can also be used synonymously with "factor that causes RNAi", "factor that causes RNAi", "RNAi factor", and the like, in some cases. For RNAi, see, for example, Zamore and Haley, 2005, Science, 309, 1519-1524; Vaughn and Martinssen, 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; Publication No. 00/01846; Mello and Fire, WO 01/29058; Deschamps-Depaillette, WO 99/07409 and Li et al., WO 00/44914; 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, as used herein, the term RNAi is intended to be synonymous with other terms used to describe sequence-specific RNA interference, such as post-transcriptional gene silencing, translational inhibition, transcriptional inhibition, and epigenetics. understood. As used herein, an "RNAi agent" can be anything as long as it causes "RNAi".

As used herein, the agent that causes RNAi includes, for example, a sequence that has at least about 70% homology to a portion of the nucleic acid sequence of the target gene or a sequence that hybridizes under stringent conditions, at least 10 Examples include, but are not limited to, RNAs containing nucleotide-long double-stranded portions or variants thereof. Here, the factor preferably comprises a 3' protruding end, more preferably the 3' protruding end can be DNA of 2 or more nucleotides in length (eg, DNA of 2-4 nucleotides in length).

Alternatively, RNAi agents for use in the present disclosure include, but are not limited to, pairs of short, inverted complementary sequences (eg, 15 bp or more, such as 24 bp, etc.).

Without being bound by theory, one possible mechanism by which RNAi works is that when a molecule that causes RNAi, such as dsRNA, is introduced into a cell, for relatively long (e.g., 40 base pairs or more) RNA, helicase A domain-bearing RNase III-like nuclease called Dicer, in the presence of ATP, cleaves the molecule approximately 20 base pairs from the 3' end, yielding short dsRNAs (also called siRNAs). As used herein, "siRNA" is an abbreviation for short interfering RNA, which is artificially chemically synthesized, biochemically synthesized, synthesized in vivo, or about 40 Refers to short-chain double-stranded RNA of 10 base pairs or more that is produced by degrading double-stranded RNA of base or more in the body, usually having a structure of 5'-phosphate, 3'-OH, and 3 The ' end protrudes by about 2 bases. A protein specific to this siRNA binds to form a RISC (RNA-induced-silencing-complex). This complex recognizes and binds to mRNA with the same sequence as the siRNA and cleaves the mRNA in the middle of the siRNA by an RNase III-like enzymatic activity. The relationship between the siRNA sequence and the targeted mRNA sequence to be cleaved is preferably 100% identical. However, mutation of bases at positions off the center of the siRNA does not completely eliminate the cleavage activity by RNAi, but partially retains the activity. On the other hand, mutation of bases in the central part of siRNA has a large effect, and the mRNA cleavage activity by RNAi is extremely reduced. Taking advantage of such properties, siRNA with mutations in the center can be synthesized and introduced into cells to specifically degrade only mRNAs containing mutations. Therefore, in the present disclosure, siRNA itself can be used as an agent that causes RNAi, or an agent that produces siRNA (eg, a dsRNA that is typically about 40 bases or longer) can be used as such an agent. can.

Also, without wishing to be bound by theory, siRNAs can be synthesized in a manner that, apart from the pathway described above, the antisense strand of the siRNA binds to mRNA and acts as a primer for RNA-dependent RNA polymerase (RdRP) to synthesize dsRNA. It is also contemplated that this dsRNA may become a substrate of Dicer again, giving rise to new siRNA and amplifying its action. Thus, siRNAs themselves and agents from which siRNAs are generated are also useful in the present disclosure. In fact, in insects, for example, 35 dsRNA molecules almost completely degrade intracellular mRNA, which has 1,000 copies or more. be done.

Double-stranded RNAs of about 20 bases in length (eg, typically about 21-23 bases in length) or less, referred to as siRNAs in this disclosure, can be used. When such siRNA is expressed in cells, it suppresses gene expression and suppresses the expression of pathogenic genes targeted by the siRNA. Therefore, it can be used for treatment, prevention, prognosis, and the like of diseases. The siRNA used in the present disclosure can take any form as long as it can induce RNAi.

In another embodiment, the agent that causes RNAi of the present disclosure can be a short hairpin structure (shRNA) with an overhang at the 3' end. As used herein, the term “shRNA” refers to a single-stranded RNA containing a partially palindromic base sequence, thereby forming a double-stranded structure within the molecule and forming a hairpin-like structure of about 20 base pairs. Molecules above. Such shRNAs are artificially chemically synthesized. Alternatively, such shRNAs can be generated by in vitro synthesis of RNA with T7 RNA polymerase from a hairpin-structured DNA in which the DNA sequences of the sense and antisense strands are linked in opposite directions. Without wishing to be bound by theory, such shRNAs are approximately 20 bases (typically, e.g., 21 bases, 22 bases, 23 bases) in length within the cell after introduction into the cell. , causing RNAi in the same manner as siRNA and having the therapeutic effects of the present disclosure. It should be understood that such effects are exhibited in a wide variety of organisms such as insects, plants, and animals (including mammals). Thus, shRNA can be used as an active ingredient of the present disclosure because it induces RNAi like siRNA. The shRNA may also preferably have a 3' overhang. Although the length of the double-stranded portion is not particularly limited, it is preferably about 10 nucleotides or longer, more preferably about 20 nucleotides or longer. Here, the 3' protruding end is preferably DNA, more preferably DNA with a length of at least 2 nucleotides or longer, and still more preferably DNA with a length of 2 to 4 nucleotides. Factors that cause RNAi used in the present disclosure can be artificially synthesized (e.g., chemically or biochemically) or naturally occurring, and the effect of the present disclosure can be obtained between the two. No essential difference occurs. Those chemically synthesized are preferably purified by liquid chromatography or the like.

Agents that cause RNAi used in this disclosure can also be synthesized in vitro. In this synthesis system, T7 RNA polymerase and T7 promoter are used to synthesize antisense and sense RNA from template DNA. When these are annealed in vitro and then introduced into cells, RNAi is triggered through the mechanisms described above to achieve the effects of the present disclosure. Here, such RNA can be introduced into the cell by any suitable method such as, for example, the calcium phosphate method. Agents that cause RNAi of the present disclosure also include agents such as single strands that can hybridize to mRNA, or all similar nucleic acid analogues thereof. Such factors are also useful in this disclosure.

As used herein, vectors include those that can be replicated and isolated and purified in common bacteria used in genetic experiments (a typical example is an E. coli strain derived from E. coli K12 strain). This is necessary for constructing a target gene to be introduced into plants. Specifically, there are commercially available constructed plasmids such as E. coli pBR322 plasmid, pUC18, pUC19, pBluescript, and pGEM-T. When a gene fragment is directly introduced into a plant cell for transformation by electroporation method, polyethylene glycol method, or particle gun method, such a commercially available general plasmid is used to transform the gene to be introduced. You can build. As a special example of a vector, when a plant cell is transformed using an Agrobacterium-mediated gene transfer method, the origin of replication of both E. coli and Agrobacterium and the border region capable of being introduced into the plant are used. Plasmids called "binary vectors" with nucleotide sequences corresponding to the border sequences (Left border and Right border) from the T-DNA shown should be used. For example, 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 Alternatively, pTH (Fukuoka H et al., Plant Cell Reports 19, 2000), pPZP (Hajdukiewicz P et al., Plant Molecular Biology 25, 989-994, 1994) and the like, but are not limited thereto. In addition, a tobacco mosaic virus vector is also exemplified as a vector that can be used in plants, but since this type of vector does not introduce the target gene into the plant chromosome, the gene-introduced plant is propagated through seeds. It is of limited use when it is not necessary to do so, but may be used in the present disclosure.

As used herein, the technique of introducing a nucleic acid molecule into a cell may be any technique, including transformation, transduction, transfection, and the like. Techniques for introducing such nucleic acid molecules are well known and commonly used in the art, and are described in, for example, Ausubel F. et al. A. (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. Gene transfer can be confirmed using the methods described herein, such as Northern blot, Western blot analysis, or other well-known and conventional techniques.

As a vector introduction method, any of the methods described above for introducing DNA into cells can be used. Examples include transfection, transduction, and transformation (e.g., calcium phosphate method, liposome method, DEAE dextran method, electroporation method, method using particle gun (gene gun), etc.), lipofection method, spheroplast method [Proc. Natl. Acad. Sci. USA, 84, 1929 (1978)], the lithium acetate method [J. Bacteriol. , 153, 163 (1983)], Proc. Natl. Acad. Sci. USA, 75, 1929 (1978).

As used herein, the term "gene transfer reagent" refers to a reagent used to promote transfer efficiency in a gene transfer method. Examples of such gene introduction reagents include, but are not limited to, cationic polymers, cationic lipids, polyamine-based reagents, polyimine-based reagents, calcium phosphate, and the like. Specific examples of reagents utilized during transfection include those commercially available from a variety of sources, such as Effectene Transfection Reagent (cat. no. 301425, Qiagen, Calif.), TransFast™ 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.), and the like.

When the present disclosure is utilized in plants, methods well known to those skilled in the art can be used to introduce plant expression vectors into plant cells, such as Agrobacterium-mediated methods and direct cell introduction methods. As an Agrobacterium-mediated method, for example, the method of Nagel et al. (Nagel et al. (1990), Microbiol. Lett., 67, 325) can be used. This method involves first transforming Agrobacterium by, for example, electroporation, with a plant-appropriate expression vector, and then transforming the transformed Agrobacterium into a plant as described in Gelvin et al. (Gelvin et al. (1994), Plant Molecular Biology Manual). (Kluwer Academic Press Publishers)). For methods of directly introducing plant expression vectors into cells, see electroporation (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). that). These methods are well known in the art, and methods suitable for plants to be transformed can be appropriately selected by those skilled in the art.

Cells introduced with the plant expression vector are first selected for drug resistance such as hygromycin resistance and kanamycin resistance. They can then be redifferentiated into plant tissues, plant organs and/or plants by methods well known in the art. Additionally, seeds may be obtained from the plant. Expression of the introduced gene can be detected by Northern blotting or PCR. If desired, the expression of the gene product protein can be confirmed by, for example, Western blotting.

Although the present disclosure has been shown to be particularly useful in plants, it can also be used in other organisms. Molecular biology techniques used in the present disclosure are well known and routine in the art, see, for example, Ausubel F. et al. A. (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;

Physical methods of transformation include 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, the polyethylene glycol method and the electroporation method have the problem that protoplasts must be used because the cell wall is an obstacle, and 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 needle thickness, tissue fixation, and the like. Even with the particle gun method using tissue, there are problems such as mutations appearing in the form of chimeras. In addition, in these physical methods, generally, the introduced foreign gene tends to be incompletely integrated into the nuclear genome as a multi-copy gene. It is known that introduction of multiple copies of a foreign gene tends to inactivate the gene.

On the other hand, methods for introducing an isolated gene using organisms include the Agrobacterium method, the viral vector method, and the method using pollen as a vector. These methods do not use protoplasts, but use plant callus, tissue, or plant bodies for gene transfer, so they do not require long-term culturing and are less susceptible to disorders such as somaclonal mutations. ing. Of these methods, pollen is used as a vector, and there are still few experimental examples, and there are many unknowns as plant transformation methods. Although the virus vector method has the advantage that the gene to be introduced spreads throughout the virus-infected plant body, it is only amplified and expressed in each cell, and there is no guarantee that it will be transmitted to the next generation. Another problem is 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 number of copies of the introduced gene being as low as a few copies, and high reproducibility. . Since Agrobacterium is outside the host range of monocotyledonous plants such as gramineous plants, introduction of exogenous genes into gramineous plants has hitherto been carried out by physical methods as described above. However, in recent years, the Agrobacterium method has come to be applied to monocotyledonous plants such as rice, for which a culture system has been established, and the Agrobacterium method is currently preferred. .

In the introduction of a foreign gene by the Agrobacterium method, when a low-molecular-weight phenolic compound such as acetosyringone synthesized by a plant acts on the Ti plasmid Vir region, the T-DNA region is excised from the Ti plasmid, and through several processes, Integrated into nuclear chromosomal DNA of plant cells. In dicotyledonous plants, since the plants themselves have a mechanism for synthesizing such phenolic compounds, exogenous genes can be easily introduced by the leaf disk method or the like, with high reproducibility. On the other hand, monocotyledonous plants do not synthesize such phenolic compounds themselves, so it has been difficult to produce transformed plants using Agrobacterium. However, by adding acetosyringone at the time of Agrobacterium infection, it is now possible to introduce foreign genes into monocotyledonous plants.

In the present disclosure, the transformant may or may not have the target nucleic acid molecule (transgene) introduced into the chromosome. Preferably, the nucleic acid molecule of interest (transgene) is introduced into a chromosome, more preferably into both chromosomes.

(plant)
Preferred plants include, but are not limited to, ornamental plants for viewing flowers, but any plant in which it is useful to prevent senescence of flowers, such as crops, trees, lawns, weeds, and the like. Unless otherwise indicated, plants include any plant body, plant organ, plant tissue, plant cell, and seed. Examples of plant organs include roots, bulbs, leaves, stems, flowers (or parts of organs or tissues that make up flowers, such as petals or similar organs (eg, perianth)), pollen, spores, and woody plants. , and mukago. Plants may also be provided as a combination of plant tissues or plant organs, for example as cut flowers or potted flowers (a combination of flowers, stems, leaves and/or roots). Examples of plant cells include callus and suspension cultured cells.

Any method of genome editing in plant cells can be used, as detailed elsewhere herein, eg, Yan, R. et al. 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 method described in Japanese Unexamined Patent Application Publication No. 2020-130054.

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

Cultivation of plants herein can be done by any method known in the art. A method for cultivating plants is described in "Making lilies" (edited by Hideo Imanishi, Nobunkyo, 2006) and the like.

Techniques and media known in the art are used for culture, dedifferentiation, differentiation and regeneration of plant cells, plant tissues and plants. Examples of such media include, but are not limited to, Murashige-Skoog (MS) medium, GaMborg B5 (B) medium, White medium, Nitsch & Nitsch (Nitsch) medium, and the like. These media are usually used after adding an appropriate amount of a plant growth regulator (plant hormone) or the like.

In order to prepare a plant cell, plant tissue, plant organ or plant body or a part thereof in which LhHFS or its homologous gene is partially or wholly mutated or deleted, the whole plant body or a part thereof is subjected to conventional mutation. It may be mutated by exposure to an inducer. Alternatively, a part of the plant is dedifferentiated to form callus or cells, and the callus or cells contain the factors of the present disclosure or the factors of the present disclosure are modified by modifying the callus or cells by genetic engineering or the like. may be obtained.

As used herein, in the case of plants, "redifferentiation" means a phenomenon in which undifferentiated cells differentiate into more differentiated entities such as functional cells, tissues, or whole individuals. For example, regeneration of callus can lead to the formation of tissue pieces such as cells (leaves, roots, etc.), and growth of these tissue pieces can lead to the formation of organs or plants.

Methods for redifferentiating transformants into plants are well known in the art. Such methods include 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) and the like, but are not limited thereto. Therefore, those skilled in the art can appropriately use the well-known method according to the target transgenic plant to regenerate. The transgenic plants thus obtained have the gene of interest introduced therein, and the introduction of such gene can be performed using methods described herein, such as Northern blot, Western blot analysis, or other methods. It can be confirmed using well-known and commonly used techniques.

(preferred embodiment)
Preferred embodiments of the present disclosure are described below. The embodiments provided below are provided for a better understanding of the disclosure, and it is understood that the scope of the invention should not be limited to the following description. Therefore, it is clear that those skilled in the art can make appropriate modifications within the scope of the present invention in light of the description in this specification.

<LhHFS gene and protein>
In a representative aspect of the present disclosure, the present disclosure provides the newly discovered LhHFS gene and the LhHFS protein encoded by this gene. LhHFS is one of the transcription factor family genes whose expression level increases during flower senescence, but as a result of producing a transformant (LNR18) in which LhHFS expression is suppressed, the period until flowers turn brown is extended. This was observed in the Liliaceae plant Liliaceae (Oriental hybrid line) (see Examples). These results indicate that LhHFS is a transcription factor family gene that regulates (promotes) flower senescence, and that suppression of LhHFS expression can delay flower senescence. In the Oriental hybrid system, the sequence homology of the LhHFS gene is as high as 99% or more between the tiara cultivar used in the examples and other cultivars (Casablanca cultivar and Acapulco cultivar). It is understood that the suppression of senescence by inhibitors of LhHFS is not cultivar-specific. In addition, it is understood that the ornamental period can be extended in other Liliaceae plants by gene editing, gene recombination, or suppressors of LhHFS such as inhibitors targeting LhHFS orthologues. In particular, the homologous gene (having the nucleotide sequence of SEQ ID NO: 4) of the LhHFS gene possessed by tulip (family Liliaceae, genus Tulipa) has about 71% sequence identity with the nucleic acid sequence (SEQ ID NO: 1) encoding lily LhHFS. Therefore, it is believed that a sequence variant with at least about 70% sequence identity can delay flower senescence by suppressing its expression. The LhHFS proteins share at least a region predicted to bind DNA conserved 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 (for example, van Doorn WG , Woltering EJ. Physiology and molecular biology of petal senescence. Journal of Experinental Botany 59:453-80, 2008), it is understood that a similar regulatory mechanism exists, and similar activity inhibition of LhHFS orthologues other than Yuri It is understood that it exerts an effect on It is understood that the present disclosure produces similar effects in plants other than those in the examples. The factor of the present disclosure may be any factor that can temporarily or permanently suppress, reduce or eliminate the function of LhHFS, and may be any protein, polypeptide, oligopeptide, peptide, polypeptide, Nucleotides, oligonucleotides, nucleotides, nucleic acids (including DNA such as cDNA, genomic DNA, RNA such as mRNA), genome editing factors, polysaccharides, oligosaccharides, lipids, small organic molecules (such as hormones , ligands, signaling substances, organic small molecules, molecules synthesized by combinatorial chemistry, small molecules that can be used as pharmaceuticals (e.g., small molecule ligands, etc.), and complex molecules thereof, but are not limited thereto. In some cases, it may be an element other than a substance (for example, a mutagenesis factor such as energy such as light, radiation, heat, or electricity).

INDUSTRIAL APPLICABILITY The present disclosure is effective for Liliaceae plants (for example, plants belonging to the genus Liliaceae and the genus Tulip), in which aging effects progress due to browning of flowers several days after flowering. Aging in Liliaceae plants (e.g., plants of the genus Lilium and Tulip) is different from the wilting and rolling (inrolling) of flowers commonly seen in other plants, so the anti-aging effect of the technology of the present disclosure is remarkably useful. For example, lily and tulip flowers are composed of three inner perianths and three outer perianths, whereas representative plant flowers other than Liliaceae plants do not have a similar configuration. It is also noteworthy that the corolla is composed of petals or a corolla of concatenated petals, and it is worth noting that aging can be suppressed even with a structure consisting of three inner and three outer perianths. Furthermore, the effect on monocotyledonous plants is also considered to be noteworthy.

Representative nucleic acid sequences of LhHFS genes include the nucleic acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:3 or the nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO:2. Accordingly, in one aspect, the present disclosure provides a nucleic acid molecule comprising the nucleic acid sequence set forth in SEQ ID NO: 1 or the nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO: 2; or (B) a sequence variant, wherein the ( B) sequence variants are (B-1) one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9) in the nucleic acid sequence of (A) , 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 , 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, (B-2) Nucleic acid sequence of (A) 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 (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) A nucleic acid molecule is provided. In a more specific aspect, said nucleic acid molecule comprises the nucleic acid sequence set forth in SEQ ID NO:1. In a more specific aspect, said nucleic acid molecule consists of the nucleic acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:3.

In one aspect, the present disclosure provides (a) a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 or a fragment thereof; (b) one or more (e.g., 1, 2) , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 , 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100) amino acids mutated wherein the mutation is a polypeptide selected from the group consisting of substitutions, additions and/or deletions, and which has a biological activity similar to that of the polypeptide of (a) , a polypeptide; (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 amino acid sequence shown in SEQ ID NO: 2 or (e) at least about 70%, preferably at least about 80%, at least about 85%, at least about 90%, at least about 95% identity to the polypeptide of any one of (a)-(d) having an amino acid sequence that is at least about 98%, or at least about 99%, and having a biological activity similar to that of the polypeptide of (a) I will provide a. In a more specific aspect, the amino acid sequence of said polypeptide comprises the sequence shown in SEQ ID NO:2. In a more specific aspect, the amino acid sequence of said polypeptide consists of the sequence shown in SEQ ID NO:2.

The present disclosure understands that suppression of LhHFS-homologous genes is effective in suppressing flower senescence not only in Liliaceae plants, but also in all plants having LhHFS-homologous genes, particularly Liliaceae plants in general. Without wishing to be bound by theory, it is believed that, at least across Liliaceae plants, the function of LhHFS homologous genes is substantially similar, although the LhHFS gene is found in the present disclosure. This is because the analysis in the present disclosure has revealed that this LhHFS gene is commonly found in all Liliaceae plants.

<Repressor of LhHFS>
In one aspect, the present disclosure provides factors that suppress the function of the nucleic acid sequences or amino acid sequences described in <LhHFS genes and proteins>, or modified sequences thereof. Said agents include proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids including RNA or DNA, polysaccharides, oligosaccharides, lipids, hormones, ligands, signaling agents, synthetic chemicals, radiation , a combination or multiple factors thereof. In a more specific aspect, the agent is selected from the group consisting of antisense molecules, RNAi agents, genome editing agents, mutagens.

In one aspect, the present disclosure provides agents that repress LhHFS or homologous genes thereof. The homologous gene is (i) a sequence variant of the 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, and the sequence variant is (B-1) SEQ ID NO: 1 or a sequence variant having one or more substitutions, additions and/or deletions in the nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 2, (B-2) the nucleic acid sequence shown in SEQ ID NO: 1 or SEQ ID NO (B-3) a sequence variant having at least about 70% sequence identity with the nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 1, or encoding the nucleic acid sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 2 A sequence variant that hybridizes under stringent conditions to the nucleic acid sequence, (B-4) an allelic variant of the 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 Sequence variant, or (B-5) 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, or a sequence variant that is a fragment of (B-1) to (B-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 the nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 2. (ii) a polypeptide consisting of (a) the amino acid sequence shown in SEQ ID NO: 2 or a fragment thereof; (b) in the amino acid sequence shown in SEQ ID NO: 2, one or more amino acids are mutated, and , substitutions, additions and/or deletions, and which has a biological activity analogous to that of the polypeptide of (a); (c) to 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) having an amino acid sequence that is at least about 70% identical to the polypeptide of any one of (d), and exhibiting a biological activity similar to that of the polypeptide of (a) can include polypeptides having. Suppression of LhHFS results in, for example, decreased gene expression levels, decreased gene copy number, decreased gene amplification, decreased RNA activity levels, decreased mRNA abundance, decreased mRNA synthesis rate, decreased mRNA stability, decreased protein activity Reduction in levels, reduction in protein synthesis, reduction in protein abundance, reduction in protein stability, reduction in protein enzymatic activity, or a combination thereof. The factor that suppresses LhHFS may be any substance that partially or completely suppresses the biological functions of the LhHFS gene or protein in plants. Said agents include proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids including RNA or DNA, polysaccharides, oligosaccharides, lipids, hormones, ligands, signaling agents, synthetic chemicals, radiation , combinations or multiple factors thereof. In a more specific aspect, the agent is selected from the group consisting of antisense molecules, RNAi agents, genome editing agents, mutagens. Herein, it is demonstrated that an LhHFS expression-suppressing line was created by RNAi factor and genome editing and flower senescence was delayed (see Examples).

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

In one embodiment, the agent is an agent that causes RNA interference of LhHFS in the form of double-stranded nucleic acid.

In certain embodiments, the factor of the present disclosure is in double-stranded form, one strand of which is (I)(A) the nucleic acid sequence shown in SEQ ID NO: 1 (LhHFS itself) or the sequence encoding SEQ ID NO: 2 or (B) a sequence variant, The other strand comprises (II) a complementary sequence or an annealing sequence of (I), and the (B) sequence variant is (B-1) one or several substitutions, additions in (A) or a sequence variant having a deletion, (B-2) at least about 70%, preferably at least about 80%, at least about 85%, at least about 90%, at least about 95% of the sequence of (A), at least A sequence variant that has about 98%, or at least about 99% sequence identity, or (B-3) a sequence variant that hybridizes under stringent conditions to the sequence of (A), (B-4 ) is a sequence variant that is an allelic variant of (A), or (B-5) is a sequence variant that is a fragment of (A) or (B-1) to (B-4), and said sequence variant The protein encoded by has the biological function of the protein encoded by (A).

In a more specific embodiment, the agent of the present disclosure is in double-stranded form and one strand thereof (I) does not comprise the 462 base sequence from bases 22-483 of SEQ ID NO:1. Without wishing to be bound by theory, the nucleotide sequence of bases 22-483 in SEQ ID NO: 1 corresponds to a DNA binding site, and targeting this site suppresses the expression of genes other than the LhHFS gene. It is desirable to target regions other than this site because of the potential for cytotoxicity. In a more specific embodiment, one strand (I) comprises the nucleic acid sequence of bases 574-864 of SEQ ID NO:1. It is understood that specific embodiments are not limited to this and that any range in length, for example, 22-483 bases may be used (Wesley et al., Plant Journal 27:581 -590). Suitable RNAi agents are also described in Wesley et al. Plant Journal 27:581-590 and the like can be referred to for design.

In plant RNAi, it is generally believed that any region sequence of a target gene transcript (mRNA) can be used to induce degradation of mRNA. From the principles of RNAi, it is clear to those skilled in the art that mRNA degradation occurs if it contains the transcribed sequence of the target gene (see, e.g., Wesley et al. Plant Journal 27:581-590). ). Any region can be selected, but preferably, when targeting sequences in highly conserved regions (sequences common among multiple genes), the same sequences are also used in addition to the gene of interest. It is advantageous to omit conserved regions from the target sequence, as they may degrade the mRNA of the gene they carry. For the LhHFS RNAi constructs of the present disclosure, the relatively highly conserved regions were removed from those carried out in the examples just in case. It is known that targeting the 5'UTR and 3'UTR (untranslated region) rather than the coding region can also induce RNAi (see, eg, Wesley et al. Plant Journal 27:581-590). It is understood that in plants, siRNAs can also be used, but preferably longer RNAi agents can also be used (plants express longer dsRNAs, but are ultimately processed into siRNAs as short as 20 bases). induced silencing).

In one aspect, the disclosure provides a vector comprising a nucleic acid that causes LhHFS RNA interference. The nucleic acid that causes LhHFS RNA interference included here is in any form exemplified by the factor that suppresses LhHFS of the present disclosure, and any nucleic acid that causes LhHFS RNA interference can be used. It is understood that

In another embodiment, the vectors of this disclosure contain promoters and/or terminators.

In one aspect, the present disclosure provides genome editing factors that cause LhHFS function and/or suppression of function. Factors for genome editing may be any factors used in techniques such as CRISPR/Cas9, TALENs, ZFNs, meganucleases.

When CRISPR / Cas9 is selected as a genome editing method, a guide RNA with a total of 23 bases including NGG (N is any base of ATGC) called PAM sequence at the 3' end is used. be done. A guide RNA sequence can be designed by a program such as CRISPR-P. The sequence of the guide RNA used for genome editing preferably targets genome editing in the nucleic acid sequence of bases 1 to 483 in SEQ ID NO: 1. The sequence of the guide RNA is, for example, the following table Examples include the sequences shown in

In one aspect, the disclosure provides a plant cell comprising or modified by an agent of the disclosure that suppresses LhHFS. In another aspect, plant tissue comprising said factor or said plant cell comprising said factor or modified by said factor is provided. In yet another aspect, a plant organ comprising or comprising said plant cell or said plant organ comprising said factor or modified by said factor is provided. In yet another aspect, there is provided a plant or part thereof containing or modified by said factor, or a plant or part thereof comprising said plant cell, said plant tissue or said plant organ.

In preferred embodiments, the plant cells of the present disclosure are plant cells of the family Liliaceae. Examples of such plant cells include Lilium, Tulipa, Erythronium, Fritillaria, Amana, Bowie, Calochortus. ), Cardiocrinum, Clintonia, Gagea, Lloydia, Medeola, Nomocharis, Notholirion, Prosartes, Examples include the genus Streptopus, the genus Tricyrtis, and the like, including, but not limited to, lilies, tulips, dogtooth violets, and black lilies. In a further preferred embodiment, the plant cells of the present disclosure are lily cells.

In one embodiment, the plant or part thereof comprising an agent that suppresses LhHFS is a bulb, seed, flower, stem, leaf, pollen, spore, tree, cell, tissue, tissue culture or germ, or including any combination of all or part of In a specific embodiment, for example, it may be provided in the form of a whole flower, or a combination of flower-composing organs such as petals, corolla, and perianth, or organs comprising a flower (for example, cut flowers or potted flowers). .

(Method for producing plant in which LhHFS or its homologous gene is suppressed)
In another aspect, the present disclosure provides plant cells in which LhHFS is suppressed relative to the natural state. "Native state" means a state in which the expression level of LhHFS at the peak expression level is equivalent to that of the housekeeping gene Elongation factor 1 alpha (EF1alpha). Here, the expression level of LhHFS is low on day 0 after flowering, but reaches a peak about 4-5 days after flowering, so the expression level on about 4-5 days after flowering is used.
In such cells, a state in which LhHFS is more suppressed than in the native state can be achieved by introducing into the plant cell a factor that suppresses LhHFS of the present disclosure. When obtaining the plant cells using a factor that suppresses LhHFS, transduction is conventionally performed using a vector containing a drug resistance gene (e.g., hygromycin resistance gene), and screening using a drug resistance marker. can be screened for plant cells into which mutations have been inserted. Alternatively, LhHFS-suppressed plants may be selected by PCR or the like for plants or cells in which LhHFS is mutated from a population in which plant bodies or plant cells are mutagenized with a mutagen. . In this case, for example, in the case of lilies, those with reduced levels of expression or activity relative to the native LhHFS disclosed in this disclosure may be selected. Other plants can be similarly selected. Alternatively, in addition to the method of introducing a suppressor factor or screening, a method such as zinc finger nuclease or gene targeting may be used to modify the endogenous LhHFS sequence to suppress expression. In the practice of the present disclosure, regarding the application of zinc finger nucleases in plants, see Keishi Osaka, Yuriko Osaka 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, see Molecular breeding of a novel herbicide-tolerant rice by gene targeting. Osaka, 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) can be referred to.

The fact that a plant cell, tissue, organ or plant body obtained by screening has LhHFS that is more suppressed than in the natural state is, for example, a decrease in gene expression level, a decrease in gene copy number, a decrease in gene amplification, a decrease in RNA Decreased activity level, decreased mRNA abundance, decreased mRNA synthesis rate, decreased mRNA stability, decreased protein activity level, decreased protein synthesis, decreased protein abundance, decreased protein stability, decreased protein enzymatic activity It can be confirmed by a method selected from the group consisting of a decrease, 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 be typically confirmed by a technique such as Western blotting. In addition, in the case of LhHFS, the decrease in protein enzymatic activity can be measured by expression levels of downstream genes, reporter assays, and the like.

Screening may be performed in any cell, tissue, organ or plant. Preferably, screening for drug resistance is performed on callus. The presence or absence of gene introduction and the presence or absence of mutation introduction by genome editing are analyzed by PCR or sequencing using DNA extracted from adventitious buds (shoots) induced from callus. LhHFS gene expression and decreased protein levels are analyzed in perianths.

A plant cell having the traits of interest of the present disclosure can be redifferentiated into a tissue, organ or plant by methods known in the art.

Once a plant having the trait of interest of the present disclosure is obtained as a transformant, the transformant is used as a pollen parent or seed parent, and a new plant having the trait of interest of the present disclosure is produced by conventional crossing methods. Another variety may be produced.

In preferred embodiments, the plant cells of the present disclosure are plant cells of the family Liliaceae. Such plant cells include, for example, Lilium, Tulipa, Erythronium, Fritillaria, Amana, Bowie, Calochortus ), Cardiocrinum, Clintonia, Gagea, Lloydia, Medeola, Nomocharis, Notholirion, Prosartes, Examples include the genus Streptopus, the genus Tricyrtis, and the like, including, but not limited to, lily, tulip, dogtooth violet, and black lily. In a further preferred embodiment, the plant cells of the present disclosure are lily cells.

In one embodiment, suppression of flower senescence in the present disclosure may be achieved by suppressing senescence of some or all of the elements that constitute the flower. The elements that make up a flower include the petals, corolla, perianth, stamens, pistils, calyx, peduncle, and stalk. Inhibition of flower senescence can be inhibition of senescence of at least one, at least two, at least three, at least four, or more of the elements that constitute the flower. In a preferred embodiment, it would be advantageous if the senescence of any of the flower-constituting elements, such as the petals, corolla, and perianth, is suppressed. In a preferred embodiment, suppression of senescence of flowers may be achieved by achieving suppression of senescence in any one of petals, corollas, perianths, etc. Alternatively, suppression of senescence in the entire flower may be achieved. Regarding suppression of senescence of flowers, stamens, pistils, etc. do not need to be inhibited from senescence. In various embodiments, inhibition of flower senescence typically refers to senescence of the “petals,” senescence of the corolla, senescence of the perianth, senescence of the perianth attached to the flower stalk. Aging, part of the whole flower excluding stamens and pistils, or the whole flower (i.e., including all the elements that make up the flower, such as perianth, flower axis corresponding to stem, stamen, pistil, etc.) There may be.

(How to extend the shelf life of flowers)
In another aspect, the present disclosure provides a method of extending flower vase life in a plant or part thereof. As a method for prolonging the vase life of flowers, 1) a cell (for example, a plant cell), a plant tissue, or a plant or a part thereof containing or modified by the factor described in <Inhibitor of LhHFS> 2) subjecting the cell, plant tissue, or plant or part thereof to conditions for flowering; and 3) after flowering in the cell, plant tissue, or plant or part thereof, and subjecting to shelf-life conditions and periods of time. The period may be a period longer than the period in which the plant body or part thereof expires under the long-lived conditions without modification by the factor. For example, when "Tiara", which is a cultivar of the Oriental hybrid line of lilies, is used, the shelf life period without modification by the factor is about 5 days. The period during which the plant body or its part lasts for a long time can be 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, and the like. In a preferred embodiment, the plant body or part thereof is a whole flower, or a combination of flower-constituting organs such as petals, corolla, and perianth, or a combination of flower-containing organs (for example, cut flowers or potted flowers). could be.

(screening)
In another aspect, the present disclosure provides methods of screening for agents that retard flower senescence using the nucleic acid molecules (LhHFS or variants thereof) of the present disclosure or the proteins they encode. As a screening method, a method known in the art can be applied. This method includes the steps of: (i) contacting LhHFS or a substance, cell, plant, etc. containing it with a test substance; A step of detecting expression is included. In step (i), the cells and the like used are those of Liliaceae plants. Agents that delay flower senescence can be selected from substances or factors that suppress the expression or activity of LhHFS in test substances. Such substances or factors can also be introduced into plants or plant cells to see if they can delay flower senescence.

(general technology)
Molecular biological techniques, biochemical techniques, and microbiological techniques used herein are well known and commonly used in the art, and are described, for example, in Sambrook J. et al. 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; 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). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; J. et al. (1999). PCR Applications: Protocols for Functional Genomics, Academic Press, Supplementary Volume Experimental Medicine "Gene Transfer & Expression Analysis Experimental Method" Yodosha, 1997 "Plant PCR Experimental Protocol" Shujunsha (1997), Model Plant Experimental Protocol Shujunsha (1996), etc., and the relevant portions (which may be all of them) are incorporated herein by reference.

DNA synthesis techniques and nucleic acid chemistry for making artificially synthesized genes are described, for example, in Gait, M.; J. (1985). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Gait, M.; J. (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; (1991). Oligonucleotides and Analogues: A Practical Approach, IRL Press; L. et al. (1992). The Biochemistry of the Nucleic Acids, Chapman &Hall; et al. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; M. et al. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; T. (I996). Bioconjugate Techniques, Academic Press, etc., the relevant portions of which are incorporated herein by reference.

All references, such as scientific articles, patents, patent applications, etc., cited herein are hereby incorporated by reference in their entireties to the same extent as if each were specifically set forth.

The present disclosure has been described above showing preferred embodiments for ease of understanding. The present disclosure will now be described with reference to the examples, which are provided for illustrative purposes only and not for the purpose of limiting the invention. Accordingly, the scope of the present invention is not limited to the embodiments or examples specifically described herein, but only by the claims.

When necessary, plants and the like used in the following examples were handled in compliance with the standards stipulated by the Japanese government, the National Agriculture and Food Research Organization, and the Niigata Prefectural Agricultural Research Institute. Further, the reagents specifically used the products described in the examples, but equivalent products from other manufacturers (SIGMA-ALDRICH, Wako Pure Chemical, Nacalai Tesque, Kanto Kagaku, etc.) can be substituted.

(Plant material and growing conditions)
In the following examples, the plant used was "Tiara", which is a cultivar of the Oriental hybrid line of lilies. "Tiara" was obtained from a domestic bulb sales company such as Yamaki Farm Co., Ltd. or Nakamura Farm Co., Ltd. The bulbs were planted in horticultural medium (conditioned peat moss, BVB) and grown in a greenhouse. The shelf life of flowers was evaluated by cutting flowers immediately after flowering from the plant body, arranging them in distilled water, and keeping them indoors (temperature 20°C, PPFD 10 μmol·m ⁻² ·s ⁻¹ , day length 12 hours). The end of shelf life was determined when at least half of the perianths turned brown, or when at least half of the perianths with browning were observed.

(Example 1: Isolation of LhHFS)
The present inventors have conducted intensive research on the lily genome, and as a result, have found novel genes that may be associated with flower senescence. This gene was isolated, 3'-RACE and 5'-RACE were performed based on the isolated partial sequence, and the base sequence including the full length ORF was determined. RNeasy plant mini kit (Qiagen) was used for RNA extraction, and 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 a full-length ORF was prepared using a full-length cloning primer set (LhHFS-Full-F9: 5′-TTCCAACTTTCAACCTTCAACTC-3′ (SEQ ID NO: 6), LhHFS-Full-R1188: 5′-TTGTCGACATAAATTGATCCAAG-3′ (SEQ ID NO: 7 )) was obtained by RT-PCR. The obtained cDNA clone was cloned into pGEM-T easy vector (Promega), the nucleotide sequence was determined, and the clone was named LhHFS.

(Example 2: Production of LhHFS expression-suppressed transformant)
A transformant in which LhHFS expression was suppressed was produced using the RNAi method. A 291 base pair LhHFS cDNA fragment (574-864 bases) was cloned into pDONR201 (Invitrogen) and then into the plant transformation vector pH7GWIWG(II) carrying the hygromycin resistance gene (HPT) (Karimi M, et al. 2002). (GATEWAY vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci 7: 193-195.) using LR ClonaseII Enzyme mix (Invitrogen). The resulting transformation vector pH7-HFS contains the LhHFS cDNA fragment in the antisense and sense directions downstream of the cauliflower mosaic virus 35S promoter and the 35S terminator downstream thereof. pH7-HFS was introduced into filament-derived cultured cells of lily cultivar "Tiara" using Agrobacterium EHA101 strain. Specifically, filaments of the lily cultivar “Tiara” were cultured in a callus induction medium (MS medium supplemented with 4 g L ⁻¹ gellan gum and 2 mg L ⁻¹ picloram and without MgSO ₄ .7H ₂ O) to induce callus. induced. The callus was subcultured to an adventitious bud regeneration medium (medium added with 0.1 mg L ^-1 picloram from the above composition and further added with 1 mg L ^-1 thidiazuron) that simultaneously induces proliferation and regeneration for 14 days. cultured. After inoculation of the Agrobacterium EHA101 strain carrying the transformation vector pH7-HFS into callus, the callus was selected on an adventitious bud regeneration medium supplemented with 15 mg L ^-1 of hygromycin. The regenerated adventitious buds were transplanted to bulb hypertrophy medium (MS medium supplemented with 4 g L ^-1 gellan gum and hormones removed) every 3 weeks to promote bulb formation. It was transplanted to the culture soil and acclimatized. The acclimatized bulbs were grown to flower. Two lines (LNR18-2 and LNR18-15) were obtained for the LhHFS expression-suppressing line (LNR18). The shelf life of flowered lilies was evaluated by the method described above. The presence or absence of the introduced gene was confirmed by PCR amplification of the HPT gene.

(Results and Discussion)
Table 1 shows the shelf life of the wild-type and LhHFS expression-suppressed line (LNR18). In Table 1, while the wild type had a shelf life of 5 days, the two LhHFS expression-suppressed strains had a shelf life of 8 days. Photographs of wild-type and LhHFS expression-suppressed lines from flowering to senescence are shown in FIG. In FIG. 1, in the wild type, part of three or more perianths turned brown 5 days after flowering, whereas in the LhHFS expression-suppressed line, browning of perianths was observed even 7 days after flowering. However, on the 8th day, part of three or more perianths turned brown. These results indicate that suppression of LhHFS delays flower cover senescence and prolongs the vase life (viewing period) of flowers by about 1.5 to 2 times compared to the wild type.

(Example 3: Analysis of gene expression by quantitative real-time RT-PCR)
RNA was extracted from the inner perianths of the flowers of the wild-type and LhHFS expression-suppressed lines on the 4th day after flowering 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 Thermal Cycler Real Time System (Model TP600, Takara bio). The LhHFS amplification primers used for PCR were LhHFS-907Fq (5′-AAGCAGGGATCAGACAGAG-3′ (SEQ ID NO:8)) and LhHFS-1013Rq (5′-TTGTTGCCCACTCCGAGTAT-3′ (SEQ ID NO:9)). The expression level was normalized using LhEF1alpha as an internal control and calculated as a relative expression level. Primers for LhEF1alpha amplification are LhEF1a-QF2-729 (5′-TGAGGTGTTCTTCTATTCATGTCC-3′ (SEQ ID NO: 10)) and LhEF1a-QR2-867 (5′-CCAAATAGAGCAGCAAAGCA-3′ (SEQ ID NO: 11)).

(Results and Discussion)
Table 2 below and FIG. 2 show the relative expression levels of the LhHFS gene in the perianth 4 days after flowering of the wild-type and LhHFS expression-suppressed lines. All LhHFS expression-suppressed lines (LNR18-2, LNR18-15) show that LhHFS gene expression was greatly reduced compared to the wild type. This result indicated that the LhHFS expression-suppressed line created in this Example suppressed the expression of LhHFS more than twice as much as the wild type.

(Example 4: Production of transformant in which mutation is introduced into LhHFS by genome editing)
Genome editing using CRISPR/Cas9 is used to introduce mutations into LhHFS to produce transformants in which the expression of LhHFS is suppressed or deleted. Plant transformation vector pIG121-Hm (Ohta et al., Plant Cell Physiol. 31: 805-813 (1990)) having a hygromycin resistance gene (HPT), gene expression cassette expressing guide RNA and Cas9 gene insert. The resulting transformation vector pIG-HFS 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 described in Table 3 downstream of the rice U6 promoter. It contains an RNA sequence, downstream of which is a gene expression cassette containing a guide RNA scaffold sequence and a poly-T sequence. pIG-HFS is introduced into filament-derived cultured cells of lily cultivar "Tiara" using Agrobacterium strain EHA101. Specifically, filaments of the lily cultivar “Tiara” were cultured in a callus induction medium (MS medium supplemented with 4 g L ⁻¹ gellan gum and 2 mg L ⁻¹ picloram and without MgSO ₄ .7H ₂ O) to induce callus. to induce The callus was subcultured to an adventitious bud regeneration medium (medium added with 0.1 mg L ^-1 picloram from the above composition and further added with 1 mg L ^-1 thidiazuron) that simultaneously induces proliferation and regeneration for 14 days. culture. After inoculation of the Agrobacterium EHA101 strain carrying the transformation vector pIG-HFS into callus, the callus is selected on adventitious bud regeneration medium supplemented with 15 mg L ^-1 of hygromycin. The regenerated adventitious buds were transplanted to bulb hypertrophy medium (MS medium supplemented with 4 g L ^-1 gellan gum and hormones removed) every 3 weeks to promote bulb formation. Transplant into soil and acclimate. Grow and flower acclimatized bulbs. The presence or absence of the introduced gene is confirmed by PCR amplification of the HPT gene using DNA extracted from adventitious buds. The presence or absence of mutation introduction is confirmed by amplifying the region containing the target sequence by PCR using DNA extracted from adventitious buds and sequencing it.

Alternatively, the introduction of mutations by genome editing, without incorporating the Cas9 gene and guide RNA expression cassette into the plant genome, a complex consisting of guide RNA and Cas9 protein by particle bombardment can also be achieved by direct injection into plant cells. . For example, Zhen Liang, Kunling Chen, Yi Zhang, Jinxing Liu1, Kangquan Yin, Jin-Long Qiu & Caixia Gao. Genome editing of bread heat using biological delivery of CRISPR/Cas9 in vitro transcripts or ribonucleoprotein. Illustrative are the techniques described in Nature Protocol 13:413-430. Specifically, guide RNA and Cas9 protein complexes are introduced into the lily shoot apical meristem using particle bombardment. The shoot apical meristem after 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 confirmed to be mutagenized were transplanted to bulb hypertrophy medium (MS medium supplemented with 4 g L ^-1 gellan gum and hormones removed) every 3 weeks to promote bulb formation, and identified after about 12 months. Transplant to horticultural soil in a netted room and acclimatize. Grow and flower acclimatized bulbs.

(Example 5: Verification of LhHFS expression in flower cover of different lily varieties)
In this example, lily cultivars other than "Tiara" are used to verify the expression of LhHFS in perianths. LhHFS amplification primers are designed based on the LhHFS gene sequence of "Tiara", and RNA extracted from the perianth of the target lily is used to perform RT-PCR to isolate the LhHFS of the target lily. After determining the base sequence, the expression of LhHFS is analyzed by real-time PCR. Specifically, RNA is extracted from perianth using RNeasy plant mini kit (Qiagen), and cDNA is synthesized using PrimeScript RT Master Mix (Takara bio). PrimeSTAR HS DNA polymerase (Takara bio) is used for PCR. The obtained cDNA clone is cloned into pGEM-T easy vector (Promega) and the nucleotide sequence is determined. For analysis of expression, RNA is extracted from the inner perianth of flowers on the 4th day after flowering using RNeasy plant mini kit (Qiagen), and cDNA is synthesized using PrimeScript RT Master Mix (Takara bio). PCR is then 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 LhHFS is normalized using LhEF1alpha as an internal control, and calculated as a relative expression level.

(Example 6: Screening of inhibitors)
Proteins that interact with the LhHFS protein are isolated and a system is developed that allows in vitro (96-well plate) monitoring of the interaction with the LhHFS protein. Specifically, a reporter such as a fluorescent protein is used to measure the degree of inhibition of the interaction by the compound. For implementation, it is possible to refer to methods for searching for molecular-targeted therapeutic drugs in cancer research. Using the developed monitoring system, chemical screening is performed using a compound library to find drugs that inhibit the activity of the LhHFS protein.

While the present disclosure has been illustrated using the preferred embodiments thereof, it is understood that the invention is to be construed in scope only by the appended claims. It is understood that the patents, patent applications and publications cited herein are hereby incorporated by reference for their contents as if the contents themselves were specifically set forth herein. understood.

The present disclosure may be used in the field of floriculture. Flowers with extended vase life (for example, lilies) have commercial value. In other Liliaceae plants such as tulips, the suppression of expression of LhHFS orthologs can also delay flower senescence.

SEQ ID NO: 1: Nucleic acid sequence corresponding to the open reading region of LhHFS.
SEQ ID NO: 2: Amino acid sequence of protein of LhHFS.
SEQ ID NO: 3: Full length nucleic acid sequence of LhHFS including untranslated region.
SEQ ID NO: 4: partial length nucleic acid sequence of the tulip LhHFS homologous gene.
SEQ ID NO: 5: Partial length amino acid sequence of homologous gene of LhHFS of tulip.
SEQ ID NO: 6: Sequence of the primer used for cloning the LhHFS gene.
SEQ ID NO: 7: Sequence of the primer used for cloning the LhHFS gene.
SEQ ID NO: 8: Sequence of primers used for RT-PCR of LhHFS gene.
SEQ ID NO: 9: Sequence of primers used for RT-PCR of LhHFS gene.
SEQ ID NO: 10: Sequence of primers used for RT-PCR of LhEF1alpha gene.
SEQ ID NO: 11: Sequence of primers used for RT-PCR of LhEF1alpha gene.
SEQ ID NOs: 12-95: Sequences of guide RNAs used for genome editing.

Claims (58)

(A) 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; or (B) a nucleic acid molecule comprising a sequence variant,
The (B) sequence variant is
(B-1) a sequence variant having one or more substitutions, additions and/or deletions in the nucleic acid sequence of (A);
(B-2) a sequence variant having at least about 90% 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),
A nucleic acid molecule, wherein the protein encoded by the sequence variant has the biological function of the protein encoded by (A). 2. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule comprises the nucleic acid sequence set forth in SEQ ID NO:1. 2. The nucleic acid molecule according to claim 1, wherein said nucleic acid molecule consists of the nucleic acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3. (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 mutations include substitutions, additions and/or deletions, and the polypeptide of (a) a polypeptide having a biological activity analogous to that of
(c) a polypeptide encoded by a splice 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) having at least about 90 identity to the polypeptide of any one of (a)-(d) % and has a biological activity similar to that of the polypeptide of (a). An agent that suppresses the expression of the nucleic acid sequence according to any one of claims 1 to 3 and/or the amount or function of said nucleic acid sequence or the protein encoded by said nucleic acid sequence or said polypeptide according to claim 4. Said agents include proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids including RNA or DNA, polysaccharides, oligosaccharides, lipids, hormones, ligands, signaling agents, synthetic chemicals, radiation , or a combination thereof. 7. The agent according to claim 5 or 6, which is selected from the group consisting of antisense molecules, RNAi agents, genome editing agents, or mutagens. A factor that represses LhHFS or its homologous gene. The homologous gene is
(i)
A sequence variant of 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, wherein the sequence variant comprises
(B-1) a sequence variant having one or more substitutions, additions and/or deletions in the 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;
(B-2) a sequence variant having at least about 70% sequence identity with the 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;
(B-3) a sequence variant that hybridizes under stringent conditions to 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;
(B-4) a sequence variant that is an allelic variant of the nucleic acid sequence shown in SEQ ID NO: 1 or the nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 2, or (B-5) 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, or a sequence variant that is a fragment of (B-1) to (B-4),
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 the amino acid sequence shown in SEQ ID NO: 2, or (ii)
(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 mutations include substitutions, additions and/or deletions, and the polypeptide of (a) a polypeptide having a biological activity analogous to that of
(c) a polypeptide encoded by a splice 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) having at least about 70 identity to the polypeptide of any one of (a)-(d) % and has a biological activity similar to that of the polypeptide of (a), comprising a polypeptide. The agent may be a protein, polypeptide, oligopeptide, peptide, polynucleotide, oligonucleotide, nucleotide, nucleic acid including RNA or DNA, polysaccharide, oligosaccharide, lipid, hormone, ligand, signaling agent, synthetic chemical, or 10. Agents according to claim 9, which are complex molecules thereof. The factor of any one of claims 8-10, wherein the factor is in nucleic acid form. The agent according to any one of claims 8 to 11, which is selected from the group consisting of antisense molecules, RNAi agents, genome editing agents, or mutagenesis agents. 9. The agent of claim 8, wherein the agent is an LhHFS RNAi agent in the form of a double-stranded nucleic acid. Said factor is in double-stranded form, one strand of which is (I)
(A) a nucleic acid sequence comprising at least about 100 bases of the 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; or (B) a sequence variant, the other strand comprising:
(II) comprising complementary or annealing sequences of (I);
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),
9. The factor according to claim 8, wherein the protein encoded by said sequence variant has the biological function of the protein encoded by (A). 15. The factor according to claim 14, wherein (I) does not contain the nucleic acid sequence of bases 22-483 of SEQ ID NO:1. 16. The factor according to claim 14 or 15, wherein (I) comprises the nucleic acid sequence of bases 574-864 of SEQ ID NO:1. The agent according to any one of claims 14 to 16, which is a vector containing the RNAi agent of LhHFS or its homologous gene. 18. The factor of claim 17, wherein said vector comprises a promoter and terminator. 9. The factor according to claim 8, wherein the factor is a genome-editing factor that suppresses the expression and/or function of LhHFS. 20. The factor according to claim 19, wherein the factor for genome editing targets the nucleic acid sequence of the following sequence in SEQ ID NO:1.

20. The factor according to claim 19, wherein the factor for genome editing targets genome editing of the nucleic acid sequence of bases 1-483 in SEQ ID NO:1. A plant cell comprising or modified by a factor according to any one of claims 8-21. A plant tissue comprising or modified by a factor according to any one of claims 8 to 21 or a plant tissue comprising cells according to claim 22. A plant organ comprising or modified by a factor according to any one of claims 8 to 21 or a plant organ comprising a cell according to claim 22 or a plant tissue according to claim 23. A plant body or part thereof containing or modified by the factor according to any one of claims 8 to 21, or a cell according to claim 22, a plant tissue according to claim 23 or a claim Item 25. A plant body or part thereof comprising the plant organ according to Item 24. 26. The plant or part thereof according to claim 25, wherein the plant belongs to the Liliaceae family. 27. The plant or part thereof according to claim 26, wherein the plant belongs to the genus Lilium. 4. The plant or part thereof is a bulb, seed, flower, stem, leaf, pollen, spore, tree, cell, tissue, tissue culture or germ, or a combination of all or part thereof. 28. The plant or part thereof according to any one of 25 to 27. 29. A plant or part thereof according to any one of claims 25 to 28, wherein said plant or part thereof comprises a flower or part thereof. A plant cell in which at least part of the LhHFS or its homologous gene possessed by the plant has been suppressed. 31. A plant tissue in which at least part of LhHFS or a homologous gene thereof possessed by the plant is suppressed, or a plant tissue comprising the cell of claim 30. A plant organ in which at least part of the LhHFS possessed by the plant or a homologous gene thereof is suppressed, or a plant organ comprising the cell according to claim 30 or the plant tissue according to claim 31. A plant in which at least a part of LhHFS or a homologous gene thereof is suppressed in the plant, or a part thereof, or the cell according to claim 30, the plant tissue according to claim 31, or the plant organ according to claim 32. A plant body or part thereof containing The cell, tissue, organ or plant body or part thereof according to claims 30 to 33, wherein the expression and/or function of at least part of said LhHFS or its homologous gene is suppressed. The suppression is below the standard of reducing expression of the vascular processing enzyme gene (VPE gene) or the cysteine protease gene (SAG12 gene) by about 50% as measured by RT-PCR method, claim 34 Cells, tissues, organs or plants or parts thereof according to . 35. The cell, tissue, organ or according to claim 34, wherein the inhibition is below the standard of reducing expression of the vascular processing enzyme gene (VPE gene) by about 50% as measured by RT-PCR. A plant or part thereof. Suppression of said LhHFS results in decreased gene expression levels, decreased gene copy number, decreased gene amplification, decreased RNA activity levels, decreased mRNA abundance, decreased mRNA synthesis rates, decreased mRNA stability, protein activity levels. reduction of protein synthesis, reduction of protein abundance, reduction of protein stability, reduction of protein enzymatic activity, or a combination thereof. or a cell, tissue, organ or plant body or a part thereof according to any one of items. The structural gene of at least part of said LhHFS or its homologous gene has mutations compared to the natural one, and/or the gene expression regulator has mutations compared to the natural one, and/or 38. A cell, tissue, organ or plant or part thereof according to any one of claims 34 to 37, comprising a non-naturally occurring factor that represses at least part of LhHFS or a homologous gene thereof. 39. The cell, tissue, organ or plant or part thereof according to claim 38, wherein said mutation or factor is provided by RNA interference or genome editing of the LhHFS gene. A plant cell, plant tissue, plant organ, plant body, or a part thereof in which the gene of LhHFS or its homologous gene is partially or wholly mutated or deleted. The plant cell, plant tissue, plant organ or plant body or part thereof according to any one of claims 30 to 40, wherein the plant is a plant of the Liliaceae family. 42. The plant cell, plant tissue, plant organ or plant body or part thereof according to claim 41, wherein said plant is a plant of the genus Lilium. 4. The plant or part thereof is a bulb, seed, flower, stem, leaf, pollen, spore, tree, cell, tissue, tissue culture or germ, or a combination of all or part thereof. The plant cell, plant tissue, plant organ or plant body or part thereof according to any one of 33 to 42. 44. The plant cell, plant tissue, plant organ or plant or part thereof according to any one of claims 33 to 43, wherein said plant or part thereof comprises a flower or part thereof. A method for producing a plant or part thereof in which flower senescence is suppressed, wherein at least part of LhHFS or a homologous gene thereof possessed by the plant or part thereof is suppressed in the plant or part thereof. a method comprising providing a state of 46. The method of claim 45, wherein said suppression comprises suppressing the expression and/or function of at least a portion of said LhHFS or its homologous gene relative to the state prior to said providing step. 47. The method according to claim 45 or 46, further comprising measuring or otherwise determining that the expression and/or function of at least a part of said LhHFS or its homologous gene is suppressed compared to the state before said providing step. described method. The measuring or otherwise determining may include the abundance of mRNA of at least a portion of said LhHFS or a homologous gene thereof, the abundance of a protein of at least a portion of said LhHFS or a homologous gene thereof, the LhHFS or a homologous gene thereof. 48. The method of claim 47, comprising measuring, or calculating from the measurements, the biological functions of at least some of the proteins of, combinations thereof. Suppression of said LhHFS or its homologous gene results in decreased gene expression level, decreased gene copy number, decreased gene amplification, decreased RNA activity level, decreased mRNA abundance, decreased mRNA synthesis rate, decreased mRNA stability. , decreased protein activity levels, decreased protein synthesis, decreased protein abundance, decreased protein stability, decreased protein enzymatic activity, or combinations thereof. The method described in . 50. The method of claim 49, wherein silencing the LhHFS or its homologous gene is accomplished by performing RNA interference or genome editing of the LhHFS gene. The method according to any one of claims 45 to 50, wherein said plant is a plant of the Liliaceae family. 52. The method of claim 51, wherein the plant is of the Lilium genus. 4. The plant or part thereof is a bulb, seed, flower, stem, leaf, pollen, spore, tree, cell, tissue, tissue culture or germ, or a combination of all or part thereof. 53. The method of any one of 45-52. 54. A method according to any one of claims 45 to 53, wherein said plant or part thereof comprises a flower or part thereof. The method includes:
A step of modifying LhHFS or a homologous gene thereof in a plant cell to introduce a mutation in part or the whole thereof, or to delete a part or the whole thereof A plant body by growing plant cells in which LhHFS or a homologous gene thereof is modified 55. A method according to any one of claims 45 to 54, comprising the step of obtaining or part thereof. 56. The method of claim 55, wherein said modification is by antisense, RNA interference, mutagens, genome editing. A method for extending flower vase life in a plant or part thereof, comprising:
1) A plant body or part thereof containing the factor according to any one of claims 8 to 21 or modified by the factor, the cell according to claim 22, or the plant tissue according to claim 23 or providing a plant body or part thereof comprising the plant organ according to claim 24;
2) subjecting the plant or part thereof to flowering conditions;
3) subjecting the plant body or part thereof to a condition and a period of keeping for a long time after flowering; 58. The method according to claim 57, wherein said period of time is longer than the period of time in which said plant or part thereof will expire in the absence of modification by said factor under said perishable conditions.

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