JP6044952B2 - Petal aging delay method - Google Patents

Description

  The present invention provides a NAC transcription factor family gene (InNALl) that regulates petal senescence and uses thereof. For example, by incorporating the InNAL1 gene or gene fragment into the plant genome, it becomes possible to delay petal senescence.

  For flowers whose flower senescence is controlled by the plant hormone ethylene, extend the flower's quality retention period by genetic recombination and treatment with inhibitors targeting the ethylene biosynthetic system or receptor / information transmission system. Has been tried. On the other hand, flowers with little involvement of ethylene in flower aging (ethylene-independent flowers) include major items such as lilies, but there is no effective quality maintenance technology.

  It is known that leaf senescence is delayed in function-deficient mutants of the Arabidopsis NAC transcription factor family genes AtNAP and OREl (Non-patent Documents 1 and 2, Non-patent Document 4). Expression of ORE1 is induced during senescence of tobacco petals (Non-patent Document 3).

  Patent Document 1 uses the Hd1 gene or a variant thereof to stabilize a short-day plant having a flowering time delayed under short-day conditions and having a trait substantially equivalent to that of a wild strain in characters other than the flowering time. Disclose the technology to obtain.

  U.S. Patent No. 6,057,028 provides a non-chemical method for controlling plant and plant flower senescence that can be easily and efficiently utilized across a wide range of plant species, particularly expressing ACC metabolic enzymes such as ACC deaminase enzyme. Thus, a technique for performing this control is disclosed.

  Patent Document 3 discloses a flowering inhibitor containing S (+)-abscisic acid as an active ingredient, and suppresses flower bud formation (flowering itself) or flowering time (period until a flower blooms). A technique for delaying the process is disclosed.

  Patent Document 4 discloses that leaf senescence is delayed in the function-deficient mutants of the NAC transcription factor family genes AtNAP and ORE1.

  Patent Document 5 describes a technique for delaying ripening of a fruit, and discloses a technique related to a NOR gene.

  Patent document 6 discloses the technique regarding the SAG12 promoter for changing an aging characteristic.

JP 2008-54512 A JP-A-9-238689 Japanese Patent No. 2855801 International Application 2007/112430 Pamphlet International Application 2009/143155 Brochure Special table hei 11-501819

Guo and Gun (2006) Plant Journal 46: 601-612 Kim et al. (2009) Science 323: 1053-1057. Balazadeh et al. (2010) Plant Journal 62: 250-264

In general, there is a need for the development of effective quality maintenance techniques, including flowers that are independent of ethylene. Therefore, as a result of intensive studies, the present invention has identified that a gene that controls the senescence of ethylene-independent flowering has been identified, and has developed a quality maintenance technique.

Accordingly, the present invention provides the following.
(1) A factor that suppresses InNAL1.
(2) The factor according to item 1, wherein the factor is in the form of a nucleic acid.
(3) The factor according to item 1 or 2, wherein the factor is a factor that causes RNA interference of InNAL1 in the form of a double-stranded nucleic acid.
(4) The factor is in double-stranded form, one strand of which is (I)
(A) the sequence shown in SEQ ID NO: 1 (InNAL1 itself) or a sequence encoding SEQ ID NO: 2; or (B)
(B-1) a sequence variant having one or several substitutions, additions or deletions in (A),
(B-2) a sequence variant having at least about 70% sequence identity to the sequence of (A), or (B-3) hybridizing under stringent conditions to the sequence of (A) A sequence variant comprising:
The sequence variant comprises a sequence having the biological function of (A), and the other strand is
(II) comprising a complementary sequence of (I),
The factor of any one of items 1-3.
(5) The factor according to item 4, wherein (I) is a sequence of 370 bases at positions 690-1059 in SEQ ID NO: 1.
(6) The factor according to any one of items 1 to 5, which is a vector containing a nucleic acid that causes RNA interference of InNAL1.
(7) The factor according to item 6, wherein the vector comprises a promoter and a terminator.
(8) The factor according to item 6 or 7, which is pH7-InNAL (SEQ ID NO: 3).
(9) A plant cell comprising the factor according to any one of items 1 to 8.
(10) The cell according to item 9, wherein the plant cell is a cell of an ethylene-independent flowering plant.
(11) The cell according to item 9 or 10, wherein the plant cell is a morning glory cell.
(12) A plant comprising the factor according to any one of items 1 to 8.
(13) The plant according to item 12, wherein the plant is an ethylene-independent flowering plant.
(14) The plant according to item 12 or 13, wherein the plant is a morning glory.
(15) Plant cells in which InNAL1 is suppressed more than in the natural state.
(16) The cell according to item 15, wherein the plant cell is a cell of an ethylene-independent flowering plant.
(17) The cell according to item 15 or 16, wherein the plant cell is a morning glory cell.
(18) A plant in which InNAL1 is suppressed more than its natural state.
(19) The plant according to item 18, wherein the plant is an ethylene-independent flowering plant.
(20) The plant according to item 18 or 19, wherein the plant is morning glory.
(21) (A) the sequence shown in SEQ ID NO: 1 or a sequence encoding SEQ ID NO: 2; or (B)
(B-1) a sequence variant having one or several substitutions, additions or deletions in (A),
(B-2) a sequence variant having at least about 70% sequence identity to the sequence of (A), or (B-3) hybridizing under stringent conditions to the sequence of (A) A sequence variant comprising:
An isolated nucleic acid molecule, wherein the sequence variant comprises a sequence having the biological function of (A).
(22) The nucleic acid molecule according to item 21, wherein the nucleic acid molecule comprises the sequence shown in SEQ ID NO: 1.
(23) The nucleic acid molecule according to item 21 or 22, wherein the nucleic acid molecule consists of the sequence shown in SEQ ID NO: 1.
(24) (a) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2 or a fragment thereof;
(B) a polypeptide having one mutation in which one or more amino acids are selected from the group consisting of substitution, addition and deletion in the amino acid sequence set forth in SEQ ID NO: 2 and having biological activity;
(C) a polypeptide encoded by a splice variant or allelic variant of the base sequence set forth in SEQ ID NO: 1;
(D) a polypeptide that is a species homologue of the amino acid sequence set forth in SEQ ID NO: 2; or (e) an amino acid sequence that is at least 70% identical to any one polypeptide of (a)-(d) And an isolated protein comprising a polypeptide having biological activity.
(25) A method for screening a drug that delays petal senescence using the nucleic acid molecule or a part thereof according to any one of items 21 to 23, or the protein encoded by the nucleic acid molecule or the protein according to item 24.

  Accordingly, these and other advantages of the invention will be apparent upon reading the following detailed description.

  By suppressing the expression of the NAC transcription factor family gene (InNAL1) according to the present invention, it is possible to delay the petal senescence and extend the viewing period for flowers in general including flowers independent of ethylene such as morning glory. Can do.

FIG. 1 shows the relative expression level of InNAL1 in the InNAL1r strain. In each of the wild type, InNAL1r-1, InNAL1r-2, and InNAL1r-3 plants, the expression level of InNAL1 in the petals at 8 hours after flowering was analyzed. As a result, it was confirmed that InNAL1 expression was suppressed in the transformed line compared to the wild type. In each plant of InNAL1r-1, InNAL1r-2, and InNAL1r-3, the expression of InNAL1 in petals 8 hours after flowering was reduced to 20%, 26%, and 22%, respectively, compared to the wild type. FIG. 2 shows the senescence of wild type and InNAL1r-1 flowers. In FIG. 2, the wild type is photographed at the upper stage, and the flowers of the InNAL1r-1 plant body are photographed every 4 hours from the flowering, respectively. In InALL1r-1 plants, senescence of petals is remarkably delayed (flower life is extended). The same applies to InNAL1r-2 and InNAL1r-3 plants. FIG. 3 shows a state 24 hours after the flowering of InNAL1r-1. Arrows indicate flowers for 24 hours (2nd day) after flowering, and triangles indicate flowers for 0 hour (1st day) after flowering. In the wild type, the petals usually wither in about 14 hours after flowering. On the other hand, in the InNAL1r-1 plant, the petals are blooming without wilt for 24 hours or more, so that the flower bloomed the day before and the flower bloomed on the same day are observed as shown in the figure. The same applies to InNAL1r-2 and InNAL1r-3 plants. The petal color changes from blue to red as time passes after flowering. FIG. 4 shows the wild-type and InNAL1r plant flowers 24 hours after flowering. From the left, wild type, InNAL1r-1, InNAL1r-2, and InNAL1r-3 are shown. In the wild type, the petals are completely deflated, whereas in the InNAL1r line, none of the petals of InNAL1r-1, InNAL1r-2, and InNAL1r-3 have yet deflated.

  The present invention will be described below. Throughout this specification, it should be understood that expression in the singular also includes the concept of the plural unless specifically stated otherwise. Thus, it should be understood that singular articles (eg, “a”, “an”, “the”, etc. in the case of English) also include the plural concept unless otherwise stated. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally 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 invention belongs. In case of conflict, the present specification, including definitions, will control.

(Definition)
In the present specification, “InNAL1” refers to a gene found in the present invention, and is intended to encompass both nucleic acid molecules and proteins. Understand what only means or both. As used herein, “InNAL1” is not only a protein having an amino acid sequence described in a specific sequence number or accession number (or a nucleic acid encoding it), but also functionally active. A derivative thereof, or a functionally active fragment thereof, or a homologue thereof, or a variant encoded by a nucleic acid that hybridizes under high or low stringency conditions to a nucleic acid encoding this protein. , Understood to mean. “Mutants” or “variants” include, but are not intended to be otherwise limited, molecules that comprise a region that is substantially homologous to the original protein, and such molecules may comprise various embodiments. At least 30%, 40%, 50%, 60%, 70%, 80 when compared to aligned sequences over the same size amino acid sequence or by alignment with computer homology programs known in the art %, 90%, 95% or 99% identical or nucleic acid encoding such a molecule can be obtained under stringent, moderately stringent or non-stringent conditions. It can hybridize to a protein coding sequence. This is the result of modification of a naturally occurring protein by amino acid substitutions, deletions and additions, respectively, and its derivatives still indicate the biological function of the naturally occurring protein, although not necessarily to the same degree. Means protein. For example, the biological function of such proteins can be examined by suitable and available in vitro assays described herein or known in the art.

  In the present invention, InNAL1 is mainly discussed as morning glory, but besides that, petunia (GenBank number: AAM34777), apple (ACI13682), grape (XP_002283811), poplar (XP_002323870), soybean (XP_003549684), etc. other than morning glory Since many plants are known to express proteins, it is understood that these plants, particularly non-ethylene dependent plants, are also within the scope of the present invention.

  As used herein, “functionally active” or “having biological function” as used herein refers to biological activity according to the embodiment to which the polypeptide of the invention, ie, a fragment or derivative, relates. Refers to a polypeptide, i.e., fragment or derivative, having a structural, regulatory, or biochemical function of a protein.

A typical nucleotide sequence of InNAL1 is:
(A) a polynucleotide having the base sequence set forth in SEQ ID NO: 1 or a fragment sequence thereof;
(B) a polynucleotide encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 or a fragment thereof;
(C) a variant polypeptide or fragment thereof in which one or more amino acids have one mutation selected from the group consisting of substitution, addition and deletion in the amino acid sequence set forth in SEQ ID NO: 2, A polynucleotide that encodes a variant polypeptide having functional activity;
(D) a polynucleotide which is a splice variant or allelic variant of the base sequence set forth in SEQ ID NO: 1 or a fragment thereof;
(E) a polynucleotide encoding a species homologue of the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 or a fragment thereof;
(F) a polynucleotide that hybridizes to any one of the polynucleotides of (a) to (e) under a stringent condition and encodes a polypeptide having biological activity; or (g) (a) to It may be a polynucleotide that consists of a base sequence that is at least 70% identical to any one polynucleotide of (e) or its complementary sequence, and encodes a polypeptide having biological activity. Here, the biological activity typically means the activity of InNAL1.

Preferably, the nucleotide sequence of InNAL1 is
(A) the sequence shown in SEQ ID NO: 1 (InNAL1 itself) or a sequence encoding SEQ ID NO: 2; or (B)
(B-1) a sequence variant having one or several substitutions, additions or deletions in (A),
(B-2) a sequence variant having at least about 70% sequence identity to the sequence of (A), or (B-3) hybridizing under stringent conditions to the sequence of (A) A sequence variant comprising:
The sequence variant can include a sequence having the biological function of (A).

As the amino acid sequence of InNAL1,
(A) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2 or a fragment thereof;
(B) a polypeptide having one mutation in which one or more amino acids are selected from the group consisting of substitution, addition and deletion in the amino acid sequence set forth in SEQ ID NO: 2 and having biological activity;
(C) a polypeptide encoded by a splice variant or allelic variant of the base sequence set forth in SEQ ID NO: 1;
(D) a polypeptide that is a species homologue of the amino acid sequence set forth in SEQ ID NO: 2; or (e) an amino acid sequence that is at least 70% identical to any one polypeptide of (a)-(d) And a polypeptide having biological activity,
It can be. Here, the biological activity typically means the activity of InNAL1.

  In the context of the present invention, “an agent that inhibits InNAL1” or “InNAL1 inhibitor” acts directly or indirectly on InNAL1, and may temporarily or permanently inhibit, decrease or eliminate the function of InNAL1. A possible factor (eg, molecule or substance). The factor that suppresses InNAL1 may be an inhibitor (substance) of InNAL1, and examples thereof include antibodies or antigenic fragments thereof, antisense oligonucleotides, RNAi factors such as siRNA, low molecular weight molecules (LMW), May include binding peptides, aptamers, ribozymes and peptidomimetics such as, for example, binding proteins or binding peptides directed against InNAL1, in particular directed against the active site of InNAL1, and InNAL1 Also included are nucleic acids directed against the gene. Nucleic acid for InNAL1 refers to double- or single-stranded DNA or RNA, or a modification or derivative thereof that inhibits, for example, InNAL1 gene expression or InNAL1 activity, and includes, without limitation, antisense nucleic acids, aptamers, siRNAs (Small interfering RNA) and ribozymes. As used herein, “binding protein” or “binding peptide” for InNAL1 refers to the type of protein or peptide that binds to InNAL1 and is directed against InNAL1, polyclonal or monoclonal antibodies, antibody fragments and Including but not limited to protein backbone.

  As used herein, “protein”, “polypeptide”, “oligopeptide” and “peptide” are used interchangeably herein and refer to a polymer of amino acids of any length. This polymer may be linear, branched, or cyclic. The amino acid may be natural or non-natural and may be a modified amino acid. The term can also encompass one assembled into a complex of multiple polypeptide chains. The term also encompasses 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 (eg, including unnatural amino acids, etc.), peptide-like compounds (eg, peptoids) and other modifications known in the art. Is done.

  In the present specification, the “amino acid” may be natural or non-natural as long as it satisfies the object of the present invention.

  As used herein, “polynucleotide”, “oligonucleotide” and “nucleic acid” are used interchangeably herein and refer to a polymer of nucleotides of any length. The term also includes “oligonucleotide derivatives” or “polynucleotide derivatives”. “Oligonucleotide derivatives” or “polynucleotide derivatives” refer to oligonucleotides or polynucleotides that include derivatives of nucleotides or that have unusual linkages between nucleotides, and are used interchangeably. 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. Derivative converted to N3′-P5 ′ phosphoramidate bond, oligonucleotide derivative in which ribose and phosphodiester bond in oligonucleotide are converted to peptide nucleic acid bond, uracil in oligonucleotide is C− Oligonucleotide derivatives substituted with 5-propynyluracil, oligonucleotide derivatives wherein uracil in oligonucleotide is substituted with C-5 thiazole uracil, cytosine in oligonucleotide is C-5 propynylcytosine Substituted oligonucleotide derivatives, oligonucleotide derivatives in which the cytosine in the oligonucleotide is replaced with phenoxazine-modified cytosine, oligonucleotide derivatives in which the ribose in DNA is replaced with 2'-O-propylribose Examples thereof include oligonucleotide derivatives in which the ribose in the oligonucleotide is substituted with 2′-methoxyethoxyribose. Unless otherwise indicated, a particular nucleic acid sequence may also be conservatively modified (eg, degenerate codon substitutes) and complementary sequences, as well as those explicitly indicated. Is contemplated. Specifically, a degenerate codon substitute creates a sequence in which the third position of one or more selected (or all) codons is replaced with a mixed base and / or deoxyinosine residue. (Batzer et al., Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8: 91-). 98 (1994)). As used herein, “nucleic acid” is also used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide. In the present specification, the “nucleotide” may be natural or non-natural.

  As used herein, “gene” refers to a factor that defines a genetic trait. 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 affects its expression is called a regulatory gene. As used herein, “gene” may refer to “polynucleotide”, “oligonucleotide”, and “nucleic acid”.

  As used herein, “homology” of a gene refers to the degree of identity of two or more gene sequences to each other, and generally “having homology” means that the degree of identity or similarity is high. Say. Therefore, the higher the homology between two genes, the higher the sequence identity or similarity. Whether two genes have homology can be examined by direct sequence comparison or, in the case of nucleic acids, hybridization methods under stringent conditions. When directly comparing two gene sequences, the DNA sequence between the gene sequences is typically at least 50% identical, preferably at least 70% identical, more preferably at least 80%, 90% , 95%, 96%, 97%, 98% or 99% identical, the genes are homologous. Thus, as used herein, “homolog” or “homologous gene product” refers to a protein in another species, preferably a plant, that performs the same biological function as the protein component of the complex further described herein. means. Such homologues may also be referred to as “ortholog gene products”. Algorithms for detecting orthologous gene pairs from certain plants or other species use the entire genome of these organisms. First, paired best hits are recovered using the full Smith-Waterman parallel of the predicted protein. To further improve reliability, paired best hits are used to form clusters of these pairs. Such an analysis is provided, for example, in Nature, 2001, 409: 860-921. Based on the sequence homology of the genes encoding the proteins provided herein to other species of genes, conventional techniques can be applied to clone each gene and express the protein from such a gene. Or by isolating other species of proteins by isolating similar complexes according to the methods provided herein or according to other suitable methods well known in the art. It is also possible to isolate homologues of the proteins described in the specification.

  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 also be referred to by a generally recognized one letter code. In this specification, the comparison of similarity, identity and homology between amino acid sequences and base sequences is calculated using default parameters using BLAST, which is a sequence analysis tool. The identity search can be performed using, for example, NCBI BLAST 2.2.9 (issued 2004.12). In the present specification, the identity value usually refers to a value when the BLAST is used and aligned under default conditions. However, if a higher value is obtained by changing the parameter, the highest value is the identity value. When identity is evaluated in a plurality of areas, the highest value among them is set as the identity value. Similarity is a numerical value calculated for similar amino acids in addition to identity.

  As used herein, “polynucleotide hybridizing under stringent conditions” refers to well-known conditions commonly used in the art. Such a polynucleotide can be obtained by using a colony hybridization method, a plaque hybridization method, a Southern blot hybridization method or the like using a polynucleotide selected from among the polynucleotides of the present invention as a probe. Specifically, hybridization was performed at 65 ° C. in the presence of 0.7 to 1.0 M NaCl using a filter on which DNA derived from colonies or plaques was immobilized, and then 0.1 to 2 times the concentration. Means a polynucleotide that can be identified by washing the filter under conditions of 65 ° C. using a SSC (saline-sodium citrate) solution (composition of 1-fold concentration of SSC solution is 150 mM sodium chloride, 15 mM sodium citrate). To do. Hybridization was performed in Molecular Cloning 2nd ed. , Current Protocols in Molecular Biology, Supplements 1-38, DNA Cloning 1: Core Techniques, A PR Applical Approach, Second Edition, Oxford Universe. Here, the sequence containing only the A sequence or only the T sequence is preferably excluded from the sequences that hybridize under stringent conditions. Therefore, the polypeptide used in the present invention (for example, a protein encoded by InNAL1) is a nucleic acid that hybridizes under stringent conditions to a nucleic acid molecule encoding the polypeptide particularly described in the present invention. Also included are polypeptides encoded by the molecule. 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 in buffer containing dextran sulfate at 40 ° C. for 18-20 hours, in buffer consisting of 2 × SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS Washing at 55 ° C. for 1-5 hours and washing in buffer consisting of 2 × SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS for 1.5 hours at 60 ° C. Including.

The functional equivalent of InNAL1 used in the present invention can be found by searching a database or the like. As used herein, “search” refers to finding another nucleobase sequence having a specific function and / or property using a nucleobase sequence electronically or biologically or by other methods. Say. As an electronic search, 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. 48:44:44). -453 (1970)) and the like. Biological searches include stringent hybridization, macroarrays with genomic DNA affixed to nylon membranes, microarrays affixed to glass plates (microarray assays), PCR and in situ hybridization. It is not limited. In the present specification, it is intended that the gene used in the present invention should include a corresponding gene identified by such an electronic search or biological search.
As a functional equivalent of the present invention, an amino acid sequence having one or more amino acid insertions, substitutions or deletions, or those added to one or both ends can be used. In the present specification, “insertion, substitution or deletion of one or a plurality of amino acids, or addition to one or both ends thereof in an amino acid sequence” means a well-known technical method such as site-directed mutagenesis. It means that a modification has been made by substitution of a plurality of amino acids to the extent that can occur naturally by a method or by natural mutation.

  As used herein, an “isolated” biological factor (eg, a gene (InNAL1), nucleic acid, protein, etc.) is one in which at least a portion of the factor that naturally accompanies the substance or biological factor is removed. Used to identify natural products. Thus, typically the purity of the biological agent in an isolated biological agent is higher (ie, enriched) than the state in which the biological agent is normally present.

  As used herein, a “purified” substance or biological factor (such as a nucleic acid or protein) refers to a product from which at least a part of the factor naturally associated with the biological factor has been removed. Thus, typically, the purity of a biological agent in a purified biological agent is higher (ie, enriched) than the state in which the biological agent is normally present. The term “purified” as used herein is preferably at least 75% by weight, more preferably at least 85% by weight, even more preferably at least 95% by weight, and most preferably at least 98% by weight, Means the presence of the same type of biological agent. The materials used in the present invention are preferably “purified” materials.

  As used herein, a “corresponding” amino acid or nucleic acid has or has the same action as a predetermined amino acid or nucleotide in a reference polypeptide or polynucleotide in a polypeptide molecule or polynucleotide molecule. For example, in the case of an enzyme molecule, it means an amino acid that is present at the same position in the active site and contributes similarly to the catalytic activity. For example, an antisense molecule can be a similar part in an ortholog corresponding to a particular part of the antisense molecule. Corresponding amino acids are, for example, cysteinylated, glutathione, SS bond formation, oxidation (eg, oxidation of methionine side chain), formylation, acetylation, phosphorylation, glycosylation, myristylation, etc. Of amino acids. Alternatively, the corresponding amino acid can be an amino acid responsible for dimerization. Such “corresponding” amino acids or nucleic acids may be regions or domains spanning a range. Thus, in such cases, it is referred to herein as a “corresponding” region or domain.

  As used herein, a “corresponding” gene (eg, a polynucleotide sequence or molecule) has, or is expected to have, in a species, the same effect as a given gene in a species to which comparison is made. It refers to a gene (for example, a polynucleotide sequence or a molecule), and when there are a plurality of genes having such an action, those having the same origin in evolution. Thus, a gene corresponding to a gene can be an ortholog of that gene. Therefore, the morning glory InNAL1 can find the corresponding InNAL1 in the lily, 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, morning glory) is a reference gene of the corresponding gene (for example, InNAL1), for example, by using the sequence such as SEQ ID NO: 1 as a query sequence. ) By searching a sequence database.

  As used herein, “fragment” refers to a polypeptide or polynucleotide having a sequence length of 1 to n−1 with respect to a full-length polypeptide or polynucleotide (length is n). The length of the fragment can be appropriately changed according to the purpose. For example, the lower limit of the length is 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50 and more amino acids, and lengths expressed in integers not specifically listed here (eg 11 etc.) are also suitable as lower limits obtain. In the case of polynucleotides, examples include 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides. Non-integer lengths (eg, 11 etc.) may also be appropriate as a lower limit. In the present specification, it is understood that such a fragment falls within the scope of the present invention as long as the full-length fragment functions as a marker, as long as the fragment itself also functions as a marker.

  As used herein, “activity” refers to the function of a molecule in the broadest sense. Activity is not intended to be limiting, but generally includes the biological function, biochemical function, physical function or chemical function of a molecule. Activity activates, promotes, stabilizes, inhibits, suppresses, or destabilizes, for example, enzyme activity, the ability to interact with other molecules, and the function of other molecules Ability, stability, and ability to localize to specific intracellular locations. Where applicable, the term also relates to the function of the protein complex in the broadest sense.

  As used herein, “biological function” refers to a specific function that a gene, nucleic acid molecule or polypeptide may have in vivo when referring to a gene or a nucleic acid molecule or polypeptide related thereto. Examples of the antibody include, but are not limited to, generation of specific antibodies, enzyme activity, and imparting resistance. In the present invention, for example, InNAL1 or the like can promote or control the senescence of petals, but is not limited thereto. As used herein, a biological function can be exerted by “biological activity”. As used herein, “biological activity” refers to activity that a certain factor (eg, polynucleotide, protein, etc.) may have in vivo, and exhibits various functions (eg, transcription promoting activity). For example, an activity in which another molecule is activated or inactivated by interaction with one molecule. When two factors interact, their biological activity depends on the binding between the two molecules and the resulting biological change, eg, when one molecule is precipitated with an antibody, the other When molecules also coprecipitate, the two molecules are considered bound. Therefore, seeing such coprecipitation is one of the judgment methods. In the case of an activity that delays senescence of petals, as in the present invention, such activity is included. For example, when a factor is an enzyme, the biological activity includes the enzyme activity. In another example, when an agent is a ligand, the ligand includes binding to the corresponding receptor. Such biological activity can be measured by techniques well known in the art. Thus, “activity” indicates or reveals binding (either direct or indirect); affects the response (ie, has a measurable effect in response to some exposure or stimulus), Refers to various measurable indicators, such as the affinity of a compound that directly binds to a polypeptide or polynucleotide of the invention, or the amount of protein upstream or downstream after some stimulation or event or other Similar measures of function are mentioned.

  In the present specification, “expression” of a gene, polynucleotide, polypeptide or the like means that the gene or the like undergoes a certain action in vivo to take another form. Preferably, a gene, polynucleotide or the like is transcribed and translated into a polypeptide form. However, transcription and production of 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 InNAL1 can be determined by any method. Specifically, the expression level of InNAL1 can be determined by evaluating the amount of InNAL1 mRNA, the amount of InNAL1 protein, and the biological activity of InNAL1 protein. The amount of InNAL1 mRNA or protein can be determined by a method as described herein.

  In this specification, the “functional equivalent” refers to any target having the same target function but a different structure from the original target entity. Therefore, in the case of “InNAL1 or a functional equivalent thereof” or “a group consisting of InNAL1 and a functional equivalent thereof”, in addition to InNAL1 itself, a mutant or variant of InNAL1 (for example, an amino acid sequence variant, etc.) And having the effect of delaying senescence of petals, and one that can be changed to InNAL1 itself or a mutant or variant of InNAL1 at the time of action (for example, InNAL1 itself or a mutant of InNAL1 or It is understood that nucleic acids encoding variants, and vectors, cells, etc. containing the nucleic acids are encompassed. In the present invention, it is understood that the functional equivalent of InNAL1 can be used in the same manner as InNAL1, even if not specifically mentioned.

  The modified amino acid sequence of InNAL1, for example, 1 to 30, preferably 1 to 20, more preferably 1 to 9, more preferably 1 to 5, particularly preferably 1 to 2 amino acid insertions or substitutions, Alternatively, it can be deleted or added to one or both ends. The modified amino acid sequence is preferably an amino acid sequence having one or more (preferably one or several, or 1, 2, 3, or 4) conservative substitutions in the amino acid sequence of InNAL1. May be. As used herein, “conservative substitution” means substitution of one or more amino acid residues with another chemically similar amino acid residue so as not to substantially alter the function of the protein. For example, when a certain hydrophobic residue is substituted by another hydrophobic residue, a certain polar residue is substituted by another polar residue having the same charge, and the like. Functionally similar amino acids that can make such substitutions are known in the art for each amino acid. Specific examples include non-polar (hydrophobic) amino acids such as alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Examples of polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Examples of positively charged (basic) amino acids include arginine, histidine, and lysine. Examples of negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

  In the present specification, “drug”, “agent” or “factor” (both corresponding to “agent” in English) are used interchangeably in a broad sense, and so long as they can achieve their intended purpose. It may also be a substance or other element (eg energy such as light, radioactivity, heat, electricity). Such substances include, for example, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (eg, DNA such as cDNA, genomic DNA, RNA such as mRNA), poly Saccharides, oligosaccharides, lipids, small organic molecules (for example, hormones, ligands, signaling substances, small organic molecules, molecules synthesized by combinatorial chemistry, small molecules that can be used as pharmaceuticals (for example, small molecule ligands, etc.)) , These complex molecules are included, but not limited thereto. As a factor specific for a polynucleotide, typically, a polynucleotide having a certain sequence homology to the sequence of the polynucleotide (for example, 70% or more sequence identity) and complementarity, Examples include, but are not limited to, a polypeptide such as a transcription factor that binds to the promoter region. Factors specific for a polypeptide typically include an antibody specifically directed against the polypeptide or a derivative or analog thereof (eg, a single chain antibody), and the polypeptide is a receptor. Alternatively, specific ligands or receptors in the case of ligands, and substrates thereof when the polypeptide is an enzyme include, but are not limited to.

  As used herein, the term “agonist” refers to a substance that causes the target entity (eg, receptor) to express the biological action of the receptor. In addition to natural agonists (also called ligands), synthesized ones and modified ones can be mentioned.

  As used herein, “antagonist” refers to a substance that inhibits the biological action of a target entity (eg, receptor). In addition to those that inhibit competitively with agonists (or ligands), there are those that inhibit non-competitively. It can also be obtained by modifying the agonist. Antagonists can be included in the concept of inhibitors or suppressors because they inhibit physiological phenomena.

(Antisense / RNAi)
As used herein, “antisense activity” refers to an activity that can specifically suppress or reduce the expression of a target gene. More specifically, depending on a certain nucleotide sequence introduced into the cell, an activity that can reduce the protein expression level by specifically reducing the mRNA level of a gene having a nucleotide sequence region complementary to that sequence. Say. As a technique, there are a method of directly introducing an RNA molecule complementary to mRNA produced from a target gene into a cell, and a method of introducing a construction vector capable of expressing RNA complementary to a target gene in the cell. Although broadly divided, the latter is more common in plants.

  Antisense activity is usually achieved by a nucleic acid sequence of at least 8 contiguous nucleotides that is complementary to the nucleic acid sequence of the gene of interest. Such a nucleic acid sequence is 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 14 contiguous nucleotide lengths, 14 contiguous nucleotide lengths, 15 contiguous nucleotide lengths, 20 contiguous nucleotide lengths, 25 contiguous nucleotide lengths, 30 contiguous nucleotide lengths, 40 contiguous nucleotide lengths It can be a nucleic acid sequence that is 50 contiguous nucleotides in length. Such nucleic acid sequences include nucleic acid sequences that are at least 70% homologous, more preferably at least 80% homologous, more preferably 90% homologous, 95% homologous to the sequences described above. Such antisense activity is preferably complementary to a sequence at the 5 'end of the nucleic acid sequence of the gene of interest. Such antisense nucleic acid sequences also include those having one, several or one or more nucleotide substitutions, additions and / or deletions relative to the sequences described above. Therefore, as used herein, “antisense activity” includes, but is not limited to, a decrease in gene expression.

  General antisense technology is described in textbooks (Murray, JAH eds., Antisense RNA and DNA, Wiley-Liss Inc, 1992). Furthermore, the latest research revealed a phenomenon called RNA interference (RNAi), which led to the development of antisense technology. When RNAi introduces a short double-stranded RNA (about 20 bases) having a sequence homologous to the target gene into the cell, the 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 organisms including plants, and the molecular mechanism by which the expression of target genes is suppressed by antisense technology is It has been elucidated that it goes through a similar process. Conventionally, one DNA sequence that is complementary to the nucleotide sequence of the target gene is linked to an appropriate promoter, and an expression vector that expresses artificial mRNA under the control of the DNA is constructed and introduced into the cell. Was done. In recent knowledge, an expression vector designed so that a double-stranded RNA can be constructed in a cell is used. The basic structure is created by linking one DNA sequence complementary to a target gene, one under the promoter, and the other in the opposite direction. In the single-stranded mRNA transcribed from this constructed gene, a pair of nucleotide sequences connected in the opposite direction are in a complementary relationship, and thus are paired to form a double-stranded RNA state having a hairpin-like secondary structure. This causes mRNA degradation of the target gene according to the RNAi mechanism. In plants, an example used in Arabidopsis thaliana has been reported (Smith, NA et al., Nature 407.319-320, 2000). Also, RNAi in general is summarized in a recent review (Morita and Yoshida, Protein / Nucleic Acid / Enzyme 47, 1939-1945, 2002). The contents described in these documents are incorporated herein by reference in their entirety.

  As used herein, “RNA interference” or “RNAi” is an abbreviation for RNA interference, an organism that is generally known in the art and that inhibits or down-regulates gene expression in cells mediated by factors that cause RNAi. Process. For example, a phenomenon in which homologous mRNA is specifically decomposed by introducing a factor causing RNAi such as double-stranded RNA (also referred to as dsRNA) into a cell, and the synthesis of a gene product is suppressed, and a technique used therefor Say. In this specification, “RNAi” can also be used interchangeably with “factor causing RNAi”, “factor causing RNAi”, and “RNAi factor” in some cases. For RNAi, see, for example, Zamore and Haley, 2005, Science, 309, 1519-1524; Vaughn and Martinsensen, 2005, Science, 309, 1525-1526; Zamore et al. 2000, Cell, 101, 25-33; Bass, 2001, Nature, 411, 428-429; Elbashirital. , 2001, Nature, 411, 494-498; and Kreutzer et al., WO 00/44895; Zernica-Goetz et al., WO 01/36646; Fire, WO 99/32619; Publication 00/01846; Melo and Fire, WO 01/29058; Deschamps-Depairlette, WO 99/07409 and Li et al., WO 00/44914; Allshire, 2002, Science, 297, 1818-1819; Volpe et al. , 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al. , 2002, Science, 297, 2232-2237; Hutvagner and Zamore, 2002, Science, 297, 2056-60; McManus et al. , 2002, RNA, 8, 842-850; Reinhart et al. , 2002, gene & Dev. 16, 1616-1626; and Reinhart & Bartel, 2002, Science, 297, 1831. ). In this specification, the term RNAi is synonymous with other terms used to describe sequence-specific RNA interference such as post-transcriptional gene silencing, translational inhibition, transcriptional inhibition, and epigenetics. Understood. In the present specification, the “factor causing RNAi” may be any as long as it causes “RNAi”.

  In the present specification, the “factor causing RNAi” includes “small interfering nucleic acid”, “siNA”, “small interfering RNA”, “siRNA”, “small interfering nucleic acid molecule”, “small interfering oligonucleotide molecule”. ”Or“ chemically modified small interfering nucleic acid molecules ”and the like, these terms inhibit or down-regulate gene expression or viral replication by mediating RNA interference“ RNAi ”or gene silencing in a sequence-specific manner. Refers to any nucleic acid molecule that can. These terms may also represent individual nucleic acid molecules, 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, the antisense region includes a nucleotide sequence that is complementary to a nucleotide sequence in a target nucleic acid molecule or a part thereof, and a sense region having a nucleotide sequence corresponding to the target nucleic acid sequence or a part thereof. These molecules can be assembled from two separate oligonucleotides, one strand is the sense strand and the other is the antisense strand. Here, the antisense strand and the sense strand are self-complementary (i.e., each strand has a nucleotide sequence in the other strand such that the antisense strand and the sense strand form a double-stranded or double-stranded structure). Complementary nucleotide sequences are included, where, for example, the double-stranded region is from about 15 to about 30, for example, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25. , 26, 27, 28, 29 or 30 base pairs, but may be longer, the antisense strand comprises a nucleotide sequence that is complementary to a nucleotide sequence in the target nucleic acid molecule or a portion thereof, The sense strand includes a nucleotide sequence corresponding to the target nucleic acid sequence or portion thereof (eg, about 15 to about 25 or more nucleotides of the molecule are present in the target nucleic acid or portion thereof. Alternatively, these molecules are assembled from a single oligonucleotide, and the self-complementary sense and antisense regions of these molecules are linked by a nucleic acid linker or a non-nucleic acid linker. These molecules can be polynucleotides having a double-stranded, asymmetric duplex, hairpin or asymmetric hairpin secondary structure comprising self-complementary sense and antisense regions, where the antisense region is A nucleotide sequence that is complementary to a nucleotide sequence in a separate target nucleic acid molecule, or a portion thereof, and a sense region that has a nucleotide sequence corresponding to the target nucleic acid sequence, or a portion thereof, the molecule comprising two or more A ring having a loop structure and a stem comprising a self-complementary sense region and an antisense region The antisense region may comprise a nucleotide sequence that is complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof, and a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The circular polynucleotide can be processed in vivo or in vitro to produce an active molecule that can mediate RNAi, these factors being present in the nucleotide sequence or part thereof in the target nucleic acid molecule. Single-stranded polynucleotides having nucleotide sequences that are complementary can also be included (eg, these factors do not require a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof to be present in these factors). Single-stranded polynucleotides are 5 ′ phosphates (eg, Martinez et al, 2002, Cell. 110, 563-574 and Schwartz et al. , 2002, Molecular Cell, 10, 537-568) and may further comprise a terminal phosphate group such as 5 ', 3'-diphosphate. In certain embodiments, an agent that inhibits InNAL1 of the present invention comprises separate sense and antisense sequences or regions. Here, the sense and antisense regions are covalently linked by nucleotide or non-nucleotide linker molecules known in the art, or ionic interactions, hydrogen bonds, van der Waals interactions, hydrophobic interactions and / or Alternately non-covalently linked by stacking interactions. In certain embodiments, an InNAL1 inhibitor of the invention comprises a nucleotide sequence that is complementary to the nucleotide sequence of the target gene. In another embodiment, an agent that suppresses InNAL1 of the present invention interacts with the nucleotide sequence of the target gene so as to inhibit expression of the target gene. In the present specification, a factor that suppresses InNAL1 is not necessarily limited to a molecule containing only RNA, but also includes chemically modified nucleotides and non-nucleotides. In certain embodiments, when the invention is a small interfering nucleic acid molecule, it may be devoid of 2'hydroxy (2'-OH) containing nucleotides. In certain embodiments, the invention can be a small interfering nucleic acid that does not require the presence of a nucleotide having a 2 'hydroxyl group to mediate RNAi. Therefore, when the present invention is a small interfering nucleic acid molecule, ribonucleotides (for example, nucleotides having a 2'-OH group) may not be contained. However, if the presence of a ribonucleotide in the factor that suppresses InNAL1 is not required to maintain RNAi, a linked linker comprising one or more nucleotides with 2′-OH groups, or other bonds or It can have associated groups, moieties or chains. In some cases, an agent that inhibits InNAL1 of the present invention may comprise ribonucleotides at about 5, 10, 20, 30, 40, or 50% of the nucleotide positions. As used herein, factors that inhibit InNAL1 include 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 oligonucleotide, small interfering nucleic acid, small interfering modified oligonucleotide, chemically modified siRNA, post-transcriptional gene silencing RNA (ptgsRNA).

  As used herein, factors that cause RNAi include, for example, a sequence having at least about 70% homology to a portion of the nucleic acid sequence of a target gene or a sequence that hybridizes under stringent conditions. Examples include, but are not limited to, RNA containing a double-stranded portion of nucleotide length or a variant thereof. Here, the factor preferably comprises a 3 'overhang, more preferably the 3' overhang can be 2 or more nucleotides long DNA (eg 2 to 4 nucleotides long DNA).

  Alternatively, examples of RNAi used in the present invention include, but are not limited to, a pair of short reverse complementary sequences (for example, 15 bp or more, for example, 24 bp).

  Without being bound by theory, one of the possible mechanisms for RNAi to work is that when a molecule that causes RNAi, such as dsRNA, is introduced into a cell, a relatively long (eg, 40 base pairs or more) RNA, helicase In the presence of ATP, an RNaseIII-like nuclease called Dicer having a domain excises the molecule from the 3 ′ end by about 20 base pairs to produce a short dsRNA (also called siRNA). As used herein, “siRNA” is an abbreviation for short interfering RNA, which is artificially chemically synthesized, biochemically synthesized, synthesized in an organism, or about 40 This refers to short double-stranded RNA of 10 base pairs or more formed by decomposing double-stranded RNA of bases or more in the body, and usually has a 5′-phosphate, 3′-OH structure. 'The end protrudes about 2 bases. A protein specific to the siRNA binds to form RISC (RNA-induced-silencing-complex). This complex recognizes and binds to mRNA having the same sequence as siRNA, and cleaves mRNA at the center of siRNA by RNaseIII-like enzyme activity. The relationship between the siRNA sequence and the mRNA sequence cleaved as a target is preferably 100% identical. However, with respect to the base mutation at a position off the center of siRNA, the cleavage activity by RNAi is not completely lost, but a partial activity remains. On the other hand, the mutation of the base at the center of siRNA has a large effect, and the cleavage activity of mRNA by RNAi is extremely reduced. Utilizing such properties, for mRNA having a mutation, only siRNA having the mutation can be specifically decomposed by synthesizing siRNA having the mutation in the center and introducing it into the cell. Therefore, in the present invention, siRNA itself can be used as a factor that causes RNAi, and a factor that generates siRNA (for example, a dsRNA typically having about 40 bases or more) can be used as such a factor. it can.

  Moreover, although not wishing to be bound by theory, siRNA is synthesized separately from the above pathway, and the antisense strand of siRNA binds to mRNA and acts as a primer for RNA-dependent RNA polymerase (RdRP). It is also contemplated that this dsRNA becomes Dicer's substrate again, generating new siRNA and amplifying the action. Thus, in the present invention, siRNA itself and factors that produce siRNA are also useful. In fact, in insects and the like, for example, 35 dsRNA molecules almost completely degrade the mRNA in a cell having 1,000 copies or more, so it is understood that siRNA itself and factors that generate siRNA are useful. Is done.

  In the present invention, double-stranded RNA having a length of about 20 bases (for example, typically about 21 to 23 bases in length) or less than that called siRNA can be used. Such siRNA can be used for treatment, prevention, prognosis, etc. of a disease because it suppresses gene expression by expressing in a cell and suppresses expression of a pathogenic gene targeted by the siRNA. The siRNA used in the present invention may take any form as long as it can cause RNAi.

  In another embodiment, the factor causing RNAi of the present invention may be a short hairpin structure (shRNA; short hairpin RNA) having an overhang at the 3 'end. As used herein, “shRNA” is a single-stranded RNA that includes a partially palindromic base sequence, and thus has a double-stranded structure within the molecule, resulting in a hairpin-like structure. The above molecules. Such shRNA is artificially chemically synthesized. Alternatively, such shRNA can be generated by synthesizing RNA in vitro with T7 RNA polymerase, which has a hairpin structure DNA in which the DNA sequences of the sense strand and antisense strand are ligated in the reverse direction. Although not wishing to be bound by theory, such shRNAs are approximately 20 bases in length (typically 21 bases, 22 bases, 23 bases, etc.) in length after being introduced into the cell. It should be understood that it is degraded to cause RNAi as well as siRNA and has the therapeutic effect of the present invention. It should be understood that such effects are exerted in a wide range of organisms such as insects, plants, animals (including mammals). Thus, since shRNA causes RNAi similarly to siRNA, it can be used as an active ingredient of the present invention. The shRNA can also preferably have a 3 'overhang. The length of the double-stranded part is not particularly limited, but may preferably be about 10 nucleotides or more, more preferably about 20 nucleotides or more. Here, the 3 'protruding end may be preferably DNA, more preferably DNA having a length of at least 2 nucleotides, and further preferably DNA having a length of 2 to 4 nucleotides. The factor causing RNAi used in the present invention can be either artificially synthesized (for example, chemical or biochemical) or naturally occurring, and the effect of the present invention can be achieved between the two. There is no essential difference. Those chemically synthesized are preferably purified by liquid chromatography or the like.

The factor that causes RNAi used in the present invention can also be synthesized in vitro. In this synthesis system, antisense and sense RNAs are synthesized from template DNA using T7 RNA polymerase and T7 promoter. When these are annealed in vitro and then introduced into cells, RNAi is caused through the mechanism described above, and the effects of the present invention are achieved. Here, for example, such RNA can be introduced into cells by any appropriate method such as the calcium phosphate method.
Factors causing RNAi of the present invention also include factors such as single strands that can hybridize to mRNA, or all similar nucleic acid analogs thereof. Such factors are also useful in the present invention.

  In the present specification, when referring to a gene, a “vector” refers to one that can transfer a target polynucleotide sequence into a target cell. Such vectors can be autonomously replicated in host cells such as prokaryotic cells, yeast, animal cells, plant cells, insect cells, individual animals and individual plants, or can be integrated into the chromosome, Examples include those containing a promoter at a position suitable for transcription of the polynucleotide of the present invention. In the present specification, the vector may be an expression vector, a recombinant vector, or the like.

  In the present specification, examples of the vector include those that can be replicated and isolated and purified by common bacteria used in genetic experiments (typically, E. coli strain derived from E. coli K12 strain). This is necessary to construct the target gene to be introduced into the plant. Specific examples include commercially available plasmids such as pBR322 plasmid of E. coli, pUC18, pUC19, pBluescript, and pGEM-T. When a gene fragment is directly introduced into a plant cell and transformed, such as electroporation, polyethylene glycol, or particle gun, transformation of the gene to be introduced using such a commercially available general plasmid is performed. Just build it. As a special example of a vector, when transforming a plant cell using an Agrobacterium-mediated gene transfer method, the replication origin of both E. coli and Agrobacterium, and the boundary region that can be introduced into the plant are defined. It is necessary to use a plasmid called a “binary vector” having a nucleotide sequence corresponding to the border sequence derived from the T-DNA (Left border and Right Border). For example, pBI101 (commercially available from Clontech), pBIN (Bevan, N., Nucleic Acid Research 12, 8711-8721, 1984), pBINPlus (van Engelen, FA et al., Transgenic Research 4, 288-290, N), 95). Examples thereof include, but are not limited to, pTH (Fukuoka H et. Al., Plant Cell Reports 19, 2000), pPZP (Hajdukiwicz P et al., Plant Molecular Biology 25, 989-994, 1994). In addition, examples of vectors that can be used in plants include tobacco mosaic virus vectors. However, since this type of vector does not introduce the target gene into the plant chromosome, the transgenic plant is propagated through seeds. The application is limited to the case where it is not necessary to make it, but can be used in the present invention.

  As used herein, “expression cassette” refers to a structural gene, a promoter sequence that regulates its expression, various regulatory elements, and a terminator sequence that terminates transcription of mRNA. 1 unit of an artificially constructed gene linked by Typical examples include a selection marker (for example, hygromycin resistance gene) expression cassette for selecting only the host cell into which the gene has been introduced, or an expression cassette for a useful protein gene to be expressed in the host cell. The The types, structures, and numbers of expression cassettes to be prepared should be properly used depending on the organism, host cell, and purpose, and combinations thereof are well known to those skilled in the art.

  In the present specification, an “expression vector” is defined as a “vector” that may contain one or more of the above “expression cassettes”. Each target gene expression cassette to be introduced into a plant may be arranged on a separate vector, or all expression cassettes may be linked on one vector. The plant expression vector used in the present invention may be of a binary vector type. Furthermore, this vector can contain a target marker expression cassette to be introduced and a selection marker (eg, hygromycin resistance gene) expression cassette suitable for the host plant.

  Examples of the selection marker used in the present specification include, but are not limited to, hygromycin phosphotransferase and mutant acetobutyrate synthase. Promoters that can be used in the selectable marker expression cassette include CaMV35S promoter and its modified promoters, ubiquitin promoters, etc., and terminators include, but are not limited to, Nos terminator, Tml terminator, 10 kDa prolamin terminator, 13 kDa prolamin terminator, and 16 kDa prolamin terminator. Not.

  As used herein, “terminator” is a sequence that is located downstream of a region encoding a protein of a gene and is involved in termination of transcription when DNA is transcribed into mRNA and addition of a poly A sequence. Examples of the terminator include, but are not limited to, CaMV35S terminator, terminator of nopaline synthase gene (Tnos), and terminator of tobacco PR1a gene. In the present invention, any sequence can be used as long as it exhibits terminator activity in plants.

  As used herein, the term “promoter” refers to a region on DNA that determines the transcription start site of a gene and directly regulates its frequency, and is a base sequence that initiates transcription upon binding of RNA polymerase. In general, the promoter region can be estimated by predicting the protein coding region in the genome 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 may be present in or downstream of the structural gene. In the present invention, any promoter that exhibits the function of a factor causing RNA interference of InNAL1 can be used.

  In the present specification, when used for expression of a gene, “site specificity” generally means a part of an organism (eg, a plant) (eg, in the case of a plant, a protein body, root, stem, stem, leaf, flower). (Petals, etc.), seeds, endosperm, embryos, embryos, fruits, etc.). “Time specificity” refers to the specificity of expression of a gene according to the developmental stage of a living organism (eg, a plant) (eg, the growth stage of a plant (eg, the time when petals bloom)). Such specificity can be introduced into a desired organism by selecting an appropriate promoter.

As used herein, “enhancer” can be used to increase the expression efficiency of a target gene. When used in plants, as an enhancer, for example, an enhancer region containing an upstream sequence in the CaMV35S promoter and an upstream region of an octopine synthase gene are preferable. A plurality of enhancers may be used, but one may be used or may not be used.
As used herein, “silencer” refers to a sequence having a function of suppressing and resting gene expression. In the present invention, any silencer may be used as long as it has the function, and the silencer may not be used.
As used herein, “operably linked” refers to a transcriptional translational regulatory sequence (eg, promoter, enhancer, etc.) or a translational regulatory sequence that has the expression (operation) of a desired sequence. That means. In order for a promoter to be operably linked to a gene, the promoter is usually placed immediately upstream of the gene, but need not necessarily be adjacent.

  In this specification, any technique may be used for introducing a nucleic acid molecule into a cell, and examples thereof include transformation, transduction, and transfection. Such a technique for introducing a nucleic acid molecule is well known in the art and commonly used. For example, AusubelF. A. (1988), Current Protocols in Molecular Biology, Wiley, New York, NY; Sambrook J, et al. (1987) Molecular Cloning: Laboratory Manual, 2nd Ed. And its 3rd ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, a separate volume of experimental medicine “Gene Transfer & Expression Analysis Experimental Method” Yodosha, 1997, and the like. Gene transfer can be confirmed using the methods described herein, such as Northern blot, Western blot analysis, or other well-known conventional techniques.

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

  In the present specification, the “gene introduction reagent” refers to a reagent used for promoting the introduction efficiency in the gene introduction method. Examples of such gene transfer reagents include, but are not limited to, cationic polymers, cationic lipids, polyamine reagents, polyimine reagents, calcium phosphate, and the like. Specific examples of reagents used in the transfection include reagents commercially available from various sources, such as Effectene Transfection Reagent (cat. No. 301 425, Qiagen, CA), TransFast ™ Transfection Reagent (E2431). , Promega, WI), TfxTM-20 Reagent (E2391, Promega, WI), SuperFect Transfection Reagent (301305, Qiagen, CA), PolyFect Transfection Reagent (301105, Qiagen, CA) AM01, Regen 168 rogen corporation, CA), JetPEI (× 4) conc. (101-30, Polyplus-translation, France) and ExGen 500 (R0511, Fermentas Inc., MD) and the like, but are not limited thereto.

  When the present invention is used in plants, plant expression vectors can be introduced into plant cells by methods well known to those skilled in the art, for example, methods involving Agrobacterium and methods for direct introduction into cells. As a method using Agrobacterium, 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 electroporation with, for example, an expression vector appropriate for a plant, and then transforming the transformed Agrobacterium into Gelvin et al. (Edited by Gelvin et al. (1994), Plant Molecular Biology Manual). (Kluwer Academic Press Publishers)). For methods of introducing plant expression vectors directly into cells, see electroporation (see Shimamoto et al. (1989), Nature 338: 274-276; and Rhodes et al. (1989) Science 240: 204-207. ), Particle gun method (see Christou et al. (1991), Bio / Technology 9: 957-962) and polyethylene glycol (PEG) method (Datta et al. (1990), see Bio / Technology 8: 736-740). ). These methods are well known in the art, and a method suitable for the plant to be transformed can be appropriately selected by those skilled in the art.

  A cell into which a plant expression vector has been introduced is first selected for drug resistance such as hygromycin resistance and kanamycin resistance. It can then be redifferentiated into plant tissues, plant organs and / or plants by methods well known in the art. Furthermore, seeds can be obtained from the plant body. Expression of the introduced gene can be detected by Northern blotting or PCR. If necessary, the expression of a protein as a gene product can be confirmed by, for example, Western blotting.

  Although the present invention has been shown to be particularly useful in plants, it can also be used in other organisms. The molecular biology techniques used in the present invention are well known and commonly used 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 third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, a separate volume of experimental medicine “Gene Transfer & Expression Analysis Experimental Method” Yodosha, 1997, and the like.

  As used herein, “transformant” refers to all or part of a living organism such as a cell produced by transformation. Examples of the transformant include prokaryotic cells, yeast, animal cells, plant cells, insect cells and the like. A transformant is also referred to as a transformed cell, transformed tissue, transformed host, etc., depending on the subject, and encompasses all of these forms herein, but may refer to a particular form in a particular context. .

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

  On the other hand, methods for introducing an isolated gene using a living organism include an Agrobacterium method, a viral vector method, and a method using pollen as a vector. These methods have the advantage that the culture does not last for a long period of time and is not susceptible to damage such as somaclonal mutation because gene transfer is performed using plant callus, tissue or plant without using protoplasts. ing. Among them, the method using pollen as a vector has few experimental examples yet, and there are many unknown parts as plant transformation methods. Although the viral vector method has the advantage that the gene to be introduced to the entire plant infected with the virus spreads, 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 that DNA of about 20 kbp or more can be introduced into a chromosome without large rearrangement, the number of copies of the introduced gene is as few as several copies, and reproducibility is high. . Since Agrobacterium is outside the host range for monocotyledonous plants such as gramineous plants, introduction of foreign genes into gramineous plants has been conventionally performed by the physical methods described above. However, in recent years, the Agrobacterium method has been applied to monocotyledonous plants such as rice, which have established culture systems, and the Agrobacterium method is now preferred. .

In the introduction of foreign genes by the Agrobacterium method, when a low molecular weight phenolic compound such as acetosyringone synthesized by plants acts on the Ti plasmid Vir region, the T-DNA region is excised from the Ti plasmid and undergoes several processes. It is integrated into the nuclear chromosomal DNA of plant cells. In the dicotyledonous plant, since the plant itself has such a phenol compound synthesis mechanism, a foreign gene can be easily introduced by the leaf disk method or the like, and reproducibility is also high. On the other hand, in monocotyledonous plants, since such plants do not synthesize such phenolic compounds, it was difficult to produce transformed plants with Agrobacterium. However, it is now possible to introduce foreign genes into monocotyledons by adding acetosyringone during Agrobacterium infection.
In the present invention, in the transformant, the target nucleic acid molecule (transgene) may or may not be introduced into the chromosome. Preferably, the nucleic acid molecule of interest (transgene) has been introduced into a chromosome, and more preferably has been introduced into both two chromosomes.

  Examples of plant cells include those described below in the present specification, and examples include cells of ethylene-independent flowering plants such as morning glory and lily. More preferably, the plant cell may be a convolvulaceae plant, more preferably a morning glory.

  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. For example, Agrobacterium Agrobacterium (JP 59-140885, JP 60-70080, WO 94/00977), electroporation method (JP 60-251887), method using particle gun (gene gun) (Japanese Patent No. 2606856) Patent No. 2517813).

  As used herein, “plant” is a general term for organisms belonging to the plant kingdom, and is characterized by chlorophyll, hard cell walls, the presence of abundant permanent embryonic tissues, and organisms that are not capable of motility. Typically, a plant refers to a flowering plant having cell wall formation and anabolism by chlorophyll. “Plant” includes both monocotyledonous and dicotyledonous plants. Preferred plants include, for example, “ethylene-independent flowering” plants. “Ethylene-independent flower” is a flower that does not involve or is a small flower of ethylene, which is a plant hormone, in senescence of petals. Such plants include morning glory, rose, lily, tulip, chrysanthemum, sunflower, gerbera, Margaret, Dahlia, Gladiolus, Hanashobu, Iris, Freesia, Thundersonia.

  More preferably, the plant may be a convolvulaceae, rose family, liliaceae, asteraceae, iridaceae plant, more preferably morning glory, rose, lily, tulip, chrysanthemum, sunflower, gerbera, margaret, dahlia, gladiolus, It can be hanashobu, iris, freesia, thundersonia.

  Preferred plants are not limited to ornamental plants for viewing flowers, but also include any plant that is useful in preventing petal senescence, such as crops, trees, lawns, weeds, and the like. Unless otherwise indicated, a plant includes any plant, plant organ, plant tissue, plant cell, and seed. Examples of plant organs include roots, leaves, stems, and flowers (eg, petals). Examples of plant cells include callus and suspension culture cells.

  Examples of “ethylene-independent flowering” plants include morning glory, rose, lily, tulip, chrysanthemum, sunflower, gerbera, margaret, dahlia, gladiolus, hanashobu, iris, freesia, thundersonia and the like.

  Examples of convolvulaceae plants include the genus Nagasea (Aniseia), the genus Convolvulus, the genus Cuscuta, the genus Dichondra, the genus Erycibe, and the genus Erycium vulus vulgaris. (Ipomoea), Jacqwmontia, Lepistemon, Merremia, Perculina, and the like. , Convolvulus, sweet potato, jorgao, rumor, and the like.

  In the present specification, the “tissue” of an organism refers to a group of cells having a certain similar action in the group. Thus, the tissue can be part of an organ. In an organ, there are many cells having the same function, but those having slightly different functions may be mixed. May be mixed.

  In this specification, “has a structure in which one or more structures are combined to form a structure that performs a specific function. Examples of the plant include, but are not limited to, callus, root, stem, stem, leaf, flower, seed, germ, embryo, fruit, and endosperm.

  In the present specification, the “organism” (or “plant” in the case of plants) is used in the broadest sense in the field and refers to an organism (or plant) that operates a biological phenomenon, and typically has a cell structure. It has various characteristics such as proliferation (self-reproduction), growth, regulation, substance metabolism, repair ability, etc., and usually has inheritance governed by nucleic acid and proliferation involved in metabolism governed by protein as basic attributes. Living organisms include prokaryotes, eukaryotes (plants, animals, etc.) and the like. Preferably, in the present invention, the organism may be a plant. As used herein, preferably such a plant may be fertile. More preferably, such a plant can produce seeds. It is contemplated that the genetic constructs, agents, compositions and methods of the present invention function not only in ethylene-independent flowering plants but also in ethylene-dependent flowering plants.

  As used herein, “transgenic” refers to an organism (for example, a plant (including morning glory)) that is incorporated into an organism in which a specific gene is present or incorporated.

  In the present specification, plant cultivation can be performed by any method known in the art. The plant cultivation method is, for example, “Asagao Edo gift” (Yoshiaki Yoneda): How to grow pp. 65-76 (A method for cultivating morning glory is described. For the actual method for cultivation, see the examples below).

  Techniques and media known in the art are used for culturing, differentiation and regeneration of plant cells, plant tissues and plants. Examples of such a medium 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 the present specification, in the case of a plant, “redifferentiation” of the plant means a phenomenon in which the whole individual is restored from a part of the individual. For example, by redifferentiation, an organ or a plant is formed from a tissue piece such as a cell (a leaf, a root, etc.).

  A method for redifferentiating a transformant into a plant is 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. Examples thereof include, but are not limited to, those described in Cold Spring Harbor Laboratory Press (1995). Therefore, those skilled in the art can use the above-mentioned well-known methods as appropriate according to the transgenic plant intended for redifferentiation. The transgenic plant thus obtained is introduced with a gene of interest, and the introduction of such a gene can be performed by a method described herein such as Northern blot or Western blot analysis or other methods. This can be confirmed using well-known conventional techniques.

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

<InNAL1 inhibitory factor>
In one aspect, the present invention provides a factor that suppresses InNAL1. InNAL1 is one of the NAC transcription factor family genes whose expression level increases during petal senescence. As a result of producing a transformant (InNAL1r) that suppresses InNAL1 expression, the period until the petal wilt is about twice as long. Was observed in the morning glory, an ethylene-independent flowering plant (see Examples). These results indicate that InNAL1 is a NAC transcription factor family gene that regulates (promotes) petal senescence in the morning glory, which is an ethylene-independent flower, and that inhibition of InNAL1 expression can delay the senescence of morning glory petals. ing. In other ethylene-independent flowers, it is understood that the viewing period can be extended by InNAL1 repressing factors such as genetic recombination or inhibitors targeting the InNAL1 ortholog. The NAC transcription factor family is also referred to as NAC domain protein, NAC family protein, NAC transcription factor. It has in common a region that is expected to bind to conserved DNA called NAC domain. More than 100 NAC domain protein genes have been found in Arabidopsis, but the function of most genes is unknown. On the other hand, since the sequence corresponding to InNAL1 is also found in other plants, it is understood that InNAL1 ortholog is involved in the control of petal senescence in other plants. In particular, although not wishing to be bound by theory, in ethylene-independent plants, programmed cell death occurs during petal senescence, and expression of a common gene cluster is induced (for example, Van Dorn WG, Woltering EJ. Physiology and molecular biology of petal sensence. Journal of Experimental Botany 59: 453-80, 2008. It is understood that the effect is similarly exerted in other cases. It will be understood that the present invention provides similar effects in plants other than the examples. The factor of the present invention may be any factor as long as it can temporarily or permanently suppress, decrease or eliminate the function of InNAL1, and it can be any protein, polypeptide, oligopeptide, peptide, poly Nucleotides, oligonucleotides, nucleotides, nucleic acids (eg, including DNA such as cDNA, genomic DNA, RNA such as mRNA), polysaccharides, oligosaccharides, lipids, small organic molecules (eg, hormones, ligands, signaling substances) , Organic small molecules, molecules synthesized by combinatorial chemistry, small molecules that can be used as pharmaceuticals (for example, small molecule ligands, etc.), and complex molecules of these, but are not limited to them. Other elements (eg light, radioactivity, heat, electricity, etc.) Ghee) may be even.

  In one embodiment, the factor is in nucleic acid form. Examples of nucleic acid inhibitors include aptamers, ribozymes, antisense oligonucleotides, and RNAi factors such as siRNA. In other embodiments, factors in protein form may also be used. Examples of such protein forms include antibodies or antigenic fragments, binding peptides, peptidomimetics and the like. Alternatively, the factor of the present invention may be another molecule such as a small molecule.

  In one embodiment, the factor is a factor that causes RNA interference of InNAL1 in the form of a double-stranded nucleic acid.

  In certain embodiments, an agent of the invention is in double stranded form, one strand of which (I) (A) a sequence shown in SEQ ID NO: 1 (InNAL1 itself) or a sequence encoding SEQ ID NO: 2; or ( B) (B-1) a sequence variant having one or several substitutions, additions or deletions in (A), (B-2) at least about 70% sequence identity to the sequence of (A) Or a sequence variant that hybridizes under stringent conditions to the sequence of (B-3) (A), the sequence variant having the biological function of (A) The other strand contains the complementary sequence of (II) (I).

  In a more specific embodiment, the agent of the present invention is in a double-stranded form, one strand of which is a sequence of 370 bases at positions 690-1059 of SEQ ID NO: 1 and the other strand is its complement. Contains an array. It is understood that the specific embodiment is not limited to this, for example, any range as long as it is 98-853 bases long (Wesley et al., Plant Journal 27: 581). -590). Suitable RNAi factors are also described in Wesley et al. It can be designed with reference to Plant Journal 27: 581-590.

  In plant RNAi, it is generally considered that mRNA degradation can be induced by using the sequence of any region of a transcript (mRNA) of a target gene. From the RNAi principle, it is clear to those skilled in the art that degradation of mRNA occurs if it contains the transcribed sequence of the target gene (see, for example, Wesley et al. Plant Journal 27: 581-590). ). Any region can be selected, but preferably, when targeting a highly conserved region sequence (a common sequence among multiple genes), the same sequence as the target gene is used. It is advantageous to remove the conserved region from the target sequence because it may degrade the mRNA of the gene it has. Also in the InNAL1 RNAi construct of the present invention, the region of comparatively high conservation was excluded from the examples performed in the examples. It is known that RNAi can be induced by targeting the 5'UTR and 3'UTR (untranslated region) rather than the coding region (see, for example, Wesley et al. Plant Journal 27: 581-590). It will be appreciated that siRNA can also be used in plants, but preferably longer RNAi factors can also be used (in plants, longer dsRNAs are expressed but eventually processed into siRNAs of about 20 bases). Will induce silencing).

  In one aspect, the present invention provides a vector comprising a nucleic acid that causes RNA interference of InNAL1. The nucleic acid that causes RNA interference of InNAL1 included here is any form exemplified by the factor that suppresses InNAL1 of the present invention, and any nucleic acid can be used as long as it is a nucleic acid that causes RNA interference of InNAL1. It is understood that

  In another embodiment, the vector of the invention comprises a promoter and / or terminator.

  In a particular embodiment, the vector of the invention is pH7-InNAL (SEQ ID NO: 3).

  In another aspect, the present invention provides a plant cell comprising an agent that suppresses InNAL1 of the present invention.

  In one embodiment, the plant cell of the present invention is an ethylene independent flowering plant cell.

  In a preferred embodiment, the plant cell of the present invention is a convolvulaceae plant cell. Examples of such plant cells include the genus Nagasea (Aniseia), the genus Convolvulus, the genus Cuscuta, the genus Dichondra, the genus Erycibe, the genus Vulgaris ul (v), Genus (Ipomoea), genus Jacqumontia, canopy moth genus (Lepistemon), genus Convolvulaceae (Merremia), genus Operculina (such as Dianthus cerevisiae), genus (Stococ) Examples include, but are not limited to, convolvulus, convolvulus, sweet potato, jorgao, and rumors. In a further preferred embodiment, the plant cell of the present invention is a morning glory cell.

  In another aspect, the present invention provides a plant comprising a factor that suppresses InNAL1 of the present invention.

  In one embodiment, the plant of the invention is an ethylene independent flowering plant.

  In a preferred embodiment, the plant of the present invention is a convolvulaceae plant. Examples of such plants include the genus Nagasea (Aniseia), the genus Convolvulus, the genus Cuscuta, the genus Dichondra, the genus Erycibe, the genus Usa vulgaris ul (Ipomoea), Jacqwmontia, Lepistemon, Merremia, Perculina, and the like. , Convolvulus, sweet potatoes, jorgao, and liquorice, but are not limited thereto. In a further preferred embodiment, the plant of the present invention is morning glory.

  In another aspect, the present invention provides a plant cell in which InNAL1 is suppressed more than its natural state. In such a cell, the state in which InNAL1 is suppressed more than the natural state may be achieved by the factor that suppresses InNAL1 of the present invention, or may be selected by screening or the like. In the screening, plants or cells having a mutation in InNAL1 may be selected by PCR or the like from a naturally occurring mutation or artificial mutation treatment group. In this case, for example, in the case of morning glory, those having a lower level of expression or activity than the natural InNAL1 disclosed in the present invention can be selected. In the case of other plants, it can be selected in the same manner. Alternatively, in addition to a method for introducing an inhibitory factor or screening, a method for inhibiting expression by altering the sequence of endogenous NAL1 using a method such as zinc finger nuclease or gene targeting may be used. In the practice of the present invention, with regard to the application of zinc finger nucleases in plants, Keishi Osaka, Yuriko Osaka and Seiichi Tokyo, Site-directed mutagenesis in Arabidopsis using cincincedgingzincedinzingingzincinzedgingzincinzedgingzincinzedgingzinc. Proc. Nat. Acad. Sci. USA (2010) June 29, 107 (26): 12034-12039. As for gene targeting, Molecular bleeding of a novel herbicide-tolerant rice by gene targeting. Endo, Masaki; Osakabe, Keishi; Ono, Kazuko; Handa, Hirokazu; Shimizu, Tsutomu; Toki, Seiichi. The Plant Journal vol. 52 issue 1 October 2007. p. 157-166; Rie Terada, Hiroko Urawa, Yoshishige Inagaki, Kazuo Tsugane, and Shigeru Iida: Efficient gene targeting by homologous rebirth. Nature Biotechnology 20 (10) 1030-1034 (2002) can be referred to.

  In one embodiment, the plant cell of the present invention is an ethylene independent flowering plant cell.

  In a preferred embodiment, the plant cell of the present invention is a convolvulaceae plant cell. Examples of such plant cells include the genus Nagasea (Aniseia), the genus Convolvulus, the genus Cuscuta, the genus Dichondra, the genus Erycibe, the genus Vulgaris ul (v), Genus (Ipomoea), genus Jacqumontia, canopy moth genus (Lepistemon), genus Convolvulaceae (Merremia), genus Operculina (such as Dianthus cerevisiae), genus (Stococ) Examples include, but are not limited to, convolvulus, convolvulus, sweet potato, jorgao, and rumors. In a further preferred embodiment, the plant cell of the present invention is a morning glory cell.

  In another aspect, the present invention provides a plant in which InNAL1 is suppressed more than in a natural state. In such a plant, the state in which InNAL1 is suppressed from the natural state may be achieved by the factor that suppresses InNAL1 of the present invention, or may be selected by screening or the like. Such a plant can also be induced or regenerated based on a plant cell in which the InNAL1 of the present invention is suppressed from its natural state. Similarly to the screening of plant cells, plant screening may be performed by selecting plants or cells that have caused mutations in InNAL1 from naturally occurring mutations or artificially mutagenized populations. . In this case, for example, in the case of morning glory, those having a lower level of expression or activity than the natural InNAL1 disclosed in the present invention can be selected. In the case of other plants, it can be selected in the same manner. Alternatively, in addition to a method for introducing an inhibitory factor or screening, a method for inhibiting expression by altering the sequence of endogenous NAL1 using a method such as zinc finger nuclease or gene targeting may be used.

  In one embodiment, the plant of the invention is an ethylene independent flowering plant.

  In a preferred embodiment, the plant of the present invention is a convolvulaceae plant. Examples of such plants include the genus Nagasea (Aniseia), the genus Convolvulus, the genus Cuscuta, the genus Dichondra, the genus Erycibe, the genus Usa vulgaris ul (Ipomoea), Jacqwmontia, Lepistemon, Merremia, Perculina, and the like. , Convolvulus, sweet potatoes, jorgao, and liquorice, but are not limited thereto. In a further preferred embodiment, the plant of the present invention is morning glory.

<InNAL1 nucleic acid molecule / protein>
In another aspect, the present invention provides the following:
(A) the sequence shown in SEQ ID NO: 1 or the sequence encoding SEQ ID NO: 2; or (B)
(B-1) a sequence variant having one or several substitutions, additions or deletions in (A),
(B-2) a sequence variant having at least about 70% sequence identity to the sequence of (A), or (B-3) hybridizing under stringent conditions to the sequence of (A) A sequence variant comprising:
The sequence variant provides an isolated nucleic acid molecule comprising a sequence having the biological function of (A). These can be InNAL1.

  In one embodiment, the nucleic acid molecule of the invention comprises the sequence shown in SEQ ID NO: 1.

  In a preferred embodiment, the nucleic acid molecule of the invention consists of the sequence shown in SEQ ID NO: 1.

In another aspect, the present invention provides (a) a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 or a fragment thereof;
(B) a polypeptide having one mutation in which one or more amino acids are selected from the group consisting of substitution, addition and deletion in the amino acid sequence set forth in SEQ ID NO: 2 and having biological activity;
(C) a polypeptide encoded by a splice variant or allelic variant of the base sequence set forth in SEQ ID NO: 1;
(D) a polypeptide that is a species homologue of the amino acid sequence set forth in SEQ ID NO: 2; or (e) an amino acid sequence that is at least 70% identical to any one polypeptide of (a)-(d) And an isolated protein comprising a polypeptide having biological activity. These can be InNAL1.

  In one embodiment, the isolated protein of the invention comprises the sequence shown in SEQ ID NO: 2.

  In a preferred embodiment, the isolated protein of the invention consists of the sequence shown in SEQ ID NO: 2.

(screening)
In another aspect, the present invention provides a method for screening an agent that delays petal senescence using the nucleic acid molecule (InNAL1 or a variant thereof) of the present invention or a protein encoded by the nucleic acid molecule. As a screening method, a method known in the art can be applied. This method comprises the steps of (i) contacting InNAL1 or a substance, cell, plant or the like containing it with a test substance; and (ii) InNAL1 in the substance, cell, plant or the like after contacting the test substance Detecting the expression. In the step (i), the cells and the like used are those of an ethylene-independent flowering plant. The agent that delays senescence of petals can be selected from substances or factors that suppress the expression or activity of InNAL1 in a test substance. Such substances or factors can be further introduced into plants or plant cells to see if petal senescence can be delayed.

(General technology)
Molecular biological techniques, biochemical techniques, and microbiological techniques used in this specification are well known and commonly used in the art, and are described in, for example, Sambrook J. et al. et
al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, F .; M.M. (1987). Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, F .; M.M. (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Innis, M .; A. (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, F .; M.M. (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, F .; M.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.M. (1999). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; Sninsky. J. et al. et al. (1999). PCR Applications: Protocols for Functional Genomics, Academic Press, Separate Experimental Medicine “Gene Transfer & Expression Analysis Experimental Method” Yodosha, 1997 “Plant PCR Experimental Protocol” Shujunsha (1997), Model Plant Experimental Protocol Shujunsha (1996), etc., which are incorporated herein by reference in their entirety (which may be all).

  For DNA synthesis technology and nucleic acid chemistry for producing artificially synthesized genes, see, for example, Gait, M. et al. J. et al. (1985). Oligonucleotide Synthesis: A Practical Approach, IRLPpress; Gait, M .; J. et al. (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F .; (1991). Oligonucleotides and Analogues: A Practical Approac, IRL Press; Adams, R .; L. et al. (1992). The Biochemistry of the Nucleic Acids, Chapman &Hall; Shabarova, Z. et al. et al. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, G .; M.M. et al. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, G .; T. T. (I996). Bioconjugate Technologies, Academic Press, etc., which are incorporated herein by reference in the relevant part.

  References such as scientific literature, patents and patent applications cited herein are hereby incorporated by reference in their entirety to the same extent as if each was specifically described.

  The present invention has been described with reference to the preferred embodiments for easy understanding. In the following, the present invention will be described based on examples, but the above description and the following examples are provided only for the purpose of illustration, not for the purpose of limiting the present invention. Accordingly, the scope of the present invention is not limited to the embodiments or examples specifically described in the present specification, but is limited only by the scope of the claims.

  When necessary, the animals used in the following examples were handled in compliance with the standards established by the Government of Japan or the National Institute of Agricultural Food Industry Research. In addition, specifically, the products described in the examples were used as reagents, but equivalent products from other manufacturers (SIGMA-ALDRICH, Wako Pure Chemicals, Nakarai Techs, Kanto Chemical, etc.) can be substituted.

Example 1: Delayed experiment of petal senescence using InNAL1 in morning glory
(Experimental materials and methods)
Plant material and growing condition plant Asagao 'purple' (Ipomoea nil cv. Violet) (distributed from Kyushu University) was used. The seeds were sown in a horticultural soil (Kureha Horticultural Culture Kureha) in a pot and fertilized with 1 g / L of Hyponex 15-30-15 (Hyponex Japan) once a week. Plants are kept in a plant culture incubator (temperature 24 ° C, relative humidity 70%, 16 hours day length, PPFD 100 µmol · m ^-2 · s ^-1 ) (FLI-2000, Tokyo Rika Instrument Co., Ltd.) for 1 month after sowing. After that, in order to induce flowering, the day length was changed to 12 hours. The beginning of the light period was defined as 0 hours after flowering. In 0 hours after flowering petals are open almost completely (Shibuya K, et al 2011. Expression of autophagy-associated ATG8 genes during petal senescence in Japanese morning glory Journal of the Japanese Society for Horticultural Science 80:.. 89-95 ).

A BLAST search was performed in the morning glory EST database using the sequence of InNAL1 isolated Arabidopsis ORE1 (At5g39610; also known as NAC6, AtNAC2) and microRNA164 (miR164). As a result, EST (contig005501) having high homology with ORE1 and containing the miR164 recognition sequence was selected. Based on EST sequence information, RT-PCR was performed using RNA extracted from morning glory 'purple' to obtain a cDNA clone containing the full-length ORF. RNA extraction was performed using Trizol reagents (Invitrogen), and SuperScript III first-strand synthesis system RT-PCR (Invitrogen) was used for cDNA synthesis. PCR uses InNAL1-F1 (5′-TTTCTTTTAACACCGCCTCTTTTT-3 ′ (SEQ ID NO: 4) and InNAL1-R1 (5′-CAGAGCAATTTAGTTTCCAAGAA-3 ′ (SEQ ID NO: 5)) primers, PrimeSTAR HS DNA polymerase (Taka) The obtained cDNA clone was cloned into pGEM-T easy vector (Promega), the nucleotide sequence was determined, and it was named InNAL1 (InNAC-like 1) .InNAL1 was 58% identical with ORE1 in the deduced amino acid sequence. did.

Production of InNAL1 expression-suppressing transformant A transformant in which the expression of InNAL1 was suppressed was produced using the RNAi method. A 370 base pair InNAL1 cDNA fragment (690-1059 bases) was cloned into pDONR201 (Invitrogen), and then a plant transformation vector having a hygromycin resistance gene (Hyg) pH7GWIWG (II) (Karimi M, et al. 2002). GATEWAY vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci 7: 193-195.) Using LR Clone II Enzyme mix (Invitrogen). The resulting transformation vector pH7-NAL1 contains an InNAL1 cDNA fragment in the antisense and sense directions downstream of the cauliflower mosaic virus 35S promoter, and a 35S terminator downstream thereof. pH7-NAL1 was introduced into cultured cells derived from immature embryos of morning glory 'purple' using Agrobacterium AGL0 strain. Specifically, morning seeds of morning glory (11-14 days after flowering) are sterilized, and immature embryos are taken out from them and liquid culture (N6 medium + MS vitamin, 3 mg / L NAA, 60 g / L sucrose) (Wako Jun) Drug). The formed callus was subcultured to a fresh medium every 1-2 weeks to grow. The grown callus was immersed in an Agrobacterium solution and co-cultured for 3 days in a co-culture medium (MS medium, 10 g / L sucrose, 3.2 g / L gellan gum) (Wako Pure Chemical Industries). After co-culture, callus was transferred to the selective medium and cultured. The composition of the selection medium is 3 mg / L NAA, 60 g / L sucrose, 3.2 g / L gellan gum, 50 mg / L hygromycin (Wako Pure Chemical Industries), 200 mg / L kraforan or 1-2 capsules / L Augmentin in MS medium. Includes 250RS (GlaxoSmithKline). Embryoid bodies that survived on the selection medium were transferred to the same fresh composition medium after one month and further cultured for 1-3 months. Embryoid bodies were transferred to fresh medium every month. Embryoid bodies were transferred to a shoot formation medium to promote shoot formation. The composition of the shoot-forming medium includes MS medium, 2 mg / L BAP, 0.2 mg / L IAA, 30 g / L sucrose, 10 g / L agar, 200 mg / L claforan or 1 g / L augmentin. The shoots were rooted for about one month in a rooting medium containing 1/2 MS medium + MS vitamin and 30 g / L sucrose, and acclimated to the horticultural soil after transplantation. The presence or absence of the transgene was confirmed by PCR amplification of the Hyg gene.

Analysis of gene expression by quantitative real-time RT-PCR RNA was extracted using Trizol reagents (Invitrogen) and treated with Cloned DNase I (Takara bio). Random hexamer primers and PrimeScript RT Master Mix (Takara bio) were used for the synthesis of cDNA, and PCR was performed using SYBR Premix EX TaqII (Takara bio) and Thermal Cycler Real Time System (between 600 and Mod 600). Primers for amplification of InNAL1 used for PCR were InNAL1-q1F (5′-TTGTGTGATCCATCAGCTCCA-3 ′ (SEQ ID NO: 6)) and InNAL1-q1R (5′-CAGAGCAATTTAGTTTTCCCAAGAA-3 ′ (SEQ ID NO: 7)). Moreover, InACTIN4 was used as an internal control. Primers for amplification of InACTIN4 are InACTqF1 (5′-AATTACTGCAATGGCCCTA-3 ′ (SEQ ID NO: 8)) and InACTqR1 (5′-CCACATCTGTTGGAATGTGC-3 ′ (SEQ ID NO: 9)).

(result)
The results are shown in FIG. In the transformant InNAL1r, it was confirmed that the expression of InNAL1 was suppressed compared to the wild type.

(Aging of wild type and InNAL1r-1 flowers)
Seed the seeds of wild-type morning glory and transformed morning glory (InNAL1r) in a horticultural soil (Kureha Horticultural Culture Kureha) in a pot, and once a week, 1g / L Hyponex 15-30-15 (Hyponex Japan) Fertilized. Plants are kept in a plant culture incubator (temperature 24 ° C, relative humidity 70%, 16 hours day length, PPFD 100 µmol · m ^-2 · s ^-1 ) (FLI-2000, Tokyo Rika Instrument Co., Ltd.) for 1 month after sowing. After that, in order to induce flowering, the day length was changed to 12 hours. The beginning of the light period was defined as 0 hours after flowering. In 0 hours after flowering petals are open almost completely (Shibuya K, et al 2011. Expression of autophagy-associated ATG8 genes during petal senescence in Japanese morning glory Journal of the Japanese Society for Horticultural Science 80:.. 89-95 ).

(result)
The results are shown in FIGS.

  FIG. 2 shows the senescence of wild type and InNAL1r-1 flowers. In FIG. 2, the wild type is photographed at the upper stage, and the flowers of the InNAL1r-1 plant body are photographed every 4 hours from the flowering, respectively. In InALL1r-1 plants, senescence of petals is remarkably delayed (flower life is extended). The same applies to InNAL1r-2 and three plants.

  FIG. 3 shows a state 24 hours after the flowering of InNAL1r-1. Arrows indicate flowers for 24 hours (2nd day) after flowering, and triangles indicate flowers for 0 hour (1st day) after flowering. In the wild type, the petals usually wither in about 14 hours after flowering. On the other hand, in the InNAL1r-1 plant, the petals are blooming without wilt for 24 hours or more, so that the flower bloomed the day before and the flower bloomed on the same day are observed as shown in the figure. The same applies to InNAL1r-2 and three plants. The petal color changes from blue to red as time passes after flowering.

  FIG. 4 shows the wild-type and InNAL1r plant flowers 24 hours after flowering. In the wild type, the petals are completely deflated, whereas in the InNAL1r line, the petals are not yet deflated in all three lines.

Table 1 shows the time (n ≧ 8) from the time of flowering until the start of petal wilting.
(Table 1 In-life period in InNAL1 expression inhibitor (InNAL1r))

  From the above results, it was found that in the 1nNAL1r line that produced a transformant (InNAL1r) in which the expression of InNAL1 was suppressed in the morning glory, the period until the petal wilt was extended about twice. These results indicate that senescence of morning glory petals can be delayed by suppressing expression of InNAL1. In other ethylene-independent flowers, it is speculated that the ornamental period can be extended by gene recombination or treatment with an inhibitor that targets the InNAL1r ortholog.

(Example 2: Example with a lily)
InNAL1 orthologs are isolated in lilies. Isolation can be performed by BLAST analysis in the lily EST database or RT-PCR using primers set to the conserved sequence of InNAL1. Using the obtained sequence, a transformation vector for inducing RNAi is prepared. A transformation vector can be prepared in the same manner as in the morning glory. A transformation vector is introduced into a lily to produce a transformed lily. Regarding the method of transformation of lilies, it can be carried out with reference to the following documents. Masami Tanaka, Transformation of Lily genus by party glans, Kumamoto Agricultural Research Center Research Report 12: 41-48, 2003; Kohei Irifune, et al. , Production of Herbicide Resistant Transgenic Lily Plants by Particulate Bombard Journal of the Japan Society for Horticultural 51

(Example 3: Screening of inhibitors)
A protein or gene sequence that interacts with InNAL1 protein is isolated, and a system that can monitor the interaction with InNAL1 protein in vitro (96-well plate) is developed. Specifically, the degree of inhibition of the interaction by the compound is measured using a reporter such as a fluorescent protein. In the implementation, it is possible to refer to the search method of molecular targeted therapeutic drugs in cancer research. Using the developed monitoring system, chemical screening using a compound library is performed to find a drug that inhibits the activity of InNAL1 protein.

  As mentioned above, although this invention has been illustrated using preferable embodiment of this invention, it is understood that the scope of this invention should be construed only by the claims. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.

  The present invention may be used in the field of flower gardening. Recombinant flowers with extended flower life (eg morning glory) have commercial value. In other ethylene-independent flower plants such as lily, senescence of petals can be delayed by suppressing the expression of InNAL1 ortholog. For example, a genetic recombination technique for the target plant species can be used.

SEQ ID NO: 1 is the nucleic acid sequence of InNAL1 (InNAL1 registration sequence 120723; InNAL1 (1214 bp) ORF: 144-992 bp).
Sequence number 2: It is a protein of InNAL1 (InNAL1 protein (282aa)).
SEQ ID NO: 3 is the nucleic acid sequence of pH7-InNAL.
SEQ ID NO: 4 is the nucleic acid sequence of primer InNAL1-F1.
SEQ ID NO: 5 is the nucleic acid sequence of primer InNAL1-R1.
Sequence number 6: It is a nucleic acid sequence of primer InNAL1-q1F.
SEQ ID NO: 7 is the nucleic acid sequence of primer InNAL1-q1R.
SEQ ID NO: 8 is the nucleic acid sequence of primer InACTqF1.
SEQ ID NO: 9: Nucleic acid sequence of primer InACTqR1.

Claims (20)

A factor that suppresses InNAL1, which is a double-stranded nucleic acid that causes RNA interference of an InNAL1 nucleic acid. Including factors. 2. The agent of claim 1, wherein the double-stranded nucleic acid is 370-853 bases in length. The factor according to claim 1 or 2, wherein the nucleic acid in the double-stranded form has 5'UTR or 3'UTR (untranslated region). A factor that suppresses InNAL1 comprising a nucleic acid in a double-stranded form comprising the nucleic acid sequence shown in SEQ ID NO: 3. A nucleic acid molecule in double-stranded form, one strand of which is (I)
(A) a nucleic acid sequence shown in SEQ ID NO: 1 (InNAL1 itself) or a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 2; or (B)
(B-1) a sequence variant having one or several substitutions, additions or deletions in (A), or (B-2) having at least 90% sequence identity to the nucleic acid sequence of (A) A sequence variant comprising:
Sequence variants encode a protein having a transcriptional activities of the N AC transcription factor comprises a sequence variants and the other strand,
(II) comprising a complementary sequence of (I),
Nucleic acid molecule. The vector containing the factor as described in any one of Claims 1-4. The vector according to claim 6, comprising a promoter and a terminator. A plant cell comprising the factor according to any one of claims 1 to 4 or the vector according to claim 6 or 7. 9. The cell according to claim 8, wherein the plant cell is a cell of an ethylene-independent flowering plant. The cell according to claim 8 or 9, wherein the plant cell is a morning glory cell. A plant comprising the factor according to any one of claims 1 to 4 or the vector according to claim 6 or 7. The plant according to claim 11, wherein the plant is an ethylene-independent flowering plant. The plant according to claim 11 or 12, wherein the plant is a morning glory. (I) (A) 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)
(B-1) a sequence variant having one or several substitutions, additions or deletions in (A), or (B-2) having at least 90% sequence identity to the nucleic acid sequence of (A) A sequence variant comprising:
Sequence variants encode a protein having a transcriptional activities of the N AC transcription factors, including complementary sequences of sequence variants or (II) (I), isolated nucleic acid molecules. 15. The nucleic acid molecule of claim 14, wherein the nucleic acid molecule comprises the sequence shown in SEQ ID NO: 1. The nucleic acid molecule according to claim 14, wherein the nucleic acid molecule consists of the sequence shown in SEQ ID NO: 1. (A) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2 ;
(B) In the amino acid sequence shown in SEQ ID NO: 2, one or several amino acids have one mutation selected from the group consisting of substitution, addition and deletion, and the transcriptional activity possessed by the NAC transcription factor Or (c) an isolated polypeptide comprising a polypeptide having an amino acid sequence that is at least 90% identical to the polypeptide of (a) and having the transcriptional activity possessed by a NAC transcription factor Protein. A nucleic acid molecule according to claim 14, or a protein encoded by the nucleic acid molecule or a method for screening a drug that delays petal senescence using the protein according to claim 17. The nucleic acid molecule of Claim 14, or the protein which it encodes, or the screening method of the substance or factor which suppresses InNAL1 using the protein of Claim 17. The substance or factor includes an antibody against InNAL1 protein or an antigenic fragment thereof, a double-stranded nucleic acid that causes RNA interference of InNAL1 nucleic acid, an antisense nucleic acid of InNAL1 nucleic acid, an aptamer of InNAL1 nucleic acid, an siRNA of InNAL1 nucleic acid (low molecular interference) 20. The method of claim 19, wherein the method is selected from the group consisting of RNA) and a ribozyme of InNAL1 nucleic acid.