KR102092406B1 - Composition for improving tolerance of plant for heavy metal stress comprising bacillus amyloliquefaciens RWL-1 and method for improving tolerance of plant for heavy metal stress - Google Patents

Abstract

The present invention relates to a composition for enhancing tolerance of a plant to heavy metal stress, comprising Bacillus amyloliquefaciens RWL-1 as an effective component, and a method for enhancing tolerance of a plant to heavy metal stress. According to the present invention, the Bacillus amyloliquefaciens RWL-1 has excellent heavy metal adsorption capacity to enhance the tolerance in a plant to heavy metal stress, can promote the growth of the plant and increase the carbohydroate and amino acid contents thereof, and can also reduce plant hormones, abscisic acid (ABA) and jasmonic acid (JA). Therefore, the Bacillus amyloliquefaciens RWL-1 of the present invention can significantly improve the growth of crops in cultivation sites contaiminated with heavy metals, thereby improving the quality and production of agricultural products, and thus is expected to be useful as an environmentally-friendly material in agricultural industry.

Description

Composition for improving tolerance of plants against heavy metal stress and method for enhancing resistance of plants against heavy metal stress, comprising the Bacillus amyloliquefaciens RWL-1 strain as an active ingredient amyloliquefaciens RWL-1 and method for improving tolerance of plant for heavy metal stress}

The present invention relates to Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) RWL-1 strain (KCTC 13637BP) as an active ingredient for improving the resistance of plants to heavy metal stress and methods for enhancing the resistance of plants to heavy metal stress, etc. .

The rapid increase in population, urbanization and industrialization intensifies global environmental pollution, causing rapid abiotic stress on crops and negatively impacting agricultural productivity. Of the various abiotic stresses, heavy metals are considered a serious and serious problem, and heavy metal contamination in soil occurs both in natural and anthropogenic activities such as weathering of rocks, mining, release of untreated industrial waste and excessive application of synthetic fertilizers. do. Heavy metal contamination is of great concern as this accumulation of harmful heavy metals causes serious metabolic and physiological disorders in organisms.

Some of the heavy metals, such as copper (Cu), zinc (Zn), manganese (Mn), and iron (Fe), are considered micronutrients essential for plants to maintain their normal functions, but are toxic at high concentrations. Among them, copper (Cu) is known to be the oldest among the heavy metals used by mankind, and it is still an important element used in various fields. In essence, the uncontaminated Cu concentration in the soil ranges from 20-100 mg kg ^-1 . However, anthropogenic agricultural activities have increased the concentration of copper in the soil to a maximum of 200 to 1000 mg kg ^{- 1} . Cu of 50 mg kg ^-1 or more usually shows toxic effects on plants and negatively affects agricultural productivity. High concentrations of copper in plants induce nutritional imbalances, photosynthetic disorders, physicochemical changes in plants, poor germination and reduced growth and yield.

Thus, many new approaches to soil environmental cleanup have been developed by many researchers and some have actually been put into practical use as an alternative to physical methods for low-cost environmental cleanup techniques. Phytoremediation is predicted to be an effective and cost-effective approach to removing metal contaminants, but most plants are slow to grow and biomass is reduced.

Therefore, the use of microorganisms is an ideal solution for reducing pollutants and is considered to be the most effective strategy for managing contaminated environments, and many studies on the use of microorganisms for plant growth development in heavy metal contaminated soils have been conducted. Among them, the growth of host plants using endogenous bacteria is an important material in that it shows an improvement effect upon stress of heavy metals and does not cause a clear disease.

On the other hand, the microorganism of the genus Bacillus is aerobic or facultative anaerobic Gram-positive bacillus, and is a strain generally known as safe (generally recognized as safe, GRAS) and has various activities such as denitrification, iron reduction, and manganese oxidation, and various natural environments. It is known to inhabit extensively.

Among them, Bacillus amyloliquefacience is an aerobic bacterium that forms spores and is known to have excellent protein and starch degrading enzyme activity. Although known as Bacillus amyloliquefaciens species, the effect of improving the heavy metal stress of the Bacillus amyloliquefaciens RWL-1 strain has not been well known.

Accordingly, the present inventors tried to use Bacillus amyloliquefaciens RWL-1 as an eco-friendly material that separates from rice seeds and purifies heavy metal soil pollution and promotes growth of crops.

Korean Registered Patent No. 1898260

The present inventors researched to develop a method for increasing resistance to heavy metal stress by using eco-friendly microorganisms. As a result, the Bacillus amyloliquefaciens RWL-1 strain has excellent copper (Cu) adsorption ability and reduces copper absorption of plants. It was confirmed to promote growth, and based on this, the present invention was completed.

Therefore, the object to be solved by the present invention is Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) RWL-1 strain (KCTC 13637BP) as an active ingredient, a composition for enhancing plant resistance to heavy metal stress and against heavy metal stress It is to provide a method for enhancing plant resistance.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned can be clearly understood by those skilled in the art from the following description. There will be.

In order to achieve the object of the present invention as described above, the present invention comprises a Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) RWL-1 strain (KCTC 13637BP) as an active ingredient, a composition for enhancing the resistance of plants to heavy metal stress Gives

As an embodiment of the present invention, the Bacillus amyloliquefaciens RWL-1 strain may include the nucleotide sequence represented by SEQ ID NO: 1.

As another embodiment of the present invention, the Bacillus amyloliquefaciens RWL-1 strain may have heavy metal adsorption capacity.

As another embodiment of the present invention, the heavy metal may be at least one selected from the group consisting of copper (Cu), chromium (Cr), lead (Pb), and cadmium (Cd).

As another embodiment of the present invention, the composition may promote plant growth.

As another embodiment of the present invention, the composition may increase the carbohydrate or amino acid content of the plant.

As another embodiment of the present invention, the composition may reduce the abscisic acid (ABA) or jasmonic acid (JA) of the plant.

In addition, the present invention (a) Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) culturing the RWL-1 strain; And

(b) treating the seedlings of plants with at least one selected from the group consisting of the cultured strain, the culture of the strain, the concentrate of the culture, the dried product of the culture, and combinations thereof. It provides a method for enhancing plant resistance to stress.

As an embodiment of the present invention, the step (a) may be to incubate Bacillus amyloliquefaciens RWL-1 strain in LB (Luria Bertani) medium at 20 ° C to 30 ° C for 1 to 10 days. have.

As another embodiment of the present invention, the LB (Luria Bertani) medium may include tryptone (tryptone), yeast extract, and sodium chloride (NaCl).

Bacillus amyloliquefaciens RWL-1 strain according to the present invention has excellent heavy metal adsorption ability to enhance plant resistance to heavy metal stress, promote plant growth as well as increase the content of carbohydrates and amino acids And the effect of reducing the plant hormones abscisic acid (ABA) and jasmonic acid (JA). Therefore, the Bacillus amyloliquefaciens RWL-1 strain of the present invention is expected to be usefully used as an eco-friendly material in the agricultural industry, such as improving the crop growth of crops contaminated with heavy metals and improving the quality and yield of crops. .

1A is a diagram showing the growth degree of Bacillus amyloliquefaciens RWL-1 according to the type of heavy metal.
1B is a diagram showing the growth rate of Bacillus amyloliquefaciens RWL-1 according to the concentration of heavy metal copper (Cu).
1C is a diagram showing the amount of copper (Cu) accumulated in Bacillus amyloliquefaciens RWL-1 according to the concentration of heavy metal copper (Cu).
1d is a diagram showing the copper (Cu) adsorption capacity of Bacillus amyloliquefaciens RWL-1 through SEM / EDS.
FIG. 2 is a diagram showing copper (Cu) content by region and growth photos of rice according to Bacillus amyloliquefaciens RWL-1 treatment.
3 is a view showing the change in the content of carbohydrates by Bacillus amyloliquefaciens RWL-1 treatment in copper (Cu) stressed rice.
4A and 4B are diagrams showing changes in amino acid content by Bacillus amyloliquefaciens RWL-1 treatment in copper (Cu) stressed rice.
FIG. 5 is a diagram showing changes in plant hormone abscisic acid (ABA) and jasmonic acid (JA) content by Bacillus amyloliquefaciens RWL-1 treatment in copper (Cu) stressed rice.

The present invention provides a composition for enhancing plant resistance to heavy metal stress, including Bacillus amyloliquefaciens RWL-1 strain (KCTC 13637BP) as an active ingredient.

In the present invention, the Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) RWL-1 strain may include the nucleotide sequence represented by SEQ ID NO: 1, as of September 06, 2018, Korea Institute of Bioscience and Biotechnology (BRC) KCTC) and was assigned an accession number KCTC 13637BP.

The strain of the present invention may be isolated from rice seeds, but is not limited thereto.

In the present invention, the Bacillus amyloliquefaciens RWL-1 strain may have heavy metal adsorption capacity.

In the present invention, "heavy metals" is a general term for metal elements having a specific gravity of about 4 or more, arsenic, antimony, lead, mercury, zinc, cadmium, chromium, tin, nickel, barium, bismuth, cobalt, vanadium, selenium, and manganese. , Copper and the like. Among heavy metals, copper, zinc, nickel, and cobalt are essential elements that are indispensable to life, but when exposed to high concentrations of heavy metals, they act as fatal toxic substances to living organisms. These heavy metals are easily adsorbed to the soil particles, causing not only heavy metal accumulation in the soil, but also secondary and tertiary pollution such as water pollution and vegetation destruction. Unlike other organic compound pollutants, heavy metals are not decomposed into non-toxic substances or converted into stable compounds, and remain in the ecosystem for a long time and contaminate soil and water. Recently, with the development of such a pollutant removal method, heavy metals are treated using ion exchange, activated carbon adsorption, and biological treatment.

In the present invention, the heavy metal may be at least one selected from the group consisting of copper (Cu), chromium (Cr), lead (Pb), and cadmium (Cd), and according to an embodiment of the present invention, the Bacillus amyl The Bacillus amyloliquefaciens RWL-1 strain was confirmed to have the best adsorption capacity of copper (Cu), but the Bacillus amyloliquefaciens RWL-1 strain has a heavy metal adsorption capacity, which is the type of heavy metal. It is not limited to.

In the present invention, "copper (Copper, Cu)" is a red and soluble metal. Adults are excreted in sweat or urinary tract when ingesting 2 to 5 mg, so there is no accumulation in the human body, so there is less risk of chronic poisoning, but excessive intake (lethal dose) 5 to 15 g) affects nausea, abdominal pain, liver, kidney failure, etc. The average daily intake per person is 1.2 to 1.4 mg.

In the present invention, the composition can promote plant growth.

In the present invention, the composition can increase the carbohydrate or amino acid content of the plant.

In the present invention, the composition can reduce the abscisic acid (ABA) or jasmonic acid (JA) of the plant.

In the present invention, "abscisic acid (ABA)" is a type of plant hormone, which is detected in any part of a plant such as a leaf, a stem, or a root, but is particularly present in immature fruits or dormant seeds. Absistic acid rapidly increases in leaves when it is subjected to moisture stress (moisture deficiency), which has a deep relationship with the opening and closing of the pores, inhibiting germination of seeds, inhibiting the collapse of winter snow, promoting sleep, promoting aging and falling It is known to be involved in many physiological phenomena such as palpation.

In the present invention, "jasmonic acid (jasmonic acid; JA)" is a plant signaling molecule derived from linolenic acid present in the membranous lipid, jasmonate and methyl jasmonate, which are a kind of signaling substances involved in the expression of plant defense mechanisms, are a plant. It is a substance that is involved in the growth, development, aging of leaves, and defense mechanisms of the entire plant as well as the increase in the content of bioactive substances.

In addition, the present invention (a) Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) culturing the RWL-1 strain; And

(b) treating the seedlings of plants with at least one selected from the group consisting of the cultured strain, the culture of the strain, the concentrate of the culture, the dried product of the culture, and combinations thereof. It provides a method for enhancing plant resistance to stress.

In the present invention, the "cultivation" refers to a series of actions to grow microorganisms under environmental conditions appropriately artificially controlled. In the present invention, the method for culturing the Bacillus amyloliquefaciens RWL-1 strain may be performed using a method well known in the art, and specifically, 20 ° C. to LB (Luria Bertani) medium. Can be cultured at 30 ° C for 1 to 10 days, and more specifically, when cultured for 5 days at 28 ° C in LB (Luria Bertani) medium, the effect of enhancing the resistance of plants to heavy metal stress may be good, but limited to the culture conditions It does not work.

In the present invention, the LB (Luria Bertani) medium may include tryptone (tryptone), yeast extract, and sodium chloride (NaCl).

In the present invention, the "culture" means that a specific microorganism is cultured in a culture medium or a culture medium, and the culture may or may not include the specific microorganism. The culture is not limited to the formulation, and may be, for example, liquid or solid.

In the present invention, the composition for enhancing the resistance of plants to heavy metal stress can be prepared by adding additives such as additives, extenders, and nutrients to the Bacillus amyloliquefaciens RWL-1 strain or its culture. At this time, additives include polycarboxylate, sodium lignosulfonate, calcium lignosulfonate, sodium dialkyl sulfosuccinate, sodium alkyl aryl sulfonate, polyoxyethylene alkyl phenyl ether, sodium tripolyphosphate, polyoxyethylene alkyl From the group consisting of aryl phosphoric esters, polyoxyethylene alkyl aryl ethers, polyoxyethylene alkyl aryl polymers, polyoxyalkylone alkyl phenyl ethers, polyoxyethylene nonyl phenyl ethers, sodium sulfonate naphthalene formaldehyde, Triton 100 and Tween 80 One or more selected may be used, and as a bulking agent and a nutritional agent, a group consisting of skim milk, yeast extract, soybean flour, rice, wheat, ocher, diatomaceous earth, bentonite, dextrin, glucose, and starch Use one or two or more selected from, and as a disintegrant, bento Sites (bentonite), can be used talc (talc), diamond light (dialite), kaolin (kaolin), calcium carbonate and one or more selected from the group consisting of (calcium carbonate).

In one experimental example of the present invention, as a result of confirming the heavy metal adsorption capacity of the Bacillus amyloliquefaciens RWL-1 strain, it was confirmed that the copper (Cu) adsorption ability is the best (see Experimental Example 1).

In another experimental example of the present invention, as a result of confirming the effect of promoting the growth of rice by treatment with Bacillus amyloliquefaciens RWL-1 strain under copper (Cu) stress conditions, fat part, underground part length, live weight, building weight, and It was confirmed that chlorophyll content increased to promote growth (see Experimental Example 2).

In another experimental example of the present invention, as a result of confirming the effect of enhancing the resistance of rice according to Bacillus amyloliquefaciens RWL-1 strain treatment under copper (Cu) stress conditions, Cu content of rice is reduced, carbohydrate and amino acid content As this increased, the contents of the plant hormones, abscisic acid (ABA) and jasmonic acid (JA), were reduced, and it was confirmed that rice tolerance was improved (see Experimental Example 3).

Hereinafter, preferred examples and experimental examples are provided to help understanding of the present invention. However, the following examples and experimental examples are provided only to more easily understand the present invention, and the contents of the present invention are not limited by the following examples and experimental examples.

Example  One. Bacillus Amyloliquefaciens RWL Isolation and identification of -1 strain

To isolate the endogenous strains of rice, the varieties of Jinsoomi were used, first washed with sterile water for surface sterilization, and then dropped one or two drops of tween 80 into a solution of 62.5 ml of 12% NaOCl and 237.5 ml of sterile water The floated solution was immersed at 20 ° C for 30 minutes. Then, the seeds were washed several times with sterile water to remove the sterile reagent. Sterilized rice seeds were placed in LB medium and cultured at 30 ° C for 3 days. This was diluted with sterilized 0.85% NaCl, and pure colony obtained by culturing in LB agar medium was stored in 50% glycerin for use in the next experiment. To identify the rice seed endogenous bacteria, 16S ribosomal RNA gene sequence analysis was performed, and based on this, similarity was confirmed through Blast analysis.

Experimental example  One. Bacillus Amyloliquefaciens RWL -1 strain of heavy metals Adsorption capacity  Confirm

50mL LB medium (tryptone 10g) with 1 mM Cu, Cr, Pb, and Cd added to Bacillus amyloliquefaciens RWL-1 (KCTC 13637BP), an endogenous strain that produces phytohormones and organic acids, to confirm the ability to remove heavy metals , 5 g of yeast extract, 10 g of NaCl, pH 7.0 ± 0.2,) were cultured at 28 ° C for 5 days, and the heavy metals Cu, Cr, Pb, and Cd were evaluated to evaluate the growth rate by Bacillus amyloliquefaciens RWL-1 inoculation. Among them, heavy metal Cu showing the highest vitality was added at various concentrations (1 mM, 2.5 mM and 5 mM) and cultured at 28 ° C. for 5 days.

In addition, the Bacillus amyloliquefaciens RWL-1 biomass was confirmed by measuring the Cu content using a plasma mass spectrometer (ICP-MS, Optima 7900DV Perkin-Elmer, USA), and energy-dispersive X-ray spectrometry (EDS) Bacillus amyloliquefaciens RWL-1 cells were fixed with 2.5% glutaraldehyde using scanning electron microscopy (SEM) combined with, washed with brine phosphate buffer (pH 7), and then dehydrated treated cells The morphology and absorbed heavy metal Cu in the cells were investigated.

As a result, Bacillus amyloliquefaciens RWL-1 showed the best growth in 1 mM Cu compared to Cr, Pb and Cd, as shown in FIG. 1A, and the growth degree of RWL-1 according to the concentration of heavy metal Cu As shown in FIG. 1B, 1mM Cu was increased than the control, but it was found that at 2.5mM and 5mM concentrations, biological water and biomass were significantly limited.

In addition, as a result of analyzing the heavy metal Cu content of Bacillus amyloliquefaciens RWL-1 through a plasma mass spectrometer, as shown in FIG. 1C, the most accumulated amount (8,200 mg) of cells cultured in LB medium containing 5 mM Cu Kg-1DW), and the cells cultured in a medium containing a concentration of 2.5 mM were 3536.33 mg Kg-1DW, and the cells cultured in a medium containing a concentration of 1 mM were examined for a Cu content of 973.66 mg Kg-1DW.

Finally, as a result of analyzing the cell morphology and the Cu content ratio of RWL-1 using the SEM with EDS combined, as shown in FIG. The adsorption capacity of heavy metal Cu was excellent.

Therefore, it was confirmed from the above results that the Bacillus amyloliquefaciens RWL-1 strain has excellent copper (Cu) adsorption capacity.

Experimental example  2. For enhancing crop resistance under heavy metal Cu stress conditions Bacillus  Amyloliquefaciens RWL -1 Check the effect of promoting growth of culture and crops

Bacillus amyloliquefaciens RWL-1 was inoculated with 1% of the seed in LB medium (tryptone 10g, yeast extract 5g, NaCl 10g, pH 7.0 ± 0.2,) and cultured at 28 ° C for 150 rpm for 5 days. This was centrifuged at 6,000 g for 10 minutes using a centrifuge to remove the supernatant and recover only the cells. The cells were diluted with sterile water and O.D. The value was adjusted to 0.5 and utilized.

Rice was used as a rice cultivar, and after disinfection and sowing the seeds, Bacillus amyloliquefaciens RWL-1 was inoculated when the number of leaves was two. The inoculation method irrigated 10% of the sterilized top soil weight per pot. To establish heavy metal stress conditions, 50 ml of copper sulfate (CuSO ₄ ) solution was treated daily for 12 days.

As a result, as shown in FIG. 2, the growth of rice was improved due to Bacillus amyloliquefaciens RWL-1 treatment under conditions according to heavy metal Cu concentrations (1 mM, 2.5 mM, 5 mM). Specifically, as shown in Table 1, the lengths of the above-ground parts (10.82%, 16.71%, 61.09%) and underground parts (88.75%, 198.87%, 71.58%) were accelerated, respectively, and in vivo weight (88.52%, 198.87%, 136.50%) and Among buildings (102.15%, 125%, 115.25%) increased. In addition, chlorophyll content (39.61%, 31.95%, 71.58%) also increased compared to the non-microbial inoculation.

Experimental example  3. Under heavy metal Cu stress conditions Bacillus Amyloliquefaciens RWL -1 Confirmation of improvement of crop resistance according to treatment

3-1. In rice Bacillus Amyloliquefaciens RWL -1 Cu content measurement by site according to treatment

The heavy metal Cu content in the upper and lower parts of the rice was measured using an inductively coupled plasma mass spectrometer (ICP-MS) to confirm the effect of enhancing the resistance of crops according to Bacillus amyloliquefaciens RWL-1 treatment under heavy metal Cu stress conditions. Analysis.

As a result, as shown in Fig. 2, Cu concentrations of the upper and lower parts of rice increased with Cu treatment at various concentrations (1 mM, 2.5 mM, 5 mM), but the Bacillus amyloliquefaciens RWL-1 microbe treated In, the Cu content of the ground (78.54%, 75.23%, 69.46%) and the underground (74.59%, 67.66%, 69.46%) decreased significantly.

3-2. In rice Bacillus Amyloliquefaciens RWL Changes in the content of carbohydrates and amino acids by -1 treatment

Carbohydrates such as glucose, sucrose, fructose, and raffinose were analyzed through HPLC (Millipore Corp., Waters Chromatography, Milford, MA, USA) analysis using a sugar-pak column and an RI detector. 8900, Hitachi, Japan). Because carbohydrates and amino acids are the main energy sources for plants, they are indicators of the beneficial effects of crop stress.

As a result of confirming the content of carbohydrates in rice, as shown in Fig. 3, in the treatment group inoculated with the useful microorganism Bacillus amyloliquefaciens RWL-1, glucose (32.14%), sucrose (50.54%), and fructose (10%) than the control group ), the content of raffinose (70%) increased, and the treatment with the Bacillus amyloliquefaciens RWL-1 in the stress treatment according to the concentration of heavy metal Cu (1mM, 2.5mM, 5mM) was glucose (54.23%, 34.21) %, 53.84%), sucrose (58.06%, 38.39%, 27.27%), fructose (40.62%, 70.58%, 89.06%), raffinose (52.63%, 76.47%, 55.0%).

In addition, in the case of amino acids, amino acids (aspartic acid, glutamic acid, alanine, threonine, serine, glycine, cystine, valine, in a sphere inoculated with Bacillus amyloliquefaciens RWL-1 under heavy metal Cu stress conditions), as shown in FIG. The content of methionine, isoleucine, leucine, phenylalanine, lysine, and proline) was higher than that of the untreated group.

3-3. In rice Bacillus Amyloliquefaciens RWL -1 by treatment Phytohormones  Content change

Phytohormonal abscisic acid (ABA) analysis was performed by lyophilizing the pulverized plants with isoprpanol and glacial acetic acid in a ratio of 95: 5, then filtering and filtering [± -3,5,5,7,7,7 -d ⁶ ] -ABA standard After adding 20ng, it was concentrated under reduced pressure, and the pH of the solution extracted with 1N NaOH was adjusted to be 12-13. Here, methylene chloride was added and mixed, followed by fractionation three times to remove the chlorophyll component, and the pH of the aqueous layer was 2.5 to 3.5 using 6N HCl. EtOAc was added to the aqueous layer to separate the layers, and the EtOAc layer was recovered 3 times and concentrated under reduced pressure. Then, after dissolving the concentrated residue with phosphate buffer (pH 8.0), 1 g of PVPP (polyvinypolypyrrolidone) was added and shaken for 1 hour to remove the phenol compound. The filtered phosphate buffer (pH 8.0) solution was adjusted to pH 2.5-3.5 with 6N HCl, and EtOAc was added to recover the EtOAc layer, and concentrated under reduced pressure. Thereafter, EtOAc was added to the concentrated residue, transferred to a vial, and concentrated. After inducing metyl ester, it was dried with nitrogen gas. In order to analyze the content of ABA by GC-MS, it was analyzed by GC-MS-SIM, and the quantity was calculated by comparing ion 190 and 194.

Plant hormones jasmonic acid (JA) assay leaving only the receiving layer was added 0.5 g of rice plant sample after the extraction with acetone was filtered, and then the internal standard of ^{50 ng [9, 10- 2 H} 2] JA and 50 ml of distilled water under reduced pressure , Concentrated. The concentrated residue was dissolved in 0.1 M potassium phosphate (pH 7.5), adjusted to pH 2.5 with 6 N HCl, and 1 g of diethylaminoethyl cellulose (DEAE, Sigma Aldrich, USA) was added and shaken for 1 hour. The filtrate shaken was filtered through filter paper, fractionated three times with chloroform, and then removed with anhydrous sodium sulfate (Na ₂ SO ₄ ) and then concentrated under reduced pressure. After dissolving the concentrated residue with a small amount of diethyl ether, pass it using NH ₂ cartridges, followed by chloroform: iso-propanol (2: 1, v / v) and diethyl ether: acetic acid (98: 2, v / v). Eluted with. The filtrate was concentrated under reduced pressure, dissolved in diethyl ether, transferred to a 1 ml reaction vial, dried with nitrogen gas at 40 ° C, and then induced with methyl ester with 60 μl ethereal diazomethane. The methyl ester-derived samples were analyzed by GC-MS-SIM.

The interaction of plant hormones such as ABA and JA plays an important role in the important physiological aspects of plants under normal and stressful conditions. As shown in the ABA analysis, as shown in FIG. 5, the ABA content of rice increased according to various concentrations of Cu (1 mM, 2.5 mM, 5 mM), but the ABA content was 17.23% in Bacillus amyloliquefaciens RWL-1 treatment, respectively. , 26.79% and 19.72%. Similarly, the JA results were reduced to 17.48%, 14.31% and 11.35%, respectively, in the Bacillus amyloliquefaciens RWL-1 treatment under heavy metal stress conditions.

Therefore, based on the results as described above, it was confirmed that the resistance of the crop to heavy metal stress was improved by the treatment of Bacillus amyloliquefaciens RWL-1 of the present invention.

The above description of the present invention is for illustration only, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the above-described embodiments are illustrative in all respects and not restrictive.

Depository name: Korea Research Institute of Bioscience and Biotechnology

Accession number: KCTC13637BP

Date of accession: 20180906

<110> Kyungpook National University Industry-Academic Cooperation Foundation <120> Composition for improving tolerance of plant for heavy metal          stress comprising bacillus amyloliquefaciens RWL-1 and method for          improving tolerance of plant for heavy metal stress <130> MP18-177 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1461 <212> RNA <213> Bacillus amyloliquefaciens RWL-1 16S rRNA <400> 1 cggcggtggc gggtgctata atgcagtcga gcggacagat gggagcttgc tccctgatgt 60 tagcggcgga cgggtgagta acacgtgggt aacctgcctg taagactggg ataactccgg 120 gaaaccgggg ctaataccgg atggttgttt gaaccgcatg gttcagacat aaaaggtggc 180 ttcggctacc acttacagat ggacccgcgg cgcattagct agttggtgag gtaacggctc 240 accaaggcga cgatgcgtag ccgacctgag agggtgatcg gccacactgg gactgagaca 300 cggcccagac tcctacggga ggcagcagta gggaatcttc cgcaatggac gaaagtctga 360 cggagcaacg ccgcgtgagt gatgaaggtt ttcggatcgt aaagctctgt tgttagggaa 420 gaacaagtgc cgttcaaata gggcggcacc ttgacggtac ctaacccaga aagccacggc 480 taactacgtg cccagcagcc gcggtaatac gtaggtggca agcgttgtcc gggaattatt 540 gggcgtaaag ggctcgcagg cggtttctta agtctgatgt gaaagccccc ggctcaaccg 600 gggagggtca ttggaaactg gggaacttga gtgcagaaga ggagagtgga attccacgtg 660 tagcggtgaa atgcgtagag atgtggagga acaccagtgg cgaaggcgac tctctggtct 720 gtaactgacg ctgaggagcg aaagcgtggg gagcgaacag gattagatac cctggtagtc 780 cacgccgtaa acgatgagtg ctaagtgtta gggggtttcc gccccttagt gctgcagcta 840 acgcattaag cactccgcct ggggagtacg gtcgcaagac tgaaactcaa aggaattgac 900 ggggggcccg cacaagcggt ggagcatgtg gtttaattcg aagcaacgcg aagaacctta 960 ccaggtcttg acatcctctg acaatcctag agataggacg tccccttcgg gggcagagtg 1020 acaggtggct gcatgattgt cgtcagctcg tgtcgtgaga tgttgggttt aagtcccgca 1080 acgagcgcaa cccttgatct tagttgccag cattcagttg ggcactctaa ggtgactgcc 1140 ggtgacaaac cggaggaagg tggggatgac gtcaaatcat catgcccctt atgacctggg 1200 ctacacacgt gctacaatgg acagaacaaa gggcagcgaa accgcgaggt taagccaatc 1260 ccacaaatct gttctcagtt cggatcgcag tctgcaactc gactgcgtga agctggaatc 1320 gctagtaatc gcggatcagc atgccgcggt gaatacgttc ccgggccttg tacacaccgc 1380 ccgtcacacc acgagagttt gtaacacccg aagtcggtga ggtaaccttt atggagccag 1440 ccgccgaagt gacagaattg t 1461

Claims (10)

Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) RWL-1 strain (KCTC 13637BP) containing as an active ingredient, copper (Cu) composition for enhancing the resistance of plants to stress.
According to claim 1,
The Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) RWL-1 strain is characterized in that it comprises a nucleotide sequence represented by SEQ ID NO: 1, copper (Cu) composition for enhancing plant resistance to stress.
According to claim 1,
The Bacillus amyloliquefaciens RWL-1 strain is characterized in that it has the ability to adsorb heavy metals, copper (Cu) composition for enhancing the resistance of plants to stress.
delete According to claim 1,
The Bacillus amyloliquefaciens RWL-1 strain is a composition for promoting plant resistance to copper (Cu) stress, characterized in that to promote the growth of plants.
According to claim 1,
The Bacillus amyloliquefaciens RWL-1 strain is characterized in that to increase the carbohydrate or amino acid content of the plant, copper (Cu) composition for promoting plant resistance to stress.
According to claim 1,
The Bacillus amyloliquefaciens RWL-1 strain is characterized by reducing abscisic acid (ABA) or jasmonic acid (JA) of plants, against copper (Cu) stress Composition for enhancing plant resistance.
(A) culturing the Bacillus amyloliquefaciens RWL-1 strain (KCTC 13637BP); And
(b) treating the seedlings of plants with at least one selected from the group consisting of the cultured strain, the culture of the strain, the concentrate of the culture, the dried product of the culture, and combinations thereof. (Cu) Method for enhancing plant resistance to stress.
The method of claim 8,
The step (a) is characterized in that the Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) RWL-1 strain is cultured in LB (Luria Bertani) medium at 20 ° C to 30 ° C for 1 to 10 days, copper (Cu) stress How to increase plant resistance to.
The method of claim 9,
The LB (Luria Bertani) medium is tryptone (tryptone), characterized in that it comprises a yeast extract, and sodium chloride (NaCl), copper (Cu) method for enhancing plant resistance to stress.

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