KR100595845B1 - Method of massive production of Gibberellic acids using Penicillium commune KNU5379 strain and the use thereof - Google Patents

Abstract

The present invention relates to a mass production method and use of gibberellin using penicillium commune KNU5379 strain, the present invention has an excellent effect of providing a penicillium commune KNU5379 strain having a gibberellin production ability isolated from brown sesame root. In addition, the present invention has an excellent effect of providing a method for mass production of biologically active GA1, GA3, GA4 or GA9 by culturing the strain in large quantities. In addition, the strain of the present invention can produce GA1, GA3, GA4, GA9 significantly higher than the conventional gibberellin production strain.

Gibberellin, Penicillium communal strain, composition for promoting plant growth

Description

Method of massive production of Gibberellic acids using Penicillium commune KNU5379 strain and the use approximately}             

1 shows the nucleotide sequence of the small subunit coding gene of rDNA of Penicillium commune KNU5379.

2 is a penicillium commune cultured in Hagem medium. The photograph shows the KNU5379 strain.

3 is Penicillium commune Electron micrograph of KNU5379 strain.

The present invention relates to a mass production method and use of gibberellin using the penicillium commune KNU5379 strain, and more particularly, the present invention is cultured penicillium commune KNU5379 strain having a gibberellin production ability has been isolated and isolated from the brown sesame root gibberellin It relates to a mass production and composition for promoting plant growth comprising the strain.

Gibberellin is a diterpenoid complex produced by green plants, fungi, and bacteria (B. Tudzynski, 1999). Plant growth, seed germination, stem elongation, leaf growth, flowering, and pulp It is known to regulate maturity (Neil Olszewski et al. 2002; Rademacher, 1997). In 1935, Yabuta isolated Gibberellia fujikuroi , the causative agent of the bakana disease of rice, and the second metabolite produced by the bacterium is Gibberellin (GAs) (Peter Heedden, 1988). 1999). To date, there are 130 known GAs, among which GA1, GA3, GA4 and GA7 are known to be highly biologically active (Hedden and Phillips, 2000; Masatomo kabayashi, 2000). These GAs are widely used commercially, most of them are GA3, soften the growth of grape seeds and the citrus skin, keep the skin in good condition, inhibit aging and prevent damage from pests and other environmental factors. It is also high. In apples, in particular, the reddish brown spots of the skin are adjusted to improve taste, and in general, flowers are used for decorative purposes by inducing flowering in the season when flowers do not bloom. Occasionally, some plants may have problems with flowering, where GAs is also applied (Lewis N. mander, 2003). The bacteria known to produce GAs include Gibberells fujikuroi (Takahashi et al, 1955; Hanson, 1967; B. Tudzynski, 1999), as well as Spaceloma manihoticola . (Rademacher and Graebe 1978), Neuro Castello La Chrysler Corporation (Neurospora crassa) (Kawanabe et al 1983.), Paae Oscar L Leah Espy (Phaeosphaeria sp.) L487 (Sassa et al, 1989;. Kawaide et al 1995), Spokane Sporisorisium reilianum (Matheussen et al, 1990), but the commercially available bacteria are not known other than Gibberells fujikuroi .

Thus, the present inventors isolated and identified GAs producing bacteria from the roots of brown sesame seeds to find new bacteria producing GAs which are very useful for agriculture and horticulture. GA1, GA3, GA4 and GA9 are known to be active among several types of GAs. And the production of GA53 was analyzed.

Accordingly, an object of the present invention is to isolate and identify microorganisms with gibberellin production from brown sesame roots.

Another object of the present invention is to provide a method for mass production of gibberellin by culturing the microorganism.

Another object of the present invention is to provide a composition for promoting plant growth, including microorganisms having the ability to separately identify and produce gibberellin.

The object of the present invention is to isolate and identify microorganisms with excellent gibberellin production ability from the roots of brown sesame seeds, of which strains with excellent production capacity of GA1, GA3, GA4 or GA9 are named penicillium commun KNU5379, and cultured in large quantities. It was achieved by mass-producing biologically active gibberellin, and producing a plant growth promoting composition comprising the strain as an active ingredient.

EMBODIMENT OF THE INVENTION Hereinafter, the structure of this invention is demonstrated concretely.

The present invention is to identify and identify the microorganisms having gibberellin production capacity from the brown sesame root; Identifying a biologically active gibberellin producing strain; Culturing the strain to mass produce gibberellin; Comprising the step of producing a plant growth promoting composition comprising the strain.

Penicillium microorganisms excellent in Gibberellin production ability isolated from the brown sesame root of the present invention is characterized in that the penicillium commune ( Penicillium commune ) strain.

Isolation and identification of penicillium strains with gibberellin-producing ability in the brown sesame root of the present invention was carried out by modifying the method of Bayman B. et al.

After treatment with surfactant Tween80 at the root of the plant, it is washed with distilled water, and the root is completely settled in bleach perchloric acid (Perchloric acid (1%)), sterilized and washed with sterile water,

The roots were cut to a certain length with sterile scissors and water was removed, followed by subcultured at 24 ° C. in Hagem medium to purely isolate the strain.

In the present invention, the most excellent strain of the biologically active gibberellin producing ability was named Penicillium commune KNU5379.

The composition of Hagem medium for propagation of the strain of the present invention is as follows:

Composed of 0.5% glucose, 0.05% KH ₂ PO ₄ , 0.05% MgSO ₄ .7H ₂ O, 0.05% NH ₄ Cl, 0.5cc of FeCl ₃ , streptomycin 80 ppm and 1.5% agar, adjusted to pH 5.6.

When the isolates of the present invention with excellent gibberellin strains were isolated and identified, the activity of gibberellin was measured by using a 100-mL flask with 40 mL of Czapek`s (1% glucose, 1% bactopeptone, 0.1% KH ₂ PO ₄ , 0.05). % KCl, 0.05% MgSO ₄ · 7H ₂ O, 0.001% FeSO ₄ · 7H ₂ O, pH 7.3 ± 0.2) was measured by incubating at 120 rpm for 7 days in a liquid medium.

In order to identify Penicillium commune strains having excellent gibberellin production ability of the present invention, after culturing the strains selected above, sequencing and analysis of small subunit coding genes of rDNA were performed.

Ability invention gibberellin producing superior strain rDNA, small sub as a strain belonging to the genus (Penicillium) penny room Solarium in Molecular Phylogenetic analysis using the nucleotide sequence of the unit-coding gene, showing the homology of 100% penny room Solarium commune (Penicillium commune ) and was named Penicillium commune KNU5379.

In the present invention, a Penicillium commune strain having excellent gibberellin production capacity is cultured in Hagem medium and shaken at 25 ° C. in Czapek's liquid medium to produce biologically active GA1, GA3, GA4 or GA9. There is a feature that provides a method.

In addition, the present invention can promote the growth of the plant by producing a composition for promoting plant growth comprising the purified or concentrated or lyophilized Penicillium commune strain culture directly or using it as an active ingredient. There is a characteristic of providing a composition.

Hereinafter, the specific method of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited only to these Examples.

EXAMPLE

Example 1 Separation Step of Microorganism with Gibberellin Production Capacity

In order to isolate and identify microorganisms having gibberellin production ability of the present invention, the Bayman B. method was modified. First, after cutting the root portion of brown sesame seeds, all the soil in the roots are removed from the flowing water, and then treated with Tween80, a surfactant for 5 minutes, stirred and washed with distilled water. Completely settle the roots in bleach perchloric acid (1%), sterilize twice for 5 minutes, and then wash three times with sterile water. After cutting about 1.5cm using sterile scissors, water was completely removed from the sterile paper, purely separated from Hagem medium, and then subcultured and incubated at 25 ° C. The growth of bacteria was Hagem (0.5% glucose, 0.05% KH ₂ PO ₄ , 0.05% MgSO ₄ · 7H ₂ O, 0.05% NH ₄ Cl, 0.5cc of FeCl ₃ , Streptomycin 80ppm, 1.5% agar, pH 5.6) 40 mL of Czapek`s (1% glucose, 1% bactopeptone, 0.1% KH ₂ PO ₄ , 0.05% KCl, 0.05% MgSO ₄₎ was used to culture GAs and 100 mL flasks were used to measure GAs activity. 7H ₂ O, 0.001% FeSO ₄ 7H ₂ O, pH 7.3 ± 0.2) incubated for 7 days at 25 ℃ 120 ℃ at liquid medium.

Example 2 Identification of Microorganisms Having Gibberellin Production Capacity of the Present Invention

KMG5379 strain isolated in Example 1 was prepared using Hagem medium (0.5% glucose, 0.05% KH ₂ PO ₄ , 0.05% MgSO ₄ .7H ₂ O, 0.05% NH ₄ Cl, 0.5cc of FeCl ₃ , streptomycin 80ppm, 1.5% agar). , pH 5.6) at 25 ° C. and incubated in 100 mL flask with 40 mL of Czapek`s (1% glucose, 1% bactopeptone, 0.1% KH ₂ PO ₄ , 0.05% KCl, 0.05% MgSO ₄ · 7H ₂ O, 0.001% FeSO ₄ · 7H ₂ O, pH 7.3 ± 0.2) The culture medium was incubated at 25 ° C. at 120 rpm for 7 days. The sequencing and analysis of the small subunit rDNA gene of cultured cells was performed by Yoon et al. , Int. J. Syst. Bacteriol. , 47 , 933, 1997.

The 404 nucleotide sequence of the small subunit rDNA gene of KMG5379 was determined, and in this molecular sequence analysis, the strain KMG5379 showed 100% homology with Penicillium commune . On the other hand, it was confirmed that the nucleotide sequence of the small subunit rDNA gene of KNU5379 is described by SEQ ID NO: 1.

The selected strains based on the above molecular phylogenetic characteristics were was identified as a penny room Solarium commune (Penicillium commune) belonging to the penny room Solarium in (Penicillium), named for the selected strain as a penny room Solarium commune (Penicillium commune) KNU5379 .

Example 3: Penicillium commune of the present invention Penicillium commune ) Measurement and Analysis of Gibberellin Production of KNU5379

Gibberellin production of the Penicillium commune KNU5379 strain isolated and identified in Examples 1 and 2 was measured. The concentration of gibberellin was measured by using a spectrophotometric method to measure absorbance at 254 nm by converting gibberellic acid to gibberellenic acid under acidic conditions developed by Holbrook et al. (Holbrook et al. , 1961). For the analysis of gibberellin, as an extraction method of endogenous gibberellin, Lee et al. (1998) was used. After incubating the bacteria for 7 days, the culture was filtered through a membrane filter, and the pH was adjusted to 2.5 using 6N HCl. As internal standard was added to the culture solution before extracting _{^{[17.17- 2 H 2] GA1,}} GA3, GA4, GA9 , and GA53 of 20ng. The same amount of ethyl acetate was added to the culture solution, divided into three portions, and then the ethyl acetate was evaporated, diluted with 60% methanol, and the pH was adjusted to 8.3 with 2N NH ₄ OH. The sample was concentrated under reduced pressure after passing through a C ₁₈ column (90-130 ㎛, 60Å, pore size, Altech). The concentrated residue was concentrated under reduced pressure in a filtrate passed through a Celite / SiO ₂ column (ethyl acetate saturated with solvent formic acid: hexan = 95: 5), dissolved in phosphate buffered solution (pH8), and the pH was adjusted to 8-9 with 2N NaOH. After fractionation three times with phosphate buffer solution, PVPP was added to the filtrate (phosphate buffer solution) and shaken for 1 hour. The pH of the filtrate was adjusted to 2.5 using 6N HCl, fractionated three times with ethyl acetate and concentrated under reduced pressure. The concentrated residue was dissolved in methanol, filtered through a 0.2 μm membrane filter, and used as an analytical sample for HPLC. HPLC column was used BondaPak C ₁₈ column (3.9 × 300 mm) and each GA was separated by concentration gradient of 28% methanol and 100% methanol solution containing 1% acetic acid. The flow rate was maintained at 1.5 mL per minute and divided into 50 fractions of 1.5 mL each. The exact retention time of each GA was determined by checking the presence or absence of ³ H-GA standard by liquid scintillation spectrometry (Beckman, LS 1801) by taking a small amount (15 μl) for each fraction. Each GA fraction dried for gas chromatography / mass spectrometer (GC-MS) analysis was dissolved in 100% methanol, combined fractions containing the same GA, transferred to 1 mL of reaction vial and dried with nitrogen gas at 40. . Fractions containing high impurity in the GA fraction were removed from GA using NH ₂ PreSep extraction cartridge and transferred to the reaction vial. Each GA was induced twice with ethereal diazomethane to methyl ester and dried with nitrogen gas. GA, which requires silylation, was reacted with 35 μl of pyridine and 35 μl of N, D-Bis (trimethyl silyl) -trifluoroacetamide (including 1% TMCS) at 65 ° C. for 30 minutes and then dried with nitrogen gas. After the sample was dissolved in anhydrous dichloromethane, 1 μl was injected into a GC-MS equipped with a 30 m × 0.25 mm (id) and 0.25 μm film thickness HP-1 Capillary Column (J & W). GC-MS with 5973 Network Mass Selective Detector (Hewlett Packard) was used for quantification of internal GAs, and data were processed using HP5970C Chemstation (Hewlett Packard). For the purpose of qualitative and quantitative analysis, KRI was calculated using a hydrocarbon standard, and three major ion masses of each GA and [2H2] GA internal standards (obtained from Prof. Lewis N. Mander, Australian National University, Canberra, Australia) were compared. Quantification by

As a result, the Penicillium commune KNU5379 strain, which produces gibberellin, has the characteristics of producing various plant growth hormones GA1, GA3, GA4, and GA9, which promote plant growth. When comparing the production of Gibberella fujikuroi and Gibberellin, it was found that GA1 produced 41%, GA3 produced 140%, GA4 produced 105%, and GA9 produced 127%. Can be used in place of Roy ( Gibberella fujikuroi ).

Example 4: Mass production of gibberellin using the strain of the present invention gibberellin

In order to mass-produce biologically active gibberellins, the strains of the present invention isolated and identified in Examples 1 to 3 were used.

To this end, the penicillium commune KNU5379 strain having excellent gibberellin production ability of the present invention is plated in Hagem medium and shaken at 25 ° C. in Czapek's liquid medium to purify, concentrate or freeze-dried. It was.

Example 5: Preparation of a plant growth promoting composition using the present invention gibberellin strains

In order to manufacture a composition for promoting plant growth using the strain having the present invention gibberellin, the strain of the present invention was cultured in a large amount according to Example 4, and then the culture solution obtained therefrom was purified, concentrated or lyophilized and directly To use or to prepare a composition for promoting plant growth comprising as an active ingredient.

As described through the above examples, the present invention relates to a mass production method and a use of gibberellin using the penicillium commune KNU5379 strain, and the present invention provides a penicillium commune KNU5379 having a gibberellin-producing ability isolated from brown sesame roots. There is an excellent effect of providing strains. In addition, the present invention has an excellent effect of providing a method for mass production of biologically active GA1, GA3, GA4 or GA9 by culturing the strain in large quantities. In addition, the strain of the present invention is able to produce significantly higher GA1, GA3, GA4, GA9 than the conventional gibberellin producing strains and is a very useful invention in the plant industry by commercializing it.

<110> Kyungbuk University <120> Method of massive production of Gibberellic acids using          Penicillium commune KNU5379 strain and the use specifically <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 404 <212> DNA <213> Penicillium commune KNU5379 <400> 1 gtaggtgaac ctgcggaagg atcattaccg agtgagggcc ctctgggtcc aacctcccac 60 ccgtgtttat tttaccttgt tgcttcggcg ggcccgcctt aactggccgc cggggggctc 120 acgcccccgg gcccgcgccc gccgaagaca ccctcgaact ctgtctgaag attgaagtct 180 gagtgaaaat ataaattatt taaaactttc aacaacggat ctcttggttc cggcatcgat 240 gaagaacgca gcgaaatgcg atacgtaatg tgaattgcaa attcagtgaa tcatcgagtc 300 tttgaacgca cattgcgccc cctggtattc cggggggcat gcctgtccga gcgtcattgc 360 tgccctcaag cccggcttgt gtgttgggcc ccgtcctccg attt 404

Claims (4)

A method of mass-producing gibberellin from the culture by culturing a Penicillium commune strain having a gibberellin-producing ability derived from brown sesame roots. The method of claim 1, wherein the gibberellin is any one of gibberellin 1 (GA1), gibberellin 3 (GA3), gibberellin 4 (GA4) or gibberellin 9 (GA9). The method of claim 1, wherein the strain culture is obtained by incubating a Penicillium commune strain having Gibberellin-producing ability derived from brown sesame root in Hagem medium and shaking in a Czapek's liquid medium at a temperature of 25 ℃ How to mass-produce gibberellins characterized by. Plant growth promoting composition comprising a culture of Penicillium commune ( Penicillium commune ) strain having a gibberellin-producing ability derived from brown sesame root.

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