WO2018066898A2 - Mtatpg2 protein having function of increasing productivity, improving resistance against stress, and delaying senescence in plant, gene thereof, and use of same protein and gene - Google Patents

Abstract

Disclosed in the present invention is an MtATPG2 protein having the function of increasing productivity, improving resistance against stress, and delaying senescence in plant, a gene MtATPG2 thereof, and use of the same protein and gene. A plant in which the gene is introduced and expressed has the feature of anti-stress resistance and senescence delay in addition to an increase in productivity.

Description

MTATPG2 protein, its genes, and uses thereof, which increase productivity, stress tolerance and delay aging of plants

The present invention relates to a MtATPG2 protein having its productivity enhancing function, stress resistance function and delaying aging function, genes thereof and uses thereof.

Plant senescence is considered to be a major step in the plant's lifecycle, and progresses in a highly sophisticated manner at the cellular, tissue, organ and individual levels through genetically regulated reactions. The aging process can occur differently in different organs of the plant. In the case of leaves, the degradation of chloroplasts is followed by the degradation of lipids, proteins and nucleic acids. Cell structures, such as cell membranes and intracellular compartments, are maintained until the end and lead to death after rearrangement of the degraded product nitrogen and nutrients (Lohman et al., Physiologia Plantarum, 1994, 92: 322-8 .; Smart, New Phytologist, 1994, 126: 419-48; Pruzinska et al., Plant Physiology, 2005, 139: 52-63). The timing at which plants determine aging is related to nutrient retention and rearrangement, so flowering and seed production are often factors that promote aging. Plants also progress the aging process as a species-wide survival strategy for seasonal or unforeseen external environmental changes.

When cultivating plants to obtain useful substances, the amount and timing of plants available for producing useful substances are limited, and the plants are sensitive to the influence of external factors such as climate and climate, leading to aging and death. The study of plant aging is very important not only for understanding biological phenomena from a biological point of view, but also for economic profit creation through enhancement of productivity, storage and transport.

The aging process is accompanied by changes in the expression of several genes, leading to a decrease in the expression of photosynthesis and basal metabolism-related genes and an increase in the expression of apoptosis, stress response genes, and hydrolase-related genes (Hopkins et al. 2007, 175: 201-14; Lim et al., Annual Review of Plant Biology, 2007, 58: 115-136). Senescence Associated Genes (SAGs) of these plants are being actively researched due to their importance in academic and industrial terms.

Introducing the antisense sequence of the SAG101 gene with acyl hydrolase activity into the plant to delay aging (He and Gan, Plant Cell, 2002, 14 (4): 805-15) As a result, a method of inhibiting the expression of an aging promoter gene has been developed, and a method of overexpressing a gene that inhibits aging during the aging process has also been developed. US Patent No. 5689042 discloses SAG12 I SAG13 There was coupled the cytokinin synthesis genes in the promoter of the gene is disclosed a method for delaying the aging process in the promoter of the IPT gene SAG12 Recombinant genes showed a 50% increase in productivity in tobacco. When introduced into the lettuce by the same method it can be seen that the shelf life after harvest significantly increased (McCabe et al., Plant Physiol. 2001, 127 (2): 505-16). In addition, there were reports of increased cytokinin levels and delayed leaf aging in tobacco expressing corn homeobox gene (knotted1) in SAG12 promoter (Naomi et al., Plant Cell, 1999, 11: 1073-1080).

Tomatoes have been reported to control the ripening of fruits by inhibiting the ethylene synthesis process (Oeller et al., Science. 1991, 254 (5030): 437-9) and also inhibit the expression of polygalacturonase genes involved in cell wall degradation. For example, Flav-O-Savor, which increases the transport and storage properties of tomatoes, may be a typical commercialized example (Giovannoni et al., 1989, Plant Cell 1 (1): 53-63).

In addition, GmSARK gene and WRKY53 , WRKY6 and AtNAP genes belonging to the NAC family have been reported as genes overexpressed during aging (Miao et al., Plant Mol. Biol. 2004, 55: 853-867; Guo and Gan et al. , Plant J. 2006, 46 (4): 601-12; Li et al., Plant Mol Biol. 2006, 61: 829-844; Besseau et al., J Exp Bot 2012, 63 (7): 2667-79 Genes such as CBF2 and CBF3 are known to inhibit aging (Sobieszczuk-Nowicka et al., Physiol Plant 2007; 130: 590-600). Controlling the expression of these genes can delay aging, resulting in improvements in crop productivity. U.S. Pat.No.8420890 discloses a method for delaying aging by inhibiting the expression of AtNAP gene. Recently, studies have been conducted for overexpressing AtNAP to facilitate the harvesting of cotton or the like or to help ripening fruit. (Kou et al., J Exp Bot. 2012, 63 (17): 6139-47).

In recent years, the regulation of IPT gene expression through the application of appropriate promoters provides agricultural traits of increased productivity as well as delayed aging in canola, and it is interesting to note that morphological and phenotypic abnormalities due to cytokinin overexpression are not observed in transgenic plants. Surya Kant et al., PLOS ONE. 2015, 10 (1): e0116349. Doi: 10.1371 / journal.pone.0116349. In addition, ATPG4, which is the AT hook gene of Arabidopsis, has been suggested to exhibit a phenotypic characteristic of delaying aging or a phenotypic characteristic of increasing productivity according to gene expression level (Korean Patent Application No. 10-2011-0110593). Contrary to the expression patterns of the ATPG4 gene, inhibition of expression of GhCKX , a cytokinin regulator gene isolated from cotton, has been shown to provide traits of increased productivity and extended melting. Severe inhibition of expression of this gene is reported to lead to traits of telogen extension, while adequate inhibition of expression leads to increased productivity of fibers and seeds (Zhao et al., Mol Breeding. 2015, 35:60). The study of gene expression control and productivity increase will be further accelerated in that the function of these genes is to provide expression of increased productivity and delayed aging through expression control.

The Tobacco Expression Atlas (TobEA) database has been recently obtained through the tobacco life cycle research (Edwards et al., BMC Genomics. 2010, 11: 142) and further accelerated genetic studies on plant aging and growth regulation. It is becoming.

The present invention also discloses a gene having a function of delaying aging and the like, which can improve the productivity of crops and the like.

SUMMARY OF THE INVENTION An object of the present invention is to provide a MtATPG2 protein having a productivity enhancing function of a plant, a stress resistance function, and a delaying aging function.

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 a yield increasing characteristic.

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

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

Other objects of the present invention will be presented below.

In one aspect, the present invention relates to a MtATPG2 protein having a productivity enhancing function of a plant, a stress resistance function, and a delaying aging function.

The inventor (s) is based on the nucleotide sequence of the AT-hook motif nuclear localized protein (GeneBank accession number NC_016414.1) of Medicago truncatula , as identified in the following examples When the gene was isolated and transformed into the Arabidopsis, the productivity increase characteristics such as the increase in the biomass and / or the seed productivity of the individual were clearly shown, and the resistance to oxidative stress was also clearly shown, and the aging of the plant was delayed. It was confirmed that the phenomenon appeared clearly.

These experimental results may mean that the genes and proteins increase plant productivity, provide resistance to stress, and also contribute to delaying aging of the plant.

We use the gene MtATPG2 ( M edicago t runcatula AT -hook p rotein of G enomine 2) was named as a gene and protein MtATPG2, these nucleotide sequences and amino acid sequences are disclosed in each of SEQ ID NO: 1 (or FIG. 14) and 2 (or FIG. 15).

The MtATPG2 protein of the 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" still retains productivity enhancement and stress tolerance and plant aging delayed functions as compared to the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: It is defined as a polypeptide comprising a portion of an amino acid sequence of SEQ ID NO: 2 sufficient to the following. The length of the polypeptide and the degree of activity of such a polypeptide is not a problem since it still needs to be sufficient to retain productivity and stress resistance and to delay plant aging. 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 is still a product having increased productivity, stress resistance, and delayed plant aging, the length of the amino acid sequence of SEQ ID NO: 2 Polypeptide comprising a substantial portion thereof. Those skilled in the art, that is, those skilled in the art, who are familiar with the related prior art known at the time of filing this application, will still be able to increase productivity and stress resistance, even if a portion of the polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2 is deleted, and It will be expected to retain the ability to delay plant aging. 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, furthermore, a polypeptide encoded by the nucleotide sequence of SEQ ID NO: 1 and consisting of the amino acid sequence of SEQ ID NO: 2 increases productivity and stress resistance of plants. In addition, the present invention discloses an embodiment confirming whether the plant has a delaying function of aging, and thus, a polypeptide having a partial deletion in the amino acid sequence of SEQ ID NO: 2 may still retain the function of the polypeptide comprising the amino acid sequence of SEQ ID NO: 2 It is very clear that those skilled in the art can fully confirm within the ordinary capability range. Therefore, in the present invention, the "polypeptide comprising a substantial part of the amino acid sequence set forth in SEQ ID NO: 2" as described above, based on the disclosure of the present specification, the productivity of a plant that can be manufactured by those skilled in the art within the range of its usual capacity can be increased. It is to be understood as meaning including all polypeptides in a deleted form having a function, a stress resistance function, and a plant aging delay function.

Also used herein, "polypeptide substantially similar to the polypeptide 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, the productivity of a plant. It refers to a polypeptide possessing augmentation and stress resistance functions and a delay in plant aging. 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 productivity of the plant, the stress resistance function, and the delayed plant aging function. In other words, even if a polypeptide comprising one or more substituted amino acids contains a large number of substituted amino acids, even if its activity is lower than that of a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, the polypeptide is a plant productivity. It is included in the present invention as long as it possesses augmentation and stress tolerance functions, and a plant aging delay 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. One skilled in the art would prepare a polypeptide that contains one or more substituted amino acids as described above, but still retains the productivity and stress resistance functions of plants comprising the amino acid sequence of SEQ ID NO. can do. One skilled in the art can also determine whether a polypeptide comprising one or more substituted amino acids still has this function. Furthermore, the present disclosure discloses the nucleotide sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2, and also the polypeptide comprising the amino acid sequence of SEQ ID NO: 2 has a function of increasing productivity and stress resistance of plants, and delaying plant aging. Since the confirmed examples are disclosed, it is evident that the "polypeptides substantially similar to the polypeptides of (a) and (b)" of the present invention can be easily implemented by those skilled in the art. Therefore, "polypeptide substantially similar to the polypeptide of (a) or (b) above" is meant to include all polypeptides that contain one or more substituted amino acids but still have plant productivity and stress resistance and delayed aging of plants. Should be understood as. As such, the term "polypeptide substantially similar to the polypeptide of (a) or (b)" includes all polypeptides that contain one or more substituted amino acids but still have plant productivity and stress resistance functions, and plant aging function. In view of the degree of activity, however, 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. Herein, "polypeptide as described above" refers to a polypeptide comprising the entire amino acid sequence of SEQ ID NO: 2, a substantial portion of the amino acid sequence of SEQ ID NO. Including polypeptides, and polypeptides substantially similar to the above polypeptides, as well as all polypeptides of the preferred embodiments as described above. Therefore, the polynucleotide of the present invention is an isolated polynucleotide encoding a polypeptide comprising all or a substantial part of the amino acid sequence of SEQ ID NO: 2 and having a function of increasing productivity and stress resistance of plants, and delaying aging of plants, and Polypeptides encoding polypeptides that are substantially similar to the polypeptides include isolated polynucleotides and, in a preferred embodiment, the sequences in the order of sequence homology as described above, with increasing plant productivity and stress resistance, and delayed plant aging. It includes an isolated polynucleotide encoding all polypeptides with homology. 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” as used herein includes both chemically synthesized polynucleotides, polynucleotides isolated from organisms, especially Medicago truncatula , and polynucleotides containing modified nucleotides, It is defined as including all polymers of stranded or double stranded RNA or DNA.

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

Method for producing a plant having a productivity enhancing feature of the present invention comprises the steps of (a) expressing 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 plant's seeds (plants) Number and / or mass of seeds per individual) is increased compared to 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 productivity gains can give useful results to humans. Therefore, the meaning of the plant includes crops (specifically, crops such as edible crops, feed crops, craft crops, and horticultural crops), forest trees, and ornamental plants. Specifically, rice, wheat, barley, corn, soybeans, potatoes, red beans, oats, sorghum, legumes (specifically knotweed, hoe belonging to Medicago truncatula ) from which the MtATPG2 gene of the present invention is isolated. Fire, stone, bird, red bean, soybean, pea, adzuki bean, peanut, lentil, kidney bean, lupine, alfalfa or clover, 칡, shamrock, lump, etc.), Chinese cabbage, bok choy, kale, cauliflower, broccoli, Young radish, radish, freshly, pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, green onion, onion, carrot, ginseng, tobacco, cotton, sesame, sugarcane, beet, perilla, peanut, It will include rapeseed, apple, pear, jujube, peach, lamb, grape, citrus, persimmon, plum, apricot, banana, and other bioenergy crops such as switchgrass, silver grass, reed, and other lygras, red clover. , Orchardgrass, Alpha Alpha, Tolsque Scue, Perennial Lig Las, roses, gladiolus, gerberas, carnations, chrysanthemums, lilies, tulips.

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 having the characteristics of increasing the productivity of the plant, etc., due to the evolutionary pathways different according to the type of plant consisting of the nucleotide sequence of SEQ ID NO: 1 and other nucleotide sequences It is meant to include all 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 the method for producing a plant having a productivity increasing characteristic of the present invention, step (a) may be performed by a genetic engineering method.

Genetic engineering method includes the steps of (i) inserting the gene of SEQ ID NO: 1 or a gene having a sequence similar to the nucleotide sequence of SEQ ID NO: 1 into an expression vector to be operably linked to a regulatory sequence capable of expressing 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 in this specification, "promoter" follows the conventional meaning known in the art, specifically located at the top (5 'side) based on the transcription initiation point of a gene, 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. This promoter may be a TATA box (usually located at the transcription start point (+1) -20 to -30 position) above the transcription initiation point if it is of eukaryotes, and a CAAT box (typically about -75 position compared to the transcription start site) Present), a 5 'enhancer, a transcription repression factor, and the like.

Usable promoters include constitutive promoters (promoters which induce expression in all plant tissues at all times), inducible promoters (expression of target genes in response to specific external stimuli) as long as they are capable of expressing 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.

Transcription termination sequence is poly (A ) addition signal ( polyadenylation As a sequence acting as a signal ), it is intended to enhance the integrity and efficiency of transcription. An example of a transcription termination sequence that can be used is nopalin Synthase (NOS) a transcription termination sequence, rice α- amylase RAmy1 A transcription termination sequence, Agrobacterium Tome Pacific Enschede of the gene of the gene Transcription termination sequence of octopine gene, transcription termination sequence of wheat heat shock protein 17, transcription termination sequence of wheat ubiquitin gene, transcription termination sequence of rice gluterin gene, transcription termination sequence of rice lactate dehydrogenase gene Etc. can be mentioned.

The expression vector may include a selection marker gene. Where "screening Marker gene "means a gene encoding a trait that enables the selection of a plant comprising such a marker gene selectable marker gene may be a number of days antibiotic resistance genes and herbicide tolerance genes. Examples of antibiotic resistance genes Examples of antibiotic resistance genes include puromycin resistance genes (eg Streptomyces). puromycin N-acetyl transferase gene derived from alboniger , neomycin resistance gene (eg Streptomyces) aminoglycoside phosphotransferase gene derived from fradiae , hygromycin resistance gene ( Streptomyces) hygroscopicus Hygromycin phosphotransferase gene derived from, bleomycin resistance gene (bleomycin binding protein derived from Streptomyces verticillus ), blasticidin resistance gene (eg Streptomyces) blasticidine S-acetyltransferase gene derived from verticillum ), hygromycin resistance gene (such as aminocyclitol phosphotransferase gene derived from Escherichia coli ), ampicillin resistance gene (β-lactamase gene), and the like. For example, the herbicide resistance gene may include a vasta herbicide resistance bar gene.

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, the MtATPG2 gene of the present invention is homologous to Medicago truncatula from which it is isolated, but is heterologous to Arabidopsis and 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. In particular, since the MtATPG2 gene of the present invention was isolated from Medicago truncatula belonging to the legumes, the MtATPG2 gene of the present invention was transformed into legumes so as to be useful for breeding the legumes. Methods of transforming such legumes are also known in the art through a number of documents. As such documents, for example, Plant Science (Shannon) 150 (1), Jan. 14, 2000, 41-49, J. of Plant Biochemistry & Biotechnology 9 (2) July, 2000, 107-110, literature (Acta Physiologiae Plantarum 22 (2), 2000, 111-119), Molecular Breeding 5 (1) 1999, 43-51, In Vitro Cellular & Developmental Biology, Animal 34 (3 part 2) March, 1998 , 53A), Plant Cell Reports 16 (8), 1997, 513-519 and 541-544, Theoretical & Applied Genetics 94 (2), 1997, 151-158, Plant Science, 117 ( 1-2), 1996, 131-138), Plant Cell Reports 16 (1-2), 1996, 32-37, and the like.

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 selection step (b) may be selected through the characteristics of the inserted gene by growing and growing the transformed plant, or, if the selection marker gene is transformed together during transformation, the selection marker gene may be selected. Can be. The characteristics of the inserted gene include the biomass of the plant and / or the productivity of the seed.

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

The method for producing a stress resistant plant of the present invention comprises the steps of: (a) expressing 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) having a stress resistant phenotype Comprising plant screening.

As used herein, "stress" means oxidative stress.

Step (a) may be performed genetically, as for the genetic engineering method, as described with reference to the method of manufacturing a plant having the productivity increasing characteristic of the present invention.

The step (b) is selected by comparing the stress resistance of the plant that is characteristic of the inserted gene (for example, the degree of progress of leaf yellowing or necrosis, the biomass of the leaves and / or stems, chlorophyll content, photosynthetic efficiency, etc.). When a selection marker gene is transformed together during transformation, it may be selected using a selection marker gene, or may be selected by mixing these methods.

In another aspect, the present invention relates to a method for producing a plant having aging delay properties.

The method for producing a plant having a aging retardation property of the present invention comprises the steps of: (a) expressing 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) Selecting the plant with the delayed phenotype.

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.

Step (a) may be performed genetically, as for the genetic engineering method, as described with reference to a method for preparing a plant having productivity enhancing characteristics of the present invention.

In the step (b), the selected plant is selected using delayed aging characteristics, which are characteristics of the gene inserted by developing and growing a transformed plant (quantitative evaluation of leaf yellowing or necrosis, chlorophyll content, photosynthetic efficiency, etc.). Method, a method of mixing the above methods, etc.), and when the selection marker gene is transformed together during transformation, the selection may be performed using the selection marker gene.

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

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 regulatory sequence capable of expressing 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 expressing it Inserting into the expression vector preferably and (b) transforming the expression vector into a plant.

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

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 expressing it. Inserting into the expression vector and (b) transforming the expression vector into a plant.

Steps (a) and (b) in the above methods are as described with reference to the method for producing a plant having the productivity enhancing properties of the present invention.

In another aspect, the present invention is 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 the productivity enhancing characteristics 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 a gene encoding the MtATPG2 protein consisting of the amino acid sequence of SEQ ID NO: 2, in particular the gene MtATPG2 having the nucleotide sequence of SEQ ID NO: 1, into the plant .

In another aspect, the present invention is a stress resistance that is expressed by 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 stress resistant 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 a stress resistance by introducing a gene encoding the MtATPG2 protein consisting of the amino acid sequence of SEQ ID NO: 2, in particular the gene MtATPG2 having the nucleotide sequence of SEQ ID NO: 1, into the plant.

In another aspect, the present invention is 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 the aging delay characteristics of the present invention The present invention relates to a transgenic plant having delayed aging characteristics.

In a preferred aspect, the plant is a transgenic plant having delayed aging characteristics by introducing a gene encoding the MtATPG2 protein consisting of the amino acid sequence of SEQ ID NO: 2, in particular the gene MtATPG2 having the nucleotide sequence of SEQ ID NO: 1, into the plant .

In the present specification, the "transformed plant" is obtained by crossing with a transformed plant as well as when the gene is introduced and transformed into a plant cell, plant tissue, or plant seed capable of developing and growing into a mature plant. Genome modified plants, plant seeds derived from the plants, plant cells, plant tissues.

As described above, according to the present invention, it is possible to provide the MtATPG2 protein and its gene, MtATPG2 , which have a productivity enhancing function and a stress resistance function of the plant, and a delaying function of aging. Since the gene provides a function of increasing productivity and stress resistance and delaying aging of the plant, when the plant is transformed with the gene, the gene may not only increase productivity but also produce a plant having a stress-resistant and aging delaying function of the plant. Can be.

Figure 1 shows the structure (schematic) of the pCSEN-MtATPG2 recombinant vector in which the MtATPG2 gene having productivity enhancement function, stress resistance function, and aging delay function was introduced in the sense direction.

Figure 2 is a photograph of the Arabidopsis grown 50 days and 70 days after germination of the Arabidopsis T ₂ line transformed with the pCSEN-MtATPG2 recombinant vector of FIG. DAG, day after germination.

Con: Baby Pole Wild Type or Control

MtATPG2-7 : Arabidopsis T ₂ line transformed with pCSEN-MtATPG2 recombinant vector

MtATPG2-8 : Arabidopsis T ₂ line transformed with pCSEN-MtATPG2 recombinant vector

MtATPG2-15 : Arabidopsis T ₂ line transformed with pCSEN-MtATPG2 recombinant vector

MtATPG2-16 : Arabidopsis T ₂ line transformed with pCSEN-MtATPG2 recombinant vector

MtATPG2-17 : Arabidopsis T ₂ line transformed with pCSEN-MtATPG2 recombinant vector

Figure 3 shows the results of analyzing the expression of the MtATPG2 gene of Arabidopsis thalass for 25 days after cotyledon generation of the Arabidopsis T ₂ line transformed with the pCSEN-MtATPG2 recombinant vector of Figure 1 through qRT-PCR TUB was used as a PCR positive control.

Con: Baby Pole Wild

MtATPG2-7 : Arabidopsis T ₂ line transformed with pCSEN-MtATPG2 recombinant vector

MtATPG2-8 : Arabidopsis T ₂ line transformed with pCSEN-MtATPG2 recombinant vector

MtATPG2-15 : Arabidopsis T ₂ line transformed with pCSEN-MtATPG2 recombinant vector

MtATPG2-16 : Arabidopsis T ₂ line transformed with pCSEN-MtATPG2 recombinant vector

MtATPG2-17 : Arabidopsis T ₂ line transformed with pCSEN-MtATPG2 recombinant vector

FIG. 4 is a diagram for increasing productivity and seed size of the Arabidopsis line of FIG. 3.

Con: Baby Pole Wild

MtATPG2-7 : Arabidopsis T ₂ line transformed with pCSEN-MtATPG2 recombinant vector

MtATPG2-8 : Arabidopsis T ₂ line transformed with pCSEN-MtATPG2 recombinant vector

MtATPG2-15 : Arabidopsis T ₂ line transformed with pCSEN-MtATPG2 recombinant vector

MtATPG2-16 : Arabidopsis T ₂ line transformed with pCSEN-MtATPG2 recombinant vector

MtATPG2-17 : Arabidopsis T ₂ line transformed with pCSEN-MtATPG2 recombinant vector

Height: height, NTS: long angle and number, FW: biomass, DW: biomass, TSW: total seed weight, TNS: total seed count, 1,000SW: 1,000 seed weight

5 is MtATPG2 CLV1 gene, histidine kinase (HK) gene family, histidine phosphotransfer proteins (HP) gene family, and MtATPG2 in the cytokinin signaling pathway of expression Arabidopsis T ₂ transformants The expression of genes was analyzed by RT-PCR, and TUB was used as a PCR positive control.

Figure 6 is a photograph of the Arabidopsis grown 50 days after germination of the Arabidopsis T ₃ or T ₄ homo line transformed with the pCSEN-MtATPG2 recombinant vector of FIG. DAG, day after germination.

Con: Baby Pole Wild Type or Control

MtATPG2 -7-15-11 : Arabidopsis T ₄ homo line transformed with pCSEN-MtATPG2 recombinant vector

MtATPG2 -7-15-12 : Arabidopsis T ₄ homo line transformed with pCSEN-MtATPG2 recombinant vector

MtATPG2 -8-7: pCSEN-MtATPG2 Transgenic Arabidopsis T ₃ homo line with a recombinant vector

MtATPG2 -8-8: pCSEN-MtATPG2 Transgenic Arabidopsis T ₃ homo line with a recombinant vector

MtATPG2 -15-3: pCSEN-MtATPG2 Transgenic Arabidopsis T ₃ homo line with a recombinant vector

MtATPG2 -15-6: pCSEN-MtATPG2 Transgenic Arabidopsis T ₃ homo line with a recombinant vector

Figure 7 is a photograph of the Arabidopsis grown 70 days after germination of the Arabidopsis T ₃ or T ₄ homo line transformed with the pCSEN-MtATPG2 recombinant vector of FIG. DAG, day after germination.

Con: Baby Pole Wild Type or Control

MtATPG2 -7-15-11 : Arabidopsis T ₄ homo line transformed with pCSEN-MtATPG2 recombinant vector

MtATPG2 -7-15-12 : Arabidopsis T ₄ homo line transformed with pCSEN-MtATPG2 recombinant vector

MtATPG2 -8-7: pCSEN-MtATPG2 Transgenic Arabidopsis T ₃ homo line with a recombinant vector

MtATPG2 -8-8: pCSEN-MtATPG2 Transgenic Arabidopsis T ₃ homo line with a recombinant vector

MtATPG2 -15-3: pCSEN-MtATPG2 Transgenic Arabidopsis T ₃ homo line with a recombinant vector

MtATPG2 -15-6: pCSEN-MtATPG2 Transgenic Arabidopsis T ₃ homo line with a recombinant vector

FIG. 8 shows the expression patterns of the MtATPG2 gene of Arabidopsis thaliana grown in 25 days after cotyledon generation using T ₃ or T ₄ homoline transformed with the pCSEN-MtATPG2 recombinant vector of FIG. 1 through qRT-PCR. FIG. One result is shown and TUB was used as a PCR positive control.

FIG. 9 is generated after the cotyledons from 16 wild-type Arabidopsis thaliana (Con) and three progress with T ₃ or T ₄ 3-4 times left lobe (rosette leaf) of homo-line observing the phenotype of the leaves to 56 days every 4 days Figure 10 is a figure of the investigation of the chlorophyll content of the leaves for this. DAE, day after emersion.

Con: Baby Pole Wild Type or Control

MtATPG2 -7-15-11 : Arabidopsis T ₄ homo line transformed with pCSEN-MtATPG2 recombinant vector

MtATPG2 -7-15-12 : Arabidopsis T ₄ homo line transformed with pCSEN-MtATPG2 recombinant vector

MtATPG2 -8-7: pCSEN-MtATPG2 Transgenic Arabidopsis T ₃ homo line with a recombinant vector

MtATPG2 -8-8: pCSEN-MtATPG2 Transgenic Arabidopsis T ₃ homo line with a recombinant vector

MtATPG2 -15-3: pCSEN-MtATPG2 Transgenic Arabidopsis T ₃ homo line with a recombinant vector

MtATPG2 -15-6: pCSEN-MtATPG2 Transgenic Arabidopsis T ₃ homo line with a recombinant vector

11 shows the phenotype of leaves up to 6 days every 2 days after detaching the left lobe 3-4 of the Arabidopsis wild-type (Con) and generation-advanced T ₃ or T ₄ homo lines after germination to maintain cancer status. FIG. 12 is a diagram illustrating the chlorophyll content of leaves. DAT, day after treatment.

Con: Baby Pole Wild Type or Control

MtATPG2 -7-15-11 : Arabidopsis T ₄ homo line transformed with pCSEN-MtATPG2 recombinant vector

MtATPG2 -7-15-12 : Arabidopsis T ₄ homo line transformed with pCSEN-MtATPG2 recombinant vector

MtATPG2 -8-7: pCSEN-MtATPG2 Transgenic Arabidopsis T ₃ homo line with a recombinant vector

MtATPG2 -8-8: pCSEN-MtATPG2 Transgenic Arabidopsis T ₃ homo line with a recombinant vector

MtATPG2 -15-3: pCSEN-MtATPG2 Transgenic Arabidopsis T ₃ homo line with a recombinant vector

MtATPG2 -15-6: pCSEN-MtATPG2 Transgenic Arabidopsis T ₃ homo line with a recombinant vector

FIG. 13 shows the leaves treated with 4 mM H ₂ O ₂ in 3 mM MES solution by detaching the left lobe No. 3-4 of the Arabidopsis wild-type (Con) and generation-advanced T ₃ or T ₄ homo lines 25 days after germination. Fig. 13A shows a phenotypic change in Fig. 13A and Fig. 13A shows a phenotypic change in leaves treated with 150 mM NaCl in a 3 mM MES solution for 6 days in a sample under the same conditions (Fig. 13B). DAT, day after treatment.

Con: Baby Pole Wild Type or Control

MtATPG2 -7-15-11 : Arabidopsis T ₄ homo line transformed with pCSEN-MtATPG2 recombinant vector

MtATPG2 -7-15-12 : Arabidopsis T ₄ homo line transformed with pCSEN-MtATPG2 recombinant vector

MtATPG2 -8-7: pCSEN-MtATPG2 Transgenic Arabidopsis T ₃ homo line with a recombinant vector

MtATPG2 -8-8: pCSEN-MtATPG2 Transgenic Arabidopsis T ₃ homo line with a recombinant vector

MtATPG2 -15-3: pCSEN-MtATPG2 Transgenic Arabidopsis T ₃ homo line with a recombinant vector

MtATPG2 -15-6: pCSEN-MtATPG2 Transgenic Arabidopsis T ₃ homo line with a recombinant vector

14 and 15 shows the amino acid sequence of the base sequence of each protein and MtATPG2 MtATPG2 gene.

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

< Example  1> Medicago truncatula from  In addition to increasing productivity and stress tolerance of plants Providing delayed aging MtATPG2 Isolation of genes

Medicago uses the MtATPG2 gene, which has increased productivity and stress resistance in plants and also delays aging. To separate from truncatula , the following process was performed.

Example 1-1 Medicago planting and cultivation of truncatula

Medicago truncatula Jemalong A17 was cultivated in a growth chamber controlled in a soil containing pots at a temperature of 22 ° C. and a cycle of 16/8 hours.

Example 1-2 RNA Extraction and Preparation of cDNA Library

Medicago To make truncatula cDNA library, RNA was extracted from whole organs of differentiation stages using RNasey Plant Mini Kit (QIAGEN, Germany), and cDNA was synthesized using Superscript III Reverse Tanscriptase (INVITROGEN, USA) from the extracted whole RNA. It was.

Example 1-3 Isolation of MtATPG2 Gene Having Increased Productivity, Stress Tolerance, and Delay in Aging of Plants

Based on the nucleotide sequence of AT-hook motif nuclear localized protein (GeneBank accession number NC_016414.1) of M. truncatula , a forward primer (BglII / MTR8g 098390-F, represented by SEQ ID NO: 3 and containing the sequence of restriction enzyme BglII) , 5TCT ATG GAT GGG AGA GAG GCT ATG G-3) and a reverse primer (BstEII / MTR8g 098390, 5GAC CTC ATC CTC TTG TCA AGT CAA AGC-3) represented by SEQ ID NO: 4 and containing the sequence of restriction enzyme BstEII were synthesized. . The primer was used to amplify and isolate full-length cDNA from M. truncatula cDNA using polymerase chain reaction (PCR).

As a result of analysis of the isolated cDNA, it has a 1,056 bp transcriptional translation frame (ORF) encoding 351 amino acids having a molecular weight of about 35.9 kDa, it was confirmed that it consists of one exon, AT -hook has a motif I and named it as MtATPG2 (M edicago t runcatula AT -hook p rotein of enomine G 2).

The isoelectric point of the MtATPG2 protein encoded by the gene was found to be 10.28 (hereinafter, the gene is called " MtATPG2 " or " MtATPG2 gene" using italics, and the protein is called "MtATPG2" or "MtATPG2 protein").

< Example  2> MtATPG2  Preparation and Characterization of Transgenic Arabidopsis with Sense Constructs for Genes

<Example 2-1> Preparation of the transgenic Arabesqueis in which the sense construct for the MtATPG2 gene was introduced

In order to confirm whether the gene has a productivity and stress resistance function of the plant and also provides a delay in aging, a transgenic Arabidopsis in which the MtATPG2 gene was introduced in the sense direction was prepared to change the expression of the MtATPG2 transcript.

MtATPG2 cDNA using PCR from cDNA of M. truncatula using a forward primer represented by SEQ ID NO: 3 and containing a sequence of restriction enzyme Bgl II and a reverse primer represented by SEQ ID NO: 4 and a sequence of restriction enzyme BstE II Was amplified. The DNA was digested with restriction enzymes Bgl II and BstE II, cloned in the sense direction into a pCSEN vector designed to be controlled by the SEN1 promoter, an inducible promoter, and the pCSEN-MtATPG2 recombinant vector, a sense construct for the MtATPG2 gene. Was produced. The SEN1 promoter has specificity for the gene expressed according to the growth stage of the plant.

1 is a diagram illustrating a pCSEN-MtATPG2 recombinant vector in which a MtATPG2 gene is introduced in a sense direction into a pCSEN vector. In FIG. 1, BAR refers to a BAR gene (phosphinothricin acetyltransferase gene) that confers resistance to Basta herbicide, RB to the right border, LB to the left border, p35S to the CaMV 35S promoter, and T35S to CaMV 35S RNA polyA, pSEN refers to the SEN1 promoter, NOA-polyA refers to the polyA of the nopaline synthase gene.

The pCSEN-MtATPG2 recombinant vector was introduced into Agrobacterium tumefaciens using an electroporation method. The transformed Agrobacterium cultures were incubated at 28 until the OD600 value was 1.0, and the cells were harvested by centrifugation at 5,000 rpm for 10 minutes at 25 ° C. 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 OD600 value was 2.0. Four week old Arabidopsis immersed in an Agrobacterium suspension in a vacuum chamber and left under vacuum at 104 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 MtATPG2 gene was used.

Example 2-2 Characterization of T2 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, during the growth of the transformed Arabidopsis, the pollen was treated 5 times with the Basta herbicide, and the transformed Arabidopsis was selected from each pollen. Surprisingly, the T ₁ Arabidopsis transformed with the pCSEN-MtATPG2 vector compared with the phenotype transformed only with a control (a vector without the MtATPG2 gene (pCSEN vector) or their wild-type Arabidopsis), surprisingly the variants were distinct. Multiple traits and biomass enhancing traits were shown.

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, T ₂ transformed seed that had been cold-treated (4 ° C.) for 3 days was cultivated in a pollen, and then T ₂ transgenic Arabidopsis was selected through the treatment of Basta herbicide. Phenotyping of selected Arabidopsis T ₂ transformation lines was performed 50 days and 70 days after germination (FIG. 2).

MtATPG2 -7 with the pCSEN-MtATPG2 construct, MtATPG2 -8, MtATPG2 -15, MtATPG2-16 and Compared to Arabidopsis control (Col-0), all of the MtATPG2 -17 variant lines showed a marked increase in biomass, such as an increase in plant populations, and an increase in long shell length and number. There was a pronounced increase. This increase in seed production seems to be attributable to the increase in the long shell length and the number of seeds.

To confirm whether these phenotypic features are induced by the expression of the transgene, we extracted the total RNA from the control and leaf of the mutant line 25 days after cotyledon formation using the RNasey Plant 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. Thereafter, the synthesized cDNA was used as a template, and PCR was performed using the primers specific to the following Table 1 for the MtATPG2 gene and the TUB 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. As a result, it was confirmed that all five variant lines showed normal expression due to the introduction of the MtATPG2 gene as compared to Arabidopsis control, and these facts prove that the variants were introduced into the MtATPG2 gene. The increase in biomass and seed production were slightly different for each transformation line due to the difference in relative expression of the introduced MtATPG2 gene. On the other hand, these variants were found in the ATPG gene group encoding the Arabidopsis AT-hook protein (Lim et al., Plant J. 2007, 52: 1140, Korean Patent Application 10-2011-0110593). Appeared in a high line of expression.

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. In particular, MtATPG2 can be used as an excellent gene source for the development of high-productivity crops, as it is easy to manufacture plants that increase productivity with the same harvesting season as Arabidopsis wild type through the regulation of gene expression.

Primer Sequence and Sequence Number for MtATPG2 and TUB Gene Expression

Gene name	Forward / Reverse Primer (SEQ ID NO)
MtATPG2	5'-TCT ATG GAT GGG AGA GAG GCT ATG G-3 '(SEQ ID NO: 5) 5'-GAC CTC ATC CTC TTG TCA AGT CAA AGC-3 '(SEQ ID NO: 6)
Tubulin	5'-AAG AGG TTC TCA GCA GTA-3 '(SEQ ID NO: 7) 5'-CCT TCT TCA TCC GCA GTT-3 '(SEQ ID NO: 8)

<Example 3> MtATPG2  Characterization of productivity increase of expression variants

MtATPG2 It is shown in Figure 2 that the plant obtained through the introduction of the gene has an expression trait for multiplicity. In order to analyze the phenotypic characteristics of these multiple traits more accurately, productivity indicators such as seed yields were investigated in five transformation lines and compared with Arabidopsis control.

The productivity indicators applied are plant height, silique number (NTS), biomass (FW), biomass (DW), total seed weight (TSW), and 1,000 seed weights (1,000 SW). Is the average value of 20 objects per line.

As a result, surprisingly MtATPG2 gene mutant lines, MtATPG2 -7, MtATPG2 -8, MtATPG2 -15, MtATPG2 -16 and All of MtATPG2 -17 increased seed production more than 1.5 times compared to Arabidopsis control, especially MtATPG2 -16 and MtATPG2 -17 more than doubled seed production compared to the control. In proportion to this increase in seed production, all variant lines had a long and numbered pattern of increase. The seed weight is 1,000 or almost've found that similar to the control group, MtATPG2 - 7 as compared to the control group It weighed about 1.4 times or more. In the detailed study of the weight of 1,000 seeds, the team observed the size of the seeds under an anatomical microscope. As a result, it was confirmed that MtATPG2-7 increased seed size compared to Arabidopsis control and other transformation lines (FIG. 4). Part of the seed size increased MtATPG2 -7 variants are expected to continue to proceed with further research. Overall, the expression of the MtATPG2 gene does not seem to have a significant effect on the seed size, but it is thought to induce an increase in the long shell length and the number, leading to an increase in seed yield. There was no significant difference in the size of the plant between the mutant and the control, but there was a marked increase in both the biomass and the dry weight, especially in the biodry weight.

Interestingly, the timing of harvesting variant lines with specific traits is not significantly different from Arabidopsis control. This fact is that the increase in productivity due to the expression of the gene is similar to the harvest time of the control can minimize the problem of harvest time.

Therefore, the application of this crop to other crops is considered to have a very high utility value in terms of productivity, such as multiplicity of traits and biomass.

< Example  4> MtATPG2  Expression Variant cytokinin  Characterization of signaling pathway

CLV1 gene involved in the separated MtATPG2 in M. truncatula increase productivity by controlling the cytokinin signaling in Arabidopsis thaliana or / and CLV1 / WUS pathway of cytokinin signaling pathway in order to ensure that provide phenotypic features of senescence delay (receptor protein MtATPG2 the histidine kinase 4, AT2G01830) expression of the gene -: kinase CLAVATA1, AT1G75820) and histidine kinase HK2 (histidine kinase 2, AT5G35750) of the genes having a function, HK3 (histidin kinase3, AT1G27320) and CRE1 (HK4 as cytokinin receptor 7, MtATPG2 -8, and It was aimed at MtATPG2 -15 (Fig. 5). RT-PCR primer information for the applied gene and MtATPG2 and the positive control tubulin are shown in Table 2. As a result, the expressions of HK2 and HK3 genes in the HK gene group were increased in all of the variants compared to the control, and the expression of the CRE1 gene showed similar or similar procedures in the variants compared to the control. On the other hand, the expression of the CLV1 gene did not have a big difference in all of the variants compared to the control.

CLV1 in the cytokinin signaling pathway Primer sequence and sequence number for RT-PCR for the gene, histidine kinase (HK) gene group , MtATPG2 , and positive control TUB gene

gene	Forward / Reverse Primer (SEQ ID NO)
CLV1	CLV1-RT-F: 5'-ACT TAC CTC TGT CTC CCT CA-3 '(SEQ ID NO: 9) / CLV1-RT-R: 5'-GAC CAC CTT TAG ATC CAT GC-3 '(SEQ ID NO: 10)
HK3	HK3-RT-F: 5'-CAA CAA CCA GCC CAT ATT CTC-3 '(SEQ ID NO: 11) / HK3-RT-R: 5'-TTC CAA TAC CCA ATC CCC TC-3 '(Dial No. 12)
CRE1	WOL-RT-F: 5'-CTG AGG AGC AGT CAT TAT CG-3 '(SEQ ID NO: 13) / WOL-RT-R: 5'-GGT TTT GTT GGG AGA GGA GA-3 '(SEQ ID NO: 14)
HK2	HK2-RT-F: 5'-GTA TGG CTC AGA AAT TGG GG-3 '(SEQ ID NO: 15) / HK2-RT-R: 5'-GCC AGA GAG GAG AGA TGA AA-3 '(SEQ ID NO: 16)
MtATPG2	5'-TCT ATG GAT GGG AGA GAG GCT ATG G-3 '(SEQ ID NO: 5) / 5'-GAC CTC ATC CTC TTG TCA AGT CAA AGC-3 '(SEQ ID NO: 6)
Tubulin	5'-AAG AGG TTC TCA GCA GTA-3 '(SEQ ID NO: 7) 5'-CCT TCT TCA TCC GCA GTT-3 '(SEQ ID NO: 8)

We describe histidine , the next step in cytokinin signaling. AHP1 from the phosphotransfer proteins (HP) gene family (AT3G21510) , AHP2 (AT3G29350) , AHP3 (AT5G39340), AHP4 (AT3G16360) , AHP5 (AT1G03430) , and AHP6 Expression patterns of the (AT1G80100) gene were also examined (FIG. 5). The expression pattern of the gene was analyzed by RT-PCR, primer information used is shown in Table 3. As a result, AHP1, AHP2, and in the case of AHP3 showed a high expression in MtATPG2 MtATPG2 -7 and -15 mutants compared to the control, in the case of AHP4 exhibited high expression in MtATPG2 MtATPG2 -7 and -8 variant compared to the control.

RT-PCR primer sequence and sequence number for histidine phosphotransfer proteins (HP) gene group, MtATPG2 , and positive control TUB genes in the cytokinin signaling pathway

gene	Forward / Reverse Primer (SEQ ID NO)
AHP1 (AT3G21510)	AHP1-RT-F: 5'-ATGGATTTGGTTCAGAAGCAGAA-3 '(SEQ ID NO: 17) / AHP1-RT-R: 5'-TCAAAATCCGAGTTCGACGGCC-3' (SEQ ID NO: 18)
AHP2 (AT3G29350)	AHP2I-RT-F: 5'-ATGGACGCTCTCATTGCTCAGC-3 '(SEQ ID NO: 19) / AHP2I-RT-R: 5'-TTA GTT AAT ATC CAC TTG AGG AAC-3' (SEQ ID NO: 20)
AHP3 (AT5G39340)	AHP3-RT-F: 5'-GGACACACTCATTGCTCAGT-3 '(SEQ ID NO: 21) / AHP3-RT-R: 5'-CTGCAAACATCTCACACACC-3' (SEQ ID NO: 22)
AHP4 (AT3G16360)	AHP4I-RT-F: 5'-ATGCAGAGGCAAGTGGCACTCA-3 '(SEQ ID NO: 23) / AHP4I-RT-R: 5'-TTACTTGGGCCTACGTGCTGTC-3' (SEQ ID NO: 24)
AHP5 (AT1G03430)	AHP5-RT-F: 5'-GGTAGTAGCTCCAGTGTCG-3 '(SEQ ID NO: 25) / AHP5-RT-R: 5'-CTAATTTATATCCACTTGAGGAAT-3' (SEQ ID NO: 26)
AHP6 (AT1G80100)	AHP6-3UTR-F: 5'-CAAGCCGACATCAACCGGCTC-3 '(SEQ ID NO: 27) / AHP6-3UTR-R: 5'-AGGGTTTCGCTTCGGTAGCTT-3' (SEQ ID NO: 28)
MtATPG2	5'-TCT ATG GAT GGG AGA GAG GCT ATG G-3 '(SEQ ID NO: 5) / 5'-GAC CTC ATC CTC TTG TCA AGT CAA AGC-3 '(SEQ ID NO: 6)
Tubulin	5'-AAG AGG TTC TCA GCA GTA-3 '(SEQ ID NO: 7) 5'-CCT TCT TCA TCC GCA GTT-3 '(SEQ ID NO: 8)

Through this analysis, we can hypothesize the following. 1) MtATPG2 is not significantly involved in the CLV1 / WUS pathway in the cytokinin pathway. 2) MtATPG2 regulates the expression of the cytokinin receptor gene family and histidine phosphotransfer proteins (HP) gene family to regulate cytokinin signaling to increase plant yield. Productivity traits such as increased biomass and delayed aging traits, and 3) these phenotypic characteristics are regulated according to the expression level of the MtATPG2 gene. That is, normal or relatively low expression of the MtATPG2 gene provides a phenotype of increased productivity, and a relatively high gene expression provides a phenotype of extended melting with a productivity-enhancing trait. The regulation of cytokinin signaling through MtATPG2 was different from that of Arabidopsis AT-hook gene and MtATPG1 . Therefore, this means that the role of this gene in cytokinin signaling is different from that of Arabidopsis AT-hook gene and MtATPG1 . But this gene is also like the Arabidopsis AT-hook gene and MtATPG1 . Since this gene has a role in cytokinin signaling, this gene may also have similar functions in major crops, thus providing many advantages in the development of a multiplicity of major crop varieties.

< Example  5> MtATPG2  Expression Variant  Homo line screening through generation progress And function  analysis

In order to confirm the phenotypic stability against pluripotency and aging delay of the variants, the homologous lines were selected by generation advancement, and then the six phenotypes were identified whether their phenotypes were the same as the previous generation phenotypes. . As shown in FIGS. 6 and 7, most of the transformant T ₃ or T ₄ lines at 50 and 70 days after germination showed distinct multiplicity and biomass enhancement traits as in the previous results. In addition, MtATPG2 -7-15-11 and MtATPG2 -7-15-12 lines in the transformation lines exhibited some aging delay traits as well as productivity enhancing traits. Investigation of the gene expression of the transformants showed that the aging delay traits appearing with this multiplicity were due to the increased expression level of the present gene (FIG. 8). This phenotypic characteristic of the transformant may be slightly different from the previous generation, which is believed to be due to changes in gene expression as the homo line is selected from the hetero line.

In order to confirm the reproducibility of the traits for increasing the productivity of generation-advanced homo lines, the team analyzed the productivity of the transformants by analyzing the productivity indicators. All of the MtATPG2 gene expression variant lines showed 1.2-fold increase in seed production compared to Arabidopsis control, especially MtATPG2 -8-8, MtATPG2 -15-3 and MtATPG2-15-6 , 1.5 times more than the Arabidopsis control. Seed production increased. Interestingly, there is a difference in seed production depending on the degree of gene expression. I.e., gene expression levels are low MtATPG2 - showed that was higher than the average seed yield of control line 7 is low In the mutant line, while the average seed yield by 15 lines the highest, the degree of gene expression higher MtATPG2. Therefore, the expression regulation of the present gene is expected to provide higher seed productivity. In proportion to this increase in seed production, most of the variant lines have had long lengths and increased numbers. Interestingly, the seed weight is 1,000, most of the mutants were similar to the control've found me, MtATPG2 - 7 lines was confirmed that it has the seed size increased significantly compared to control Arabidopsis thaliana and other transgenic lines. This fact is consistent with the results of the previous generation, and part of the seed size increased MtATPG2 -7 mutant lines are expected to continue to proceed with further research. In addition, both the variants in the biomass and biomass showed a marked increase compared to the control (Table 4). This result is the same as the result of the productivity increase of the previous generation. In view of this fact, MtATPG2 Gene expression is expected to provide many advantages for stable generation of productivity-enhancing traits when applied to crops in that the expression of genes maintains the stabilization of phenotypic traits for productivity-enhancing traits.

Meanwhile, as shown in the previous results, MtATPG2 Expression T ₃ or T ₄ are transformed variants of the melt extending from the gene group that appears coding for the Arabidopsis thaliana are AT-hook protein did not appear very strong, but still in the mutant lines such as gene expression levels are high MtATPG2 -7 variants with the trait of delayed senescence I could see that.

In order to accurately analyze the traits for the prolongation of the melting of the gene, the characteristics of age-dependent aging and cancer-induced aging were investigated in T ₃ or T ₄ homo variant lines obtained through generation evolution. To identify age-dependent aging delayed traits of MtATPG2 expressing variants, phenotypic observations were made on the left leaf 3 to 4 times 56 days after the cotyledon production from 16 days after generation of cotyledons in T ₃ or T ₄ homo generations. Leaf chlorophyll content was measured and compared with Arabidopsis control. As a result, in the Arabidopsis control group, the leaves were yellowed after 32 days, and the leaves entered necrosis at 40 days. MtATPG2-8 and MtATPG2 -15 mutant lines age of wild - I've found similar age-dependent phenomenon MtATPG2 -7 mutant lines were found to have the traits of aging delay compared to the control (Fig. 9). In order to examine these phenotypic features in more detail, chlorophyll content was measured during age-dependent aging. 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 with the phenotypic characteristics of the variant MtATPG2 -8 and MtATPG2 -15 mutant lines or've found a similar trend with changes in the chlorophyll content wild MtATPG2 -7 mutant lines are shown to be chlorophyll degradation is significantly delayed compared to the control (Fig. 10). These results were similar to the phenotypic features of aging delay in the previous generation.

A second study of aging delayed trait analysis examined the characteristics of cancer-induced aging. To detach the T ₃ or T ₄ after germination in three homo 3-4 times 21 days left lobe (rosette leaf) in order to analyze the characteristics of aging delay traits of the leaves of the mutant expression MtATPG2 for cancer treatment known to promote aging factor 3 mM After floating in MES buffer (2- [N-morpholino] -ethanesulfonic acid, pH 5.8), the phenotype was observed and leaf chlorophyll content was measured every 6 days until 6 days every 2 days, and compared with Arabidopsis control. . As a result, in the Arabidopsis control group, the leaves were yellowed 4 days after the cancer treatment, and the leaves entered necrosis (necrosis) on the 6th day. MtATPG2 -8 and MtATPG2 -15 variant of the control lines are cancer-induced aging and appear nateu or MtATPG2 -7 mutant lines were found to have similar traits of aging delay by keeping the degree to melt even after 6 days (Fig. 11). In order to identify these phenotypic features, chlorophyll content was investigated during cancer-induced aging, and the chlorophyll content decrease pattern was proportional to the phenotypic features (FIG. 12). In view of this fact, MtATPG2 Since gene expression maintains the stabilization of phenotypic traits against pluripotent traits and aging delayed traits even in generational development, it may be possible to develop multiply crops through stable generation of agricultural traits when applied to crops.

To investigate the resistance of MtATPG2 expression variants to external stress, we examined the responses to oxidative and salt stress. In order to investigate resistance to oxidative stress, 4 mM H ₂ O ₂ was added to 3 mM MES buffer, and after germination, the leaves were detached and floated 25 and 3 days old. Changes in chlorophyll content were investigated to determine the degree of resistance to oxidative stress. As compared to control Arabidopsis MtATPG2 - 7 lines H ₂ O ₂ Delaying of the yellowing of leaves with treatment (FIG. 13A) and also a decrease in chlorophyll content was observed (data not shown). On the other hand, as a result of examining the resistance to salt stress through the 150 mM NaCl treatment, the mutants showed little delay in leaf sulfation compared to Arabidopsis control (FIG. 13B), and little delay in decrease in chlorophyll content (data not shown). This suggests that high expression of MtATPG2 provides resistance to oxidative stress in plants, while resistance to salt stress provides little to no difference in gene expression. Thus MtATPG2 The resistance of genes to oxidative stress is thought to provide many advantages in the development of productivity-enhancing crops, along with the existing traits that increase plant productivity.

Claims (14)

1. An MtATPG2 protein having 90% or more sequence homology with the amino acid sequence set forth in SEQ ID NO: 2 and having a productivity enhancing function, a stress resistance function, and an aging delay function of a plant.
2. MtATPG2 gene encoding the protein of claim 1.
3. (a) inserting the gene of claim 2 into an expression vector operably linked to a regulatory sequence capable of expressing it,

   (b) transforming the expression vector into a plant, and

   (c) a method for producing a plant having a productivity increasing characteristic, comprising selecting a plant having the productivity increasing characteristic.
4. The method of claim 3,

   The gene is a method for producing a plant having productivity enhancement characteristics, characterized in that the gene consisting of the nucleotide sequence of SEQ ID NO: 1.
5. (a) inserting the gene of claim 2 into an expression vector operably linked to a regulatory sequence capable of expressing it,

   (b) transforming the expression vector into a plant, and

   (c) selecting a plant having a stress resistance characteristic comprising the steps of producing a plant having a stress resistance characteristic.
6. The method of claim 5,

   The gene is a method for producing a plant having stress resistance characteristics, characterized in that the gene consisting of the nucleotide sequence of SEQ ID NO: 1.
7. (a) inserting the gene of claim 2 into an expression vector operably linked to a regulatory sequence capable of expressing it,

   (b) transforming the expression vector into a plant, and

   (c) selecting a plant having a aging retardation property.
8. The method of claim 7, wherein

   The gene is a method for producing a plant having a aging delay characteristics, characterized in that the gene consisting of the nucleotide sequence of SEQ ID NO: 1.
9. (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 expressing it, and

   (b) transforming the plant with the expression vector.

   How to increase plant productivity.
10. (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 expressing it, and

    (b) transforming the plant with the expression vector.

    How to increase the stress resistance of plants.
11. (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 expressing it, and

    (b) transforming the plant with the expression vector.

    How to delay the aging of plants.
12. A transgenic plant obtained by the method according to claim 3, wherein the gene encoding the amino acid sequence of SEQ ID NO: 2 is introduced into a plant and expressed, thereby exhibiting productivity enhancing properties.
13. 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 into and expressed in the plant and has stress resistance properties.
14. A transgenic plant obtained by the method of claim 7, wherein the gene encoding the amino acid sequence of SEQ ID NO: 2 is introduced into and expressed in the plant, thereby exhibiting aging delay characteristics.

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