WO2012148121A2 - Atpg7 protein having functions of increased productivity, delayed aging, and resistance to stress of plant, gene thereof, and use thereof - Google Patents

Abstract

Disclosed in the present invention are an ATPG7 protein having functions of increased productivity, delayed aging, and resistance to stress of a plant, a gene thereof, and a use thereof. The transformed and overexpressed plant body not only shows a feature of increased productivity, but also features of delayed aging and resistance to stress.

Description

AtpX7 protein, its genes, and uses thereof, which have a function of increasing productivity, delaying aging, and stress resistance of plants

The present invention relates to an ATPG7 protein, a gene thereof, and a use thereof having a productivity enhancing function, a aging delay function, and a stress resistance function of a plant.

Plant aging is the final stage of plant development, an age-dependent process of decay at the cellular, tissue, organ, or organismal level, leading to the lethal stage through the growth and development stages. As aging progresses, the plant gradually loses its ability to synthesize and loses its homeostasis as its intracellular structures and macromolecules break down sequentially (Thomas et al., 1993). The aging of these plants is genetically planned as a series of consecutive biochemical and physiological phenomena that are very sophisticated and active at the level of cells, tissues and organs.

The initial phenomenon of aging in cell structure is the degradation of chloroplasts, which are organelles containing more than 70% of leaf protein. In metabolic terms, this means that carbon assimilation in plants is converted to catabolism of chlorophyll and macromolecules such as proteins, membrane lipids, and RNA. Increased catabolic activity through aging induces the conversion of cellular components accumulated in the assimilated leaves during growth into ventilated cellular components supplied for the development of seeds or other storage organs. Thus, aging of plants is thought to be a process of cell degeneration as well as a genotype actively acquired to adapt to the environment during evolution (Buchanan-Wollaston et al., 2003; Lim and Nam, 2005; Nam, 1997).

Aging of such plants is influenced by internal environmental factors such as plant hormones and external environmental factors such as drought, nutrient restriction, pathogen penetration, and the like. Among the plant hormones, cytokinin is a physiologically delayed aging hormone and many aging control techniques have been reported. The Amasino group developed a method for regulating aging-specific cytokinin synthesis by recombining the IPT gene into a senescence-specific SAG12 gene promoter, which showed a 50% increase in productivity in cigarettes that delayed aging. When introduced into the lettuce in the same way it can be seen that the shelf life after harvest significantly increased (McCabe et al., 2001). In addition, there was a report that the cytokinin level was increased and leaf aging was delayed in tobacco expressing corn homeobox gene (knotted1) in SAG12 promoter. Tomatoes have been reported to control the ripening of fruit through ethylene control.Flav-O-Savor, which has been shown to increase the transport and storage of tomatoes by inhibiting the expression of polygalacturonase genes related to cell wall degradation, is commercialized. This can be an example. In the case of apples, it is reported that one of the domestic varieties bred to delay aging is accompanied by a mutation of the ACC oxidase gene, a ethylene synthetic gene.

Recently, many studies have been conducted on the isolation of genes induced in aging and analysis of their expression patterns in order to investigate aging regulation. Analysis of genes with increased expression in aging was studied in Arabidopsis, radish, tomato, etc. Analysis of expression patterns suggests that pathways of aging form a very complex network. Recently, subtractive hybridization and microarray By using the method, genes induced during aging are separated in large quantities. Among them, expression analysis is performed by targeting genes such as transcription factor or receptor-like kinase, which are presumed to be aging regulator genes. Many of the 96 transcription factors with increased expression during the aging process were proteins with NAC, WRKY, C2H2-type zinc fingers, Ap2 / EREBP, and MYB domains (Lim et al., 2007). Inhibition of expression of WRKY53 gene among WRKY transcription factors caused delayed aging in plants, whereas increased expression caused early aging of plants. The WRKY53 gene thus appears to be a positive regulator of plant aging (Miao et al., 2004). In addition, the NAC transcription factor, AtNAP gene, is reported to be a positive regulator of plant aging as well as the gene (Guo and Gan, 2006). On the other hand, the expression of GmSARK in soybean, one of the receptor-like kinases, is up-regulated not only in naturally occurring aging but also in the artificial aging process by cancer treatment. Inhibition of this gene is known to cause delay in leaf aging (Li et al. al., 2006).

Recently, reactive oxygen species (ROS) are known to play an important role in plant aging. A catalase derived from a particular isoform are peroxysome to the Arabidopsis thaliana that control the aging of the plant with APX1 from a material proposed in Zentgraf group (Zimmermann et al., 2006) .

On the other hand, from an agricultural point of view, aging of a plant may limit crop productivity due to limitations on the growth stage of the plant, and may also cause quality loss rates such as leaf yellowing and nutrient loss in vegetable crops. Therefore, research on plant aging can not only increase the understanding of plant growth processes but also provide plant aging control, which can lead to improvement of agricultural traits such as crop productivity and shelf life. Gan et al . (1995) have been able to increase productivity by up to 50% by controlling aging in tobacco, and also by increasing control of aging in grains such as soybeans (Guiamett et al ., 1990). Could. However, research on increasing productivity through regulating plant aging is still very limited.

For this reason, plant biotechnologists are trying to find genes and proteins that are involved in life extension in plants.

An object of the present invention is to provide an ATPG7 protein which has a function of delaying aging of plants and a function of increasing productivity.

Another object of the present invention is to provide a gene encoding the protein.

Still another object of the present invention is to provide a method for producing a plant having aging delay characteristics.

Still another object of the present invention is to provide a method for producing a plant having a yield increasing characteristic.

Still another object of the present invention is to provide a method for producing a plant having stress resistance.

Other objects of the present invention will be presented below.

In one aspect, the present invention relates to an ATPG7 protein which has a function of delaying aging of plants and a function of increasing productivity.

The inventor (s) is to isolate the gene of the protein based on the nucleotide sequence of the DNA binding protein-related protein (DNA-binding protein-related, GeneBank accession number NP 194012.1) as confirmed in the following Examples When the gene is overexpressed by transforming in the Arabidopsis, the aging retardation phenomenon of the plant is apparent, and the productivity increase characteristics such as the increase in the biomass and / or the seed productivity of the individual are also apparent, and in addition to the drought stress or the oxidative stress It was also confirmed that the resistance characteristics were also apparent.

These experimental results can be said to mean that the genes and proteins delay the aging of the plant, increase the productivity of the plant and participate in the resistance to stress.

The present inventors have named the gene to gene and protein ATPG7 ATPG7 (AT -hook p rotein of G enomine 7), these nucleotide sequences and amino acid sequences are disclosed in SEQ ID NOS: 1 and 2, respectively.

The ATPG7 protein of the present invention is one of the polypeptides of (a), (b) and (c) below.

(a) a polypeptide comprising the entire amino acid sequence of SEQ ID NO: 2;

(b) a polypeptide comprising a substantial portion of the amino acid sequence set forth in SEQ ID NO: 2; And

(c) a polypeptide substantially similar to the polypeptide of (a) or (b) above.

As used herein, the terms "protein" are used interchangeably with each other in the same sense as a polypeptide, and the term "gene" is used interchangeably with each other in the same sense as a polynucleotide.

As used herein, a "polypeptide comprising a substantial portion of the amino acid sequence set forth in SEQ ID NO: 2" is still sufficient to retain plant aging delay and productivity enhancing functions as compared to the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: And a polypeptide comprising a portion of the amino acid sequence of SEQ ID 2 of. The length of the polypeptide and the degree of activity of such a polypeptide does not matter since it still needs to be sufficient to retain the function of delaying aging and increasing productivity of the plant. That is, even if the activity is lower than that of the polypeptide comprising the amino acid sequence of SEQ ID NO: 2, if the polypeptide still has the aging delay function and productivity enhancing function, the length of the "including the substantial portion of the amino acid sequence of SEQ ID NO: 2 Polypeptide ". Those skilled in the art, that is, those who are familiar with the related prior art known on the basis of the present application, even if a portion of the polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2 is still deleted, such polypeptide still exhibits delayed aging function and increased productivity. Expect to retain. As such a polypeptide, the polypeptide which deleted the N-terminal part or C-terminal part in the polypeptide containing the amino acid sequence of SEQ ID NO: 2 is mentioned. This is because it is generally known in the art that even if the N- or C-terminal portion is deleted, such a polypeptide has the function of the original polypeptide. Of course, in some cases, the N-terminal or C-terminal moiety is necessary for maintaining the function of the protein, so that a polypeptide deleted with the N-terminal or C-terminal moiety does not exhibit this function. It is within the ordinary skill of one of ordinary skill in the art to distinguish and detect inactive polypeptides from active polypeptides. Furthermore, the deletion of the N-terminal or C-terminal moiety as well as other moieties may still have the function of the original polypeptide. Here too, one of ordinary skill in the art will be able to ascertain whether such deleted polypeptide still has the function of the original polypeptide within the scope of its usual ability. In particular, the present specification discloses the nucleotide sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2 and further, the polypeptide encoded by the nucleotide sequence of SEQ ID NO: 1 and consisting of the amino acid sequence of SEQ ID NO: 2 increases the delaying function and productivity of the plant In the present disclosure, the present invention confirms whether a polypeptide having a partial deletion in the amino acid sequence of SEQ ID NO: 2 still retains the function of the polypeptide including the amino acid sequence of SEQ ID NO: 2. It becomes very clear that it can fully confirm within a normal capability range. Therefore, in the present invention, the "polypeptide comprising a substantial part of the amino acid sequence as set forth in SEQ ID NO: 2", as defined above, delays the aging of a plant that a person skilled in the art can manufacture within the range of its usual capacity based on the disclosure herein. It is to be understood as meaning including all polypeptides in a deleted form that have function and productivity enhancing function.

Also herein, "polypeptide substantially similar to the polypeptides of (a) and (b)" includes a function comprising one or more substituted amino acids, but comprising the amino acid sequence of SEQ ID NO: 2, ie aging of a plant. It refers to a polypeptide having a delaying function and a productivity increasing function. Here too, the degree of activity or amino acid substitution of the polypeptide is not a problem as long as the polypeptide containing at least one substituted amino acid retains the function of delaying aging and increasing productivity of the plant. In other words, even if the polypeptide comprising one or more substituted amino acids contains a large number of substituted amino acids, even if the activity is low compared to the polypeptide comprising the amino acid sequence of SEQ ID NO: 2, such polypeptides may be used in plant aging. It is included in the present invention as long as it has a delay function and a productivity increase function. Even if one or more amino acids are substituted, if the amino acid before substitution is chemically equivalent to the substituted amino acid, the polypeptide comprising such substituted amino acid will still retain the function of the original polypeptide. For example, even if the hydrophobic amino acid alanine is substituted with another hydrophobic amino acid such as glycine or a more hydrophobic amino acid such as valine, leucine or isoleucine, the polypeptide having such substituted amino acid (s) may be It will still retain the function of the original polypeptide. Likewise, even if a negatively charged amino acid such as glutamic acid is replaced with another negatively charged amino acid such as aspartic acid, a polypeptide having such substituted amino acid (s) will still retain the function of the original polypeptide even if its activity is low. Also, even if a positively charged amino acid such as arginine is replaced with another positively charged amino acid such as lysine, the polypeptide having such substituted amino acid (s) will still retain the function of the original polypeptide even if its activity is low. will be. In addition, a polypeptide comprising amino acid (s) substituted at the N-terminal or C-terminal portion of the polypeptide will still retain the function of the original polypeptide. Those skilled in the art can produce a polypeptide comprising at least one substituted amino acid as described above, while still retaining the aging delaying function and productivity enhancing function of the polypeptide comprising the amino acid sequence of SEQ ID NO: 2. One skilled in the art can also determine whether a polypeptide comprising one or more substituted amino acids still has this function. Moreover, the present specification discloses an example in which the base sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2 are disclosed, and the polypeptide comprising the amino acid sequence of SEQ ID NO: 2 has a function of delaying aging and increasing productivity of plants. It is clear that the "polypeptide substantially similar to the polypeptide of (a) and (b)" of the present invention can be easily implemented by those skilled in the art. Therefore, a "polypeptide substantially similar to the polypeptide of (a) or (b) above" should be understood as meaning including all polypeptides that contain one or more substituted amino acids but still have the ability to delay aging and increase productivity of plants. As such, "polypeptide substantially similar to the polypeptide of (a) or (b)" is meant to include all polypeptides that contain one or more substituted amino acids but still have the ability to delay aging and increase productivity of plants, but nevertheless active In view of the degree, the polypeptide is preferably higher in sequence homology with the amino acid sequence of SEQ ID NO. Preferably, the polypeptide has at least 60% sequence homology at the lower end of sequence homology, while at the upper limit of sequence homology, it is preferred that the polypeptide has 100% sequence homology. More specifically, the above sequence homology is 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% in order of higher. And "polypeptides substantially similar to the polypeptides of (a) and (b) above" of the present invention are not only "polypeptides substantially similar to polypeptides comprising the entire amino acid sequence of SEQ ID NO: 2", All descriptions above are intended to include "substantially similar to polypeptides comprising the entire amino acid sequence of SEQ ID NO: 2" as well as "amino acids of SEQ ID NO: 2", since the polypeptide comprising substantially part thereof is included. The same applies to polypeptides that are substantially similar to polypeptides comprising substantial portions of the sequence.

In another aspect, the invention is directed to an isolated polynucleotide encoding a polypeptide as described above. "Polypeptide as described above" herein refers to a polypeptide comprising the entire amino acid sequence of SEQ ID NO: 2, a polypeptide comprising a substantial portion of the amino acid sequence of SEQ ID NO: 2, having a function of delaying aging and increasing productivity of a plant, and In addition to including polypeptides substantially similar to the above polypeptides, it is meant to include all polypeptides of the preferred embodiments as described above. Therefore, the polynucleotide of the present invention is substantially isolated from the isolated polynucleotide and the polypeptides encoding the polypeptide including all or a substantial part of the amino acid sequence described in SEQ ID NO: 2, while having a function of delaying aging and increasing productivity of plants. Encoding an isolated polypeptide includes an isolated polynucleotide, and furthermore, in a preferred embodiment, an isolation that encodes all polypeptides having the sequence homology in the sequence sequence homology as described above, with the ability to delay aging and increase productivity of plants. Polynucleotides. When amino acid sequences are found, those skilled in the art can readily prepare polynucleotides encoding such amino acid sequences based on those amino acid sequences.

Meanwhile, the term “isolated polynucleotide” herein includes both chemically synthesized polynucleotides, polynucleotides isolated from organisms, especially Arabidopsis thaliana , and polynucleotides containing modified nucleotides, which are single-stranded or double-stranded. It is defined as including all polymers of stranded RNA or DNA.

In another aspect, the present invention relates to a method for producing a plant with delayed aging.

The method for producing a delayed aging plant of the present invention comprises the steps of: (a) overexpressing a gene having a nucleotide sequence of SEQ ID NO: 1 or a gene having a sequence similar to the nucleotide sequence of SEQ ID NO: 1 in the plant; and (b) a phenotype of delayed aging. It comprises a step of selecting a plant having a.

As used herein, the term "aging delay" refers to a property of prolonged plant life as compared to wild-type plants, and specifically, the yellowing and / or necrosis of leaves and / or stems is delayed compared to wild-type plants or the chlorophyll content of plants is wild-type. It is more characteristic than plants or photosynthetic efficiency of plants is higher than wild type plants.

In addition, in the present specification, "plant" is meant to include mature plants, immature plants (plants), plant seeds, plant cells, plant tissues and the like. When plant cells or plant tissues are used for transformation, the transformed plant cells or plant tissues are described in European Patent EP0116718, European Patent EP0270822, International Patent WO 84/02913, Gould et al. 1991, Plant Physiol 95,426-434, etc., can be used to develop and grow into mature plants.

Also herein, "plant" includes all plants for which delayed aging can give useful results to humans. The delay in aging is directly related to an increase in production, i.e. an increase in seed productivity and / or biomass of an individual, so in the sense of the above plants, productivity is primarily useful for human crops such as rice, wheat, barley, corn, soybeans, potatoes and red beans. , Oats, sorghum, cruciferous vegetables (cabbage, bok choy, kale, cauliflower, broccoli, young radish, radish, fresh, etc.), pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, green onion, onion Includes carrots, ginseng, tobacco, cotton, sesame seeds, sugarcane, beets, perilla, peanuts, rapeseed, apple trees, pears, jujube trees, peaches, yolk, grapes, citrus fruits, persimmons, plums, apricots, bananas And other bioenergy crops such as switchgrass, silver grass, reed, and other lygras, red clover, orchardgrass, alphaalpha, tall pescue, perennial lice, rose, gladiolus, gerbera, carnation, chrysanthemum, lily, tulip This will be included.

In addition, in the present specification, "gene consisting of a sequence similar to the nucleotide sequence of SEQ ID NO: 1" is a gene encoding the first amino acid of SEQ ID NO: 2 while having a base sequence different from that of the gene of SEQ ID NO: 1 due to codon degeneracy And, as a homologue of genes consisting of the nucleotide sequence of SEQ ID NO: 1, all of the nucleotide sequence of SEQ ID NO: 1 and other nucleotide sequences due to the evolutionary pathways different according to the type of plant, having the aging delay function of the plant It is meant to include genes. Herein, the gene consisting of a sequence similar to the nucleotide sequence of SEQ ID NO: 1 is preferably higher in sequence homology with the nucleotide sequence of SEQ ID NO: 1, and most preferably, having 100% sequence homology. On the other hand, in the lower limit of sequence homology, it will be preferable that the gene has a sequence homology of 60% or more with the nucleotide sequence of SEQ ID NO: 1. More specifically, the above sequence homology is 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73 %, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, Higher in order of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% is preferred.

In addition, in the present specification, "overexpression" means expression above the level expressed in wild-type plants. Such "overexpression" can be directly determined by quantifying the gene of SEQ ID NO: 1 or a gene consisting of a sequence similar to the nucleotide sequence of SEQ ID NO: 1, or indirectly by quantifying the protein encoded by the gene. have.

In the method for producing a plant in which aging is delayed according to the present invention, the step (a) may be performed by a genetic engineering method.

Genetic engineering method includes the steps of: (i) inserting a gene having the sequence of SEQ ID NO: 1 or a sequence similar to the sequence of SEQ ID NO: 1 into an expression vector to be operably linked to a regulatory sequence capable of overexpressing it, and ( ii) transforming the expression vector into a plant.

As used herein, "operably" means that the transcription and / or translation of a gene is linked to be affected. For example, if a promoter influences the transcription of a gene linked to it, the promoter and the gene are operably linked.

In addition, in the present specification, "regulatory sequence" is meant to include all sequences whose presence may affect the transcription and / or translation of a gene linked thereto, and such regulatory sequences include a promoter sequence and a polyadenylation signal. ), The replication start point.

Also herein, "promoter" follows the conventional meaning known in the art, specifically located upstream (5 'side) based on the transcription initiation point of a gene and binding to DNA-dependent RNA polymerase. By nucleic acid sequences having the function of controlling transcription of one or more genes, including sites, transcriptional initiation sites, transcription factor binding sites, and the like. Such a promoter may be a TATA box upstream of the transcription initiation point (usually at the transcription initiation point (+1) -20 to -30 position), CAAT box (usually approximately -75 position relative to the transcription initiation site if it is of eukaryotes Present), a 5 'enhancer, a transcription repression factor, and the like.

Usable promoters include constitutive promoters (promoters which induce constant expression in all plant tissues), inducible promoters (expression of target genes in response to specific external stimuli) as long as they are promoters capable of overexpressing the gene of SEQ ID NO. 1 linked thereto. Promoters that induce expression or promoters that specifically induce expression in specific developmental periods or specific tissues). Representative examples of constitutive promoters that can be used include the promoter of the 35S RNA gene of cauliflower mosaic virus (CaMV), and the ubiquitin family of promoters (Christensen et al., 1992, Plant Mol). Biol. 18, 675-689; EP0342926; Cornejo et al., 1993, Plant Mol. Biol. 23, 567-581), rice actin promoter (Zhang et al. 1991, The Plant Cell 3, 1155-1165), etc. Can be mentioned. Examples of inducible promoters that can be used include the yeast metallothionein promoter (Mett et al., Proc. Natl. Acad. Sci., USA, 90: 4567, 1993), which is activated by copper ions, substituted by substituted benzenesulfonamides. In2-1 and In2-2 promoters (Hershey et al., Plant Mol. Biol., 17: 679, 1991), GRE regulatory sequences regulated by glucocorticoids (Schena et al., Proc. Natl. Acad. Sci., USA, 88 : 10421, 1991), ethanol regulating promoters (Caddick et al., Nature Biotech., 16: 177, 1998), light regulating promoters derived from bovine subunits of ribulose bis-phosphate carboxylase (ssRUBISCO) (Coruzzi et al. , EMBO J., 3: 1671, 1984; Broglie et al., Science, 224: 838, 1984), mannino synthase promoter (Velten et al., EMBO J., 3: 2723, 1984), nopalin synthase (NOS) Promoter, octopin synthase (OCS) promoter, heat shock promoter (Gurley et al., Mol. Cell. Biol., 6: 559, 1986; Severin et al., Plant Mol. Biol., 15: 827, 1990) And a gluten promoter, a soy-derived lectin promoter, a cabbage-derived napin promoter, and the like.

The transcription termination sequence is a sequence that acts as a poly (A) addition signal (polyadenylation signal) to enhance the integrity and efficiency of transcription. Examples of transcription termination sequences that may be used include the transcription termination sequence of the nopaline synthase (NOS) gene, the transcription termination sequence of the rice α-amylase RAmy1 A gene, and the transcription termination of the Octopine gene of Agrobacterium tumefaciens. A sequence, a transcription termination sequence of wheat heat shock protein 17, a transcription termination sequence of wheat ubiquitin gene, a transcription termination sequence of rice gluterin gene, a transcription termination sequence of rice lactate dehydrogenase gene, and the like.

The expression vector may include a selection marker gene. As used herein, "marker gene" refers to a gene encoding a trait that enables the selection of a plant comprising such a marker gene. The marker gene may be an antibiotic resistance gene or may be a herbicide resistance gene. Examples of suitable selectable marker genes include genes of adenosine deaminase, genes of dihydrofolate reductase, genes of hygromycin-B-phosphortransferase, genes of thymidine kinase, genes of xanthine-guanine phosphoribosyltransfer Laze gene, phosphinnothricin acetyltransferase gene, etc. are mentioned.

As used herein, the term "transformation" refers to a modification of the genotype of a host plant by the introduction of a hereditary gene, and regardless of the method used for the transformation, the herb gene is a host plant, more precisely a cell of the host plant. Introduced into and integrated into the genome of a cell. Here, the hereditary genes include homologous and heterologous genes, wherein "homologous genes" refer to endogenous genes of a host organism or the same species, and "heterologous genes" are genes that do not exist in the organism to which they are transformed. Say. For example, Arabidopsis derived genes are homologous to Arabidopsis plants, but heterologous to tomato plants.

Meanwhile, a method of transforming a plant with an exogenous gene may use a method known in the art, such as a direct gene transfer method using a gene gun, an in planta transformation method using a floral dip, pollen mediation, and the like. Transformation methods, protoplast transformation methods, viral mediated transformation methods, liposome mediated transformation methods, and the like can be used. In addition, it is also possible to select and use a transformation method suitable for a specific plant, for example, a method for transforming corn is described in US Pat. No. 6,140,553, Fromm et al, 1990, Bio / Technology 8, 833-839, Gordon- Kamm et al, 1990, The Plant Cell 2, 603-618) and the like can be used, methods for transforming rice are described in Shimamoto et al, 1989, Nature 338, 274-276, Datta et al 1990, Bio / Technology 8, 736-740), international patent WO 92/09696, international patent WO 94/00977, international patent WO 95/06722 and the like. Also, in tomato or tobacco transformation (An G. et al., 1986, Plant Physiol. 81: 301-305), Horsch RB et al, 1988, In: Plant Molecular Biology Manual A5, Dordrecht, Netherlands, Kluwer Academic Publishers, pp 1-9, Koornneef M. et al, 1986, In: Nevins DJ. And RA Jones, eds. Tomato Biotechnology, New York, NY, USA, Alan R. Liss, Inc. pp 169 -178) and the like can be used.

Generally used in transforming plants is a method of infecting seedlings, plant seeds and the like with the transformed Agrobacterium.

Such Agrobacterium mediated transformation methods are well known in the art (Chilton et al., 1977, Cell 11: 263: 271; European Patent EP 0116718; US Patent US 4,940,838), and methods suitable for particular plants are also known in the art. Known in See, for example, US Pat. No. 5,159,135 for cotton, US Pat. No. 5,824,877 for soybean, US Pat. No. 5,591,616 for corn, and the like. The Agrobacterium mediated transformation method uses Ti-plasmid, which will contain left and right border sequences that allow the integration of T-DNA into the genome of plant cells.

On the other hand, the (b) screening step is to develop and grow the transformed plant, and to visually select through the progress of the leaf yellowing or the progression of leaf necrosis, or when the selection marker gene is transformed When transformed together, the selection may be performed using a selection marker gene, and further, may be selected through a method of quantifying chlorophyll content, photosynthetic efficiency, and the like, and a method of mixing the above methods.

In another aspect, the present invention relates to a method for producing a plant having productivity enhancing properties of the present invention.

Method for producing a plant having a productivity enhancing feature of the present invention comprises the steps of (a) overexpressing a gene having a nucleotide sequence of SEQ ID NO: 1 or a gene having a sequence similar to the nucleotide sequence of SEQ ID NO: 1 in the plant and (b) increased productivity And selecting the plant having the characteristic.

As used herein, "productivity enhancing properties" means that the biomass (size and / or mass) of the whole, stem, root and / or leaves of the plant is increased compared to wild type plants and / or the productivity of the seed of the plant (plant 1). Number and / or mass of seeds per individual) is increased compared to wild type plants.

Step (a) may be carried out genetically, as described above with respect to the method for producing a aging delayed plant of the present invention for this genetic engineering method.

The step (b) may be selected by comparing the biomass and / or seed productivity of the plant, or when the selection marker gene is transformed together at the time of transformation, may be selected using the selection marker gene, or a method thereof It may be selected by mixing.

In another aspect, the present invention relates to a method for producing a stress resistant plant.

The method of producing a stress resistant plant of the present invention comprises the steps of: (a) overexpressing a gene having the nucleotide sequence of SEQ ID NO: 1 or a gene having a sequence similar to the nucleotide sequence of SEQ ID NO: 1 in the plant, and (b) having a stress resistant phenotype Comprising plant screening.

As used herein, "stress" refers to drought stress and / or oxidative stress.

Step (a) may be performed genetically, as described above with respect to the method for producing an aging delayed plant of the present invention for this genetic engineering method.

The step (b) is selected by comparing the stress resistance of the plant (e.g., the progress of leaf sulfidation, the progression of leaf necrosis, the biomass of the leaves and / or stems, chlorophyll content, photosynthetic efficiency, etc.) When the selection marker gene is transformed together at the time, the selection marker gene may be used for selection, or a combination thereof may be used for selection.

In another aspect, the present invention relates to a method for delaying aging of a plant of the present invention.

The method for delaying aging of a plant of the present invention is to (a) operably link a gene having a nucleotide sequence of SEQ ID NO: 1 or a gene having a sequence similar to that of SEQ ID NO: 1 to a regulatory sequence capable of overexpressing it. Inserting into the expression vector and (b) transforming the expression vector into a plant.

In another aspect, the present invention relates to a method for increasing productivity of a plant of the present invention.

The method for increasing the productivity of a plant of the present invention includes (a) an expression vector such that a gene having a nucleotide sequence of SEQ ID NO: 1 or a gene having a sequence similar to that of SEQ ID NO: 1 is operably linked to a control sequence capable of overexpressing it And (b) transforming the expression vector into a plant.

In another aspect, the present invention relates to a method for increasing stress resistance of a plant.

The method of increasing the stress resistance of a plant of the present invention is (a) operably linking a gene having a nucleotide sequence of SEQ ID NO: 1 or a gene having a sequence similar to that of SEQ ID NO: 1 to a regulatory sequence capable of overexpressing it Inserting into the expression vector preferably and (b) transforming the expression vector into a plant.

Steps (a) and (b) in the above methods are the same as those described in connection with the method for producing the delayed aging plant of the present invention.

In another aspect, the present invention is the delayed aging of the gene having a nucleotide sequence of SEQ ID NO: 1 or a gene having a sequence similar to the nucleotide sequence of SEQ ID NO: 1 obtained by the method for producing an aging delayed plant of the present invention The present invention relates to a transgenic plant having characteristics.

In a preferred aspect, the plant is a transgenic plant having delayed aging characteristics by being introduced into a gene encoding the ATPG7 protein consisting of the amino acid sequence of SEQ ID NO: 2, in particular, a gene having a nucleotide sequence of SEQ ID NO: 1 and overexpressed.

In another aspect, the present invention is an overexpression of a gene having a nucleotide sequence of SEQ ID NO: 1 or a gene having a sequence similar to the nucleotide sequence of SEQ ID NO: 1 obtained by the method for producing a plant having a productivity enhancing feature of the present invention The present invention relates to a transgenic plant having improved productivity.

In a preferred aspect, the plant is a transgenic plant having productivity enhancing properties by introducing and overexpressing a gene encoding the ATPG7 protein consisting of the amino acid sequence of SEQ ID NO: 2, in particular, the gene ATPG7 having the nucleotide sequence of SEQ ID NO: 1.

In still another aspect, the present invention provides an increase in productivity in which a gene having a nucleotide sequence of SEQ ID NO: 1 or a gene having a sequence similar to the nucleotide sequence of SEQ ID NO: 1 obtained by the method for preparing a stress resistant plant of the present invention is overexpressed. The present invention relates to a transgenic plant having characteristics.

In a preferred aspect, the plant is a transgenic plant having stress resistance by introducing and overexpressing a gene encoding the ATPG7 protein consisting of the amino acid sequence of SEQ ID NO: 2, in particular, the gene ATPG7 having the nucleotide sequence of SEQ ID NO: 1.

In the present specification, the "transformed plant" refers to a plant cell, a plant tissue, or a plant seed capable of developing and growing as a mature plant, when the gene is introduced and transformed, as well as by mating with the transformed plant. Genomes include altered plants, plant seeds, plant cells.

As described above, according to the present invention, it is possible to provide an ATPG7 protein and its gene having a function of delaying aging and increasing productivity of plants. Since the gene has a function of delaying aging and has a function of increasing productivity, when transforming a plant with this gene, the gene may be delayed in aging of the plant and have a function of increasing productivity of the plant.

Fig. 1 shows the structure (schematic diagram) of the pCSEN-ATPG7 recombinant vector in which the ATPG7 gene, which has a function of delaying aging of plants and has a function of increasing productivity, is introduced in the sense direction.

Figure 2 is a photograph of the Arabidopsis grown 60 days after germination of T ₁ plants transformed with the pCSEN-ATPG7 recombinant vector of Figure 1 above.

Con: Baby Pole Wild Type or Control

AT7-4 : Arabidopsis T ₁ plant transformed with pCSEN-ATPG7 recombinant vector

AT7-8 : Arabidopsis T ₁ plant transformed with pCSEN-ATPG7 recombinant vector

Figure 3 shows the results of analyzing the expression of the ATPG7 gene in Arabidopsis thalass cultivated for 20 days after cotyledon generation T ₂ line transformed with the pCSEN-ATPG7 recombinant vector of Figure 1 through qRT-PCR will be.

Wt: Baby Pole Wild

ATPG7 ox-4 : Arabidopsis T ₂ line transformed with pCSEN-ATPG7 recombinant vector

ATPG7 ox-5 : Arabidopsis T ₂ line transformed with pCSEN-ATPG7 recombinant vector

ATPG7 ox-6 : Arabidopsis T ₂ line transformed with pCSEN-ATPG7 recombinant vector

Figure 4 shows the results of analyzing the expression of the ATPG7 gene in the various plant organs of Arabidopsis wild type through qRT-PCR.

S: seedling, R: root, Ar: arial region, GL: green leaf, YL: yellow leaf, St: stem, F: inflorescence organ

Figure 5 is a photograph of the Arabidopsis grown 50 days and 70 days after germination of the Arabidopsis T ₂ line transformed with the pCSEN-ATPG7 recombinant vector of FIG.

Con: Baby Pole Wild Type or Control

ATPG7 ox-4 : Arabidopsis T ₂ line transformed with pCSEN-ATPG7 recombinant vector

ATPG7 ox-5 : Arabidopsis T ₂ line transformed with pCSEN-ATPG7 recombinant vector

ATPG7 ox-6 : Arabidopsis T ₂ line transformed with pCSEN-ATPG7 recombinant vector

6 is a diagram for increasing the productivity of the Arabidopsis line of FIG.

Con: Baby Pole Wild Type or Control

ATPG7 ox-4 : Arabidopsis T ₂ line transformed with pCSEN-ATPG7 recombinant vector

ATPG7 ox-5 : Arabidopsis T ₂ line transformed with pCSEN-ATPG7 recombinant vector

ATPG7 ox-6 : Arabidopsis T ₂ line transformed with pCSEN-ATPG7 recombinant vector

FIG. 7 shows the Arabidopsis wild type (Con), delayed aging-induced variants ATPG7 ox-4, ATPG7 ox-5 , and ATPG7 ox-6 from day 3 and 12, respectively. A picture of the phenotype of a leaf observed up to 40 days per day.

FIG. 8 shows every 4-4 leaves of the Arabidopsis wild type (Con), delayed aging-induced variants ATPG7 ox-4, ATPG7 ox-5 , and ATPG7 ox-6 from day 12 after cotyledon production. Figure shows the chlorophyll content of leaves for up to 40 days per day.

9 shows that every 12 to 4 leaves of Arabidopsis wild-type (Con), ATPG7 ox-4, ATPG7 ox-5 , and ATPG7 ox-6 , 3-4 leaves, from cotyledon generation after 12 days The photosynthetic efficiency of leaves up to 40 days per day was investigated by Fv / Fm.

FIG. 10 shows every 4-4 leaves of the Arabidopsis wild-type (Con), delayed aging-induced variants ATPG7 ox-4, ATPG7 ox-5 , and ATPG7 ox-6 from day 12 after cotyledon production. Expression of senescence marker genes in leaves up to 40 days per day was analyzed by qRT-PCR, and ACT was used as a PCR-positive control. CAB2 is a chlorophyll a / b binding protein gene, SEN4 and SAG12 are aging genes and aging marker genes.

11 shows detachment of the left lobe of the Arabidopsis wild-type (Con), ATPG7 ox-4, ATPG7 ox-5 , and ATPG7 ox-6 3-4 days after germination, to maintain cancer A picture of the phenotype of the leaves every 12 days until 12 days.

Figure 12 detaches the left lobe of the Arabidopsis wild-type (Con), delayed-induced mutants ATPG7 ox-4, ATPG7 ox-5 , and ATPG7 ox-6 3-4 days after germination to maintain cancer status. A picture of the chlorophyll content of leaves every 12 days until 12 days.

FIG. 13 shows detachment of the left lobe of the Arabidopsis wild-type (Con), delayed-induced mutants ATPG7 ox-4, ATPG7 ox-5 , and ATPG7 ox-6 3-4 days after germination to maintain cancer status. This is a picture of Fv / Fm of photosynthetic efficiency of leaves up to 12 days every 2 days.

14 shows detachment of the left lobe of the Arabidopsis wild-type (Con), ATPG7 ox-4, ATPG7 ox-5 , and ATPG7 ox-6 3-4 days after germination to maintain cancer status. The expression patterns of aging marker genes on the leaves every 12 days were analyzed by qRT-PCR, and ACT was used as a PCR positive control. CAB2, SEN4, and SAG12 are aging marker genes.

FIG. 15 shows a 12-day drought treatment of Arabidopsis wild-type or control (Con) and aging-induced variants ATPG7 ox-4, ATPG7 ox-5 , and ATPG7 ox-6 at 30 days after germination , The figure shows the phenotypic change of.

FIG. 16 shows a 12-day drought treatment of Arabidopsis wild-type or control (Con) and aging-induced variants ATPG7 ox-4, ATPG7 ox-5 , and ATPG7 ox-6 at 30 days after germination , The figure shows the change in the weight of a leaf.

FIG. 17 shows a 6-day H ₂ detachment of the Arabidopsis wild-type or control group (Con) and the left lobe 3-4 of the variants ATPG7 ox-4, ATPG7 ox-5 , and ATPG7 ox-6 at 25 days after germination. Figure shows the phenotype change of leaves treated with O ₂ .

Figure 18 day 25 Arabidopsis thaliana wild-type or control (Con) and aging delays induced mutant ATPG7 ox-4, ATPG7 ox- 5, and to detach the left lobe of three to four times ATPG7 ox-6 6 days after germination H ₂ Figure shows changes in chlorophyll content of leaves treated with O ₂ .

19 is to detach the germinated after 25 days Arabidopsis thaliana wild-type or control (Con) and aging delays induced mutant ATPG7 ox-4, ATPG7 ox- 5, and 3-4 times ATPG7 left lobe of ox-6 6 H ₂ ilgan The figure shows the change in photosynthetic efficiency of leaves treated with O ₂ in Fv / Fm.

Hereinafter will be described with reference to embodiments of the present invention. However, the scope of the present invention is not limited to this embodiment.

<Example 1> It has a function of delaying aging of the plant from the Arabidopsis and increasing productivity ATPG7  Isolation of genes

In order to separate the ATPG7 gene having a function of delaying aging and increasing productivity of the plant from the Arabidopsis, the following process was performed.

Example 1-1 Cultivation and Cultivation

Arabidopsis cultivars were grown in pots containing soil or Petri dishes containing MS (Murashige and Skoog salts, Sigma, USA) medium containing 2% sucrose (pH 5.7) and 0.8% agar. . When cultivated in a pollen, it was grown in a growth chamber controlled at a light cycle of 16/8 hours at a temperature of 22 ° C.

Example 1-2 RNA Extraction and Preparation of cDNA Library

RNA was extracted from Arabidopsis whole organs of differentiation stages using RNasey Plant Mini Kit (QIAGEN, Germany) to create a Arabidopsis cDNA library, and Superscript III Reverse Tanscriptase (INVITROGEN, USA) was extracted from the extracted RNA. cDNA was synthesized.

<Example 1-3> ATPG7 gene separation having a function of delaying aging and increasing productivity of plants

Based on the nucleotide sequence of DNA binding protein-related protein (DNA-binding protein-related, GeneBank accession number NP 194012.1) of Arabidopsis, the forward primer (PacI / AT4G22810 SOE), which is represented by SEQ ID NO: 3 and contains the sequence of restriction enzyme PacI -F, 5'-TTA ATT AAA TGG ATC CAG TAC AAT CTC ATG G -3 ') and reverse primer (XbaI / AT4G22810 SOE-R, 5'-), represented by SEQ ID NO: 4 and containing the sequence of restriction enzyme XbaI TCT AGA TCA ATA CGG TGG TCG TCC CGT-3 ′) was synthesized. The two primers were used to amplify and isolate full-length cDNA from polymerase chain reaction (PCR) from Arabidopsis cDNA prepared in Example 1-2.

As a result of analysis of the isolated cDNA, it has a 975 bp transcriptional translation frame (ORF) encoding 324 amino acids having a molecular weight of about 34.3 kDa, it was confirmed that it consists of one exon, AT- It has a hook domain I was named them as ATPG7 (AT -hook p rotein of G enomine 7). The isoelectric point of the ATPG7 protein encoded by the gene was found to be 6.8 (hereinafter, the gene is called " ATPG7 " or " ATPG7 gene" using italics, and the protein is called "ATPG7" or "ATPG7 protein").

<Example 2> ATPG7  Characterization of Manufacturing and Aging of Transgenic Arabidopsis Transplanted with Sense Constructs for Genes

<Example 2-1> Preparation of the transgenic Arabidopsis in which the sense construct for the ATPG7 gene was introduced

In order to confirm whether the gene has a aging delay function of the plant, a transgenic Arabidopsis in which the ATPG7 gene was introduced in the sense direction was prepared to change the expression of the ATPG7 transcript.

ATPG7 cDNA was amplified by PCR from Arabidopsis cDNA using a forward primer represented by SEQ ID NO: 3 and a sequence of restriction enzyme PacI and a reverse primer represented by SEQ ID NO: 4 and a sequence of restriction enzyme XbaI . The DNA was digested with restriction enzymes PacI and XbaI and cloned in the sense direction into a pCSEN vector prepared to be controlled by the SEN1 promoter, an inducible promoter, to prepare a pCSEN-ATPG7 recombinant vector, a sense construct for the ATPG7 gene. It was. The SEN1 promoter has specificity for the gene expressed according to the growth stage of the plant.

1 is a diagram showing a pCSEN-ATPG7 recombinant vector in which the ATPG7 gene is introduced in the sense direction into the pCSEN vector. In FIG. 1, BAR indicates a phosphinothricin acetyltransferase gene ( BAR gene) that confers resistance to a Vaster herbicide, RB is a right border, LB is a left border, and P35S is a CaMV 35S promoter, 35S-A CaMV 35S RNA polyA, PSEN is the SEN1 promoter, Nos-A refers to the polyA of the nopaline synthase gene.

The pCSEN-ATPG7 recombinant vector was introduced into an Agrobacterium tumefaciens using an electroporation method. The transformed Agrobacterium cultures were incubated at 28 ° C. until the OD ₆₀₀ value was 1.0, and the cells were harvested by centrifugation at 25 ° C. at 5,000 rpm for 10 minutes. Harvested cells were suspended in Infiltration Medium (IM; 1X MS SALTS, 1X B5 vitamin, 5% sucrose, 0.005% Silwet L-77, Lehle Seed, USA) medium until the final OD ₆₀₀ value was 2.0. Four week old Arabidopsis immersed in an Agrobacterium suspension in a vacuum chamber and placed under vacuum at 10 ⁴ Pa for 10 minutes. After immersion, the Arabidopsis was placed in a polyethylene bag for 24 hours. Thereafter, the transformed Arabidopsis cultivars continued to grow to harvest seeds (T ₁ ). As a control group, a non-transformed wild type Arabidopsis or a Arabidopsis transformed with only a vector (pCSEN vector) containing no ATPG7 gene was used.

Example 2-2 Characterization of T ₁ and T ₂ Transgenic Arabidopsis

Seeds harvested from the transformed Arabidopsis larvae as in <Example 2-1> were selected by immersing and incubating for 30 minutes in a 0.1% Bassta herbicide (light, South Korea) solution. Thereafter, the pollen was treated 5 times with the Basta herbicide during the growth of the transformed Arabidopsis larvae, and the Arabidopsis growth change in each pollen was investigated.

T ₁ Arabidopsis AT7-4 and AT7-8 transformed with pCSEN-ATPG7 vector were treated with a control group (a vector without the ATPG7 gene (pCSEN vector) transformed with a Arabidopsis or wild-type Arabidopsis), and their germination 60 days after germination. Surprisingly, the AT7-4 and AT7-8 variants showed distinct aging delay characteristics, and the difference in aging delay characteristics in transgenic individuals was due to the overexpression of genes. (FIG. 2).

In order to more accurately identify the phenotypic changes of the transgenic Arabidopsis, the phenotypes of these lines were examined by receiving T ₂ transgenic seeds from the T ₁ transgenic Arabidopsis. First, the T ₂ transformed Arabidopsis thawed for 3 days at low temperature (4 ℃) T ₂ transformed seedlings were grown in pollen, and transgenic Arabidopsis was selected through the treatment of Basta herbicide.

To analyze the expression patterns of the ATPG7 gene of the mutant with the selected aging delayed phenotype, the RNasey Plant was extracted from the leaves of Arabidopsis wild type and ATPG7 ox-4, ATPG7 ox-5 and ATPG7 ox-6 mutants , which were grown for 20 days after cotyledon production. Total RNA was extracted using Mini Kit (QIAGEN, Germany), respectively. 1 μg of RNA each as a template and 5 minutes at 65 ° C. using Superscript III Reverse Tanscriptase (INVITROGEN, USA); 60 minutes at 50 ° C; And cDNA was synthesized at 70 ° C. for 15 minutes. Subsequently, the synthesized cDNA was used as a template, and PCR was performed using the primers specific to the following [Table 1] for the ATPG7 gene and the ACT gene used as a PCR positive control. PCR denatured template DNA by heating at 94 ° C. for 2 minutes and then 1 minute at 94 ° C .; 1 minute 30 seconds at 55 ° C; And 1 cycle at 72 ℃ one cycle was performed a total of 30 times, followed by a final reaction for 15 minutes at 72 ℃. Then, the PCR product was confirmed by 1% agarose gel electrophoresis, the results are shown in FIG. ATPG7 ox-4, ATPG7 ox-5 and ATPG7 ox-6 variants showed significantly increased expression of ATPG7 genes compared to the Arabidopsis wild - type, and this fact proves that these variants are overexpressions of the ATPG7 gene.

Table 1 Primer sequence and sequence number for <i> ATPG7 </ i> gene and <i> ACT </ i> gene expression No. Gene name Forward / Reverse Primer (SEQ ID NO) One ATPG7 TGCAACATCAGCAACAAGCTATG (SEQ ID NO: 5) / GCTGCAACTGAACCGAACCA (SEQ ID NO: 6) 2 ACT ATGGCCGATGGTGAGGATATTC (SEQ ID NO: 7) / CACCAGCAAAACCAGCCTTC (SEQ ID NO: 8)

Meanwhile, in order to analyze plant organ expression patterns of ATPG7 gene of Arabidopsis wild type, cDNA was synthesized by extracting RNA from organs at various stages of development of Arabidopsis wild-type ATPG7 gene and ACT gene used as PCR positive control. PCR was performed using the specific primers shown in Table 1 below. As a result, as shown in Figure 4, it was confirmed that the expression of the ATPG7 gene is mainly made in the stem, it can be seen that the expression is also made in the leaves and young leaves during the aging process. On the other hand, gene expression was significantly lower in seedlings, roots and flowers in early development. These facts suggest that the gene has a function mainly in the stem of the plant and is involved in regulating the aging of the plant, whereas it has little function in the early stages of plant development such as roots and early seedlings.

Phenotyping of selected Arabidopsis T ₂ transformation lines was performed 50 days and 70 days after germination (FIG. 5).

The ATPG7 ox-4, ATPG7 ox-5 and ATPG7 ox-6 variant lines with pCSEN-ATPG7 constructs showed a marked delay in aging of plants as compared to the T ₁ variant. In addition, these variants resulted in not only a delayed aging phenotype but also a marked increase in individual size and seed yield during aging delay. The delay in aging and the increase in productivity were slightly different for each line. This is because the overexpression of genes is slightly different for each line as shown in FIG. 3. Interestingly, ATPG7 ox-5 , which has a strong aging trait, does not have a large phenotypic difference in productivity, whereas ATPG7 ox-4, which does not have a strong aging trait, has a large phenotypic characteristic compared to a control group in productivity. Appeared. Therefore, the expression level control of the present gene is thought to be able to arbitrarily produce plants with phenotypic characteristics for increased productivity and delay aging.

<Example 3> ATPG7  Characterization of productivity increase of overexpressed variants

In order to determine whether delayed aging of plants obtained through overexpression of the ATPG7 gene can lead to increased productivity of crops, productivity improvement indicators such as seed yield by line of the variants ATPG7 ox-4, ATPG7 ox-5 and ATPG7 ox-6 are provided. The application was compared with the Arabidopsis control.

Productivity indicators applied include plant height, silique number (NTS), biomass (Wet-W), dry-W, total seed weight (TSW), total seed number (TNS), And 1,000 seed weights (1,000 SW), and the result is the average of 20 individuals per line.

The ATPG7 ox-4, ATPG7 ox-5 and ATPG7 ox-6 variant lines all increased in seed weight and seed count more than 1.5 times compared to Arabidopsis control, and the weight of 1,000 seeds was significantly different from the control. There was no. And in the biomass and biodry weight, the overexpressed mutant was found to increase about 2.8 times more than the control. This fact is believed that the ATPG7 gene causes an increase in crop productivity, such as individual size, seed yield, and the like, with aging delay (FIG. 6). Therefore, the application of other crops of this gene is considered to be very valuable in terms of productivity.

<Example 4> ATPG7  Characterization of Aging Control of Overexpressing Variants

To identify the aging delayed traits of ATPG7 overexpressing variants, the phenotypic observations, leaf chlorophyll content, and photosynthetic activity were observed every 4 days from the _2nd generation T- ₄ generation after 12 days after cotyledon generation. Compared to the wild cephalopod.

Example 4-1 Phenotypic Changes in Leaves with Age-Dependent Aging of ATPG7 Overexpressing Variants

After 12 days after cotyledon production, the leaf phenotype of the left lobe 3-4 times was observed every 40 days until 40 days. As a result, in the Arabidopsis wild-type, the yellowing of leaves rapidly occurred after 24 days, and the leaves entered necrosis state from day 32. On the other hand, in the case of ATPG7 ox-4, ATPG7 ox-5 and ATPG7 ox-6 , the yellowing of the leaves proceeded after 36 days and the necrosis of the leaves did not occur almost 40 days (Fig. 7). These facts suggest that the ATPG7 gene may play an important role in delaying plant aging.

Example 4-2 Changes in Chlorophyll Content with Age-Dependent Aging of ATPG7 Overexpressing Variants

Chlorophyll was extracted from each sample leaf using 80% (V / V) acetone to measure chlorophyll content. Chlorophyll content was measured according to the method of Lichtenthaler and Wellburn ( Biochemical Society Transduction 603: 591 ~ 592, 1983) using extinction coefficients of 663.2 nm and 664.8 nm. As a result, as shown in Figure 8, the chlorophyll content in the wild species showed a sharp decrease from 20 days after the cotyledon production and the chlorophyll content was almost 0% at 32 days, but ATPG7 ox-4, ATPG7 ox-5 and ATPG7 ox-6 showed more than 80% of chlorophyll content at the beginning of measurement even when 36 days after cotyledon production.

Example 4-3 Changes in Photosynthetic Efficiency According to Age-Dependent Aging of ATPG7 Overexpressing Variants

Photosynthetic efficiency was measured using Oh et al . ( Plant Mol. Biol. 30: 939, 1996). First, the leaves of each DAE (day after emersion) were treated with cancer for 15 minutes, and then the fluorescence of chlorophyll was measured using a Plant Efficiency Analyzer (Hansatech). Photosynthetic efficiency was expressed by the photochemical efficiency of PSII (photosystem II) using chlorophyll fluorescence properties, which was the maximum variable fluorescence (Fv) versus the maximum value of fluorescence (Fm). It is expressed as the ratio of (Fv / Fm). Higher values indicate better photosynthetic efficiency.

As a result, as shown in Figure 9, wild species began to decrease rapidly after 28 days after cotyledon production and most of the activity disappeared after 32 days, but in the case of ATPG7 ox-4, ATPG7 ox-5 and ATPG7 ox-6 There was little change in activity up to 36 days after cotyledon formation, but there was a loss of activity up to about 20%, depending on the line at day 40. From the above results, the ATPG7 overexpressed mutants had a much longer phenotype of leaves than the wild species, and the effect of the life extension was on the biochemical effects of aging expressed by a decrease in chlorophyll content and photosynthetic efficiency by the ATPG7 gene. It is thought that the change is caused by the delay.

Example 4-4 Expression Changes of Aging-related Genes According to Age-Dependent Aging of ATPG7 Overexpressing Variants

To compare the expression of senescence associated genes ( SAGs) in wild species and ATPG7 ox-4, ATPG7 ox-5, and ATPG7 ox-6 variants, the ATPG7 gene and each aging associated over time during leaf development. Expression patterns of genes were confirmed by qRT-PCR analysis.

Total RNA was isolated using WelPrep ^TM Total RNA Isolation Reagent (JBI), and then treated with DNase I (Ambion), and 0.75ug was quantified to synthesize first cDNA using ImProm-II ^TM Reverse Transcription System (Promega). It was.

Quantitative analysis of the ATPG7 gene and marker genes for aging was confirmed by Quantitative real-time PCR (qRT-PCR) using Applied Bio-systems' 7300 Real Time PCR System. Aging marker gene was used as a SAG12, SEN4 and CAB2 genes, as qRT-PCR positive control was used the ACT gene. The primers used are shown in Table 2 below.

TABLE 2 Primer Sequence Number for Expression of Aging-Related Genes No. Gene name Forward / Reverse Primer (SEQ ID NO) One CAB2 CCGAGGACTTGCTTTACCCC SEQ ID NO: 9) / AACTCAGCGAAGGCCTCTGG (SEQ ID NO: 10) 2 SEN4 CGTCGATGACACACCCATTAGAG (SEQ ID NO: 11) / CATCGGCTTGTTCTTTGGAAAC (SEQ ID NO: 12) 3 SAG12 ACGATTTTGGCTGCGAAGG (SEQ ID NO: 13) / TCAGTTGTCAAGCCGCCAG (SEQ ID NO: 14) 4 ACT ATGGCCGATGGTGAGGATATTC (SEQ ID NO: 7) / CACCAGCAAAACCAGCCTTC (SEQ ID NO: 8) 5 ATPG7 TGCAACATCAGCAACAAGCTATG (SEQ ID NO: 5) / GCTGCAACTGAACCGAACCA (SEQ ID NO: 6)

In wild species, expression of photosynthetic genes such as CAB2 (chlorophyll a / b binding protein) decreased in proportion to aging over time. However, in the ATPG7 overexpressed variants, the expression of these genes was delayed, although there was a degree of difference. On the other hand, aging-related genes such as SAG12 and SEN4 are known to increase their expression in the aging stage. SEN4 and SAG12 increased rapidly after 28 days of cotyledon production in wild species and had the maximum expression value on day 32, whereas ATPG7 ox-4, ATPG7 ox-5, and ATPG7 ox-6 variants all expressed SEN4 and SAG12 cotyledons There was no change until day 36 post-production. On the other hand, the expression patterns of the ATPG7 gene showed that the expression levels of the ATPG7 gene overexpressed were significantly higher than those of the wild type, and gradually decreased, although there was a slight difference in the lines during aging. However, despite this reduction, it was found to maintain a high expression sequence compared to the wild type (Fig. 10). Taken together, the ATPG7 gene delays the onset of aging at the molecular level and subsequently regulates physiological phenomena such as chlorophyll content and photosynthetic efficiency, resulting in phenotypically prolonged leaf life.

Example 4-5 Analysis of Aging Characteristics According to Cancer Treatment of ATPG7 Overexpressing Variants

To detach the (rosette leaf) left lobe 21 days 3-4 times after germination in the T ₂ generation, to analyze the characteristics of the aging of the leaves of transformed delayed ATPG7 overexpression mutant of the known factor in cancer treatment that promotes aging 3mM MES buffer solution After floating in (2- [N-morpholino] -ethanesulfonic acid, pH 5.8), the cancer was maintained so that phenotypic observation, leaf chlorophyll content, photosynthetic efficiency and aging-related gene expression were observed every 2 days. 1 to 4-4> was measured in the same manner as compared to wild species Arabidopsis.

As a result, in the case of Arabidopsis wild-type, the yellowing of the leaves progressed 4 days after the cancer treatment, and the leaves entered the necrosis state (necrosis) on the 8th day. On the other hand, in the case of ATPG7 ox-4, ATPG7 ox-5 and ATPG7 ox-6 , the yellowing of leaves appeared after 12 days (Fig. 11). In wild species, chlorophyll content decreased sharply after 6 days of cancer treatment, which was about 10% of untreated cancer, but ATPG7 ox-5, ATPG7 ox-5 and ATPG7 ox- In the case of 6, most showed chlorophyll content of about 80% even on day 6 (Fig. 12). Changes in photosynthetic efficiency by cancer treatment were found to significantly delay activity reduction in ATPG7 overexpressing variants, such as chlorophyll content changes (FIG. 13).

In addition, the expression of the senescence index genes SEN4 and SAG12 , and the photo-dependent gene CAB2 were examined in the same manner as in <Example 4-4>. As a result, as shown in FIG. 14, wild-type significantly increased the expression of SAG12 on the 4th day after cancer treatment and SEN4 on the 8th day, whereas the expression of SAG12 did not appear during the pre-cancer treatment in the overexpressed variants. The expression of SEN4 increased slightly for 6 days, but the increase was significantly lower than that of wild type. On the other hand, the ATPG7 gene showed almost no expression in the wild type, whereas the overexpressed mutant showed an increase in the treatment of cancer, showing a peak at 6 days and then decreasing. However, the overall expression level was significantly higher than the wild type. These facts suggest that ATPG7 gene delays aging by slowing down the expression of aging marker genes or suppressing the expression rate.

Example 5 ATPG7  Characterization of Stress of Overexpressing Variants

Example 5-1 Characterization of Drought Stress of ATPG7 Overexpressing Variants

Drought tolerance analysis of the overexpressed variants of the ATPG7 gene drought for 12 days after 30 days of germination, compared to the phenotypic changes of the entire plant and the change in the weight of leaves per plant. The degree of resistance to was confirmed. The results are shown in FIGS. 15 and 16. In the wild type Arabidopsis, the yellowing of leaf rapidly progressed due to drought, and it was also found that the weight of leaf decreased significantly by drought. On the other hand, the overexpression of the ATPG7 gene showed that the greening of the leaves was still progressing during drought treatment, and the weight of the leaves was much higher than that of the wild type. This means that ATPG7 provides maximum resistance to plant moisture retention even under drought stress, providing resistance to drought stress.

Example 5-2 Characterization of H ₂ O ₂ Stress in ATPG7 Overexpressing Variants

To investigate the resistance to oxidative stress of the ATPG7 overexpressing mutant, 50mM H ₂ O ₂ was added to 3mM MES solution, detached and floated 25 and 3 leaves 4 days after germination, and then chlorophyll for 6 days every 2 days. Content and photosynthetic efficiency were measured to investigate the degree of resistance to H ₂ O ₂ stress. Compared with the Arabidopsis wild-type, ATPG7 overexpressed mutants were found to be delayed in retarding the yellowing of leaves and also in decreasing chlorophyll content and photosynthetic efficiency (FIGS. 17, 18 and 19). This fact means that ATPG7 provides resistance to oxidative stress in plants.

Therefore, the ATPG7 gene is expected to provide many advantages in developing stress-resistant crops by providing resistance to drought and oxidative stress in plants.

Has sequence list

Claims (14)

An ATPG7 protein having at least 90% sequence homology with the amino acid sequence set forth in SEQ ID NO: 2 and having a aging delay function, productivity increase function, and stress resistance function of the plant. ATPG7 gene encoding the protein of claim 1. (a) inserting the gene of claim 2 into an expression vector operably linked to a regulatory sequence capable of overexpressing it, (b) transforming the expression vector into a plant, and (c) selecting a plant with a delayed phenotype. The method of claim 3, The gene is a method for producing a delayed aging plant, characterized in that the gene comprising the nucleotide sequence of SEQ ID NO: 1. (a) inserting the gene of claim 2 into an expression vector operably linked to a regulatory sequence capable of overexpressing it, (b) transforming the expression vector into a plant, and (c) selecting a plant having a productive phenotype with increased productivity. The method of claim 5, The gene is a method for producing a plant having a productivity increase characteristic, characterized in that the gene comprising the nucleotide sequence of SEQ ID NO: 1. (a) inserting the gene of claim 2 into an expression vector operably linked to a regulatory sequence capable of overexpressing it, (b) transforming the expression vector into a plant, and (c) selecting a plant having a productive phenotype with increased productivity. The method of claim 7, wherein The gene is a method for producing a stress-resistant plant, characterized in that the gene comprising the nucleotide sequence of SEQ ID NO: 1. (a) inserting a gene encoding the amino acid sequence of SEQ ID NO: 2 into an expression vector so as to be operably linked to a regulatory sequence capable of overexpressing it, and (b) transforming the plant with the expression vector. How to delay the aging of plants. (a) inserting a gene encoding the amino acid sequence of SEQ ID NO: 2 into an expression vector so as to be operably linked to a regulatory sequence capable of overexpressing it, and (b) transforming the plant with the expression vector. How to increase the productivity of the plant. (a) inserting a gene encoding the amino acid sequence of SEQ ID NO: 2 into an expression vector so as to be operably linked to a regulatory sequence capable of overexpressing it, and (b) transforming the plant with the expression vector. How to increase the stress resistance of plants. A transgenic plant obtained by the method of claim 3, wherein the gene encoding the amino acid sequence of SEQ ID NO: 2 is introduced and overexpressed to have aging delay characteristics. A transgenic plant obtained by the method according to claim 5, wherein the gene encoding the amino acid sequence of SEQ ID NO: 2 is introduced and overexpressed to have productivity enhancing properties. A transgenic plant obtained by the method according to claim 7, wherein the gene encoding the amino acid sequence of SEQ ID NO: 2 is introduced and overexpressed to have stress resistance characteristics.

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