KR101611537B1 - Enterobacter ludwigii SJR3 strain promoting the growth of plants and the microbial agent containing the same - Google Patents

Abstract

The present invention relates to a novel strain Enterobacterium strain SJR3 having plant growth promoting activity and a microorganism preparation containing the culture as an active ingredient and a method for promoting plant growth using the strain, And separating and identifying the same; Measuring ACC deaminase activity, IAA, zeatin and gibberellin production ability of the strain obtained in the above step; Culturing the microorganism in a microorganism-grown soil to investigate the IAA concentration; The novel Enterobacterium strain SJR3 obtained through the step of comparing and evaluating the amounts of DREB and ACS gene expression using RNA and cDNA of the tomato grown in the above step is excellent in the activity of producing plant growth promoting hormone, Insoluble phosphoric acid immobilized on the soil can be solubilized, and when the culture medium of the strain is treated with a plant, the activity of promoting the growth of the plant is excellent.

Description

[0001] The present invention relates to a microorganism preparation containing Enterobacter ludwigii strain SJR3 for promoting plant growth and its culture as an active ingredient,

The present invention relates to a novel strain Enterobacterium strain SJR3 having plant growth promoting activity and a microorganism preparation containing the culture thereof as an active ingredient and a method for promoting plant growth using the strain.

In agriculture, fertilizer is an important agricultural material that determines crop yield, or excessive use of fertilizer causes eutrophication of river water, water pollution of ground water, imbalance of soil nutrients and poor growth of crops, It may cause a quality deterioration. However, due to the development and diversification of industry, the use of fertilizer to increase the productivity of crops is inevitable because of the decrease in the area of agricultural land. Therefore, interest in the development of environmentally friendly functional farming materials is increasing.

On the other hand, cultivation of crops using soil microorganisms is widely carried out in soybean plants, and the most frequently used microorganisms are Rhizobium (rhizobium) having a symbiotic relationship with soybean and crops. Among the microorganisms in soil, microorganisms that promote plant growth without complete symbiotic relationship with the host plant are plant growth-promoting rhizobacteria (PGPR), which include azo-furylum spp. Azospirrilum the like), Pseudomonas spp. (Pseudomonas), Bacillus spp. (Bacillus) include the representative microorganisms. These plant growth-promoting microorganisms are known to help plant growth by various functions such as fixation of air nitrogen, production of plant growth-promoting hormone, promotion of nutrient absorption of plants, and inhibition of plant disease.

The microorganism Bacillus megaterium KR076, which fixes air nitrogen and fixes on the root surface and inside of plants, and promotes the growth of plants, is disclosed in Korean Registered Patent Publication No. 10-0577717, Or a microbial agent containing the culture liquid as an active ingredient has been disclosed in Korean Patent Publication No. 10-2010-0053743 and it has been reported that a strain belonging to the genus Enterobacteria which is resistant to high salt stress and promotes the growth of the plant is registered It is disclosed in Publication No. 10-1091151. However, none of the above references discloses or suggests any effect of rhizobia bacteria on the amount of gene expression of tomato exposed to a dry environment.

It is therefore an object of the present invention is Enterobacter Ruud Cha child having a rRNA sequence of SEQ ID NO: 1 to promote the growth of plants (Enterobacter ludwigii ) SJR3 strain (KACC91932P).

Another object of the present invention is to provide a culture obtained by culturing the strain according to the present invention.

Yet another object of the present invention is to provide a microorganism preparation for promoting plant growth comprising the strain or the culture thereof according to the present invention as an effective ingredient.

The above object of the present invention is achieved by a method of isolating and identifying an Enterobacteriaceae line SJR3 strain; Measuring ACC deaminase activity, IAA, zeatin and gibberellin production ability of the strain obtained in the above step; Culturing the microorganism in a microorganism-grown soil to investigate the IAA concentration; The RNA and cDNA of the tomatoes grown in the above step were used to compare and evaluate the amounts of DREB and ACS gene expression.

The novel Enterobacter wild spot SJR3 according to the present invention is excellent in activity to produce plant growth promoting hormone and can solubilize the insoluble phosphoric acid immobilized on the soil. In addition, when the culture medium of the strain is treated with a plant, Is effective in promoting the activity.

FIG. 1 is a graph showing IAA-producing ability of Enterobacterium strain SJR3 according to the present invention.
FIG. 2 is a graph showing the ability of the Enterobacter strain SJR3 strain according to the present invention to produce zeatin; FIG.
FIG. 3 is a graph showing gibberellin-producing ability of the Enterobacter sp. SJR3 strain according to the present invention.
FIG. 4 is a graph (?) Showing the solubilization ability of insoluble phosphoric acid of the Enterobacter strain SJR3 strain according to the present invention. Acinetobacter guillouiae SW5 (?) and Pseudomonas brassicacearum subsp. neoaurantiaca BD3-35 (◇).
FIG. 5 is a photograph showing tomato cultivation experiments treated with strains SJR3, SW5 and BD3-35 according to the present invention.
6 is a graph showing root lengths of tomatoes treated with rhizospheric bacteria according to the present invention.
Fig. 7 is a graph showing shoots: root ratio of tomatoes treated with rhizospheric bacteria according to the present invention.
8 is a graph showing the average root weight of tomatoes treated with rhizospheric bacteria according to the present invention.
9 is a graph showing the average total weight of tomatoes treated with rhizospheric bacteria according to the present invention.
10 is a DGGE comparative photograph of DNA extracted from soil in a small scale cultivation experiment and DNA extracted from E. ludwigii SJR3 culture medium.
FIG. 11 is a graph showing the IAA concentration of tomato rhizosphere treated with Enterobacterium strain SJR3 ()) according to the present invention. Acinetobacter guillouiae SW5 (□).
Fig. 12 is a photographic view showing RNA (A) and cDNA (B) bands of tomato treated with rhizobacteria according to the present invention. 1: untreated, 2: E. gudwigii SJR3, 3: Acinetobacter guillouiae SW5, 4: Pseudomonas brassicacearum subsp. neoaurantiaca BD3-35, 5: bacterial IAA, 6: synthetic IAA.
13 is a graph showing relative mRNA levels of tomato Le-DREB2 treated with rhizobia according to the present invention.
14 is a graph showing relative mRNA levels of tomato Le-DREB3 treated with rhizobacteria according to the present invention.
15 is a graph showing relative mRNA levels of tomato ACS4 treated with rhizobia according to the present invention.
16 is a graph showing relative mRNA levels of tomato ACS6 treated with rhizobacteria according to the present invention.

Hereinafter, the present invention will be described in detail with reference to examples. However, these examples are intended to further illustrate the present invention, and the scope of the present invention is not limited to these examples.

Example  1: Isolation and identification of rhizobacteria

The rhizosphere soil of Equisetum arvense in the Hoanhwa area of Hwacheon - gun, Gangwon - do was sampled and used as a sample for rhizosphere bacterium isolation. 1 g of soil sample was diluted in 40 mL of physiological saline (0.85% NaCl), stirred for 15 minutes (150 rpm, 30 ° C), diluted continuously with physiological saline to 10 ^-5 , and diluted with Luria-Bertani agar (LA: 5 g of tryptone, 5 g of yeast extract, 15 g of agar, and 1 L of distilled water). Five sheets of each were plated on a plate medium and cultured at 30 ° C for 3 days (cancer condition). Subsequently, strains isolated to isolate the strains were cultured in LA medium (30 ° C, 3 days, under the dark condition), and 16s rRNA gene sequencing analysis was requested by Macrogen.

PCR included the universal forward primer 27F; 5'-AGAGTTTGATCMTGGCTCAG-3 'and 1492R; 5'-TACGGY TACCTTGTTACGACTT-3 'was used. The result of 16s rRNA gene sequence of about 1400bp obtained from PCR was compared with the NCBI (http://www.ncbi.nlm.nih.gov) registered strains and the identified strain Were registered with NCBI GenBank.

As a result, the 16s rRNA base sequence of the strain showed the highest homology with 99% of Enterobacter ludwigii EN-119 (Genbank accession number NR042349), and the nucleotide sequence of the strain identified in the present invention is shown in SEQ ID NO: In addition, Gram stain showed Gram-negative organisms, and microscopic examination showed bacterium with low motility (Table 1). The present inventors named the strain identified by the above examples as Enterobacter strain IJS3 and deposited it on the National Institute of Agricultural Science (KACC) on Jan. 28, 2014 (Accession No .: KACC 91932P).

Acinetobacter isolated by Kim and Song (2011) guillouiae SW5 and Pseudomonas isolated by Seo and Song (2012) brassicacearum subsp. neoaurantiaca BD3-35 was used as a comparative rhizobacteria.

Example  2: Rhizobium ACC deaminase  Activity

The strains selected in Example 1 were grown in Luria-Bertani broth (LB: tryptone 10 g, yeast extract 5 g, distilled water 1 L), and the culture was centrifuged (3400 × g, 4 ° C., 15 minutes) The precipitate was washed 2-3 times with DF salt medium. The washed precipitate was again dissolved in 7.5 mL of DF salt medium, and 3 mL of ACC was added and cultured for one day (200 rpm, 30 ° C.). The cultured strains were centrifuged (3400 × g, 4 ° C., 20 min), and the precipitates were washed 2-3 times with 0.1 M Tris-HCl (pH 7.6) and resuspended in 0.1 M Tris-HCl (13000 g, RT, 5 min). Subsequently, the precipitate collected after centrifugation was dissolved in 600 μL of 0.1 M Tris-HCl (pH 8.5), and 30 μL of toluene was added thereto. ACC was added to the resulting suspension at a concentration of 0.5 M, and the mixture was incubated for 15 minutes in a constant-temperature water bath. Then, 0.56 M HCl was added to the suspension, and the mixture was centrifuged (13000 g, RT, 5 minutes). 300 μL of 2,4-dinitrophenylhydrazine Was added and incubated for 30 minutes at 30 ° C in dark. 2M NaOH was added and colorimetry was performed. Then, the absorbance was measured at 540 nm using a spectrophotometer (Infinite 200 PRO, Austria).

The protein content of the strains present in the analyzed culture was measured by the Bradford method. In the previous experiment, the precipitate obtained in the centrifugation of the culture broth was dissolved in 100 mL of distilled water. 100 μL of the turbid solution was reacted with 1 mL of Bradford reagent (Sigma, USA) for 30 minutes at room temperature and absorbance was measured at 595 nm using a spectrophotometer Respectively.

On the third day, E. ludwigii SJR3 was found to be 13.76 mu mol alpha-ketobutyrate h ^-1 mg ^-1 protein, P. brassicacearum subsp. Neoaurantiaca BD3-35 showed activity as 20.26 μmol α-ketobutyrate h ^-1 mg ^-1 protein and ACC deaminase activity was not detected in A. guillouiae SW5.

Example  3: IAA , My Heart ( zeatin ) And Gibberellin ( gibberellin ) Generation

In order to investigate the production of IAA, zeatin and gibberellin (GA ₃ ) in the isolated strains, the strain was treated with brain heart broth (BHB; peptone 27.5 g, D (+) glucose 2.0 g, NaCl 5.0 g, Na ₂ HPO ₄ 2.5 g, 1 L distilled water), the absorbance was adjusted so that the culture broth was 1 at OD ₆₀₀ , and the cells were inoculated in a BHB medium to a final volume of 100 mL at a rate of 10% for 24 hours 130 rpm, 30 < 0 > C). 100 mL of the cultured bacterial culture was centrifuged (3400 × g, 4 ° C., 30 min) and 1 mg of butylated hydroxy toluene (BHT) was added to the supernatant to prevent the oxidation of the hormone. The pH of the supernatant was then adjusted to 2.5 with 7 M NaOH and 7 M HCl, the supernatant was transferred to a separatory funnel, and 15 mL of ethyl acetate was added to the supernatant. The extraction shaker (Recipro shaker RS-1, Jeio Tech ) For 30 minutes at 300 rpm. After shaking, the mixture was allowed to stand for 15 minutes, and the ethyl acetate layer was recovered. This procedure was repeated three times to collect a total of 45 mL of ethyl acetate. The recovered mixture was evaporated under reduced pressure at 40 ° C using a vacuum evaporator (Eyela, Japan), and 5 mL of methanol was added to recover the hormone. The recovered hormones were filtered (PTFE, pore size 0.2 μm) and used as analytical samples. For analysis, HPLC (Waters, USA) was used and analyzed using a Gemini 5u C18 column (250 × 4.6 mm) (Phenomenex, USA). The sample injection volume was set at 20 μL and the mobile phase was 35% methanol (1% acetic acid) for IAA, 70% methanol for zeatin and 30% methanol (pH adjusted to 3.0 using 0.1MH ₃ PO ₄₎ for gibberellin And analyzed at a flow rate of 1 mL min ^-1 at 280, 254 and 265 nm wavelength for 30 minutes (Karadeniz et al ., 2006).

As shown in Table 2, IAA produced 45.79 and 118.1 mg L ^-1 of E. ludwigii SJR3 and A. guillouiae SW5, respectively, and P. brassicacearum subsp. Neoaurantiaca was not detected in BD3-35. In addition, zeatin contains E. ludwigii SJR3, A. guillouiae SW5 and P. brassicacearum subsp. neoaurantiaca BD3-35 were 57.9, 53.27 and 72.21 mg L ^{-1, respectively,} and P. brassicacearum subsp. The production of neoaurantiaca BD3-35 was the highest. In the case of gibberellin, 115.18, 4.5 and 109.82 mg L ^-1 were produced, respectively, and the production of E. ludwigii SJR3 was the highest (Figs. 1, 2 and 3).

Example  4: Rhizobium Insoluble phosphoric acid Solubility

The isolated strains were preincubated for 3 days in LB medium (150 rpm, 30 ° C), and the culture was centrifuged (3400 × g, 4 ° C, 15 min). 5 g of Ca ₃ (PO ₄ ) _2, 5 g of MgCl ₂ 6H ₂ O, 0.25 g of MgSO ₄ 7H ₂ O, 0.2 g of KCl and 0.2 g of NH ₄ ) ₂ SO ₄ 0.1 g and 1 L distilled water) (Nautiyal, 1999), and the absorbance was corrected so that the OD ₆₀₀ became 1, and NBRIP medium supplemented with 0.5% Ca ₃ (PO ₄ ) ₂ (200 rpm, 30 < 0 > C) with a final volume of 300 mL. The absorbance of the supernatant was measured at 470 nm using a spectrophotometer in a 24 hour cycle. The supernatant was centrifuged (13000 × g, 4 ° C, 10 min), and 1 mL of the supernatant was reacted with 1 mL of vanadomolybdophosphoric acid.

As a result, E. ludwigii SJR3, A. guillouiae SW5 and P. brassicacearum subsp. Neoaurantiaca BD3-35 solubilized 132.85, 122.67 and 118.62 mg L ^-1 insoluble phosphate in one day, respectively, and E. ludwigii SJR3 was the highest. The insoluble phosphate solubilization ability remained similar until about 6 (FIG. 4).

Experimental Example  1: Small scale soil cultivation experiment

Soil was collected from the hill behind the 2nd building of Kangwon National University, Chuncheon city, Kangwon province in order to carry out the small - scale soil cultivation experiment. 250 g of soil was placed in a plastic pot and germinated in similar lengths ( Lycopersicon esculentum Mill.) Seed [Sekwang Tomato, Danon Co., Ltd.] was seeded in 13 pots with 3 seeds per pot. Tomato seeds were surface - sterilized in 70% ethanol, and then sterilized in 5 mL of sterilized distilled water and tomato seeds. The seeds were incubated in a plant growth chamber for 14 hours under light conditions (24 ℃) and light conditions (30 ℃, 97.5 μmol photons m ^-2 s ^{- 1} ) for 10 hours (Fig. 5). For strain inoculation, each strain was incubated in LB medium for 3 days (150 rpm, 30 ° C) and centrifuged (13000 g, 4 ° C, 20 minutes) and suspended in distilled water. Suspended strains were counted by hemocytometer and then inoculated into pots at 10 ⁶ cell g ^-1 and cultivated in a plant growth chamber under the same conditions for 21 days. Drying conditions were adjusted to 15.22%, which is very strong drying stress level based on water holding capacity (WHC). After the experiment, stem length, root length, dry weight and wet weight of tomato were measured.

As a result, 16 of the 39 seedlings (about 41%), E. ludwigii In the SJR3 vaccination group, 5 out of 39 (about 13%), A. guillouiae In the SW5 vaccination group, 8 seedlings (about 20%) out of 39 seedlings and P. brassicacearum subsp. neoaurantiaca Of the 39 seedlings in BD3-35 group, 9 seedlings (about 23%) died. Root lengths were increased by 26.45, 25.57, and 26.11%, respectively ( p <0.001). SJR3 and BD3-35 were increased to similar values, stem / root ratio 61.45, 46.65, 48.3, and 47.49%, respectively. In all three experimental groups, SJR3 p <0.001, SW5 and BD3-35 p <0.05 were lower than those of the control group and E. ludwigii SJR3 and P. brassicacearum subsp. neoaurantiaca BD3-35 showed a similar low value (Figs. 6 and 7).

The dry weight of the tomato was averaged by measuring the total weight of each tomato group as the tomato tissue was well broken. As a result, the total dry weight was increased by about 34.2, 12.52 and 18.08% compared to the control group was the most significant increase in E. ludwigii SJR3, root dry weight was increased from about 15.37, 10.93 and 15.72%, respectively compared to the control group E. ludwigii SJR3 And P. brassicacearum subsp. neoaurantiaca BD3-35 increased to a similar value (Figs. 8 and 9).

Experimental Example  2: Soil bacterial community analysis

G-DNA was extracted from the culture medium of each strain cultured in LB medium according to the manual of G-spin ^TM genomic DNA extraction for bacteria (Intron, Korea). The soil collected from the small-scale cultivation experiment was analyzed with Powersoil DNA isolation kit BIO, USA) according to the manufacturer's instructions. Nested PCR was performed using each extracted g-DNA. The first PCR to amplify the 16s rRNA region included the forward primer 27F; 5'-AGAGTTT GATCMTGGCTCAG-3 'and reverse primer 515R; 5'-ACCGCGGCTGCTGGCAC-3 'was used. For the second PCR, forward primer F352TA; 5'-CGCCCGCCGCGCGCGGGCGGGGCGGGGGCACGGGGGGACTCCTACGGG AGGC-3 'and reverse primer 515R; 5'-ACCGCGGCTGCTGGCAC-3 'was used. For the PCR, g-DNA extracted previously was used as a template, and 2 μL of 10 × taq polymerase buffer, 2 μL of 2.5 mM dNTP, 1 μL of 10 pmol primer, 0.125 μL of Ex-taq polymerase (Takara, Japan) and 2 μL of template were added and distilled water was added And finally corrected to 25 mL. The first PCR was repeated 25 cycles of initial denaturation (94 ℃ for 5 min), denaturation (94 ℃ for 30 sec), annealing (58 ℃ for 30 sec) and extension (72 ℃ for 40 sec) Finally, final extension (72 ° C, 3 min) was performed. Second PCR was repeated for 40 cycles consisting of initial denaturation (94 ℃ for 5 min), denaturation (94 ℃ for 30 sec), annealing (58 ℃ for 30 sec) and extension (72 ℃ for 30 sec) Finally, final extension (72 ° C, 5 min) step was performed. In order to confirm the amplification of DNA through PCR, PCR product and 6 × DNA loading dye were mixed at 5: 1 and electrophoresed on 1% agarose gel at 130 V for 30 ~ 40 minutes to analyze the band. The product was used in DGGE (denaturing gradient gel electrophoresis).

To perform DGGE, 20 μL of PCR product of rhizosphere soil was mixed with 20 μL of 2x loading dye and used as a sample. DGGE gels were prepared with 45% and 65% gel solutions, and 10 μL of N, N, N ', N'-tetramethylethylenediamine (TEMED; Sigma, USA) and APS (2 g of ammonium persulfate, 10 mL of dH 2 O ) Were mixed with a gel solution to prepare a gel. Each well of the gel was washed with 0.5x TAE buffer, and the samples obtained from the strain and rhizosphere were injected into a DCode ^TM Universal Mutation Detection System (Bio-Rad, USA) used as a DGGE device. The temperature of the buffer in the device was maintained at 60 ° C. The voltage was maintained at 20 V for the first 20 minutes and then the voltage was increased to 60 V for 20 hours and electrophoresed. After the electrophoresis, the gel was stained with 0.5x TAE buffer containing ethidium bromide for 20 minutes. With distilled water after after washing three times using a UV transilluminator (SL-20 High Performance DNA Image Visualizer TM, South Korea), thereby observing the intensity and band pattern.

As a result of the experiment, the band of the inoculum was observed to remain intact until the 8th day after the inoculation (FIG. 10), and the band appeared to be blurred until the 16th day. The band intensity of the inoculum strain SJR was measured up to 16 days (Table 3).

Experimental Example  3: Microorganisms secrete from rhizosphere IAA  density

Germination, seeding and inoculation of tomato seeds were carried out under the same conditions as in the small - scale cultivation experiments. IAA was measured for 3 days at 24 - hour intervals after strain inoculation. First, tomato seedlings were removed from the soil and 300 mL of 70% methanol was added to extract IAA (100 rpm, 12 ° C, 2 days). The recovered extract was evaporated under reduced pressure at 40 ° C using a vacuum evaporator (Eyela, NE, Rikakai Co. Ltd., Japan), and 5 mL of methanol was added to recover the hormone. The recovered hormones were filtered with filter (PTFE, pore size 0.2) and used as analytical samples. For analysis, high performance liquid chromatography (HPLC; Waters Co., USA) was used and a Gemini 5u C18 column (250 × 4.6 mm) (Phenomenex, USA) was used. The sample injection volume was set at 20 μL and the mobile phase was analyzed with a flow rate of 1 mL min ^-1 at 280 nm wavelength for 30 min using 35% methanol (1% acetic acid) (Karadeniz et al ., 2006). Standard curves were prepared using IAA standards (Sigma, USA) dissolved in ethanol and the concentrations were quantified.

Experimental results showed that E. ludwigii SJR3 and A. guillouiae SW5 had the highest concentrations on day 2 and produced about 1.2 and 2.0 nM, respectively (FIG. 11).

Experimental Example  4: Investigation of tomato gene expression

The tomato seeds were surface-sterilized in 70% ethanol for 5 minutes, and then sterilized 5 mL of sterilized distilled water and tomato seeds were added to the sterilized Petri dishes. The plants were grown in a plant growth chamber for about 3 days for 14 hours at dark (24 ° C) Germination conditions. The germinated tomatoes were adjusted to the same conditions as the small - scale cultivation experiments and the dry stress was adjusted to close to 15% WHC. For strain inoculation, each strain was cultured in LB medium for 3 days (150 rpm, 30 ° C, 3 days) and suspended in distilled water. Suspected strains were counted by hemocytometer and then inoculated into pots at 10 ⁶ cell g ^-1 . Synthetic IAA was dissolved in distilled water at 4 nM and 10 nM. Culturing E.ludwigii SJR3 strain in LB medium to process Bacterial IAA by removing the cell by centrifugation (13000g, 4 ℃, 20 bun) 0.5 mL of the supernatant and _{Salkowski reagent (H 2 SO 4 150} mL, H 2 O 250 mL, 1.5 M FeCl ₃ · 6H ₂ O 7.5 mL) were mixed and reacted at room temperature for 30 minutes. The reactants were measured for absorbance at OD ₅₃₀ using an Infinite 200 PRO multimode reader (Tekan, Germany). The standard curves were prepared using IAA standards (Sigma, USA) dissolved in ethanol. The concentrations were quantified and adjusted to 4 nM and 10 nM of bacterial IAA, respectively, and treated for 7 days.

Ten grams of tomato seedlings cultivated were ground with liquid nitrogen. 0.1 g of crushed tomatoes were placed in 1 mL TRIzol reagent (Invitrogen, USA), vortexed (5000 rpm, RT, 15 sec) and allowed to react at room temperature for 5 min. To the reacted samples, 200 mL of chloroform was added, and the mixture was mixed up and down for 15 seconds and centrifuged (10000 g, 4 ° C, 15 minutes) to recover the supernatant. 500 μL of isopropyl alcohol stored at 4 ° C was added to the recovered supernatant, and the mixture was reacted in ice for 15 minutes. After centrifugation (10000 g, 4 ° C, 10 minutes), the supernatant was removed and the precipitated RNA pellet was recovered. Finally, to wash the RNA pellet, 1 mL of a mixture of diethyl pyrocarbonate (DEPC; Sigma, USA) 30% water and 70% ethanol was added and the supernatant was removed by centrifugation (10000 g, 4 ° C, Lt; / RTI &gt; Then, 44 μL of DEPC water was added to dissolve the RNA. The cDNA was synthesized using the TOPscript ™ cDNA synthesis kit (Enzynomics, Korea). The extracted tomato RNA was quantitated using Infinite 200 PRO multimode reader (Tekan, Germany) and 2 μg of the quantified RNA was used as a template Synthesized and confirmed by electrophoresis (Fig. 12).

The concentration of cDNA synthesized above was quantitated using Infinite 200 PRO multimode reader (Tekan, Germany) and the cDNA concentration was corrected to 20 ng μL ^-1 using RNase free water. To quantify this, the size and concentration of the plasmid DNA were assigned to the URI Genomics & Sequencing Center web site (http://www.uri.edu/research/gsc/resources/cndna.html) to obtain the copy number and 2 × ^{10 10} The DNA concentration was corrected. The concentration of each sample was used as a template, and 12.5 μL of Maxima ^™ SYBR Green qPCR Master Mix 2X (Fermentas, Glen Burnie, USA), 1 μL of each 10 p mol primer, and 5 μL of template were mixed and distilled water was added to finally 25 mL Respectively. The PCR conditions were 40 cycles of holding stage (50 ℃, 2 min / 95 ℃, 10 min) and cycling stage (95 ℃, 15 sec / 60 ℃, 1 min) and finally melt curve stage Lt; 0 &gt; C, 1 minute / 95 DEG C, 15 seconds). Each sample was set to triplicate, and the resulting cycle threshold (CT) values were quantified using a standard curve and then converted to mRNA-based quantities compared to the control (Chang et al . , 2008). Real-time PCR was performed using StepONE ^™ Real-Time PCR Systems (AB Applied Biosystems, USA). The standard curve was prepared by diluting the samples at 2 × 10 ¹⁰ concentrations by 10 times and diluting them to 2 × 10 ⁶ .

The primers used in the experiments were cited in the previous studies. For the analysis of the expression level of the DREB2 gene, forward primer; 5'-TCACATTTACTGCCCCGACC-3 'and reverse primer; 5'-CCGCAGAGGTTTCGG GTAAT-3 'was used. To analyze the expression level of DREB3 gene, forward primer; 5'-CGATTAGGTTGCCGTTGTGG-3 'and reverse primer; 5'-CCCGCCTTTGGCTTGGT-3 '(Lu et al ., 2011). For analysis of the expression level of ACS4 gene, forward primer; 5'-GGAGTCATGAAGAACAAGCA-3 'and reverse primer; 5'-AA CTATGTTGGGCCCGTGCT-3 'was used (Balbi and Lomax, 2003) and forward primer to analyze the expression level of ACS6 gene; 5'-GGAGCAACAGGAGCTCATGAAAGTT TG-3 'and reverse primer; 5'-CTTTGATATTTGATTCTTGAGCTTTAC-3 'was used (Chang et al . , 2008).

As a result of the analysis of the relative expression level of DREB2 gene, E. ludwigii SJR3, A. guillouiae SW5 and P. brassicacearum subsp. neoaurantiaca The expression of BD3-35 was decreased by about 98, 94 and 97%, respectively, compared to the control group. In the bacterial IAA treated group of 4 nM, the amount of expression was decreased by about 77% compared with that of the control group. In the bacterial IAA treated group of 10 nM and the synthetic IAA treated group of 4 nM and 10 nM, the expressions were 550, 123 and 582% (Fig. 13). And DREB3 The relative expression levels of E. ludwigii SJR3, A. guillouiae SW5 and P. brassicacearum subsp. neoaurantiaca The expression of BD3-35 was decreased by about 98, 99 and 98%, respectively, compared to the control group. In the bacterial IAA treated group, the expression level was decreased by about 58% compared to the control group. In the bacterial IAA treated group of 10 nM and the synthetic IAA treated group of 4 nM and 10 nM, the expressions were 126, 14 and 117% (Fig. 14).

Analysis of the relative expression of ACS4 gene revealed that E. ludwigii SJR3, A. guillouiae SW5 and P. brassicacearum subsp. neoaurantiaca The expression of BD3-35 was decreased by about 99, 53 and 69%, respectively, compared to the control group. The expression levels of bacterial IAA treated with 4 nM and 10 nM and synthetic IAA treated with 4 nM and 10 nM, respectively, increased by about 2, 1015, 591, and 993%, respectively (FIG. 15). ACS6 The relative expression levels of E. ludwigii SJR3, A. guillouiae SW5 and P. brassicacearum subsp. neoaurantiaca The expression of BD3-35 was decreased by about 99, 97 and 96%, respectively, compared to the control group. In the bacterial IAA treated group of 4 nM, the amount of expression was decreased by about 52% as compared with that of the control group. In the bacterial IAA treated group of 10 nM and the synthetic IAA treated group of 4 nM and 10 nM, the expressions were 456, 57 and 474% (Fig. 16).

Agricultural Biotechnology Research Institute KACC91932 20140128

<110> KNU-Industry Cooperation Foundation <120> Enterobacter ludwigii SJR3 promoting the growth of plants and the          microbial agent containing the same <130> P5468 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 1572 <212> RNA <213> Enterobacter ludwigii <400> 1 acacccggca gagaagtttt tttttaggtt ttttccccac tcagattgaa cgctggcggc 60 aggcctaaca catgcaagtc gagcggtaac acagggagct tgctcctggg tgacgagcgg 120 cggacgggtg agtaatgtct gggaaactgc ctgatggagg gggataacta ctggaaacgg 180 tagctaatac cgcataacgt cgcaagacca aagaggggga ccttcgggcc tcttgccatc 240 agatgtgccc agatgggatt agctagtagg tggggtaacg gctcacctag gcgacgatcc 300 ctagctggtc tgagaggatg accagccaca ctggaactga gacacggtcc agactcctac 360 gggaggcagc agtggggaat attgcacaat gggcgcaagc ctgatgcagc catgccgcgt 420 gtatgaagaa ggccttcggg ttgtaaagta ctttcagcgg ggaggaaggt gttgaggtta 480 ataacctcag caattgacgt tacccgcaga agaagcaccg gctaactccg tgccagcagc 540 cgcggtaata cggagggtgc aagcgttaat cggaattact gggcgtaaag cgcacgcagg 600 cggtctgtca agtcggatgt gaaatccccg ggctcaacct gggaactgca ttcgaaactg 660 gcaggctaga gtcttgtaga ggggggtaga attccaggtg tagcggtgaa atgcgtagag 720 atctggagga ataccggtgg cgaaggcggc cccctggaca aagactgacg ctcaggtgcg 780 aaagcgtggg gagcaaacag gattagatac cctggtagtc cacgccgtaa acgatgtcga 840 cttggaggtt gtgcccttga ggcgtggctt ccggagctaa cgcgttaagt cgaccgcctg 900 gggagtacgg ccgcaaggtt aaaactcaaa tgaattgacg ggggcccgca caagcggtgg 960 agcatgtggt ttaattcgat gcaacgcgaa gaaccttacc tactcttgac atccagagaa 1020 cttagcagag atgctttggt gccttcggga actctgagac aggtgctgca tggctgtcgt 1080 cgctcgtgt tgtgaaatgt tgggttaagt cccgcaacga gcgcaaccct tatcctttgt 1140 tgccagcggt tcggccggga actcaaagga gactgccagt gataaactgg aggaaggtgg 1200 ggatgacgtc aagtcatcat ggcccttacg agtagggcta cacacgtgct acaatggcgc 1260 atacaaagag aagcgacctc gcgagagcaa gcggacctca taaagtgcgt cgtagtccgg 1320 attggagtct gcaactcgac tccatgaagt cggaatcgct agtaatcgta gatcagaatg 1380 ctacggtgaa tacgttcccg ggccttgtac acaccgcccg tcacaccatg gggagtgggt 1440 tgcaaaagaa gtaggtagct taaccttccg ggagggcgct taccactttg tgattcatga 1500 ctggggtgaa ttacaaaaaa aaaaacccac acacagaaaa aaaagggggg gggaaccccg 1560 gggtgttttt tt 1572

Claims (4)

Enterobacter ludwigii SJR3 strain (KACC 91932P) having the 16S rRNA nucleotide sequence shown in SEQ ID NO: 1 promoting plant growth in a dry environment. The method of claim 1, wherein the Enterobacter ludwigii ) SJR3 strain. A microorganism preparation for promoting plant growth in a dry environment containing as an active ingredient a culture of the Enterobacter ludwigii strain SJR3 (KACC 91932P) of claim 1 or the Enterobacter strain of SjR3 strain of claim 2 as an active ingredient. A method for promoting plant growth, characterized in that the microorganism preparation of claim 3 is inoculated into a soil or plant seed to produce a plant growth hormone and solubilize insoluble phosphoric acid in the soil.

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