KR101438738B1 - Gene Implicated in Abiotic Stress Tolerance and Growth Accelerating and Use Thereof - Google Patents

Abstract

The present invention provides a composition for promoting non-biological stress tolerance and a composition for promoting growth, which comprises a nucleotide sequence encoding an AtSRP (Arabidopsis thaliana stress related protein) protein of a plant.
The nucleotide sequence of the present invention is involved in resistance to abiotic stresses such as drying, low temperature and salt stress of plants, and transgenic plants overexpressing them are excellent in tolerance to these biotic stresses, And can be usefully used as new functional crops regardless of the plant.
In addition, when the nucleotide sequence of the present invention is transformed, the growth ability of the plant is greatly improved, and thus it can be usefully used as a new functional plant and biomass capable of cultivating properties.

Description

Gene Implicated in Abiotic Stress Tolerance and Growth Accelerating and Use Thereof

The present invention relates to a composition for promoting abiotic stress tolerance and a composition for promoting growth of a plant including the gene involved in abiotic stress tolerance and growth promotion, and a plant comprising the same.

Because higher plants are immobile, they face various environmental factors throughout their lifetimes such as drought, high salt, heavy metals, cold damage, thermal shock and ozone. Such abiotic stress is a limiting factor for the growth and development of crops. Among these stresses, lack of moisture is the most serious environmental factor that is considered to be the main cause of crop production decline. The consumption of water around the world has been continuously increasing, and the availability of clean water can be an important problem not only for humans but also for higher plants. In order to increase resistance to short-term or long-term water shortages, plants are operating their own cellular or genetic defense mechanisms (Shinozaki and Yamaguchi-shinozaki, 2007). However, there is still a lack of knowledge about the biological functions of stress-related genes involved in resistance or sensitivity to stress in higher plants. Therefore, it is important to study the functions of stress response genes to increase the productivity of crops.

Plants are constructing various defense strategies that enhance resistance to adverse environments by stimulating signaling networks and inducing stress-responsive genes. The present inventors that Arabidopsis (Arabidopsis thaliana) rubber tree (Hevea brasiliensis) SRPP (small rubber particle protein) like protein three (Arabidopsis thaliana stress related protein) AtSRP for encoding of the gene is rapidly induced by dehydration (dehydration) I found it. Natural rubber is a cis -1,4-polyisoprene produced through the mevalonate pathway in the cytosolic fraction of latex tubular tissue (Oh et al. 1999; Sookmark et al. 2002). ; Kim et al. 2004; Chow et al. 2007). Since Arabidopsis thaliana does not produce natural rubber, the fact that the dry-stress gene of Arabidopsis thaliana shares a base sequence with a rubber biosynthetic gene is unusual.

SRPP, originally known as latex allergin, is a protein tightly bound to small rubber particles in the latex of the rubber tree (Hevea brasiliensis).

The bark texture of the rubber tree is continuously peeled to collect rubber latex, which is known as the tapping process. Therefore, plugging of the latex tube is essential to prevent loss of cytoplasmic substances such as primary metabolites and to prevent pathogen infection (Wititsuwannakul et al., 2008b). In the process of sealing the latex tube, hevein or HLL (Hevea latex lectin) reacts with the rubber particle (RP) protein to form a rubber latex coagulum (Gidrol et al., 1994; Wititsuwannakul et al., 2008a). Recently, SRPP was purified to HLL-binding RP glycoprotein and named as HLLBP (HLL-binding protein) (Wititsuwannakul et al., 2008c). The interaction between the N-acetylglucosamine portion of SRPP and HLL can control the degree of coagulation of latex in response to taping and mechanical wounds.

However, the role of SRPP in relation to abiotic stress such as dryness, high salt and cold damage has not been known yet.

Throughout this specification, a number of papers and patent documents are referenced and citations are indicated. The disclosure contents of cited papers and patent documents are incorporated by reference in this specification as a whole, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly described.

The present inventors have made extensive research efforts to ultimately improve the productivity of plants by discovering genes that can improve the resistance to abiotic stress of plants, including drought, high salt and cold damage. As a result, when the expression of three AtSRP ( Arabidopsis thaliana stress related protein) genes encoding similar proteins of Hevea brasiliensis small rubber particle protein (SRPP) in plants is increased, the above-described abiotic stress is enhanced. By confirming that the increased resistance can be obtained, the present invention has been completed.

Accordingly, an object of the present invention is to provide a composition for enhancing abiotic stress tolerance of plants.

Another object of the present invention is to provide a composition for promoting plant growth.

Other objects and advantages of the present invention will become more apparent by the following detailed description, claims and drawings.

According to one aspect of the present invention, the present invention provides a composition for enhancing abiotic stress tolerance of a plant comprising a nucleotide sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NOS: 4 to 6 in Sequence Listing.

The present inventors have made extensive research efforts to ultimately improve the productivity of plants by discovering genes that can improve the resistance to abiotic stress of plants, including drought, high salt and cold damage. As a result, when the expression of three AtSRP ( Arabidopsis thaliana stress related protein) genes encoding similar proteins of Hevea brasiliensis small rubber particle protein (SRPP) in plants is increased, the above-described abiotic stress is enhanced. By confirming that the increased resistance can be obtained, the present invention has been completed.

According to a preferred embodiment of the present invention, the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 4, 5 and 6 of the present invention is the nucleotide sequence of SEQ ID NO: 1, 2 and 3, respectively. It consists of a sequence.

According to the present invention, the amino acid sequences of SEQ ID NOs: 4, 5, and 6 are sequences of SRPP-like proteins present in Arabidopsis thaliana, and these proteins are AtSRP (Arabidopsis thaliana stress related protein) 1, AtSRP 2, respectively. And AtSRP 3. As shown in detail in the following examples, the present inventors believe that the expression of these proteins or their coding genes is increased by various abiotic stresses, and when these genes are overexpressed, resistance to abiotic stress is increased. Found.

It is clear to those skilled in the art that the nucleotide sequence used in the present invention is not limited to the nucleotide sequence described in the attached sequence listing.

In some cases, variations in nucleotides do not result in changes in proteins. Such nucleic acids include functionally equivalent codons or codons encoding the same amino acid (e.g., by the degeneracy of codons, there are six codons for arginine or serine), or codons encoding biologically equivalent amino acids. It includes a nucleic acid molecule.

Considering the above-described mutation having biologically equivalent activity, it is interpreted that the nucleic acid molecule used in the present invention also includes a sequence exhibiting substantial identity with the sequence listed in the sequence listing. The actual identity of the above is at least 60% when the sequence of the present invention and any other sequence described above are aligned to correspond as much as possible, and the aligned sequence is analyzed using an algorithm commonly used in the art. It means a sequence that exhibits homology, more preferably 70% homology, even more preferably 80% homology, and most preferably 90% homology. Alignment methods for sequence comparison are known in the art. Various methods and algorithms for alignment are described in Smith and Waterman, Adv. Appl. Math. 2:482 (1981) ; Needleman and Wunsch, J. Mol. Bio. 48:443 (1970); Pearson and Lipman, Methods in Mol. Biol. 24: 307-31 (1988); Higgins and Sharp, Gene 73:237-44 (1988); Higgins and Sharp, CABIOS 5:151-3 (1989); Corpet et al., Nuc. Acids Res. 16:10881-90 (1988); Huang et al., Comp. Appl. BioSci. 8:155-65 (1992) and Pearson et al., Meth. Mol. Biol. 24:307-31 (1994). NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10 (1990)) can be accessed from NBCI (National Center for Biological Information), etc. It can be used in conjunction with sequence analysis programs such as blastx, tblastn and tblastx. The BLSAT is accessible at http://www.ncbi.nlm.nih.gov/BLAST/. A method for comparing sequence homology using this program can be found at http://www.ncbi.nlm.nih.gov/BLAST/blast_help.html.

According to a preferred embodiment of the present invention, the abiotic stress to which tolerance is enhanced with the composition of the present invention is selected from the group consisting of dry stress, low temperature stress and salt stress.

According to another aspect of the present invention, the present invention provides a plant cell transformed with the composition of the present invention with improved abiotic stress tolerance.

According to another aspect of the present invention, the present invention provides a plant transformed with the composition of the present invention with improved abiotic stress tolerance.

In order to prepare the transgenic plant cells and transgenic plants of the present invention , it can be carried out according to a method generally known in the art (Methods of Enzymology , Vol. 153, (1987)). Plants can be transformed by inserting foreign polynucleotides into carriers such as plasmids or viruses, etc., and Agrobacterium bacteria can be used as mediators (Chilton et ai. Cell 11:263:271 (1977)), Plants can be transformed by directly introducing foreign polynucleotides into plant cells (Lorz et ai. Mol. Genet. 199:178-182; (1985)). For example, when a vector not containing a T-DNA site is used, an electroporation method, a microparticle bombardment method, and a polyethylene glycol-mediated uptake method may be used.

In general, it is a method of infecting plant cells or seeds with Agrobacterium tumefaciens transformed with foreign polynucleotides that are widely used in transforming plants (see: U.S. Patent No. 5,004,863, 5,349,124 and 5,416,011). Those skilled in the art can develop a plant by culturing or cultivating transformed plant cells or seeds under known appropriate conditions.

In the present specification, the term “plant (body)” is understood to include not only mature plants, but also plant cells, plant tissues, and seeds of plants that can develop into mature plants.

Plants subject to transformation in the present invention include rice, wheat, barley, corn, beans, potatoes, wheat, red beans, food crops including oats and sorghum; Vegetable crops including Arabidopsis, Chinese cabbage, radish, pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, green onion, onion and carrot; Specialty crops including ginseng, tobacco, cotton, sesame, sugar cane, sugar beet, perilla, peanut and rapeseed; Fruit trees including apple tree, pear tree, jujube tree, peach, parsley, grapes, tangerines, persimmons, plums, apricots and bananas; Flowers including roses, gladiolus, gerberas, carnations, chrysanthemums, lilies and tulips; And feed crops including ryegrass, red clover, orchardgrass, alpha alpha, tolpescue, and perennial ryegrass, but are not limited thereto.

According to another aspect of the present invention, the present invention provides the above-described nucleotide sequence of the present invention; (b) a promoter operatively linked to the nucleotide sequence and forming RNA molecules in plant cells; And (c) a recombinant vector for plant expression comprising a poly A signal sequence that acts on the plant cell to cause polyadenylation of the 3'-end of the RNA molecule. .

In the present specification, the term “operably linked” refers to a functional linkage between a nucleic acid expression control sequence (eg, a promoter, a signal sequence, or an array of transcriptional regulatory factor binding sites) and another nucleic acid sequence, whereby the control sequence Controls the transcription and/or translation of the other nucleic acid sequences.

The vector system of the present invention can be constructed through various methods known in the art, and specific methods for this are described by Sambrook et al. Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory Press (2001).

Promoter suitable for the present invention may be any commonly used in the art for gene introduction into plants, for example, SP6 promoter, T7 promoter, T3 promoter, PM promoter, ubiquitin promoter of corn, cauliflower Mosaic virus (CaMV) 35S promoter, nopaline sintase (nos) promoter, Pigwort mosaic virus 35S promoter, Sugacrane Basiliform virus promoter, Commelina Yellow Mottle virus promoter, ribulose-1,5-bis-phosphate car Including the photoinducible promoter of the boxylase small subunit (ssRUBISCO), the rice cytosol triosphosphate isomerase (TPI) promoter, the adenine phosphoribosyltransferase (APRT) promoter from Arabidopsis, and the octopine synthase promoter. do. Most preferably, the promoter in the present invention is a cauliflower mosaic virus (CaMV) 35S promoter.

According to a preferred embodiment of the present invention, the poly A signal sequence causing polyadenylation at the 3'-end suitable for the present invention is derived from the nopaline synthase gene of Agrobacterium tumefaciens (NOS 3'end ) (Bevan et al. Nucleic Acids Research , 11(2):369-385(1983)), derived from the octopine synthase gene of Agrobacterium tumerfaces, protease inhibitor I or II gene of tomato or potato The 3'end portion of, CaMV 35S terminator and OCS terminator (octopine synthase terminator) sequence. Most preferably, the 3'-end poly A signal sequence that causes polyadenylation suitable for the present invention is a (NOS 3'end) sequence derived from the nopaline synthase gene of Agrobacterium tumefaciens.

Optionally, the vector may additionally carry a gene encoding a reporter molecule (eg, luciferase and β-glucuronidase). In addition, the vector of the present invention is an antibiotic (eg, neomycin, carbenicillin, kanamycin, spectinomycin, hygromycin, etc.) resistance genes (eg, neomycin phosphotransferase ( nptII ), hygromycin, etc.) Mycin phosphotransferase ( hpt ), etc.).

According to a preferred embodiment, a recombinant vector for plant expression of the invention is Agrobacterium (Agrobacterium) binary vector.

In the present specification, the term "binary vector" is a plasmid having a left border (LB) and a right border (RB), which are parts necessary for movement in a Ti (tumor inducible) plasmid, and a plasmid having a gene required to transfer the target nucleotide. Say vector. The Agrobacterium for transformation of the present invention may be any one suitable for the expression of the nucleotide sequence of the present invention, and in particular, the Agrobacterium strain for plant transformation in the present invention is usually Agrobacterium tumefaciens (Agrobacterium tumefaciens). ) GV3101 is preferred.

The method of introducing the recombinant vector of the present invention into Agrobacterium can be carried out through various methods known to those skilled in the art, for example, particle bombardment, electroporation, and transfection. ), lithium acetate method, and heat shock method. Preferably, electroporation is used.

According to another aspect of the present invention, the present invention provides a composition for promoting the growth of a plant comprising a nucleotide sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 to 6 in Sequence Listing.

According to the present invention, a transgenic plant overexpressing the AtSRPs gene of the present invention has longer roots and wider leaves than wild species after the same time elapses after germination under normal conditions, and inflorescence stems are faster. By observing the growth, as a result, it was experimentally confirmed that it showed faster growth compared to the wild species. By using another useful characteristic of the AtSRPs gene, it can be applied to new functional plants and biomass capable of rapid cultivation by improving the growth rate of plants.

According to another aspect of the present invention, the present invention provides the above-described nucleotide sequence of the present invention; (b) a promoter operatively linked to the nucleotide sequence and forming RNA molecules in plant cells; And (c) a recombinant vector for plant expression comprising a poly A signal sequence that acts on the plant cell to cause polyadenylation of the 3'-end of the RNA molecule.

Since the amino acid sequence, the nucleotide sequence, and the recombinant vector for plant expression including the amino acid sequence used in the present invention have been described above, description thereof is omitted in order to avoid excessive duplication.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a composition for promoting abiotic stress tolerance of plants and a composition for promoting growth.

(b) The nucleotide sequence of the present invention is involved in resistance to abiotic stresses such as drying, low temperature and salt stress of plants, and transgenic plants overexpressing them have excellent resistance to these abiotic stresses, It can be usefully used as a new functional crop regardless of climate or environment.

(c) In addition, when the nucleotide sequence of the present invention is transformed, the growth capacity of the plant is greatly improved, and thus it can be usefully used as a new functional plant and biomass capable of rapid cultivation.

1 is a diagram showing that AtSRPs are expressed in various tissues of Arabidopsis thaliana. 1A is a diagram showing the results of analyzing AtSRP s expression by RT-PCR at the 7th stage of development. 18s rRNA was used as a loading control.
WS; whole seedling, RT; root, RL; rosette leaf, ST; stem, F; Flower, SQ; sillique, SD; seed.
1B is a diagram showing the results of analyzing AtSRP s expression in various tissues of Arabidopsis thaliana by real-time RT-PCR. The results are expressed as the mean±standard deviation value of 4 repeated experiments.
2 is a diagram showing that AtSRP s is up-regulated by ABA and abiotic stress. Total RNA was obtained from 7-day-old Arabidopsis seedlings to which 100 mM ABA (1.5-3 hours), dry (1.5-3 hours), cold sea (4.5-24 hours at 4° C.) or high salt (300 mM NaCl for 3 hours) was added. I did. The induction pattern of AtREFP s was investigated by RT-PCR (FIG. 2A) or real-time RT-PCR (FIG. 2B). RAB18 and RD29A genes were used as positive controls for ABA and abiotic stress, respectively. AtACT8 transcription level was used as a loading control. Data are expressed as the mean±standard deviation value of 4 independent experiments.
3 is a diagram showing that the membrane-binding protein AtSRP1 has lectin-binding properties. 3A shows the results of biochemical analysis through affinity chromatography using concanavalin A. Figure 3b is a diagram showing the results of the membrane association test (Membrane association test). After temporarily overexpressing AtSRPs using a tobacco transient assay, membrane precipitates of the total protein were 1% (v/v) Triton X-100, 2 M urea, 1 M NaCl, or 0.1 M Na ₂ Resuspended in _{CO 3.} The suspension was centrifuged again at 125,000 g to separate the dissolved fraction (S) and the insoluble fraction (P), and then analyzed by immunoblotting using a Flag antibody. * Stands for AtSRP1
Figure 4 is a diagram showing the phenotypic characteristics of T3 35S:AtSRPs transgenic Arabidopsis thaliana and knock-out mutants. AtSRPs -overexpressors grew faster than wild-type, and knock-out mutations showed the opposite phenotype. Figure 4a is a picture comparing the morphological characteristics of wild-type, transformed seedlings and knock-out mutants grown for 3 days in light conditions. 4B is a diagram showing the difference in growth patterns in the initial roots of wild type, transformed and knock-out mutant seedlings. Error bars represent the mean ± standard deviation (n = 70). Figure 4c is a picture showing the three-dimensional parameters of the second leaf 2 weeks after germination. Second leaves were separated from wild type, transformant and knock-out mutant, respectively, and scanned for image analysis using the SCIONIMAGE program, and their leaf width and length, petiole length, and leaf area were calculated. . Error bars represent the mean ± standard deviation (n = 35). 4D is a diagram showing the growth patterns of wild type, transformant and knock-out mutant 28 days after germination under normal growth conditions (left panel). The right panel is a picture showing the difference in the growth patterns of wild-type and transgenic Arabidopsis inflorescence stems 20-28 days after germination. Error bar represents mean ± standard deviation (n = 80)
Figure 5 is a diagram showing the rapid growth phenotype of 35S:AtSRPs transformed Arabidopsis thaliana associated with the promotion of cell cycle progression. Figure 5a is a diagram showing the vertical shape of each root tip of a 3-day-old wild-type, AtSRP s-overexpressing transformant and knock-out mutant seedlings. The cross section of the root was stained with propidium iodide and observed with a confocal microscope. Figure 5b is a picture showing the results of observation with a confocal microscope of epidermal cells and palisade mesophyll cells collected from the second leaf of wild-type, AtSRP s-overexpressing transformants and knock-out mutants. Scale bar = 20 μm.
6 is a diagram showing that AtSRPs have a salt sensitive phenotype when they lose their function, and show the morphology of seedlings of 12-day-old At SRP s-overexpressing transformants and knock-out mutations cultured in 100 mM NaCl medium. .
7 is a diagram showing that AtSRPs play a role as a positive factor in the dry defense reaction. 7A is a diagram showing the survival rate of each of wild-type, AtSRP s-overexpressing transformants, and knock-out mutants after applying dry stress to each other. 25-day-old wild-type, 3-week-old transformant and 26-day-old knock-out mutant grown in light conditions were cultured for 8 days without water. Thereafter, the survival rate was measured 3 days after re-watering the plants. The ability to continue growing when returning to normal moisture conditions after 8 days of dry stress was considered as the survival rate. Each survival rate is as follows: wild type, 26.97% (24 of 89 subjects); 35S:AtSRP1 transformant, 64.84% (59 of 91 individuals); 35S:AtSRP2 transformant, 66.29% (59 of 89 individuals); 35S:AtSRP3 transformant, 58.02% (47 of 81 individuals); atsrp1 mutant, 3.90% (3 of 77 individuals); atsrp3 mutant, 0.00% (0 of 101 subjects). 7B is a diagram showing the result of measuring the ratio of cut rosette water loss (CRWL). Left lobes (rosette leaves) were harvested from wild-type, 35S:AtSRPs transgenic plants and knock-out mutants grown in light conditions, respectively, placed on open-lid petri dishes, and incubated for 0-5 hours at room temperature. Thereafter, the decrease in fresh weight was measured, and the loss of moisture was expressed as a percentage of the initial bioweight of the harvested leaves. Error bars represent the mean ± standard deviation (n = 20). Figure 7c is a diagram showing the amount of chlorophyll a + chlorophyll b in wild-type, 35S:AtSRPs transgenic plants and knock-out mutant leaves. Error bar represents mean ± standard deviation (n = 3)
FIG. 8 is a diagram showing the results of examining the content of proline in a leaf disc before and after drying treatment. Data were obtained through each independent experiment, and the bars represent the mean ± standard deviation (n = 3). Transgenic plants replenish leaf moisture more efficiently under dry conditions than wild type, whereas knock-out mutants showed the opposite phenotype.
9 is a diagram showing a schematic diagram of AtSRP1 , AtSRP2 and AtSRP3 cDNA clones.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Cloning and analysis of AtSRPs genes

RT-PCR was performed by extracting total mRNA from 5-day-old Arabidopsis seedlings (Seo et al., 2008). The primer combinations used in PCR to specifically amplify only the AtSRP1 , AtSRP2 , and AtSRP3 genes among cDNA synthesized by reverse transcription reaction are from the database of The Arabidopsis Information Resource (TAIR, http://www.arabidopsis.org) at AtSRP1 and AtSRP2. , After searching the nucleotide sequence of each cDNA of the AtSRP3 gene, the 5'side of the coding site (CDS) was tagged with BamHI and the 3'side was tagged with SacI to prepare primers. The cDNA of each of the amplified AtSRPs genes was ligated to pGEM-T Easy vector (Promega, http://www.promega.com) and cloned, and then the nucleotide sequence was confirmed through a sequencing reaction (Table 1). The identified nucleotide sequence was exactly the same as that retrieved from TAIR (http://www.arabidopsis.org), and At1g67360 ( AtSRP1 ) was 723 bp, At2g47780 ( AtSRP2 ) was 708 bp, At3g05500 ( AtSRP3 ) was 741 bp. It only contained the CDS.

AtSRPs gene expression analysis

RT-PCR and real-time RT-PCR were performed to analyze the expression of AtSRPs at the transcriptional level in various tissues (Seo et al. , 2009). The primers used in the analysis are shown in Table 1.

Preparation of cloning and transgenic plants, primers for protein expression gene Forward primer Reverse primer Cloning of AtSRPs ORF, RT-PCR and production of transgenic plants AtSRP1 5’-gcggatccatggagacagagaagaaaaatag-3’ 5’-gcgagctcctactccgaatcagacgatg-3’ AtSRP2 5’-gcggatccatggctgaagatgaaatagtagtc-3’ 5’-gcgagctctcaatcagctcgacactgatc-3’ AtSRP3 5’-gcggatccatggctactcaaacggatctc-3’ 5’-gcgagctctcaatcaagtggatggaactc-3’ 18s rRNA 5’-gcatttgccaaggatgtttt-3’ 5’-gtacaaagggcagggacgta-3’ AtACT8 5'-tactgattacctcatgaagatccttac-3' 5'-aaacgatgtctctttagtttagaagc-3' Real-time quantitative RT-PCR primer AtSRP1 5'-tgattctctcctggtctttc-3' 5'-ttctctgtcgccttgtagat-3' AtSRP2 5'-ttttcgtcgatcgtaagg-3' 5'-aatgctcttggtagtgtcca -3' AtSRP3 5'-ccctgttgaggtccttaaat-3' 5'-gccacaatgggtgctat-3'

Expression and purification of AtSRPs protein

AtSRPs were fused with a 2xFlag epitope, expressed in BL21-CodonPlus (DE3) RIL (Stratagene), and then purified. Protein was quantified using BSA as a standard protein (Bradford, 1976).

Measurement of lectin binding properties of AtSRPs

To investigate whether AtSRPs have the property of binding to lectins, proteins purified by expressing 2xFlag-AtSRPs in E. coli and proteins temporarily overexpressing 35S:2XFlag-AtSRPs in tobacco were used. Concanavalin A binding experiment for both proteins was performed according to the manufacturer's method (Con A Sepharose 4B-GE-71707700AG; GE Healthcare Life Sciences). The reaction product was immunoblotted using the Flag antibody. As a result, only the 35S:2XFlag-AtSRPs lane using the tobacco system was identified as a band, indicating that only AtSRP1 in which glycosylation occurred in the plant system was bound to the lectin, and AtSRP1 was found to be a glycoprotein (Fig. 3a). ).

Measurement of membrane binding properties of AtSRPs

To determine whether AtSRPs are bound to the membrane, a protein transiently overexpressing 35S:2xFlag-AtSRPs in tobacco was extracted and used. Thereafter, a membrane binding experiment was performed with reference to Kim and Bassham (Kim and Bassham, 2011), and the reaction product was immunoblotted using a Flag antibody. _{As a result, in the treatment section in which 0.1 M Na 2} CO ₃ was treated with the membrane precipitate, it was found that the supernatant band was thicker than that in the precipitate (Fig. 3B). This is because the AtSRP1 protein was bound to the membrane and then eluted into the supernatant due to weak binding by _{Na 2} CO ₃ , it was found that the AtSRPs protein is a membrane-binding protein.

AtSRPs overexpressed transformants and knock-out mutation identification

In order to overexpress AtSRPs in Arabidopsis, cDNA of AtSRPs was recombined into pBI121 binary vector. The primers used are shown in Table 1. In order to express this in plants, Agrobacterium tumefaciens was transformed, and Arabidopsis thaliana transformation was performed by a floral dip method (Joo et al ., 2004). Seeds were selected for independent transgenic individuals in 0.5 X MS, 25 mg/l kanamycin medium. For atsrp1 and atsrp3, the T-DNA insertion line was ordered from ABRC (www.arabidopsis.org) (atsrp1: GABI_3095G05, atsrp3 : WiscDSLOXHS192) and used. Atsrp2 was used to select individuals with low expression at the transcription level by producing knock-down lines using the RNAi method (Lee and Kim, 2010).

Phenotypic in-depth analysis of transformants overexpressing AtSRPs

After preparing by adjusting the pH of the medium containing 0.5 x MS (including vitamin B5), 1% sucrose, and 0.8% agar (Life Technology, Rockvile, MD, USA) to 5.7, it is excessive with wild species. The seed surface of the expression body was disinfected and planted, and it was grown in a light condition of 22 degrees. The release of radicals was observed by day until the 5th day after inhibition. In order to observe root growth, seeds were observed until the 5th day after planting. While the roots were growing, the root apex was marked on the outside of the culture dish, and then the length of the roots was measured using ScionImage software (Scion Corp., Frederic, MD, USA). In order to observe the size of the leaves, seeds were planted in sterilized soil, and at the 12th day, wild species, overexpressors, and knock-out individuals were picked from cotyledon to third leaf and compared. In addition, the second leaf was measured for leaf length, leaf width, and length of petiole using ScionImage software (Scion Corp., Frederic, MD, USA).

In order to observe when the flower stalks come up, seeds of wild species and overexpressing organisms were planted in sterilized soil and grown in a culture room under continuous light conditions at 22℃. From the 19th day after planting, the height after the flower stalks were raised was measured. As a result, there was no difference in germination ability between wild species and AtSRPs overexpression, but when comparing root growth, leaf size, and height, it was confirmed that the overexpression of AtSRPs grew rapidly in all experiments. This demonstrates that AtSRPs act as positive regulators in plant growth (FIGS. 4A to 4D ).

In-depth analysis of phenotype using a microscope

5-day-old wild-type and transgenic Arabidopsis leaves were washed with the method reported in chloral hydrate solution (Kwon et al. 2009). The cell layer of the leaf was observed under a bright field microscope (BX51 fluorescence microscope, Olympus, Japan). To observe the longitudinal section of the roots, 5-day-old roots were stained with propidium iodide and Seo et al. An image was obtained using a confocal microscope by the method of (2008) (Fig. 5).

Survival measurement of wild species and transgenic Arabidopsis plants after applying high salt stress

Five-day-old wild species and 35S:AtSRPs transgenic plants grown under normal conditions and each knock-out individual were grown for 7 days in a high salt medium containing 100 mM NaCl. After that, the length of the roots of each individual was examined. As a result, it can be seen that each knock-out individual has a phenotype that is much more sensitive to high salt stress, thus demonstrating that AtSRP1s is a positive regulator in the high salt stress response (FIG. 6).

Survival measurement of wild species and transgenic Arabidopsis plants after applying drying stress

Three-week-old wild species and 35S:AtSRPs transgenic plants grown under normal conditions and knock-out subjects were subjected to dry stress without water for 8 days. Afterwards, the plants were again watered and the phenotype was examined 3 days later. The survival rate was defined as the ability to resume growth upon return to normal conditions after drying stress (Cho et al. 2008). Each survival rate is as follows (Fig. 7A):

Wild type, 26.97% (24 of 89 individuals); 35S:AtSRP1 transformant, 64.84% (59 of 91 individuals); 35S:AtSRP2 transformant, 66.29% (59 of 89 individuals); 35S:AtSRP3 transformant, 58.02% (47 of 81 individuals); atsrp1 mutant, 3.90% (3 of 77 individuals); atsrp3 mutant, 0.00% (0 of 101 subjects). In addition, it was confirmed that the water content of the transformant over time was significantly reduced compared to the wild-type and knock-out mutant (FIG. 7b).

Measurement of chlorophyll and proline content of wild species and transgenic Arabidopsis plants after drying stress

Bae et al. Chlorophyll was extracted using 80% acetone by the method of (2009). The remaining leaf extract was dried at 105° C. for 16 hours, and the weight of the dried extract was measured (Welti et al. 2002). The proline content of the leaves was measured by the method of Claussen (2005). As a result of the measurement, it was confirmed that the transgenic plants had higher chlorophyll and proline content than the wild type, indicating that they could more efficiently replenish leaf moisture under dry conditions, whereas the knock-out mutant showed the opposite phenotype. (Fig. 8).

As described above, a specific part of the present invention has been described in detail, and it is obvious that this specific technology is only a preferred embodiment for those of ordinary skill in the art, and the scope of the present invention is not limited thereto. Accordingly, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

<110> Industry-Academic Coopreaton Foundation Yonsei University <120> Gene Implicated in Abiotic Stress Tolerance and Growth Accelerating and Use Thereof <130> PN120347 <160> 6 <170> KopatentIn 2.0 <210> 1 <211> 723 <212> DNA <213> Arabidopsis thaliana <400> 1 atggagacag agaagaaaaa tagcaaggag gtagcgctaa aacacctagc gttcgtgaga 60 attgcaacga tccatattct ggcttccgtc tcaaatctct acgaatacgc taaacaaaac 120 tccggtcctc tcaaatccgc cgtggaaaag gtcgaaggag ccgtcaccac cgtcgtcacc 180 cctgtctatc agaaattcaa agatgttcct gattctctcc tggtctttct agatcacaag 240 gtgggtgaag tttcgtacaa gtttgatgag catgctcctc caatggctaa gaaagtggtg 300 aatcaagcac atgtattgat ctacaaggcg acagagaaag ctcaaagctt tgtgaaagag 360 gctcgtaccg gtggtcccaa agctgcgttt aactatgctg caactgagta caagtttttc 420 gttgtgacca actcggttaa agtctgggct aaacttaacc agtataaacc aatccatgca 480 atgggtgaca aagctttgcc tgtggctgca cacttttcca gtcggtacaa cgatttggtg 540 actgatatga ctaatatggg ttactctttg gttggttatc ttccgttggt tcctgttgat 600 gacattgtta aggcttatga aaaggaagat gcaaggagaa agaaaggagg ggatacggct 660 ggaaagaaag gagagactac tgatgcggct gatggtgata aatcatcgtc tgattcggag 720 tag 723 <210> 2 <211> 708 <212> DNA <213> Arabidopsis thaliana <400> 2 atggctgaag atgaaatagt agtcgaagaa gagcaatcac agccacagga gattactcca 60 gttccaccat cttcttcgtc ttcgccttcg ttagtggtgg aagacgacga tgagatgaag 120 ctcaagcact tggaattcat tcaagtcgcg gcggtttact tcgccgcctg tttctcgacc 180 ctctacgagt tggctaaaga caacgctggt ccactcaaac tcggcgtcga gaacattgaa 240 gattgcgttc gaaccgtgct tgctccatta tacgaaaaat tccacgacgt tcctttcaag 300 cttctccttt tcgtcgatcg taaggtggac gatgtgttct tcgatgttga gacatatgtt 360 ccgtcattgg tgaagcaagc ttctagccaa gcgctcacgg tggccacgga ggtgcagcgc 420 accggcgttg tggacactac caagagcatt gcaagaagcg tccgggacaa gtacgagcca 480 gcggcagagt attacgcagc gacgctgtgg cgtttactga atcaactgcc gttgttccct 540 gaagttgcgc atttagtgat ccccacggcg ttttactggt cagagaagta caacgatgcg 600 gttcgttacg ttggcgacag agactacttt ggggcagagt atctacccat gattcctatt 660 gagaagatct ctgatatttt ggaacaagat cagtgtcgag ctgattga 708 <210> 3 <211> 741 <212> DNA <213> Arabidopsis thaliana <400> 3 atggctactc aaacggatct cgctcagccc aagcttgata tgaccaagga ggagaaagag 60 aggttgaagt atttgcaatt cgtgcaagct gctgctgtgg aagctctgct tcgctttgct 120 cttatttacg ctaaggcaaa ggacaagtct ggtcctttga aacctggtgt tgaatctgtt 180 gaaggagctg tcaagactgt cgttggtcct gtctacgaga aataccacga cgtccctgtt 240 gaggtcctta aatacatgga ccagaaggtt gatatgtctg tgactgagct tgaccgtcgt 300 gtcccaccag tcgtcaagca agtgtctgcc caagccatct ccgctgctca gatagcaccc 360 attgtggcac gtgcgttggc ctctgaggtt cgacgtgctg gtgttgttga aaccgcttct 420 ggaatggcta aatccgtcta ctccaagtac gagcctgctg ctaaggagtt gtatgcaaac 480 tatgagccaa aagcagagca gtgtgccgtt tcagcttgga agaagcttaa ccagcttcct 540 ctattcccaa ggctggctca agttgctgta ccaacagctg ctttctgctc tgagaagtac 600 aatgatactg tggttaaggc tgcagagaaa gggtacagag tcacatcgta catgccattg 660 gttccaacag agaggatctc aaaaatcttc gctgaggaga aagctgagac cgagcctttg 720 gagttccatc cacttgattg a 741 <210> 4 <211> 240 <212> PRT <213> Arabidopsis thaliana <400> 4 Met Glu Thr Glu Lys Lys Asn Ser Lys Glu Val Ala Leu Lys His Leu 1 5 10 15 Ala Phe Val Arg Ile Ala Thr Ile His Ile Leu Ala Ser Val Ser Asn 20 25 30 Leu Tyr Glu Tyr Ala Lys Gln Asn Ser Gly Pro Leu Lys Ser Ala Val 35 40 45 Glu Lys Val Glu Gly Ala Val Thr Thr Val Val Thr Pro Val Tyr Gln 50 55 60 Lys Phe Lys Asp Val Pro Asp Ser Leu Leu Val Phe Leu Asp His Lys 65 70 75 80 Val Gly Glu Val Ser Tyr Lys Phe Asp Glu His Ala Pro Pro Met Ala 85 90 95 Lys Lys Val Val Asn Gln Ala His Val Leu Ile Tyr Lys Ala Thr Glu 100 105 110 Lys Ala Gln Ser Phe Val Lys Glu Ala Arg Thr Gly Gly Pro Lys Ala 115 120 125 Ala Phe Asn Tyr Ala Ala Thr Glu Tyr Lys Phe Phe Val Val Thr Asn 130 135 140 Ser Val Lys Val Trp Ala Lys Leu Asn Gln Tyr Lys Pro Ile His Ala 145 150 155 160 Met Gly Asp Lys Ala Leu Pro Val Ala Ala His Phe Ser Ser Arg Tyr 165 170 175 Asn Asp Leu Val Thr Asp Met Thr Asn Met Gly Tyr Ser Leu Val Gly 180 185 190 Tyr Leu Pro Leu Val Pro Val Asp Asp Ile Val Lys Ala Tyr Glu Lys 195 200 205 Glu Asp Ala Arg Arg Lys Lys Gly Gly Asp Thr Ala Gly Lys Lys Gly 210 215 220 Glu Thr Thr Asp Ala Ala Asp Gly Asp Lys Ser Ser Ser Asp Ser Glu 225 230 235 240 <210> 5 <211> 235 <212> PRT <213> Arabidopsis thaliana <400> 5 Met Ala Glu Asp Glu Ile Val Val Glu Glu Glu Gln Ser Gln Pro Gln 1 5 10 15 Glu Ile Thr Pro Val Pro Pro Ser Ser Ser Ser Ser Pro Ser Leu Val 20 25 30 Val Glu Asp Asp Asp Glu Met Lys Leu Lys His Leu Glu Phe Ile Gln 35 40 45 Val Ala Ala Val Tyr Phe Ala Ala Cys Phe Ser Thr Leu Tyr Glu Leu 50 55 60 Ala Lys Asp Asn Ala Gly Pro Leu Lys Leu Gly Val Glu Asn Ile Glu 65 70 75 80 Asp Cys Val Arg Thr Val Leu Ala Pro Leu Tyr Glu Lys Phe His Asp 85 90 95 Val Pro Phe Lys Leu Leu Leu Phe Val Asp Arg Lys Val Asp Asp Val 100 105 110 Phe Phe Asp Val Glu Thr Tyr Val Pro Ser Leu Val Lys Gln Ala Ser 115 120 125 Ser Gln Ala Leu Thr Val Ala Thr Glu Val Gln Arg Thr Gly Val Val 130 135 140 Asp Thr Thr Lys Ser Ile Ala Arg Ser Val Arg Asp Lys Tyr Glu Pro 145 150 155 160 Ala Ala Glu Tyr Tyr Ala Ala Thr Leu Trp Arg Leu Leu Asn Gln Leu 165 170 175 Pro Leu Phe Pro Glu Val Ala His Leu Val Ile Pro Thr Ala Phe Tyr 180 185 190 Trp Ser Glu Lys Tyr Asn Asp Ala Val Arg Tyr Val Gly Asp Arg Asp 195 200 205 Tyr Phe Gly Ala Glu Tyr Leu Pro Met Ile Pro Ile Glu Lys Ile Ser 210 215 220 Asp Ile Leu Glu Gln Asp Gln Cys Arg Ala Asp 225 230 235 <210> 6 <211> 246 <212> PRT <213> Arabidopsis thaliana <400> 6 Met Ala Thr Gln Thr Asp Leu Ala Gln Pro Lys Leu Asp Met Thr Lys 1 5 10 15 Glu Glu Lys Glu Arg Leu Lys Tyr Leu Gln Phe Val Gln Ala Ala Ala 20 25 30 Val Glu Ala Leu Leu Arg Phe Ala Leu Ile Tyr Ala Lys Ala Lys Asp 35 40 45 Lys Ser Gly Pro Leu Lys Pro Gly Val Glu Ser Val Glu Gly Ala Val 50 55 60 Lys Thr Val Val Gly Pro Val Tyr Glu Lys Tyr His Asp Val Pro Val 65 70 75 80 Glu Val Leu Lys Tyr Met Asp Gln Lys Val Asp Met Ser Val Thr Glu 85 90 95 Leu Asp Arg Arg Val Pro Pro Val Val Lys Gln Val Ser Ala Gln Ala 100 105 110 Ile Ser Ala Ala Gln Ile Ala Pro Ile Val Ala Arg Ala Leu Ala Ser 115 120 125 Glu Val Arg Arg Ala Gly Val Val Glu Thr Ala Ser Gly Met Ala Lys 130 135 140 Ser Val Tyr Ser Lys Tyr Glu Pro Ala Ala Lys Glu Leu Tyr Ala Asn 145 150 155 160 Tyr Glu Pro Lys Ala Glu Gln Cys Ala Val Ser Ala Trp Lys Lys Leu 165 170 175 Asn Gln Leu Pro Leu Phe Pro Arg Leu Ala Gln Val Ala Val Pro Thr 180 185 190 Ala Ala Phe Cys Ser Glu Lys Tyr Asn Asp Thr Val Val Lys Ala Ala 195 200 205 Glu Lys Gly Tyr Arg Val Thr Ser Tyr Met Pro Leu Val Pro Thr Glu 210 215 220 Arg Ile Ser Lys Ile Phe Ala Glu Glu Lys Ala Glu Thr Glu Pro Leu 225 230 235 240 Glu Phe His Pro Leu Asp 245

Claims (9)

A composition for promoting abiotic stress tolerance of a plant comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 6.
2. The composition of claim 1, wherein the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 6 comprises a nucleotide sequence of SEQ ID NO: 3.
2. The composition of claim 1, wherein the abiotic stress is selected from the group consisting of dry stress, cold stress and salt stress.
A plant cell having enhanced abiotic stress tolerance transformed with the composition of claim 1 or 2.
A plant having enhanced abiotic stress tolerance transformed with the composition of claim 1 or 2.
(a) the nucleotide sequence of claim 1 or 2; (b) a promoter that is operatively linked to the nucleotide sequence and that forms RNA molecules in plant cells; And (c) a recombinant vector for plant expression comprising a poly A signal sequence that acts in plant cells to cause polyadenylation of the 3'-end of the RNA molecule.
A composition for promoting growth of a plant comprising a nucleotide sequence encoding an amino acid sequence of SEQ ID NO: 6.
8. The composition according to claim 7, wherein the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 6 is the nucleotide sequence of SEQ ID NO: 3.
(a) the nucleotide sequence of claim 7 or 8; (b) a promoter that is operatively linked to the nucleotide sequence and that forms RNA molecules in plant cells; And (c) a recombinant vector for plant expression comprising a poly A signal sequence that acts in plant cells to cause polyadenylation of the 3'-terminal of the RNA molecule.

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