KR101898392B1 - Method for increasing regeneration efficiency of plant using atx4 gene and plant thereof - Google Patents

Abstract

The present invention relates to a method for increasing regeneration efficiency of a plant using an ATX4 gene derived from Arabidopsis thaliana, a method for manufacturing a transgenic plant having increased regeneration efficiency, a transgenic plant manufactured thereby, and a composition for increasing plant regeneration efficiency. The present invention enhances the low regeneration efficiency of the plant transformation process, thereby enabling the regeneration of various plant species.

Description

TECHNICAL FIELD The present invention relates to a method for increasing the regeneration efficiency of a plant using the ATX4 gene derived from Arabidopsis thaliana and a method for increasing plant regeneration efficiency using the ATX4 gene derived from Arabidopsis thaliana.

The present invention relates to a method for increasing the regeneration efficiency of a plant using the Arabidopsis thaliana ATX4 gene, a method for producing a transgenic plant having an increased regeneration efficiency, a transgenic plant produced thereby, and a composition for increasing plant regeneration efficiency.

Plants have a remarkable ability to stimulate reprogramming of cells from differentiated somatic cells, and subsequent regeneration processes induce new plants from pluripotent calli. The proper ratio of auxin and cytokinin is essential for callus formation as well as for regeneration of the organs. Likewise, genetic elements such as hormone biosynthesis genes and signals are associated with plant regeneration from various tissue grafts.

In particular, the shift in organizational identity is an essential process for promoting efficient cell depletion and tracheal regeneration. Callus tissues are similar to those of root division tissues, and molecular characteristics are also similar in that the induced calli express ectopically the root cleavage genes. However, the callus can be efficiently induced from all the tissue grafts, even at the ground level, by an in vitro tissue culture method. Subsequently, the induced pluripotent callus is preferentially subjected to shoot formation for successful whole plant regeneration. Thus, the two-step tissue identity change, which consists of leaf-to-callus dedifferentiation from the leaves and callus-to-leaf regeneration from the leaves, is an in vitro tissue culture derived from leaf grafts .

Accumulated studies suggest that organizational identity is largely established by pre-dielectrophoretic chromatin composition. In general, differentiated cells have a closed chromatin state that induces a stable gene expression profile that identifies tissue identity, while pluripotent stem cells have an open chromatin state capable of differentiating into potentially diverse cellular identities. As a basis, PcG (Polycomb group) and TrxG (Trithorax group) proteins are involved in broad cell depletion and eukaryotic organ regeneration. However, despite the remarkable ability of plants to regenerate, a comprehensive postural mechanism to induce tissue identity conversion due to large-scale gene expression changes during plant regeneration is not yet known.

1. P. Che, S. Lall, D. Nettleton, S. H. Howell, Gene expression programs during shoot, root, and callus development in Arabidopsis tissue culture. Plant Physiol. 141, 620-637 (2006). 2. J. Duclercq, B. Sangwan-Norreel, M. Catterou, R. S. Sangwan, De novo shoot organogenesis: from art to science. Trends Plant Sci. 16, 597-606 (2011). 3. M. Fan, C. Xu, K. Xu, Y. Hu, LATERAL ORGAN BOUNDARIES DOMAIN transcription factors direct callus formation in Arabidopsis regeneration. Cell Res. 22, 1169-1180 (2012). 4. B. Bai, Y. H. Su, J. Yuan, X. S. Zhang, Induction of somatic embryos in Arabidopsis requires local YUCCA expression mediated by down-regulation of ethylene biosynthesis. Mol. Plant 6,1247-1260 (2013). 5. W. Ckurshumova, T. Smirnova, D. Marcos, Y. Zayed, T. Berleth, Irrepressible MONOPTEROS / ARF5 promotes de novo shoot formation. New Phytol. 204, 556-566 (2014). 6. K. Sugimoto, Y. Jiao, E. M. Meyerowitz, Arabidopsis regeneration from multiple tissues occurs via a root development pathway. Dev. Cell 18, 463-471 (2010). 7. A. Kareem, K. Durgaprasad, K. Sugimoto, Y. Du, AJ Pulianmackal, ZB Trivedi, PV Abhayadev, V. Pinon, EM Meyerowitz, B. Scheres, K. Prasad, PLETHORA genes control regeneration by a two- step mechanism. Curr. Biol. 25, 1017-1030 (2015). 8. T. Katsuyama, R. Paro, Epigenetic reprogramming during tissue regeneration. FEBS Lett. 585, 1617-1624 (2011). 9. P. Delgado-Olguin, F. Recillas-Targa, Chromatin structure of pluripotent stem cells and induced pluripotent stem cells. Brief. Funct. Genomics 10, 37-49 (2011). 10. NC Sheffield, RE Thurman, L. Song, A. Safi, JA Stamatoyannopoulos, B. Lenhard, GE Crawford, TS Furey, Patterns of regulatory activity across diverse human cell types predictive tissue identity, transcription factor binding, and long-range interactions. Genome Res. 23,777-788 (2013). 11. L. Serrano, B. N. Vazquez, J. Tischfield, Chromatin structure, pluripotency and differentiation. Exp. Biol. Med. 238, 259-270 (2013). 12. T. Chen, S. Y. Dent, Chromatin modifiers and remoders: regulators of cellular differentiation. Nat. Rev. Genet. 15, pp. 93-106 (2014). 13. V. M. Shchuka, N. Malek-Gilani, G. Singh, L. Langroudi, N. K. Dhaliwal, S. D. Moorthy, S. Davidson, N. N. Macpherson, J. A. Mitchell, Chromatin dynamics in lineage commitment and cellular reprogramming. Genes 17,641-661 (2015). 14. D. C. Kraushaar, K. Zhao, The epigenomics of embryonic stem cell differentiation. Int. J. Biol. Sci. 9, 1134-1144 (2013). 15. C. He, X. Chen, H. Huang, L. Xu, Reprogramming of H3K27me3 is critical for acquisition of pluripotency from cultured Arabidopsis tissues. PLoS Genet. 8, e1002911 (2012). 16. Z. Avramova, Evolution and pleiotropy of TRITHORAX function in Arabidopsis. Int. J. Dev. Biol. 53, 371-381 (2009). 17. C. E. Bita, T. Gerats, Plant tolerance to high temperature in a changing environment: scientific fundamentals and production of heat stress-tolerant crops. Front. Plant Sci. 4,273 (2013). 18. F. Pontvianne, T. Blevins, C. S. Pikaard, Arabidopsis histone lysine methyltransferases. Adv. Bot. Res. 53, 1-22 (2010). 19. R. Alvarez-Venegas, Regulation by polycomb and trithorax group proteins in Arabidopsis. Arabidopsis Book 8, e0128 (2010). 20. R. J. DiDonato, E. Arbuckle, S. Buker, J. Sheets, J. Tobar, R. Totong, P. Grisafi, G. R. Fink, J. L. Celenza, Arabidopsis ALF4 encodes a nuclear-localized protein required for lateral root formation. Plant J. 37, 340-353 (2004). 21. B. Shang, C. Xu, X. Zhang, H. Cao, W. Xin, Y. Hu, Very-long-chain fatty acids and their ability to restrict regeneration by pericycle competence for callus formation in Arabidopsis. Proc. Natl. Acad. Sci. USA 3, 5101-5106 (2016). 22. D. E. Schones, X. Chen, C. Trac, R. Setten, P. J. Paddison, G9a / GLP-dependent H3K9me2 patterning alters chromatin structure at CpG islands in hematopoietic progenitors. Epigenetics Chromatin 7, 23 (2014). 23. M. J. Boland, K. L. Nazor, J. F. Loring, Epigenetic regulation of pluripotency and differentiation. Circ. Res. 115, 311-324 (2014). 24. P. Venkatesh, I. V. Panyutin, E. Remeeva, R. D. Neumann, I. G. Panyutin, Effect of chromatin structure on the extent and distribution of DNA double strand breaks produced by ionizing radiation; comparative study of hESC and differentiated cells lines. Int. J. Mol. Sci. 17, pii: E58 (2016). 25. T. Hruz, O. Laule, G. Szabo, F. Wessendorp, S. Bleuler, L. Oertle, P. Widmayer, W. Gruissem, P. Zimmermann, Genevestigator v3: analysis of transcriptomes. Adv. Bioinformatics 2008, 420747 (2008). 26. Y. Tamada, J. Y. Yun, S. C. Woo, R. M. Amasino, ARABIDOPSIS TRITHORAX-RELATED 7 is required for methylation of lysine 4 of histone H3 and for transcriptional activation of FLOWERING LOCUS C. Plant Cell 21, 3257-3269 (2009).

The greatest hurdle in plant transformation is known to be low regeneration efficiency. In order to cultivate high value-added crops agronomically, transgenic processes are required. The regeneration process after the callus formation of the tissues is very inefficient, and the transformation of the crops is very limited. The present invention aims to provide a technology which can substantially improve the regeneration efficiency of plants by identifying and introducing regeneration promoting genes.

However, the problems to be solved by the present invention are not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

The first aspect of the present invention can provide a method for increasing plant regeneration efficiency, including transforming a plant cell with a recombinant vector comprising a gene encoding Arabidopsis thaliana ATX4 protein.

The second aspect of the present invention can provide a method of producing a transgenic plant having an increased regeneration efficiency, which comprises transforming a plant using a recombinant vector comprising a gene encoding Arabidopsis thaliana ATX4 protein .

A third aspect of the present invention provides a transgenic plant having a gene encoding Arabidopsis thaliana ATX4 protein, or a seed thereof, produced by the method of the second aspect of the present invention.

The fourth aspect of the present invention can provide a composition for increasing plant regeneration efficiency, which comprises, as an active ingredient, a gene encoding ATX4 protein derived from Arabidopsis thaliana.

The above-described task solution is merely exemplary and should not be construed as limiting the present invention. In addition to the exemplary implementations described above, there may be additional implementations and embodiments described in the drawings and detailed description of the invention.

The ATX4 gene, which promotes the regeneration of the plant topology, was first discovered by the present invention, and the ATX4 protein efficiently induced superficial tissues from dedifferentiated cells by positively controlling various genes that determine the topological identity. In particular, it has been found that chromatin regulator in ATX4 (ARABIDOPSIS TRITHORAX 4) / SDG16 (SET DOMAIN GROUP 16) that dramatically controlled in this process in vitro tissue culture prior to an appropriate cell dedifferentiation and regeneration.

Using these findings, it is possible to improve the low regeneration efficiency of the plant transformation process, thereby enabling regeneration of various plant species, thereby contributing to broader transformation of agronomic crops, shortening breeding period, It is expected that it will be possible to improve the efficiency of the system.

Brief Description of the Drawings Figure 1 is a plot showing improved cell depolarization ability of the atx4 -2 mutation. FIG. 1A is a photograph showing callus formation. Leaf grafts from the third leaf of two-week-old wild type and atx4-2 seedlings were used for callus induction on callus induction medium (CIM) (n> 30). Plates were incubated for 2 weeks under continuous dark conditions and photographed (scale bar = 5 mm). FIG. 1B is a graph showing the result of living body weight measurement. Thirty of the calluses were collected to measure live weight, and statistically significant differences between wild type and mutant were marked with an asterisk (Student's t-test, * P < 0.05). The bar represents the standard error of the mean.
Figure 2 is a graph showing the ability of ATX4 to massively regulate the over-expression gene. Figure 2a shows a two-way hierarchical clustering heat map. A total of 1,569 genes were standardized using Z-score calculation. Differentially expressed transcripts were clustered in wild type and atx4-2 callus. The color key at the upper right corner represents the color of the heat map. 2B is a graph showing the number of genes that are differentially expressed in ATX4-2 (ATX4 mutant). Figure 2c shows as a tissue-specific expression of genes that are down-regulated in atx4-2, analyzed the top 80 genes in Genevestigator. Figure 2d is a graph showing the expression of the leaf identity gene in the atx4-2 callus. Transcript accumulation was analyzed by RT-qPCR and the eIF4a gene was used as an internal control. Averages of three replicates were used and statistically significant differences between wild-type and mutant calli were marked with an asterisk (Student's t-test, * P <0.05). The bar represents the standard error of the mean. (DAC: days after incubation on CIM). Figure 2e is a graph showing the expression of genes in the root identity atx4 -2 callus.
Figure 3 is a plot showing that ATX4 promotes H3K4me3 deposition in the superficial identity gene during callus formation. Figure 3a is an immunoblot showing the overall results of the accumulation in the main histone marks atx4 -2. Immature calli were collected for immunoblot analysis and the modified major histone proteins (arrows) were detected immunologically using the corresponding antibodies. A portion of the Coomassie blue stained gel was labeled as loading control. FIG. 3B is a graph showing H3K4me3 accumulation in the superficial identity gene at atx4-2 . FIG. Concentration of precipitated DNA was analyzed by ChIP-qPCR, and statistically significant differences were marked by an asterisk (Student's t-test, * P < 0.05). The bar represents the standard error of the mean.
FIG. 4 is a graph showing the ability of the ATX4 to promote the regeneration ability of the above-ground part. FIG. 4A is a graph showing the expression of ATX4 during topical regeneration (DAS: days after incubation on SIM). 4B is a photographic image showing the overgrowth of the land surface. (N> 30) using a callus cultured on CIM for 2 weeks, and photographed (scale bar = 5 mm) for 2 weeks under continuous light conditions. . 4C is a graph showing the quantification of the overgrowth of the ground surface. The number of regenerated leaflets from the callus was measured (n> 30) and statistically significant differences between wild type and atx4-2 were marked with an asterisk (Student's t-test, * P <0.05). Figure 4d is a graph showing the expression of the gene in the above-ground identity atx4 -2 regeneration of aerial parts. Figure 4e shows a working diagram. The expression of ATX4 , CLF and SWN dynamically fluctuates during the regeneration process to properly establish tissue identity, and the complementary expression of ATX4 and PRC2 elements ensures balanced plant regeneration.
5 is a graph showing the effect of promoting regeneration efficiency of a plant transformed to overexpress ATX4.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those skilled in the art can easily carry out the present invention. It should be understood, however, that the present invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. In order to clearly explain the present invention in the drawings, portions not related to the description are omitted.

Throughout this specification, when an element is referred to as "including " an element, it is understood that the element may include other elements as well, without departing from the other elements unless specifically stated otherwise.

The terms "about "," substantially ", etc. used to the extent that they are used throughout the specification are intended to be taken to mean the approximation of the manufacturing and material tolerances inherent in the stated sense, Accurate or absolute numbers are used to help prevent unauthorized exploitation by unauthorized intruders of the referenced disclosure. The word " step (or step) "or" step "used to the extent that it is used throughout the specification does not mean" step for.

Throughout this specification, the term "combination thereof" included in the expression of the machine form means one or more combinations or combinations selected from the group consisting of the constituents described in the expression of the machine form, And the like.

Throughout this specification, the description of "A and / or B" means "A, B, or A and B".

Hereinafter, a method for increasing the regeneration efficiency of a plant, a method for producing a transgenic plant having an increased regeneration efficiency, a plant produced by the method or a seed thereof, and a composition for increasing the regeneration efficiency of a plant Will be described in detail with reference to embodiments, examples and drawings. However, the present invention is not limited to these embodiments and examples and drawings.

The first aspect of the present invention can provide a method for increasing plant regeneration efficiency, including transforming a plant cell with a recombinant vector comprising a gene encoding Arabidopsis thaliana ATX4 protein.

According to one embodiment of the present invention, the gene encoding the ATX4 protein may include, but is not limited to, a nucleic acid sequence of SEQ ID NO: 1. For example, the ATX4 protein-encoding gene may be a variant of the nucleic acid sequence of SEQ ID NO: 1 and is at least about 70%, at least about 80%, at least about 90%, or at least about 95% Or more of the sequence homology. Here, the percentage (%) of sequence homology can be ascertained by comparing the comparison region with two optimally arranged sequences, and some of the sequences in the comparison region include reference sequences (addition or deletion) of the optimal sequence of the two sequences (I. E., Gap) relative to &lt; / RTI &gt;

For example, the "recombinant vector" may be a recombinant expression vector and may refer to a bacterial plasmid, a phage, a yeast plasmid, a plant cell virus, a mammalian cell virus, or other vector. The expression vector may include, but is not limited to, one or more of the elements consisting of a replication origin, a promoter, a marker gene, and a translation control element.

For example, the expression vector may comprise one or more selectable markers. The marker is typically a nucleic acid sequence having a property that can be selected by a chemical method, and includes all genes capable of distinguishing a transformed cell from a non-transformed cell. Examples include herbicide resistance genes such as glyphosate or phosphinothricin, antibiotics such as kanamycin, G418, Bleomycin, hygromycin, chloramphenicol, Resistant genes, but may not be so limited.

The recombinant vector comprising the gene encoding the ATX4 protein can be constructed by methods known to those skilled in the art. Such methods may include, but are not limited to, in vitro recombinant DNA technology, DNA synthesis techniques, and in vivo recombination techniques.

For example, the plant may be a dicotyledonous plant such as Diapensiaceae, Clethraceae, Pyrolaceae, Ericaceae, Myrsinaceae, Primulaceae, , Plumbaginaceae, Ebenaceae, Styracaceae, Symplocaceae, Oleaceae, Loganiaceae, Gentianaceae, and pomegranate and pomegranate. Menyanthaceae, Apocynaceae, Asclepiadaceae, Rubiaceae, Polemoniaceae, Convolvulaceae, Boraginaceae, Verbenaceae, Labiatae, , Solanaceae, Scrophulariaceae, Bignoniaceae, Acanthaceae, Pedaliaceae, Orobanchaceae. It has been reported that the plants of the genus Gesneriaceae, Lentibulariaceae, Phrymaceae, Plantaginaceae, Caprifoliaceae, Adoxaceae, Valerianaceae, Dipsacaceae, Campacalaceae, Compositae, Myricaceae, Juglandaceae, Salicaceae, Betulaceae, Fagaceae, Ulmaceae, Moraceae, , Lepidoptera, Lepidoptera, Lepidoptera, Lepidoptera, Lepidoptera, Urticaceae, Santalaceae, Loranthaceae, Polygonaceae, Phytolaccaceae, Nyctaginaceae, Aizoaceae, (Caryophyllaceae), Chenopodiaceae, Amaranthaceae, Cactaceae, Magnoliaceae, Illiciaceae, Lauraceae, Cercidiphyllaceae, Cercopitaceae, Ranunculac eae), Berberidaceae, Lardizabalaceae, New sand vines (Menispermaceae), Nymphaeaceae, Ceratophyllaceae, Cabombaceae, Saururaceae, , Piperaceae, Chloranthaceae, Aristolochiaceae, Actinidiaceae, Theaceae, Guttiferae, Droseraceae, Papaveraceae, and so on. ), Capparidaceae, Cruciferae, Platanaceae, Hamamelidaceae, Crassulaceae, Saxifragaceae, Quercus mongolica, and Chamaecyparis obtusa. Eucommiaceae; Pittosporaceae; Rosaceae; Leguminosae; Oxalidaceae; Geraniaceae; Tropaeolaceae; Zygophyllaceae; Linaceae; Euphorbiaceae), star moss (Callitrichaceae), Rutaceae, Simaroubaceae, Meliaceae, Polygalaceae, Anacardiaceae, Aceraceae, Sapindaceae, Chickadee, Hippocastanaceae, Sabiaceae, Balsaminaceae, Aquifoliaceae, Celastraceae, Staphyleaceae, Buxaceae, Empetraceae, and the like. It is also known as Rhamnaceae, Vitaceae, Elaeocarpaceae, Tiliaceae, Malvaceae, Sterculiaceae, Thymelaeaceae, Elaeagnaceae, (Flacourtiaceae), Violaceae, Passifloraceae, Tamaricaceae, Elatinaceae, Begoniaceae, Cucurbitaceae, Lythraceae, Pomegranates, Trees (Punicac It is known that the eucalyptus species is the most abundant species in the genus Araliaceae and the genus Umbelliferae (Apiaceae) in the genus Eae, Onagraceae, Haloragaceae, Alangiaceae, Cornaceae, Or a plant such as alfalfa, soybean, broccoli, cabbage, canola, carrot, cauliflower, celery, cabbage, cotton, cucumber, eggplant, lettuce, melon, pea, pepper, peanut, potato, But are not limited to, pumpkin, radish, rapeseed, spinach, soybean, zucchini, sugar beet, sunflower, tobacco, tomato, and watermelon.

For example, the plant may be cultivated from monocotyledons such as Brachypodium, corn, onion, rice, millet, wheat, rye, millet, sorghum, oats, lime, switchgrass, May be selected, but may not be limited thereto.

According to an embodiment of the present invention, the ATX4 protein may include, but not limited to, the amino acid sequence of SEQ ID NO: 2. For example, the ATX4 protein may be a variant of the amino acid sequence of SEQ ID NO: 2 and may comprise at least about 70%, at least about 80%, at least about 90%, or at least about 95% sequence identity with the amino acid sequence of SEQ ID NO: &Lt; / RTI &gt;

For example, the ATX4 protein may be overexpressed in the process of regeneration from a callus to a leaf-like tissue, and the expression thereof may be suppressed during the process of dedifferentiation from a plant graft to a callus, but the present invention is not limited thereto. Induction and inhibition of such expression can be achieved by selecting and controlling the type of promoter to be inserted into the vector, but the present invention is not limited thereto.

For example, the ATX4 protein-encoding gene can be overexpressed at any time by a promoter such as CaMV 35S. In this case, the dedifferentiation process can be suppressed somewhat, but the regeneration process is significantly promoted, Can be obtained. Alternatively, the ATX4 protein-encoding gene may be used together with a promoter specifically expressed in the regeneration process to overexpress only the regeneration process, thereby remarkably increasing the regeneration efficiency without restraining the dedifferentiation process.

The second aspect of the present invention can provide a method of producing a transgenic plant having an increased regeneration efficiency, which comprises transforming a plant using a recombinant vector comprising a gene encoding Arabidopsis thaliana ATX4 protein .

Transformation of a plant may mean any method of transferring DNA to a plant. Such transformation methods do not necessarily have a regeneration and / or tissue culture period. Any transformation method known in the art can be used for transformation according to the present invention. For example, the calcium / polyethylene glycol method for protoplasts (Krens, FA et al., 1982, Nature 296, 72-74; Negrutiu I. et al., June 1987, Plant Mol. Biol. 8, 363-373) (Shillito RD et al., 1985 Bio / Technol. 3, 1099-1102), microinjection into plant elements (Crossway A. et al., 1986, Mol. Gen. Genet. (Klein et al., 1987, Nature 327, 70), infiltration of plants or the transformation of Agrobacterium species by transformation of mature pollen or microparticles But not limited to, infections caused by (non-integrative) viruses in Tuomorphisense mediated gene transfer (EP 0 301 316), and the like.

According to one embodiment of the present invention, the gene encoding the ATX4 protein may comprise, but not be limited to, the nucleic acid sequence of SEQ ID NO: 1.

A third aspect of the present invention provides a transgenic plant having a gene encoding Arabidopsis thaliana ATX4 protein, or a seed thereof, produced by the method of the second aspect of the present invention.

The fourth aspect of the present invention can provide a composition for increasing plant regeneration efficiency, which comprises, as an active ingredient, a gene encoding ATX4 protein derived from Arabidopsis thaliana. Transformation of a plant using the composition of the present invention can increase plant regeneration efficiency.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[Example]

Plant materials and growth conditions

Unless otherwise indicated, all experiments were carried out with Arabidopsis thaliana , Columbia-0 ecotype) was used. Plants were grown under long-term (LD) conditions (16 hours light / 8 hours cancer cycle) using white fluorescent lamps (120 μmol photons m-2 s-1) at 22-23 ° C.

For callus induction, leaf sections of 2-week-old plants were cultured in callus induction medium (CIM) (0.5 / / mL 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.05 / / And cultured in a dark room. For shoot regeneration, the induced calli were cultured in a topical induction medium (SIM) (B5 medium supplemented with 0.9 μmol / L 3-indoleacetic acid, 2.5 μmol / L 2-isopentenyl adenine) Lt; RTI ID = 0.0 &gt; 23 C &lt; / RTI &gt; for 18 days.

Production of Transgenic Plants

ATX4 deficient mutant (atx4 - 2) is Tamada, etc., (2009), "ARABIDOPSIS TRITHORAX -RELATED 7 is required for methylation of lysine 4 of histone H3 and for transcriptional activation of FLOWERING LOCUS," C. Plant Cell 21 (10): 32573269. &lt; / RTI &gt;

For the production of mutant plants overexpressing ATX4, the ATX4 gene was fused to the cauliflower mosaic virus (CaMV) 35S promoter, which was accomplished by subcloning into the pBA002 vector. The structure was introduced into Agrobacterium and transformed into a plant (Arabidopsis thaliana) by the floral dip method through it.

mRNA-Seq data

To construct the RNA library using the TruSeq RNA library kit, 1 μg of total RNA was used. The method included polyA-selected RNA extraction, RNA fragmentation, random hexamer primed reverse transcription and 100 nt pair-end sequencing by Illumina HiSeq2000. The library was quantified using qPCR according to the qPCR Quantification Protocol Guide and validated using the Agilent Technologies 2100 Bioanalyzer.

To assess expression levels, RNA-Seq readings are mapped to the Arabidopsis reference genome (ftp://ftp.arabidopsis.org/home/tair) using TopHat, which can report split-read alignment across split junctions . The transfer counts were calculated and the relative abundance of transcripts was measured in FPKM (Fragments Per Kilobase of exon per Million fragments mapped) using Cufflink.

Statistical analysis of gene expression levels

The raw data was calculated as the FPKM of each transcript of each sample using Cufflinks software. Transcripts with concentrations of FPKM greater than 1 for the entire sample were excluded. The FPKM value of the filtered transcript was added to 1 to facilitate log2 conversion. The filtered data was algebraically transformed and normalized using the logarithmic normalization method. For each transcript, the change in drainage between the case and the control was calculated. The transcripts that are differentially expressed may have at least one or more of the total comparisons | &Gt; 2.

Gene Ontology (GO) Term Enrichment Analysis

Gene function annotation analysis for a list of differentially expressed genes (DEG) was performed using the DAVID tool (http://david.abcc.ncifcrf.gov/). The DAVID tool provides functional annotations in over 40 annotation categories, including GO terms, protein-protein interactions, protein function domains, disease associations, bio-pathways, sequence common features, homology, gene function summary, gene expression, to provide. In the DAVID annotation system, the gene richness was measured in annotation terms using the modified Fisher Exact p value (EASE score). If the EASE score is lower than 0.05 for a particular GO-term, it is interpreted that the given gene list is related to the GO term rather than by a random probability. All data analysis and visualization of the differentially expressed genes was performed using R 3.1.2 software (www.r-project.org).

Quantitative real-time RT-PCR analysis

Total RNA was extracted according to the manufacturer's recommended method using TRI reagent (TAKARA Bio, Singa, Japan). First strand cDNA was synthesized from 2 μg of total RNA by reverse transcription using Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase (Dr. Protein, Seoul, Korea) with oligo (dT20). The cDNA was diluted to 100 μL with TE buffer, and 1 μL of the diluted cDNA was used for PCR amplification.

Quantitative RT-PCR reactions were performed in 96-well blocks using a Step-One Plus Real-Time PCR system (Applied Biosystems). The values of each primer set were standardized with respect to the eIF4A (EUKARYOTIC TRANSLATION INITIATION FACTOR 4A1) gene (At3g13920). All RT-qPCR reactions were performed in triplicate using total RNA samples extracted from three independent replicate samples. The relative ΔΔC _T method was used to evaluate the relative amounts of each amplified product in the sample. The critical period (C _T ) was automatically determined for each reaction set in the analysis software using the basic parameters. The specificity of the RT-qPCR reaction was determined by melting curve analysis of the amplification product.

Chromatin Immunoprecipitation (ChIP)

Anti-H3K4me3 (Millipore, 07-473) and anti-H3K27me3 antibody (Cell Signaling Technology, C36B11) and salmon sperm DNA / protein A agarose beads (Millipore, 16-157) were used for chromatin immunoprecipitation. The DNA was purified using phenol / chloroform / isoamyl alcohol and sodium acetate (pH 5.2). Levels of precipitated DNA fragments were quantified by quantitative real-time PCR using specific primer sets. The values were normalized to the input DNA level. Control plant values were set to 1 after standardization for eIF4a for quantitative PCR analysis.

Immunoblot analysis

The harvested plant material was pulverized in liquid nitrogen and whole cell extracts were suspended in SDS-PAGE sample loading buffer. Protein samples were analyzed by SDS-PAGE (10% gel) and blotted on Hybond-P + membrane (Amersham-Pharmacia). The epitope-tagged proteins were detected using anti-H3K4me3 (Millipore, 07-473), anti-H3K9me3 (Millipore, 07-442), anti-H3K27me3 (Cell Signaling Technology, C36B11), or anti-H3K36me3 antibody (Abcam, ab9050) Were immunologically detected.

result

From the initial screening, we have found that PRCs (Polycomb Repressive Complexes) and some TrxG proteins are associated with cellular de-differentiation processes in Arabidopsis . Indeed, the deposition of the inhibited H3K27me3 mark by the PRC2 complex is broadly increased in the leaf identity gene during the transition from leaf to callus, thereby eliminating the bud identity and establishing universality. clf The swn (curly leaf swinger ) and emf2 (embryonic flower 2 ) mutants exhibit reduced callus formation due to the suppression of leaf identity genes. Consistently, PcG members do not play an important role in root graft-derived callus formation.

Of the five ATX (ARABIDOPSIS TRITHORAX) proteins that precipitate the H3K4me3 activation mark, the ATX4 protein was mainly involved in callus formation. The genetic mutation of ATX4 was used to confirm callus formation ability on callus-induced medium (CIM) and accelerated callus formation rate when induced from leaf graft (FIG. 1A). The biomass results demonstrate that the callus formation capacity of atx4 - 2 leaf grafts is increased by a factor of two compared to the wild type (Fig. 1b). Consistent with its negative role in callus formation, ATX4 transcript accumulation was substantially reduced during transition from leaf to callus. In this embodiment also it was tested whether or not depends on the type of the tissue used to form the improved callus atx4 -2 mutants. All seedlings were cut into root and upper part, and root graft was used for callus induction. While the callus forming from the leaf significantly improved in atx4 -2 (Fig. 1a and b), root explants showed callus formation in the extent that it is non-distinct from the wild-type root explants. Consistent with this, ATX4 transcript levels were implicitly lower in the root than in the ground. These observations suggest that ATX4 negatively regulates cell depletion by imparting a topoisomeric identity.

Pre-transcript RNA-Seq analysis was performed to investigate the signaling network associated with ATX4 during callus formation. Wild-type and were cultured for 7 days on a CIM leaf explants from the plant atx4 -2, derived immature callus were collected for total RNA isolation (Figure 2a). As compared with wild-type callus, was transcript of 1,569 species different from that stored in atx4 -2 callus, was up-regulated transcription of the body 612 of these genes were down-regulated transcription of the body 957 gene (Fig. 2b). Gene ontology (GO) analysis shows that various biological and molecular processes are regulated by ATX4 without a particularly enriched category. These results demonstrate the possibility that ATX4 may have been implicated in the development or institutional identity implementation generally associated with a broader range of physiological and molecular changes than some selected processes. In fact, atx4 were down-regulated genes of the top 80 genes -2 callus is strongly expressed in aerial part, particularly a leaf, which is suggestive of a role in the establishment ATX4 aerial part identity (Fig. 2c). On the other hand, the top 20 genes upregulated in atx4 -2 were moderately tended to be expressed in root. Since ATX4 trxG are the member of the family that controls gene expression in both grades, and is up-regulated genes in atx 4-2 may be indirect targets, which may explain the relatively low grouping.

In order to validate the RNA-Seq assay, hormone- and tissue-identity-related genes were selected for analysis of their transcript accumulation during RT-PCT (RT-qPCR) during callus formation, Seq data (Figs. 2d and 2e). In particular, ATH1 ( ARABIDOPSIS THALIANA HOMEOBOX GENE1 ), LHCA2 (PHOTOSYSTEM I LIGHT HARVESTING COMPLEX GENE 2 ), LHCB2.2 (LIGHT - HARVESTING CHLOROPHYLL B - BINDING 2.2 ), SAW1 ( SAWTOOTH 1 ), SAW2 The expression of over-the-ground identity genes such as TCP10 (TCP FAMILY TRANSCRIPTION FACTOR 10 ) decreased in wild-type leaf grafts after culturing on CIM, but the decrease in overexpression genes within atx4 -2 was even faster during the early stages of callus induction 2d). Meanwhile, TMO6 (TARGET OF MONOPTEROS 6), WOX4 (4 WUSCHEL -RELATED HOMEOBOX), and divide the center stop (quiescent center, QC) comprises a WOX11 - root meristem identity gene expression is increased in later stages of callus formation in atx4 -2 , Indicating that ATX4 mainly controls leaf identity and indirectly controls root identity (FIG. 2e).

The ATX4 protein is a member of the trxG family of proteins, which generally induce gene transcription by inducing the deposition of H3K4me3. To confirm the biochemical activity of ATX4, this example analyzed the accumulation of several major histone marks in the ATX4 mutant callus. As expected, while other significant epigenetic marks are not changed significantly, the total H3K4me3 levels were significantly reduced in comparison with the wild type callus atx4 -2 (Fig. 3a). In this embodiment then questioned on whether or not to the above-ground identity gene suppression in atx4 -2 that are modulated by ATX4- mediated chromatin changes. To solve this problem, while the wild-type callus formation and atx4 -2 leaf identity gene ATH1, LHCA2 in, LHCB2 .2, SAW1, SAW2, and TCP1 We compared the accumulation of H3K4me3, found that low levels of accumulation in the H3K4me3 atx4 -2 callus locus (Fig. 3b) in the. However, hormone-associated gene identity and roots, as you do not change the level of H3K4me3 at the locus of the gene, the gene in callus atx4 -2 were not directly targeted by the ATX4.

PRC2 is involved in inhibiting leaf identity during callus formation and shares regulation with ATX4, competing with ATX4 activity. In this example, expression kinetics of PRC2 and ATX2 were investigated in order to derive an operational scheme in cell depletion control. The genes encoding PRC2 elements increased in late stages of callus formation, whereas ATX4 was rapidly suppressed suggesting that PcG-dependent deposition of H3K27me3 occurs in leaf identity genes following a decrease in H3K4me3 levels. While transcript accumulation is independent of each other, ATH1 , SAW1 and TCP10 Accumulation of H3K27me3 in locus was substantially increased in later stages of callus formation in the atx4 -2 callus, which means that the balance between the PcG and TrxG activity that the backbone of the tissue and cellular dedifferentiation identity conversion.

ATX plays an important role in establishing top-floor identity. Therefore, we assume that ATX4 need from all-purpose callus on de novo (de novo) aboveground regeneration. Consistent with this hypothesis, ATX4 expression increased during callus-to-leaf regeneration (Fig. 4A). For additional confirmation, atx4 -2 callus was subsequently used for the above-ground regeneration. Above-ground induced following the incubation on medium (SIM), atx4 -2 callus showed the ability to form aerial parts reduced as compared with the wild type callus, which is due to the above-ground identity established injury (Fig. 4b and c). Was thwarted in the leaf callus of identity expression also atx4 -2 gene (Fig. 4d). Thus, the PRC2 urea genes were suppressed during topoisomer regeneration. Since the PRC2 mutations were unable to form testable calli, we could confirm that they did not test the role of PRC2 in shoot regeneration but caused a balanced change in cell fate during plant regeneration from the ATX4-PRC2 module.

Collectively, tissue identity has changed dynamically during cell depletion and tracheal regeneration, and a stable molecular process is required to establish a major change in the gene profile that defines tissue identity. In particular, ATX4 is an important haplotype gene that controls the establishment of shoot identity with PRC2, which acts as an antagonist. During callus formation, ATX4 transcript levels were rapidly reduced, promoting the loss of bud identity and helping to obtain universality in pericycle-like cells. Subsequently, by transfer of the universal callus to the SIM, ATX4 is activated and promotes re-establishment of the superficial identity (Fig. 4E).

ATX4-overexpressing plants were prepared by the method described above in order to confirm whether the actual regeneration efficiency was improved in the transgenic plants to overexpress ATX4. As a result of sequential cultivation of leaf slices from plants overexpressing ATX4 and CIM and SIM medium, the regeneration efficiency was enhanced as compared with the control group using wild-type leaf slices (FIG. 5).

A two-step sequential change in tissue identity is essential for the suppression of bud identity during callus formation during in vitro tissue culture, the associated activation of root identity, and the re-activation of the superficial identity for topoisomerization from the universal callus. In the first step, impairment of root identity induced callus formation defects. Specific elimination of the in vivo function defects both lateral root formation and callus induction. Moreover, ALF4 (Aberrant Lateral Root Formation 4), which primarily regulates entanglement formation, is also involved in establishing the in vivo ability to form CIM-induced calli. Chromatin remodeling is an important regulatory scheme that promotes stable changes in organ specific gene profiles observed in cell lineage differentiation. Consistent with the role of PRC2 in suppressing topographic identity, ATX4, a clear regulator establishing leaf identity, was suppressed on CIM to improve callus formation. Overfeeding is the most difficult step in the success of in-vitro breakfast cultivation. ATX4 is the only empirically proven posterior regulator associated with the re-establishment of the superficial identity, and this single locus makes a significant contribution to cell division as well as cell division. If step-wise chromatin modification that identifies tissue identity is the basis of the plant regeneration process, the programmed manipulation of ATX4 expression will be a direct way to improve plant transformation efficiency.

It will be understood by those of ordinary skill in the art that the foregoing description of the embodiments is for illustrative purposes and that those skilled in the art can easily modify the invention without departing from the spirit or essential characteristics thereof. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive. For example, each component described as a single entity may be distributed and implemented, and components described as being distributed may also be implemented in a combined form.

The scope of the present invention is defined by the appended claims rather than the detailed description, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be interpreted as being included in the scope of the present invention.

<110> Research and Business Foundation SUNGKYUNKWAN UNIVERSITY <120> METHOD FOR INCREASING REGENERATION EFFICIENCY OF PLANT USING ATX4          GENE AND PLANT THEREOF <130> R-2017-0375-KR-1 <160> 2 <170> KoPatentin 3.0 <210> 1 <211> 3084 <212> DNA <213> Arabidopsis sp. <400> 1 atgattatca aacggaaatt taaaacccag ataccgagtt tggaacggtg caaactcggt 60 aatgagtcaa ggaagaagaa gagaaagttg aacttgggag gaggtggtta ctactatcct 120 ctcaatcttc tcggagaaat cgccgccggg attgttccag gtaatggacg caatggattc 180 tctgcttcct ggtgcactga agttacaaaa cctgttgagg tggaagagtc attgtctaag 240 agaagatctg attccggtac tgttagagat tctcctccgg cggaagtctc acgtccaccg 300 cttgttcgga catctagggg aagaattcag gtgcttcctt ctcgattcaa tgattcggtg 360 cttgataatt ggaggaagga tagtaaaagt gattgtgatt tggaggaaga agaaatagaa 420 tgcaggaatg agaaagttgt tagttttcgg gttcctaaag ctactaattt gaaaagcaag 480 gaacttgata ggaagagtaa gtatagtgca ttgtgtaaag aagaaaggtt tcacgagcaa 540 cataatgatg aggctcgtgc tcgtgttgat gagaagcttc cgaataaaaa gggtactttt 600 gggccagaga acttctattc aggtgacttg gtttgggcaa agtctgggag aaatgagccg 660 ttttggccgg ctattgttat cgatccaatg acacaagcac ccgagcttgt tttgcgttct 720 tgtatacccg atgcagcctg tgttgtgttc tttggtcact ctgggaacga gaatgaaagg 780 gactatgcat gggtgaggag agggatgatc ttcccttttg tggactatgt tgccaggttc 840 caggaacagc ctgaactgca agggtgcaag cctgggaatt ttcagatggc attggaggaa 900 gcatttttgg cagatcaggg attcacggag aagttgatgc atgatattca cttggctgct 960 gggaactcga cttttgacga ttctttctac agatggattc aagaaactgc ggtctcaaat 1020 caggagctca ataacaatgc tccaagacag gggctgttaa agaaacacag aaatccacta 1080 gcatgtgcag gatgtgaaac agtcatttct tttgagatgg caaaaaaaat gaaagatctg 1140 attcctggag atcaattgtt gtgtaaacca tgttcaaggt taacaaaatc aaaacacatt 1200 tgtggtatat gcaagaagat aaggaatcat ttggacaata aaagctgggt gcgctgtgat 1260 ggttgtaaag tccggataca cgcagaatgt gaccaaatat cagataggca tttgaaggat 1320 ttgcgggaaa cagattacta ctgtcctact tgcagagcaa aatttaactt tgacctatca 1380 gattcagaaa aacaaaactc aaagtcaaaa gttgcgaaag gcgatggtca gatggttctt 1440 cctgacaagg ttattgttgt atgcgctgga gtagaaggag tttatttccc gaggctccat 1500 ttagtggtat gtaaatgtgg ctcctgtgga cctaaaaaga aagcccttag cgaatgggaa 1560 aggcataccg gatcaaagtc aaaaaattgg aagaccagtg tgaaagtcaa aagctcgaag 1620 ctggcactag aagattggat gatgaactta gcagagttac atgcaaatgc tactgcagct 1680 aaagttccta aacgaccttc cataaagcag agaaagcaga gattgcttgc ttttctatca 1740 gagacatatg agccggtcaa tgcgaagtgg acaacagaac gatgtgcagt ttgcagatgg 1800 gttgaagatt gggactacaa caagattatt atctgcaaca gatgccaaat agcagtgcat 1860 caggaatgct atggcgctag acatgttcgt gatttcacct catgggtctg taaagcatgc 1920 gagagaccag atatcaagcg ggagtgttgc ctctgccctg taaaaggagg tgcattgaag 1980 ccaactgatg tggaaacgtt gtgggttcat gtgacatgtg cttggtttca gcctgaagta 2040 tgttttgcaa gtgaggaaaa aatggaacct gcagttggga ttttgagtat tccatcaact 2100 aattttgtga agatatgtgt aatatgcaaa caaatacatg gctcgtgtac tcagtgttgc 2160 aagtgttcta catactatca tgccatgtgt gcctcccgag ctggttatag aatggagttg 2220 cactgcttgg agaaaaatgg tcagcagata acaaagatgg tgtcctattg tgcttatcac 2280 agggctccca acccagataa cgtgttaatt atacaaactc cttcaggagc cttctctgca 2340 aagagtcttg tccaaaacaa gaagaaaggt ggttccaggc ttatttcatt aatcagagaa 2400 gacgatgaag cacctgcaga gaataccatt acatgtgatc cattttctgc tgctcgttgt 2460 cgagtattta aaagaaagat caatagcaag aagaggattg aagaagaagc cattcctcac 2520 cacacaaggg gaccacgcca tcatgcatca gcagcaatcc aaactttaaa cacattcaga 2580 catgtgccag aagaacccaa atcgttttct tctttcaggg aacgacttca ccacttgcag 2640 aggacagaaa tggatcgggt ctgctttggg cgatctggaa ttcatggatg gggtcttttt 2700 gcaagaagaa atatccaaga aggagaaatg gttcttgaat acagagggga acaggtgaga 2760 ggtagcattg cagacttgag agaagcacgt tatcgcagag taggaaagga ttgctatctt 2820 ttcaagatca gtgaagaagt agtggttgat gctacagata aagggaatat agctcggctc 2880 atcaatcatt cgtgtacacc aaactgctat gcgaggatta tgagtgtagg agatgaggaa 2940 agcaggattg tcctgatagc caaggccaat gtagctgtgg gagaggagtt aacgtatgat 3000 tacttatttg atccagacga ggcagaggaa ctcaaggtgc catgcttatg taaagcccct 3060 aactgcagga aattcatgaa ttag 3084 <210> 2 <211> 1027 <212> PRT <213> Arabidopsis sp. <400> 2 Met Ile Ile Lys Arg Lys Phe Lys Thr Gln Ile Pro Ser Leu Glu Arg   1 5 10 15 Cys Lys Leu Gly Asn Glu Ser Arg Lys Lys Lys Arg Lys Leu Asn Leu              20 25 30 Gly Gly Gly Gly Tyr Tyr Tyr Pro Leu Asn Leu Leu Gly Glu Ile Ala          35 40 45 Ala Gly Ile Val Pro Gly Asn Gly Arg Asn Gly Phe Ser Ala Ser Trp      50 55 60 Cys Thr Glu Val Thr Lys Pro Val Glu Val Glu Glu Ser Leu Ser Lys  65 70 75 80 Arg Arg Ser Asp Ser Gly Thr Val Arg Asp Ser Pro Pro Ala Glu Val                  85 90 95 Ser Arg Pro Pro Leu Val Arg Thr Ser Arg Gly Arg Ile Gln Val Leu             100 105 110 Pro Ser Arg Phe Asn Asp Ser Val Leu Asp Asn Trp Arg Lys Asp Ser         115 120 125 Lys Ser Asp Cys Asp Leu Glu Glu Glu Glu Ile Glu Cys Arg Asn Glu     130 135 140 Lys Val Val Ser Phe Arg Val Pro Lys Ala Thr Asn Leu Lys Ser Lys 145 150 155 160 Glu Leu Asp Arg Lys Ser Lys Tyr Ser Ala Leu Cys Lys Glu Glu Arg                 165 170 175 Phe His Glu Gln His Asn Asp Glu Ala Arg Ala Arg Val Asp Glu Lys             180 185 190 Leu Pro Asn Lys Lys Gly Thr Phe Gly Pro Glu Asn Phe Tyr Ser Gly         195 200 205 Asp Leu Val Trp Ala Lys Ser Gly Arg Asn Glu Pro Phe Trp Pro Ala     210 215 220 Ile Val Ile Asp Pro Met Thr Gln Ala Pro Glu Leu Val Leu Arg Ser 225 230 235 240 Cys Ile Pro Asp Ala Ala Cys Val Val Phe Phe Gly His Ser Gly Asn                 245 250 255 Glu Asn Glu Arg Asp Tyr Ala Trp Val Arg Arg Gly Met Ile Phe Pro             260 265 270 Phe Val Asp Tyr Val Ala Arg Phe Gln Glu Gln Pro Glu Leu Gln Gly         275 280 285 Cys Lys Pro Gly Asn Phe Gln Met Ala Leu Glu Glu Ala Phe Leu Ala     290 295 300 Asp Gln Gly Phe Thr Glu Lys Leu Met His Asp Ile His Leu Ala Ala 305 310 315 320 Gly Asn Ser Thr Phe Asp Asp Ser Phe Tyr Arg Trp Ile Gln Glu Thr                 325 330 335 Ala Val Ser Asn Gln Glu Leu Asn Asn Ala Pro Arg Gln Gly Leu             340 345 350 Leu Lys Lys His Arg Asn Pro Leu Ala Cys Ala Gly Cys Glu Thr Val         355 360 365 Ile Ser Phe Glu Met Ala Lys Lys Met Lys Asp Leu Ile Pro Gly Asp     370 375 380 Gln Leu Leu Cys Lys Pro Cys Ser Arg Leu Thr Lys Ser Lys His Ile 385 390 395 400 Cys Gly Ile Cys Lys Lys Ile Arg Asn His Leu Asp Asn Lys Ser Trp                 405 410 415 Val Arg Cys Asp Gly Cys Lys Val Arg Ile His Ala Glu Cys Asp Gln             420 425 430 Ile Ser Asp Arg His Leu Lys Asp Leu Arg Glu Thr Asp Tyr Tyr Cys         435 440 445 Pro Thr Cys Arg Ala Lys Phe Asn Phe Asp Leu Ser Asp Ser Glu Lys     450 455 460 Gln Asn Ser Lys Ser Lys Val Ala Lys Gly Asp Gly Gln Met Val Leu 465 470 475 480 Pro Asp Lys Val Ile Val Val Cys Ala Gly Val Glu Gly Val Tyr Phe                 485 490 495 Pro Arg Leu His Leu Val Val Cys Lys Cys Gly Ser Cys Gly Pro Lys             500 505 510 Lys Lys Ala Leu Ser Glu Trp Glu Arg His Thr Gly Ser Lys Ser Lys         515 520 525 Asn Trp Lys Thr Ser Val Lys Val Lys Ser Ser Lys Leu Ala Leu Glu     530 535 540 Asp Trp Met Met Asn Leu Ala Glu Leu His Ala Asn Ala Thr Ala Ala 545 550 555 560 Lys Val Pro Lys Arg Pro Ser Ile Lys Gln Arg Lys Gln Arg Leu Leu                 565 570 575 Ala Phe Leu Ser Glu Thr Tyr Glu Pro Val Asn Ala Lys Trp Thr Thr             580 585 590 Glu Arg Cys Ala Val Cys Arg Trp Val Glu Asp Trp Asp Tyr Asn Lys         595 600 605 Ile Ile Cys Asn Arg Cys Gln Ile Ala Val His Gln Glu Cys Tyr     610 615 620 Gly Ala Arg His Val Arg Asp Phe Thr Ser Trp Val Cys Lys Ala Cys 625 630 635 640 Glu Arg Pro Asp Ile Lys Arg Glu Cys Cys Leu Cys Pro Val Lys Gly                 645 650 655 Gly Ala Leu Lys Pro Thr Asp Val Glu Thr Leu Trp Val His Val Thr             660 665 670 Cys Ala Trp Phe Gln Pro Glu Val Cys Phe Ala Ser Glu Glu Lys Met         675 680 685 Glu Pro Ala Val Gly Ile Leu Ser Ile Pro Ser Thr Asn Phe Val Lys     690 695 700 Ile Cys Val Ile Cys Lys Gln Ile His Gly Ser Cys Thr Gln Cys Cys 705 710 715 720 Lys Cys Ser Thr Tyr Tyr His Ala Met Cys Ala Ser Arg Ala Gly Tyr                 725 730 735 Arg Met Glu Leu His Cys Leu Glu Lys Asn Gly Gln Gln Ile Thr Lys             740 745 750 Met Val Ser Tyr Cys Ala Tyr His Arg Ala Pro Asn Pro Asp Asn Val         755 760 765 Leu Ile Ile Gln Thr Pro Ser Gly Ala Phe Ser Ala Lys Ser Leu Val     770 775 780 Gln Asn Lys Lys Lys Gly Gly Ser Arg Leu Ile Ser Leu Ile Arg Glu 785 790 795 800 Asp Asp Glu Ala Pro Ala Glu Asn Thr Ile Thr Cys Asp Pro Phe Ser                 805 810 815 Ala Ala Arg Cys Arg Val Phe Lys Arg Lys Ile Asn Ser Lys Lys Arg             820 825 830 Ile Glu Glu Glu Ala Ile Pro His His Thr Arg Gly Pro Arg His His         835 840 845 Ala Ser Ala Ala Ile Gln Thr Leu Asn Thr Phe Arg His Val Pro Glu     850 855 860 Glu Pro Lys Ser Phe Ser Ser Phe Arg Glu Arg Leu His His Leu Gln 865 870 875 880 Arg Thr Glu Met Asp Arg Val Cys Phe Gly Arg Ser Gly Ile His Gly                 885 890 895 Trp Gly Leu Phe Ala Arg Arg Asn Ile Gln Glu Gly Glu Met Val Leu             900 905 910 Glu Tyr Arg Gly Glu Gln Val Arg Gly Ser Ile Ala Asp Leu Arg Glu         915 920 925 Ala Arg Tyr Arg Arg Val Gly Lys Asp Cys Tyr Leu Phe Lys Ile Ser     930 935 940 Glu Glu Val Val Val Asp Ala Thr Asp Lys Gly Asn Ile Ala Arg Leu 945 950 955 960 Ile Asn His Ser Cys Thr Pro Asn Cys Tyr Ala Arg Ile Met Ser Val                 965 970 975 Gly Asp Glu Glu Ser Arg Ile Val Leu Ile Ala Lys Ala Asn Val Ala             980 985 990 Val Gly Glu Glu Leu Thr Tyr Asp Tyr Leu Phe Asp Pro Asp Glu Ala         995 1000 1005 Glu Glu Leu Lys Val Pro Cys Leu Cys Lys Ala Pro Asn Cys Arg Lys    1010 1015 1020 Phe Met Asn 1025

Claims (9)

1. A method for increasing the regeneration efficiency of a plant, comprising transforming a plant cell with a recombinant vector comprising a gene encoding a ATX4 protein of Arabidopsis, which is represented by the nucleotide sequence of SEQ ID NO: 1. delete The method according to claim 1,
Wherein the ATX4 protein comprises the amino acid sequence of SEQ ID NO: 2.
The method according to claim 1,
Wherein the ATX4 protein is overexpressed in plant regeneration.
The method according to claim 1,
Wherein the expression of said ATX4 protein is suppressed in the process of dedifferentiation of the plant.
1. A method for producing a transformed plant having an increased efficiency of regeneration, which comprises transforming a plant using a recombinant vector represented by the nucleotide sequence of SEQ ID NO: 1 and comprising a gene encoding Arabidopsis thaliana ATX4 protein. delete delete 1. A composition for increasing the regeneration efficiency of a plant, which comprises the gene encoding the ATX4 protein of Arabidopsis thaliana as an active ingredient, which is represented by the nucleotide sequence of SEQ ID NO: 1.

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