KR20130048431A - Casd1 gene from capsicum annuum and uses thereof - Google Patents

Abstract

PURPOSE: A Capsicum annuum-derived CaSD1(Capsicum annuum senescence-delaying 1) gene is provided to enhance expression of the gene by pathogen inoculation and treatment of defense-related signal transduction molecule, and to delay aging. CONSTITUTION: A Capsicum annuum-derived CaSD1 protein which is related to disease resistance enhancement or aging delay contains an amino acid sequence of sequence number 2. A gene for encoding the protein contains a base sequence of sequence number 1. A recombinant vector contains the gene. A host cell is transformed with the recombinant vector. A composition for enhancing diseases resistance in a plant contains the gene. The plant diseases are viral diseases or bacterial diseases. A composition for delaying aging in a plant contains the gene.

Description

CaSD1 gene derived from red pepper and its use {CaSD1 gene from Capsicum annuum and uses approximately}

The present invention CaSD1 derived from pepper It relates to a gene and use thereof, and more particularly, red pepper (Capsicum annuum) promote disease resistance or aging delay derived from the relevant CaSD1 (Capsicum annuum senescence - delaying 1 ) protein, gene encoding the protein, recombinant vector comprising the gene, host cell transformed with the recombinant vector, transforming the recombinant vector to plant cells overexpressing the CaSD1 gene A method for improving disease resistance or delaying aging of a plant, a method for improving plant disease resistance or delaying aging of a plant using the recombinant vector, a transformed plant having improved disease resistance or delayed aging produced by the method and seeds thereof And it relates to a composition for enhancing disease resistance or delaying aging of plants comprising the gene.

Sessile plants face a variety of stresses, such as pathogens, droughts, and changes in temperature. Compared to animals, plants have been developed with special functions and structures to overcome biological or abiotic stresses. One unique feature of plant cells is the presence of cell walls. Plant cell walls are made up of polysaccharides such as cellulose, hemicellulose and pectin and account for up to 90% of plant dry weight (Cassab GI and Varner JE 1988. Annu Rev Plant Phys 39: 321 -353). However, certain proteins in the cell wall play an important role in maintaining the function and structure of the plant cell wall.

A group of proteins released through the secretory pathway and located in the cell wall or extracellular space is called secretome. Secretories can be divided into two groups: proteins secreted by the classical pathway and proteins secreted by the atypical pathway (Agrawal GK et al., 2010. Proteomics. 10 (4): 799-827). ). Secretory secreted via a typical pathway is secreted via the Endoplasmic reticulum / Golgi pathway with signal peptides in the N-terminal sequence and approximately 17 genes in the Arabidopsis . Occupies% (approximately 5,000 genes) (Jamet E et al., 2006. Trends Plant Sci 11 (1): 33-39). On the other hand, recent studies have shown that there are no protein peptides that bypass the Golgi during the secretion process (Agrawal GK et al., 2010. Proteomics. 10 (4): 799-827).

Plant secretions play various roles in plant development and environmental stress response (Lee SJ et al., 2004. Plant Physiol Biochem. 42 (12): 979-988). As well as proteins that affect polysaccharides, structural proteins are primarily involved in plant growth and development. For example, extensin or proline-rich proteins are linked to the cell wall matrix and maintain cellular structure. Xyloglucan endotransglucosylase / hydrolase and expansin play a role in regulating cell proliferation and differentiation in cell wall structures. The proteins are active during normal growth as well as in response to external factors (Wei G and Shirsat AH, 2006. Mol Plant Pathol. 7 (6): 579-592).

The cell wall is the first structure that recognizes and responds to external stimuli, whereby defense-related secretions in the plant cell wall are constantly expressed or induced in response to wound or pathogen infection (Veronese P et al., 2003. Plant Physiol. 131 (4): 1580-1590). Cell wall-related kinases or leucine-rich extensin proteins, which are important for signal recognition and transfer, have been widely studied (Humphrey TV et al., 2007. New Phytol. 176 (1)). : 7-21). In addition, some proteins contribute to cell wall enrichment, lipid metabolism, and protective responses by apoplastic oxidative bursts (Oh IS et al., 2005. Plant Cell. 17 (10): 2832-2847). However, the function and mechanism of the protein is not clear.

In previous studies, 101 unique secretions that may be involved in plant cell defense and development were isolated from peppers using the yeast secretion trap system (Yeom SI et al., 2011. Mol Plant Microbe Interact. 24 (6): 671-684). CaSD1 ( Capsicum) , one of the secretes annuum senescence - delaying1 ) has been selected for specific studies because of its unique properties. The inventors have shown that the new gene has a repetitive region between CaSD1 is present only in species of the genus capsicum (Capsicum), and capsicum (Capsicum) species and varieties. In addition, gene expression and gain-of-function has shown that CaSD1 has a variety of functions in defense response, aging, and trichome formation.

Korean Laid-Open Patent Publication No. 2009-0049668 discloses CaNAC2 gene derived from red pepper which is involved in environmental stress and pathogen resistance of plants, and Korean Laid-Open Patent Publication No. 2004-0022472 uses ORE8 leaf life regulation gene of Arabidopsis-derived plant. A method of delaying aging of a plant is disclosed.

The present invention is derived from the above requirements, the inventors of CaSD1 derived from pepper The present invention was completed by confirming that the plant transformed with the gene exhibited disease resistance or delayed aging.

In order to solve the above problems, the present invention pepper ( Capsicum annuum) related to disease resistance promote or aging delay resulting CaSD1 (Capsicum annuum senescence - delaying 1) Provide protein.

The present invention also provides a gene encoding the CaSD1 protein.

The present invention also provides a recombinant vector comprising the gene.

The present invention also provides a host cell transformed with the recombinant vector.

The present invention also provides a method for enhancing disease resistance or delaying aging of a plant, the method comprising transforming the recombinant vector into plant cells, thereby overexpressing the CaSD1 gene.

In addition, the present invention provides a method for producing disease-promoting or delayed aging of a plant comprising the step of transforming the plant cell with the recombinant vector.

In addition, the present invention provides a transgenic plant and its seed, which have improved disease resistance or delayed aging of the plant produced by the above method.

The present invention also provides a composition for enhancing disease resistance or delaying aging of a plant comprising the gene.

The pepper-derived CaSD1 gene of the present invention was increased in expression by pathogen inoculation and defense-related signaling molecules, and delayed aging effect was observed in the leaves of plants overexpressing the gene. It is expected to be useful for developing aging delayed transgenic plants.

1,CaSD1Sequence analysis is shown. (a) is red pepper (Capsicum annuum) In Bukhan and CM334 varietiesCaSD1It shows the sequence alignment of. Black box is between the sinus and CM334 varietiesCaSD1Indicates a matched sequence of. (b) isCaSD1The amino acid sequence structure of the is shown. Black box: signal peptide, light gray: non-repeat region (1), white: repeat region, dark gray: non-repeat region (2).
2 shows various capsicums (CapsicumSpecies originCaSD1 PCR amplification results of the gene are shown. (a) isCaSD1 The structure of genomic DNA is shown schematically. Black lines represent 5 'and 3' untranslated regions (UTRs) and introns. Gray boxCaSD1Exon. Black arrows indicate primers for PCR amplification including repeat regions. (b) shows four genomic DNAs derived from capsicum species used for each PCR amplification. PCR products were electrophoresed on a 1% agarose gel, stained with ethidium bromide, and photographed under ultraviolet light.
Figure 3 shows the results of comparative genomic analysis of known plant genome syntenic segments.CaSD1Synthene segments comprising were compared to other known plant genomes. ORFs of pepper BAC 529L23 were predicted using the FGENESH program, and each ORF was presented in the order of the FGENESH numbers. Identical colors indicate sequence similarity.
Figure 4CaSD1The results of organ-specific expression of and expression after treatment with pathogens or defense-related signaling molecules are shown. (a) from a pepper plantCaSD1Organ-specific expression of (b) isXav(X. axonopodis pv.vesicatoria) In pepper ECW30R inoculated with race 1 or race 3CaSD1It shows the expression. (c) is TMV (tobacco mosaic virusFrom the varieties of the resistant varieties of the sinus and susceptible varieties ECW30RCaSD1of Expression is shown. (d) is a nonhost bacterial pathogenXag 8ra (Xanthomonas axonopodis pv. glycines 8ra) in the red pepper varietiesCaSD1It shows the expression. Real time PCRCaSD1-Specific primers were used and the values were normalized to CaActin and calculated as relative to leaf (a) or uninfected control (b-d). Bars represent standard deviation of 4 repetitions. Cayenne pepper varieties were infiltrated with 3 mM ethephon (ET), sprayed with 5 mM salicylic acid (SA), or infiltrated with 60 μM methyl jasmonate (MeJA) (e to g ).CaAccOx (C. annuum 1-aminocyclopropane-1-carboxylate), CaBPR1 (C. annuum basic pathogenesis - related protein 1)  AndCaPINII (C. annuum proteinase inhibitor II ) Expression levels of genes were monitored with positive markers for ET, SA and MeJA treatment, respectively. Expression levels of CaUBQ and CaActin genes were used as controls. IMG: immature green, MG: mature green.
5 in the cell wallCaSD1It shows the position of. Agrobacterium containing pMBP1: smGFP or pMBP1: CaSD1-smGFP was infiltrated to tobacco leaves. Photographs were taken with a confocal laser microscope (LSM510, Carl Zeiss, Wetzlar, Germany) one day after infiltration. DAPI (4 ′, 6-diamidino-2-phenylindole) was used for nuclear staining.
6,CaSD1Transient overexpression may delay aging and regulate the expression of aging-related genes. Agrobacterium-mediated overexpression of pMBP1 or pMBP1: CaSD1 in tobacco leaves. Photos were taken 20 days after inoculation (a) and the same leaves were taken 8 days after separation (b). Pictures were also taken 45 days after inoculation (c). Total RNA was isolated from leaves at specific time points after transient overexpression (TOE) and semiquantitative RT-PCR was performed using a gene-specific primer set (d). NbActin gene was used as a control. C: control plant,NtHin1 (N. tabaccum Harpin - induced One), NtSgr (N. tabaccum Staygreen ), NtCAB (N. tabaccum Chlororoll a / b binding protein gene ).
7 isCaSD1-Increased trichome density in overexpressed cigarettes. Vector overexpression (left) andCaSD1The surface of the overexpressed tobacco leaf (right) was observed using an optical microscope 10 days after the TOE (a). White bar: 1 mm. pMBP orCaSD1Surfaces of overexpressed leaves were observed using field-emission scanning electron microscopy at specific time points after the TOE (b). The black box is an enlarged cluster of adjacent tricoms. Black bar, 200 μm.
8 isCaSD1Cell expression and cell division-related gene expression levels in overexpressed tobacco. pMBP orCaSD1The surface of the overexpressed leaves was observed by field-emission scanning electron microscopy 7 days after inoculation (a). Each 20 cell areas were calculated using the ImageJ program (b). Bars represent standard deviations of the repetition. Two asterisks indicate a significant difference in cell size (b) (T-test, P <0.01). Expression patterns of cell division-related genes in overexpressed plants were examined (c). Black bars: 100 μm,NtCYCD (N. tabaccum D- type cyclin ), NtCYCA (N. tabaccum Cyclin A), NtCDKA (N. tabaccum Cyclin - dependent kinase A), NtCDKB (N. tabaccum Cyclin - dependent kinase B).

In order to achieve the object of the present invention, the present invention consists of an amino acid sequence represented by SEQ ID NO: 2, Capsicum CaSD1 ( Capsicum) related to disease resistance or aging delay in plants derived from annuum ) annuum senescence - delaying 1 ) provides protein.

The range of CaSD1 protein according to the present invention includes a protein having an amino acid sequence represented by SEQ ID NO: 2 isolated from red pepper and a functional equivalent of the protein. Is at least 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 90% or more, more preferably 90% or more, Quot; refers to a protein having a homology of at least 95% with a physiological activity substantially equivalent to that of the protein represented by SEQ ID NO: 2. By "substantially homogeneous physiological activity" is meant activity that enhances disease resistance or delays aging of plants.

The invention also includes fragments, derivatives and analogues of CaSD1 protein. As used herein, the terms “fragment”, “derivative” and “analogue” refer to a polypeptide that possesses the same biological function or activity as the CaSD1 polypeptide of the invention. The fragments, derivatives, and analogs of the present invention may be used in conjunction with (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted (the substituted amino acid residues are encoded Or (ii) a polypeptide having substituent (s) at one or more amino acid residues, or (iii) another compound (a compound capable of extending the half-life of the polypeptide, such as polyethylene glycol) (Iv) an additional amino acid sequence (e. G., A leader sequence, a secretory sequence, a sequence used to purify the polypeptide, a proteinogen sequence or a fusion protein) and a polypeptide derived from the mature polypeptide Or a polypeptide derived from said polypeptide. Such fragments, derivatives and analogs as defined herein are well known to those skilled in the art.

The present invention also provides a gene encoding the CaSD1 protein. The gene of the present invention may be DNA or RNA encoding CaSD1 protein. DNA includes cDNA, genomic DNA or artificial synthetic DNA. DNA can be single stranded or double stranded. DNA may be a coding strand or a non-coding strand.

 Preferably, the gene of the present invention may include the nucleotide sequence shown in SEQ ID NO: 1.

Polynucleotides encoding a mature polypeptide represented by SEQ ID NO: 2 include a coding sequence encoding only a mature polypeptide; Sequences encoding mature polypeptides and various additional coding sequences; Mature polypeptides (and any additional coding sequences) and sequences encoding noncoding sequences.

The term "polynucleotide encoding a polypeptide" refers to a polynucleotide encoding a polypeptide, or a polynucleotide further comprising additional coding and / or noncoding sequences.

The invention also relates to variants of said polynucleotides encoding polypeptides comprising the same amino acid sequences as described herein, or fragments, analogs, and derivatives thereof. Polynucleotide variants can be naturally occurring allelic variants or non-naturally occurring variants. Such nucleotide variants include substitutional variants, deletional variants, and insertional variants. As is known in the art, an allelic variant is an alternative to a polynucleotide, which may comprise one or more substituted, deleted or inserted nucleotides and which does not result in a substantial functional change in the polypeptide encoded by the variant Do not.

In addition, the present invention provides a poly-hybridized hybridization with a sequence having at least 50%, preferably at least 70%, more preferably at least 80% identity with a nucleotide sequence of SEQ ID NO. It relates to nucleotides. The present invention particularly relates to polynucleotides that hybridize to the polynucleotides described herein under stringent conditions. In the present invention, "stringent conditions" are (1) hybridization and washing under lower ionic strength and higher temperature such as 0.2 x SSC, 0.1% SDS, 60 ° C; Or (2) in the presence of a denaturant such as 50% (v / v) formamide, 0.1% bovine serum / 0.1% Ficoll and 42 ° C; Or (3) at least 80%, preferably at least 90%, more preferably at least 95%. In addition, the biological function and activity of the polypeptide encoded by the hybridizable polynucleotide is the same as the biological function and activity of the mature polypeptide represented by SEQ ID NO: 2.

In accordance with an embodiment of the invention, it should be understood that the CaSD1 gene is preferably derived from pepper. However, embodiments of the invention have high homology (eg, 60% or more, ie, 70%, 80%, 85%, 90%, 95%, even 98% sequence identity) with the Cayenne CaSD1 gene and other It also includes other genes derived from plants. Methods for sequencing, and means for determining sequence identity or homology (e.g., BLAST) are well known in the art.

The present invention also provides a recombinant vector comprising the CaSD1 gene according to the present invention.

The term "recombinant" refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, heterologous peptide or heterologous nucleic acid. The recombinant cell can express a gene or a gene fragment that is not found in the natural form of the cell in one of the sense or antisense form. In addition, the recombinant cell can express a gene found in a cell in its natural state, but the gene has been modified and reintroduced intracellularly by an artificial means.

In the present invention, the polynucleotide sequence encoding the CaSD1 protein can be inserted into a recombinant expression vector. The term "recombinant expression vector" means a bacterial plasmid, a phage, a yeast plasmid, a plant cell virus, a mammalian cell virus, or other vector. In principle, any plasmid and vector can be used if it can replicate and stabilize within the host. An important characteristic of the expression vector is that it has a replication origin, a promoter, a marker gene and a translation control element.

Expression vectors comprising CaSD1 protein-encoding DNA sequences and appropriate transcriptional / translational control signals can be constructed by methods well known to those skilled in the art. Such methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence can be effectively linked to appropriate promoters in the expression vector to drive mRNA synthesis. The expression vector may also include a ribosome binding site and a transcription terminator as a translation initiation site.

A preferred example of the recombinant vector of the present invention is a Ti-plasmid vector capable of transferring a so-called T-region to a plant cell when present in a suitable host, such as Agrobacterium tumefaciens. Other types of Ti-plasmid vectors (see EP 0 116 718 B1) are currently used to transfer hybrid DNA sequences to plant cells or protoplasts in which new plants capable of properly inserting hybrid DNA into the plant's genome can be produced have. A particularly preferred form of the Ti-plasmid vector is a so-called binary vector as claimed in EP 0 120 516 B1 and U.S. Patent No. 4,940,838. Other suitable vectors that can be used to introduce the DNA according to the invention into the plant host include viral vectors such as those that can be derived from double-stranded plant viruses (e. G., CaMV) and single- For example, from non -complete plant virus vectors. The use of such vectors may be particularly advantageous when it is difficult to transform the plant host properly. Preferably, the recombinant vector may be, but is not limited to, a pKBS1-1 vector, a pBI101 vector, and a pCAMBIA vector.

The expression vector will preferably comprise one or more selectable markers. The marker is typically a nucleic acid sequence having properties that can be selected by chemical methods, and all genes that can distinguish transformed cells from non-transformed cells. Examples include herbicide resistance genes such as glyphosate or phosphinothricin, antibiotics such as kanamycin, G418, Bleomycin, hygromycin, chloramphenicol, Resistant genes, but are not limited thereto.

In the recombinant vector of the present invention, the promoter may be CaMV 35S, actin, ubiquitin, pEMU, MAS, or histone promoter, but is not limited thereto. The term "promoter " refers to the region of DNA upstream from the structural gene and refers to a DNA molecule to which an RNA polymerase binds to initiate transcription. A "plant promoter" is a promoter capable of initiating transcription in plant cells. A "constitutive promoter" is a promoter that is active under most environmental conditions and developmental conditions or cell differentiation. Constructive promoters may be preferred in the present invention because the choice of transformants can be made by various tissues at various stages. Thus, the constitutive promoter does not limit the selection possibilities.

In the recombinant vector of the present invention, conventional terminators can be used. Examples thereof include nopaline synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, Agrobacterium tumefaciens (Agrobacterium tumefaciens ) Octopine gene terminator, but the present invention is not limited thereto. Regarding the need for terminators, it is generally known that such regions increase the certainty and efficiency of transcription in plant cells. Therefore, the use of a terminator is highly desirable in the context of the present invention.

The present invention also provides a host cell transformed with the recombinant vector of the present invention. Any host cell known in the art may be used as the host cell capable of continuously cloning and expressing the vector of the present invention in a stable and prokaryotic cell, for example, E. coli JM109, E. coli BL21, E. coli RR1 , E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus tulifene, and Salmonella typhimurium, Serratia marcesensus and various Pseudomonas species And enterobacteria and strains such as the species.

When the vector of the present invention is transformed into eukaryotic cells, yeast ( Saccharomyce cerevisiae), and the like insect cells, human cells (e.g., CHO cells (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cell lines) and plant cell may be used. The host cell is preferably a plant cell.

The method of carrying the vector of the present invention into a host cell is performed by using the CaCl ₂ method or one method (Hanahan, D., J. Mol. Biol., 166: 557-580 (1983)) when the host cell is a prokaryotic cell. And the electroporation method. When the host cell is a eukaryotic cell, the vector is injected into the host cell by microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, and gene bombardment .

Transformation of a plant means any method of transferring DNA to a plant. Such transformation methods do not necessarily have a regeneration and / or tissue culture period. Transformation of plant species is now common for plant species, including both terminal plants as well as dicotyledonous plants. In principle, any transformation method can be used to introduce the hybrid DNA according to the present invention into suitable progenitor cells. Method is calcium / polyethylene glycol method for protoplasts (Krens, FA et al., 1982, Nature 296, 72-74; Negrutiu I. et al., June 1987, Plant Mol. Biol. 8, 363-373), protoplasts Electroporation (Shillito RD et al., 1985 Bio / Technol. 3, 1099-1102), microscopic injection into plant elements (Crossway A. et al., 1986, Mol. Gen. Genet. 202, 179-185 ), (DNA or RNA-coated) particle bombardment of various plant elements (Klein TM et al., 1987, Nature 327, 70), Agrobacterium tumulopasis by plant infiltration or transformation of mature pollen or vesicles And infection with (incomplete) virus (EP 0 301 316) in en mediated gene transfer. A preferred method according to the present invention comprises Agrobacterium mediated DNA delivery. Particularly preferred is the use of so-called binary vector techniques as described in EP A 120 516 and U.S. Pat. No. 4,940,838.

In addition, the present invention provides a method for enhancing disease resistance or aging delay of a plant comprising the step of transforming the recombinant vector into plant cells, overexpressing the CaSD1 gene. The plant disease may be a viral disease or a bacterial disease, but is not limited thereto.

In addition, the present invention comprises the steps of transforming plant cells with the recombinant vector; And

It provides a method for producing disease-promoting or delayed sensitized transgenic plant of the plant comprising the step of regenerating the plant from the transformed plant cell.

The method of the present invention comprises transforming plant cells with a recombinant vector according to the present invention, which transformation can be mediated by, for example, Agrobacterium tumefiaciens. In addition, the method of the present invention comprises regenerating a transgenic plant from the transformed plant cell. Any of the methods known in the art can be used for regeneration of transgenic plants from transgenic plant cells.

Transformed plant cells must be regenerated into whole plants. Techniques for the regeneration of mature plants from callus or protoplast cultures are well known in the art for a number of different species (Handbook of Plant Cell Culture, Vol. 1-5, 1983-1989, Momillan, N. Y.).

The present invention also provides a transgenic plant and its seeds for enhancing disease resistance or delaying aging of the plants produced by the above method. Preferably, the plant may be a monocotyledonous plant or a dicotyledonous plant, but is not limited thereto.

The monocotyledonous plants include Alismataceae, Hydrocharitaceae, Juncaginaceae, Schuchzeriaceae, Pomomotontonaceae, Najadaceae, Zosteraceae, Liliaceae, Haemodoraceae, Agavaceae, Amaryllidaceae, Dioscoreaceae, Pontederiaceae, Iridaceae, Burmanniaceae, Juncaceae, Commelinaceae , Eriocaulaceae, Panaxaceae (Gramineae, Gramineae, Poaceae), Araceae, Lemnaceae, Sparganiaceae, Typhaceae, Cycloaceae, Cyperaceae, Musaceae ), Ginger (Zingiberaceae), Cannaceae (Cannaceae) or Orchidaceae (Orchidaceae), but is not limited thereto.

The dicotyledonous plants are Asteraceae (Dolaceae, Diapensiaceae), Asteraceae (Clethraceae), Pyrolaceae, Ericaceae, Myrsinaceae, Primaceae (Primulaceae), Plumbaginaceae, Persimmonaceae (Ebenaceae) , Styracaceae, Stink bug, Symplocaceae, Ash (Oleaceae), Loganiaceae, Gentianaceae, Menyanthaceae, Oleaceae, Apocynaceae , Asclepiadaceae, Rubiaceae, Polemoniaceae, Convolvulaceae, Boraginaceae, Verbenaceae, Labiatae, Solanaceae, Scrophulariaceae , Bignoniaceae, Acanthaceae, Sesame (Pedaliaceae), Fructose (Orobanchaceae). Gesneriaceae, Lentibulariaceae, Phrymaceae, Plantaginaceae, Caprifoliaceae, (Perox Adoxaceae), Valerianaceae, Dipsacaceae, Campanaceae ( Campanulaceae, Compositae, Myricaceae, Sapaceae, Juglandaceae, Salicaceae, Birchaceae, Beechaceae, Fagaceae, Elmaceae, Moraceae , Urticaceae, Santalaceae, Mistletoe, Lothanthaceae, Polygonaceae, Landaceae, Phytolaccaceae, Nyctaginaceae, Pomegranate, Azizaceae (Portulacaceae), Caryophyllaceae, Chinopodiaceae, Amaranthaceae, Cactaceae, Magnoliaceae, Illiciaceae, Lauraceae, Cassia family, Cecidiphyllaceae, Ranunculus eae), Berberidaceae, Lardizabalaceae, Bird breeze (Mentaceae, Menispermaceae), Nymphaeaceae, Ceratophyllaceae, Cabombaceae, Saururaceae , Piperaceae, Chloranthaceae, Aristolochiaceae, Actinidiaceae, Camellia, Theaceae, Guttaiferae, Droseraceae, Papaveraceae ), Capparidaceae, Cruciferaceae (Mustaceae, Cruciferae), Planeaceae (Plataceae, Platanaceae), Verruaceae, Hamamelidaceae, Pheasant (Snaphaceae, Crassulaceae), Panaxaceae (Saxifragaceae) Eucommiaceae, Pittosporaceae, Rosaceae, Leguminosae, Oxalidaceae, Geraniaceae, Tropaeolaceae, Zygophyllaceae, Linaceae Euphorbiaceae), star moss (Callitrichaceae), Rutaceae, Simaroubaceae, Meliaceae, Polygalaceae, Anacardiaceae, Mapleaceae, Aceraceae, Sapindaceae, Mapleaceae Hippocastanaceae, Sabiaceae, Balsam, Balsaminaceae, Aquifoliaceae, Nova, Celastraceae, Staphyleaceae, Buxaceae, Empetraceae, Rhamnaceae, Vitaceae, Elaeocarpaceae, Tiliaceae, Malvaceae, Sterculiaceae, Adenaceae, Thymelaeaceae, Eraeagnaceae (Flacourtiaceae), Violaceae, Passifloraceae, Tamaricanaceae, Elatinaceae, Begoniaceae, Cucurbitaceae, Buddha (Lythraceae), Pomegranate Punica ceae), Onnagraceae, Haloragaceae, Bataceae (Alangiaceae), Dogwood (Hornaceae, Cornaceae), Arboraceae (Agaraceae) or Umbelliferae (Apiaceae) It may be, but is not limited thereto.

The present invention also provides a composition for enhancing disease resistance or delaying aging of a plant comprising the CaSD1 gene of the present invention. In the composition of the present invention, the CaSD1 gene may be composed of the nucleotide sequence of SEQ ID NO: 1. The composition of the present invention contains CaSD1 gene derived from red pepper as an active ingredient, and by transforming the gene CaSD1 into a plant, it is possible to enhance disease resistance or delay aging of the plant. The disease of plants and plants is as described above.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Materials and methods

Plant material

Pepper plants were used for cloning and gene expression analysis. Bukang, a red pepper ( Capsicum annuum ) variety, has a battlefield CaSD1 ( Capsicum annuum senescence - delaying 1 ) Cloning cDNA and Xag 8ra ( Xanthomonas axonopodis pv. glycines 8ra) or after inoculation with TMV to assess CaSD1 expression. Pepper varieties ECW30R (Early Calwonder 30R) are Xav ( X. axonopodis pv. vesicatoria ) was used for inoculation with Race 1, Race 3 or TMV. Four varieties of each of the five different Capsicum species were randomly selected by PCR amplification of the repeat region (Table 1). Tobacco ( Nicotiana) benthamiana ) plants were used for transient overexpression of CaSD1 . All plants were grown in a walk-in chamber maintained at 22-25 ° C. and placed under 16 hours photoperiod for 4-6 weeks.

Cloning and DNA  Sequencing

Full-length sequence of the CaSD1 was isolated from the pepper varieties of BAC (bacterial artificial chromosome) of CM334 sequence (BAC number 529L23) (Yoo EY et al, 2003. Theor Appl Genet 107 (3):.. 540-543). Gene-coding regions and 3 ′ untranslated regions (UTRs) were predicted using the FGENESH program (http://linux1.softberry.com/berry.phtml), and gene-specific primers based on the BAC sequences were CaSD1 Was designed for PCR amplification (Table 2). PCR uses CaSD1 The amplification products were cloned into pJET vectors using the pJET ^™ PCR Cloning Kit (Fermentas Canada Inc., Burlington, Canada). The CaSD1 sequence was confirmed by DNA sequencing using ABI 3730 xl (Applied Biosystems INC, Foster City, CA, USA) at the National Instrumentation Center for Environmental Management, Seoul, Korea (NICEM). The sequencing analysis can be performed using ExPasy translation tool (http://us.expasy.org/tools/dna.html), NCBI blast search (National Center for Biotechnology Information Blast search), SignalP signal peptide prediction (http://www.cbs). .dtu.dk / services / SignalP /) and the TCoffee multi-alignment tool (http://www.ebi.ac.uk/Tools/msa/tcoffee/) (Altschul SF et al., 1997. Nucleic Acids Research. 25 (17): 3389-3402).

Primer List for PCR Amplification Primer Name Forward sequence (5 '→ 3') Reverse sequence (5 '→ 3') CaSD1_full CGGGATCCCGATGGTGTCAGGTAGTTTCAATATT
(SEQ ID NO: 3) GGAGCTCGTATTGTTTCATTTGTGAGGAAAGA
(SEQ ID NO: 4) CaSD1_repeat TCCAGAACAAAATCATAAGAGAAGC
(SEQ ID NO: 5) AGCACGATTATGTAGAATCCAAAAG
(SEQ ID NO: 6) CaSD1_RT AAAGGACTATGAGCGCAAGC
(SEQ ID NO: 7) TTGTGAGGAAAGACCGATCA
(SEQ ID NO: 8) CaAccOx AGAAAGCTGCAGAGGAAAGCAAAC
(SEQ ID NO: 9) TGAGATGCAACCGTTACTCCTATAC
(SEQ ID NO: 10) CaBPR1 TCACAATGCAGCTCGTAGACA
(SEQ ID NO: 11) TTTCATTGACCCACATCTTCAC
(SEQ ID NO: 12) CaPINII TTCATAATATGGCTGTTCACAAAGA
(SEQ ID NO: 13) AGACGCTCAAGCATATTACAGAGAT
(SEQ ID NO: 14) CaUBQ CGTGGTGGTTTTTAAGATGATG
(SEQ ID NO: 15) AAAGAAACACAAGGGAACAGAAA
(SEQ ID NO: 16) CaActin CCACCTCTTCACTCTCTGCTCT
(SEQ ID NO: 17) ACTAGGAAAAACAGCCCTTGGT
(SEQ ID NO: 18) NtHin1 GAGCTATGTATaCAAGGCCAGAG
(SEQ ID NO: 19) GCTTCACTTCCATCTCAAAAAC
(SEQ ID NO: 20) NtSgr AAAGAACTCCCTGTGGTTCTGA
(SEQ ID NO: 21) AATCCCAGTTGCTATTGCTTGT
(SEQ ID NO: 22) NtCAB ACAGAGCTCTTGAGGTTATCCA
(SEQ ID NO: 23) CACAACTTGAAAACCTAGCACA
(SEQ ID NO: 24) NbActin CCAGGTATTGCTGATAGAATGAG
(SEQ ID NO: 25) CTGAGGGAAGCCAAGATAGAG
(SEQ ID NO 26) NtCYCD3; 1 AAATGATTCATGGGATTTGGAG
(SEQ ID NO 27) CAACCACCAGCTAAGCTCTTCT
(SEQ ID NO 28) NtCYCA TTCAAAACCAGTCCAATCACTG
(SEQ ID NO 29) AAGCCTTCACATTTCCACCTAA
(SEQ ID NO: 30) NtCDKA; 4 TCCTGTTGATGTGTGGTCAG
(SEQ ID NO 31) AATGGAACATCAGAGCATTGTG
(SEQ ID NO 32) NtCDKB1-1 AATGGACGAAGAAGGGATTC
(SEQ ID NO: 33) TGAGAGAAGGAGGGAGAGGT
(SEQ ID NO 34)

Pathogen Inoculation

For inoculation of pathogens, Xag 8ra ( X. axonopodis pv. glycines 8ra), Xav Race 1 and Xav Race 3 were shake incubated at 200 rpm for 18 hours at 30 ° C. in YEP (Yeast extract peptone) liquid medium. After centrifugation of the culture, the bacterial cells were sterilized 10 mM MgCl ₂ Resuspended in solution. Each pathogen was diluted to OD ₆₀₀ 0.2 and infiltrated the plant leaves using a needleless syringe. Tobacco infected with TMV P ₀ strain 1 g of leaves were harvested and ground in 10 ml of 0.1 M phosphate buffer (pH 7.0) to make TMV inoculum (Kang WH et al., 2010. heor TAppl Genet. 120 (8): 1587-1596) . The inoculum along with carborundum (Hayashi Pure Chemical Ind., Japan) was rubbed into the pepper leaves and washed with water after 10 minutes. All treated leaves were harvested at specific time points and frozen in liquid nitrogen for RNA extraction.

Processing of Defense-Related Signaling Molecules

For the processing of signaling molecules, 5 week old pepper leaves were treated with 3 mM ethephon (ET) or 60 μM methyl jasmonate (MeJA) in 10% acetone using a needleless syringe as described above. (Yeom SI et al., 2011. Mol Plant Microbe Interact. 24 (6): 671-684). Salicylic acid (SA) treatment was performed by spraying pepper leaves with 5 mM SA. Pepper plants treated with SA were stored in plastic bags for 3 hours. The leaves were collected at certain time points after treatment and frozen in liquid nitrogen for RNA extraction.

RNA  Extraction and Gene Expression Analysis

Total RNA was extracted from frozen pepper and tobacco using TRIZOL reagents for gene expression investigation (Molecular Research Center, Cincinnati, OH, USA). First strand cDNA was synthesized from 5 μg total RNA using Oligo (dT) and SuperScript II reverse transcriptase (Invitrogen, Carlsbad, Calif., USA). Quantitative or semiquantitative RT-PCR was performed using gene specific primers designed with Primer3 software (http://frodo.wi.mit.edu/primer3) (Table 2). Quantitative RT-PCR was performed with Roter-Gene 2000 (Qiagen, Chatsworth, CA, USA) using Syto 9 (Invitrogen) for analysis of gene expression levels. Fluorescence was performed at 72 ° C. for 60 cycles and the expression level of each sample was normalized to Actin. Semi-quantitative RT-PCR was performed using MyCycler (Biorad, Hercules, CA, USA), PCR products were electrophoresed on 1% agarose gel, stained with ethidium bromide, and then under ultraviolet light The picture was taken.

CaSD1 - smGFP ( CaSD1 - soluble modified form of green fluorescent protein ) or smGFP of Intracellular  location( subcellular localization )

pMBP1: CaSD1-smGFP (pBI121-modified) and pMBP1: smGFP were constructed using a LI-independent (LI) method and transformed into the Agrobacterium tumefaciens GV2260 strain (Oh SK et al. , 2010. Mol Cells. 30 (6): 57-562). The transformed cells were shaken overnight at 30 ° C., 200 rpm in YEP medium, then centrifuged and then 10 mM MES (pH 5.5) / 10 mM MgCl ₂ Was resuspended in infiltration buffer containing x. The cell suspension (OD ₆₀₀ = 0.5) was incubated with 200 μM acetosyringone for 3 hours at room temperature. The cell suspension was then infiltrated on the back of the tobacco ( N. benthamiana ) leaves using a needleless syringe. After one day of infiltration, the epidermal cell layers of the hypocotyls were peeled off and GFP fluorescence was observed by confocal laser microscopy (LSM510, Carl Zeiss, Wetzlar, Germany). DAPI (4 ′, 6-diamidino-2-phenylindole) compound was used for nuclear staining.

tobacco( N.  benthamiana )in  TOE ( Transient overexpression )

The CaSD1 ORF (3 'UTR) containing 3' UTR was amplified with primers containing Bam HI and Sac I recognition sequences at the 5 'and 3' ends, respectively. The PCR product was cloned into pMBP1 vector treated with Bam HI and Sac I (Suh MC et al., 1998. Mol Cells. 8 (6): 678-684). The cloning product was transformed into Agrobacterium GV2260 strain. Agrobacterium transform-pMBP1: CaSD1 and pMBP1 controls were shaken overnight at 30 ° C., 200 rpm in YEP medium. The cells were centrifuged and resuspended in infiltration buffer containing 10 mM MES (pH 5.5) / 10 mM MgCl ₂ . The bacterial suspension (OD ₆₀₀ = 0.5) was incubated in 200 μM acetosyringone for 3 hours at room temperature. After incubation, the bacterial suspension was infiltrated on the back of the tobacco leaf using a needleless syringe. The infiltrated leaves were used for RNA extraction and observed microscopically at specific time points.

Optical microscope and Field  Emission scanning electron microscope

Temporarily overexpressed tobacco leaf surfaces were monitored and photographed under a light microscope (Siwon Optical Technology, Suwon, Korea) for trichome observation 10 days after the TOE. For analysis of tricom density and cell size, field emission scanning electron microscopy (SUPRA 55VP, Carl Zeiss, Wetzlar, Germany, NICEM at Seoul National University) was used in low vacuum mode (low-vacuum) at 2, 7 and 10 days after TOE. mode was used without treatment of the sample. The sample was monitored with a 15-kV accelerating voltage and the picture was taken digitally.

Example  One: Capsicum ( Capsicum ) Is a new protein that exists only in species of the genus CaSD1

To identify secreted proteins involved in plant-pathogen interactions, the C. annuum secretome (CaS) gene was identified using Phytophthora . Capsici using the YST system . After inoculation, it was isolated from pepper roots (Yeom SI et al., 2011. Mol Plant Microbe Interact. 24 (6): 671-684). Of these genes, CaS113, which showed increased expression during pathogen infection, was selected for further study. Using the partial sequence of CaS113, putative full length cDNA was identified from the BAC sequence of the pepper variety CM334 (Yoo EY et al., 2003. Theor Appl Genet. 107 (3): 540-543). The predicted gene encodes 493 amino acids with a 58 kDa molecular weight consisting of signal peptide, non-repeat region, multi-repeat region and another non-repeat region (FIG. 1). The gene was then CaSD1 ( C. annuum senescence - delaying gene 1 ). In the multi-repeat region, one repeat unit consists of 15 different amino acids (KPPIHNHKPTDYDRS), often with one or two amino acids. CaSD1 derived from the 358 amino acid varieties was cloned (SEQ ID NO: 2) and the number of repetitions was less than that of the CM334 variety. Except for the repeat region, the other regions were identical (FIG. 1A). Further studies using genomic DNA PCR and sequencing showed that the number of repeat units varied in species and varieties of the genus Capsicum (FIG. 2). In addition, two genes were expected in the BAC sequence around CaSD1 , which had conservative signal peptides and non-repeat regions but no repeat regions (FIG. 3). Comparative genomic analysis using known plant genomes revealed that genes without repeat regions were present in the Solanacea family , whereas CaSD1 homologs with repeat regions were present only in Camcicum (FIG. 3). Further studies using the GenBank database did not identify any matching genes (data not shown). The results indicate that CaSD1 is a capsicum -specific gene.

Example  2: CaSD1 Organ-specific and pathogen-reactive expression of

CaSD1 To investigate organ-specific expression, real-time RT-PCR was performed using RNA samples derived from other pepper organs. CaSD1 constitutively expressed about 16-fold higher transcription levels in roots than in leaves (FIG. 4A). In previous studies, the expression level of CaSD1 increased in red pepper roots and then rapidly decreased after inoculation with Phytoprosora capsaish (Yeom SI et al., 2011. Mol Plant Microbe Interact. 24 (6): 671-684). Transcription level was CaSD1 pie Saratov shoot significantly higher in the resistant varieties susceptible varieties of CM334 than chilseongcho (Chilsungcho) when inoculated upon between the cap in the same amount. The transcription level of CaSD1 in red pepper leaves also increased after infection by other pathogens. In addition, similar expression patterns of CaSD1 were observed after viral and bacterial bacterial inoculation. Pepper varieties ECW30R were infected with Xav race 1 ( AvrBs3 / AvrBs3 ) and Xav race 3 ( avrbs3 / avrbs3 ), which are pepper spot bacteria. Xav race 1 and the mutual expression of CaSD1 after action has been increased compared to the interaction with Xav race 3 (Fig. 4B). Similarly, TMV P ₀ strains were inoculated into the leaves of the sinus cultivars (TMV resistant) and the ECW30R cultivars (TMV sensitive), and the expression of CaSD1 was induced only in the sinus cultivars (FIG. 4C). In addition, CaSD1 is a non-host pathogen Xag Induced by inoculation of 8ra, which induces hypersensitivity reactions in pepper leaves (FIG. 4D). In all cases, the transcription level of CaSD1 was significantly higher during incompatible pathogen interactions than during compatible pathogen interactions.

To investigate the effect of abiotic elicitors on the expression of CaSD1, pepper leaves were treated with SA, ET and MeJA. Expression of CaSD1 was induced within 1 hour after SA and ET treatment and remained high until 6 hours, while MeJA inhibited the expression of CaSD1 (FIGS. 4E-G). Taken together, the results indicate that CaSD1 may play a role in the pepper plant's protective response to pathogen infection.

Example  3: CaSD1 of Intracellular  location

To determine the intracellular location of CaSD1 , targeting experiments in plants were performed by a TOE using Agrobacterium delivering CaSD1 fused with the fluorescent marker smGFP (Davis SJ and Vierstra RD, 1998. Plant Mol Biol. 36 (4): 521-528). smGFP was fused at the C-terminus of CaSD1 and cloned into the pMBP1 vector for the TOE (Suh MC et al., 1998. Mol Cells. 8 (6): 678-684). Agrobacterium suspensions delivering either pMBP1: CaSD1-smGFP or pMBP1: smGFP were infiltrated with tobacco leaves. On day 1 after the TOE, the axial epidermal cell layers of the leaves were observed by confocal laser scanning microscopy. GFP fluorescence of pMBP1: CaSD1-smGFP was observed in the extracellular matrix including the cell wall, whereas pMBP1: smGFP was observed throughout the cell (FIG. 5). The results indicate that CaSD1 is secreted from and located therein the cell wall or extracellular space of tobacco plants.

Example  4: delay aging and Tricom ( trichome ) Causing formation CaSD1 of TOE

To investigate the function of CaSD1 in plants, pMBP1 (pBI121-modified) or Agrobacterium-mediated TOE of pMBP1: CaSD1 was performed in tobacco (Suh MC et al., 1998. Mol Cells. 8 (6): 678 -684). Significant differences between CaSD1- and empty vector-expressing plants were observed 7 days after inoculation. The CaSD1 -overexpression region showed aging delay, whereas aging was promoted in the empty vector pMBP1-expression region (FIG. 6A). In addition, the aging-delay region remained green spots after the leaves were separated (FIG. 6B), and the intact plant leaves were still pale green until the pMBP1-control leaves died (FIG. 6C). To further understand the effect of CaSD1 on aging, gene profiling experiments were performed. In plants, S1 ( Harpin - induced 1 ), a marker of cell death as well as cell death, and Sgr (Staygreen), which regulates chlorophyll degradation, were monitored (Park SY et al., 2007. Plant Cell. 19 (5): 1649-1664 ). The expression levels of these two genes were downregulated in CaSD1 -overexpressed plants compared to vector-control plants (FIG. 6D). On the other hand, the expression level of chlorophyll a / b binding protein gene (CAB) associated with photosynthesis progression was maintained. The results indicate that CaSD1 influences the expression level of certain aging-related genes.

Another unique phenotype of CaSD1 -overexpressing plants is the induction of tricom formation. Tricom are specific epidermal cells that are regularly distributed in plant leaves. Triome initiation sites (TIS) significantly increased approximately 10-fold at 10 days post-TOE in CaSD1 -overexpressing leaves compared to the empty vector (FIG. 7A). Compared to the tricom isolated from control leaves, some CaSD1 -overexpressing leaves generated more than one tricom per TIS, which resulted in adjacent tricomes and clusters (FIG. 7B). In addition, the size of epidermal cells in CaSD1 -overexpressing plants was significantly expanded before tricom cells developed compared to control plants (FIGS. 8A and 8B).

Tricom development and cell size are associated with cell cycle regulation, including endogenous replication (Dewitte W et al., 2007. Proc Natl Acad Sci US A. 104 (36): 14537-14542). To explain the molecular mechanism of cell cycle regulation in CaSD1 -overexpressing plants, semiquantitative RT-PCR of cell differentiation-related genes such as cyclin or cyclin-dependent kinase was performed. The gene expression levels were downregulated during aging in pMBP1-control plants, while still expressed in CaSD1 -overexpressing plants (FIG. 8C). The results indicate that CaSD1 may play a role in cell fate determination and cell division regulation.

<110> SNU R & DB FOUNDATION <120> CaSD1 gene from Capsicum annuum and uses <130> PN11188 <160> 34 <170> Kopatentin 2.0 <210> 1 <211> 1077 <212> DNA <213> Capsicum annuum <400> 1 atggtgtcag gtagtttcaa tattctgctg ctagtcctat ttgccttgat ggtcgttgcc 60 tccgcaggag gaaccagaaa attgttagtg aaacaagagt atatggttga tgatatttca 120 ggagatgcta gatttaattc aatgaagacc tctcatatga atgctcaggt ggatagccag 180 atgttcaaga agaatgaacg aaacttcctc aacatgaatc agtttccaga acaaaatcat 240 aagagaagca agaaagaaga atcaaaggag tttgttgatg tagaaaatgg agtaccaaag 300 cttataagca cagacgacca tggccacaaa ccgcccatcc ataatctcaa gcctacagac 360 tatgaccgca gcaagccgcc catccataat cacaagccta cagactatga ccgcagcaag 420 ccaccaatcc ataatcacaa ccctacagac tatgaccgca gcaagccacc aatccataat 480 cacaacccta cagactatga ccgcagcaag ccgcccatcc ataatcacaa gcctacagac 540 tatgaccgca gcaagccgcc catccataat cacaagccta cagactatga ccgcagcaag 600 ccaccaatcc ataatcacaa ccctacagac tatgaccgca gcaagccacc aatccataat 660 cacaagccta cagactatga ccgcagcaag ccgcccatcc ataatcacaa gcctacagac 720 tatgaccgca gcaagccgcc aatccataat cacaagccta cagactatga ccgcagcaag 780 ccgcccatcc ataatcacaa gcctacagac tatgaccgca gcaagccacc aatccataat 840 gacaacccta cagactatga ccgcagcaag ccgcccatgc ataatcacaa gcctacagac 900 tatgaccgca gcaagccgcc catccataat cacaagccta cagactatga ccgcagcaag 960 ccaccaatcc ataattacaa gcctacagac tatgaccgca attcgcccat caataattac 1020 aagccaaagg actatgagcg caagccacca atcaagaatc acaaacttac cgattag 1077 <210> 2 <211> 358 <212> PRT <213> Capsicum annuum <400> 2 Met Val Ser Gly Ser Phe Asn Ile Leu Leu Leu Val Leu Phe Ala Leu   1 5 10 15 Met Val Val Ala Ser Ala Gly Gly Thr Arg Lys Leu Leu Val Lys Gln              20 25 30 Glu Tyr Met Val Asp Asp Ile Ser Gly Asp Ala Arg Phe Asn Ser Met          35 40 45 Lys Thr Ser His Met Asn Ala Gln Val Asp Ser Gln Met Phe Lys Lys      50 55 60 Asn Glu Arg Asn Phe Leu Asn Met Asn Gln Phe Pro Glu Gln Asn His  65 70 75 80 Lys Arg Ser Lys Lys Glu Glu Ser Lys Glu Phe Val Asp Val Glu Asn                  85 90 95 Gly Val Pro Lys Leu Ile Ser Thr Asp Asp His Gly His Lys Pro Pro             100 105 110 Ile His Asn Leu Lys Pro Thr Asp Tyr Asp Arg Ser Lys Pro Pro Ile         115 120 125 His Asn His Lys Pro Thr Asp Tyr Asp Arg Ser Lys Pro Pro Ile His     130 135 140 Asn His Asn Pro Thr Asp Tyr Asp Arg Ser Lys Pro Pro Ile His Asn 145 150 155 160 His Asn Pro Thr Asp Tyr Asp Arg Ser Lys Pro Pro Ile His Asn His                 165 170 175 Lys Pro Thr Asp Tyr Asp Arg Ser Lys Pro Pro Ile His Asn His Lys             180 185 190 Pro Thr Asp Tyr Asp Arg Ser Lys Pro Pro Ile His Asn His Asn Pro         195 200 205 Thr Asp Tyr Asp Arg Ser Lys Pro Pro Ile His Asn His Lys Pro Thr     210 215 220 Asp Tyr Asp Arg Ser Lys Pro Pro Ile His Asn His Lys Pro Thr Asp 225 230 235 240 Tyr Asp Arg Ser Lys Pro Pro Ile His Asn His Lys Pro Thr Asp Tyr                 245 250 255 Asp Arg Ser Lys Pro Pro Ile His Asn His Lys Pro Thr Asp Tyr Asp             260 265 270 Arg Ser Lys Pro Pro Ile His Asn Asp Asn Pro Thr Asp Tyr Asp Arg         275 280 285 Ser Lys Pro Pro Met His Asn His Lys Pro Thr Asp Tyr Asp Arg Ser     290 295 300 Lys Pro Pro Ile His Asn His Lys Pro Thr Asp Tyr Asp Arg Ser Lys 305 310 315 320 Pro Pro Ile His Asn Tyr Lys Pro Thr Asp Tyr Asp Arg Asn Ser Pro                 325 330 335 Ile Asn Asn Tyr Lys Pro Lys Asp Tyr Glu Arg Lys Pro Pro Ile Lys             340 345 350 Asn His Lys Leu Thr Asp         355 <210> 3 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> CaSD_full forward primer <400> 3 cgggatcccg atggtgtcag gtagtttcaa tatt 34 <210> 4 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> CaSD1_full reverse primer <400> 4 ggagctcgta ttgtttcatt tgtgaggaaa ga 32 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> CaSD1_repeat forward primer <400> 5 tccagaacaa aatcataaga gaagc 25 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> CaSD1_repeat reverse primer <400> 6 agcacgatta tgtagaatcc aaaag 25 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CaSD1_RT forward primer <400> 7 aaaggactat gagcgcaagc 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CaSD1_RT reverse primer <400> 8 ttgtgaggaa agaccgatca 20 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> CaAccOx forward primer <400> 9 agaaagctgc agaggaaagc aaac 24 <210> 10 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> CaAccOx reverse primer <400> 10 tgagatgcaa ccgttactcc tatac 25 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CaBPR1 forward primer <400> 11 tcacaatgca gctcgtagac a 21 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CaBPR1 reverse primer <400> 12 tttcattgac ccacatcttc ac 22 <210> 13 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> CaPINII forward primer <400> 13 ttcataatat ggctgttcac aaaga 25 <210> 14 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> CaPINII reverse primer <400> 14 agacgctcaa gcatattaca gagat 25 <210> 15 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CaUBQ forward primer <400> 15 cgtggtggtt tttaagatga tg 22 <210> 16 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> CaUBQ reverse primer <400> 16 aaagaaacac aagggaacag aaa 23 <210> 17 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CaActin forward primer <400> 17 ccacctcttc actctctgct ct 22 <210> 18 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CaActin reverse primer <400> 18 actaggaaaa acagcccttg gt 22 <210> 19 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> NtHin1 forward primer <400> 19 gagctatgta tacaaggcca gag 23 <210> 20 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> NtHin1 reverse primer <400> 20 gcttcacttc catctcaaaa ac 22 <210> 21 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> NtSgr forward primer <400> 21 aaagaactcc ctgtggttct ga 22 <210> 22 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> NtSgr reverse primer <400> 22 aatcccagtt gctattgctt gt 22 <210> 23 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> NtCAB forward primer <400> 23 acagagctct tgaggttatc ca 22 <210> 24 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> NtCAB reverse primer <400> 24 cacaacttga aaacctagca ca 22 <210> 25 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> NbActin forward primer <400> 25 ccaggtattg ctgatagaat gag 23 <210> 26 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> NbActin reverse primer <400> 26 ctgagggaag ccaagataga g 21 <210> 27 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> NtCYCD3; 1 forward primer <400> 27 aaatgattca tgggatttgg ag 22 <210> 28 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> NtCYCD3; 1 reverse primer <400> 28 caaccaccag ctaagctctt ct 22 <210> 29 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> NtCYCA forward primer <400> 29 ttcaaaacca gtccaatcac tg 22 <210> 30 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> NtCYCA reverse primer <400> 30 aagccttcac atttccacct aa 22 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NtCDKA; 4 forward primer <400> 31 tcctgttgat gtgtggtcag 20 <210> 32 <211> 22 <212> DNA <213> Artificial Sequence <220> NtCDKA; 4 reverse primer <400> 32 aatggaacat cagagcattg tg 22 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NtCDKB1-1 forward primer <400> 33 aatggacgaa gaagggattc 20 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NtCDKB1-1 reverse primer <400> 34 tgagagaagg agggagaggt 20

Claims (20)

Capsicum , consisting of the amino acid sequence of SEQ ID NO: 2 annuum) related to disease resistance promote or aging delay resulting CaSD1 (Capsicum annuum senescence - delaying 1) protein. A gene encoding the protein of claim 1. 3. The gene according to claim 2, wherein the gene consists of the nucleotide sequence of SEQ ID NO: 1. A recombinant vector comprising the gene of claim 2. A host cell transformed with the recombinant vector of claim 4. 6. The transformed host cell according to claim 5, which is a plant cell. A method of enhancing disease resistance of a plant comprising the step of transforming the recombinant vector of claim 4 into plant cells. 8. The method of claim 7, wherein said plant disease is a viral disease or a bacterial disease. A method for delaying aging of a plant, comprising the step of transforming the recombinant vector of claim 4 into plant cells, overexpressing the CaSD1 gene. Transforming a plant cell with the recombinant vector of claim 4; And
Method for producing a plant disease resistance enhanced transgenic plant comprising the step of regenerating the plant from the transformed plant cells. A transgenic plant having enhanced plant disease resistance prepared by the method of claim 10. The transgenic plant of claim 11, wherein the plant disease is a viral disease or a bacterial disease. Seeds of plants according to claim 11. Transforming a plant cell with the recombinant vector of claim 4; And
Method for producing a delayed aging transformed plant comprising the step of regenerating the plant from the transformed plant cells. A transgenic plant with delayed aging produced by the method of claim 14. 16. The transgenic delayed plant according to claim 15, wherein the plant is a dicotyledonous plant. 15. A plant seed according to claim 15. A composition for enhancing disease resistance of a plant, comprising the gene of claim 2. 19. The composition of claim 18, wherein the plant disease is a viral disease or a bacterial disease. A composition for delaying aging of a plant, comprising the gene of claim 2.

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