KR100561508B1 - Novel Bacillus cereus MJ-1 and Method for Preparing a Gibberellin Using the Same - Google Patents

Novel Bacillus cereus MJ-1 and Method for Preparing a Gibberellin Using the Same Download PDF

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KR100561508B1
KR100561508B1 KR1020030079828A KR20030079828A KR100561508B1 KR 100561508 B1 KR100561508 B1 KR 100561508B1 KR 1020030079828 A KR1020030079828 A KR 1020030079828A KR 20030079828 A KR20030079828 A KR 20030079828A KR 100561508 B1 KR100561508 B1 KR 100561508B1
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주길재
이인중
김원찬
김영목
이인구
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Abstract

본 발명은 작물생육촉진 호르몬인 지베렐린을 생산하는 신규 미생물인 바실러스 시리우스(Bacillus cereus) MJ-1 및 이를 배양하는 것을 특징으로 하는 지베렐린(GA)의 제조방법에 관한 것이다.The present invention relates to a Bacillus cereus MJ-1, which is a novel microorganism producing gibberellins, which are crop growth promoting hormones, and a method of preparing gibberellins (GA), which is cultured.

본 발명에 따른, 바실러스 시리우스 MJ-1균주는 GA1, GA3, GA4, GA7 , GA9, GA12, GA19, GA20, GA24, GA34, GA36, GA44, GA53 등 다양한 지베렐린을 생성할 수 있고, 상기 지베렐린 생성 균주로 작물의 묘를 처리할 경우, 작물의 생장이 현저하게 촉진되는 효과가 있다.According to the present invention, Bacillus sirius MJ-1 strain GA 1 , GA 3 , GA 4 , GA 7 , GA 9 , GA 12 , GA 19 , GA 20 , GA 24 , GA 34 , GA 36 , GA 44 , GA 53 It is possible to produce a variety of gibberellin, and when the seedling of the crop is treated with the gibberellin producing strain, there is an effect that the growth of the crop is significantly promoted.

지베렐린, 작물성장 호르몬, 바실러스 시리우스Gibberellin, Crop Growth Hormone, Bacillus Sirius

Description

신규 미생물 바실러스 시리우스 ＭＪ-1 및 이를 이용한 지베렐린의 제조방법 {Novel Bacillus cereus MJ-1 and Method for Preparing a Gibberellin Using the Same}Novel Bacillus cereus MJ-1 and Method for Preparing a Gibberellin Using the Same}

발명의 분야Field of invention

본 발명은 작물생육촉진 호르몬인 지베렐린을 생산하는 신규 미생물인 바실러스 시리우스(Bacillus cereus) MJ-1 및 이를 배양하는 것을 특징으로 하는 지베렐린의 제조방법에 관한 것이다.The present invention relates to a Bacillus cereus MJ-1, which is a novel microorganism that produces gibberellins, which are crop growth promoting hormones, and a method for producing gibberellins, comprising culturing them.

발명의 배경Background of the Invention

자연에서 작물 뿌리와 많은 종류의 미생물과는 서로 상호작용을 하며, 토양이나 기후조건에 따라 이러한 상호작용들이 작물의 생육이나 신장하는 범위를 결정하는데 주요한 요인이 된다. 토양미생물 중에 몇몇 세균들은 오옥신류나 다른 화학물을 생산하여 작물 주위를 보호하거나 뿌리의 증식을 자극한다. 이러한 미생물들을 작물생육 촉진 미생물(Plant Growth Promoting Rhizobacteria, PGPR)이라고 한 다.In nature, crop roots and many types of microorganisms interact with each other, and depending on soil or climatic conditions, these interactions are key factors in determining the extent of crop growth or extension. Some soil microorganisms produce oxins and other chemicals to protect crops and stimulate root growth. These microorganisms are called Plant Growth Promoting Rhizobacteria (PGPR).

PGPR은 작물호르몬을 방출하거나, 근권에서 질소를 고정하거나, 인산 같은 영양원을 가용화시키거나, 근류균의 기능을 촉진시키거나, 또는 뿌리의 에틸렌의 생산을 조절하여 직접적으로 작물의 생육을 증가시키는 잠재력을 가지고 있다. 더욱이 몇몇 PGPR은 작물의 주요 병원균의 생장을 억제시키는 능력을 가지고 있는 것으로 알려져 있다. PGPR has the potential to directly increase crop growth by releasing crop hormones, fixing nitrogen in the rhizosphere, solubilizing nutrients such as phosphate, promoting the function of mycorrhizal fungi, or regulating the production of ethylene in the roots. Have. Moreover, some PGPRs are known to have the ability to inhibit the growth of major pathogens in crops.

미생물이 생산하는 작물호르몬으로는 오옥신(auxin, Indoleacetic acid, IAA), 지베렐린(gibberellin, GA), 사이토키닌(cytokinin), 에틸렌(ethylene) 등이 보고되어 있고, 어떤 질소 고정균은 오옥신, 지베렐린, 사이토키닌을 모두 생성한다는 보고가 있으며, Barea 등(J. Appl Bacteriol. 40:129-34. 1976)은 여러 작물의 근권에서 분리한 50종의 미생물에서 각각 86%, 58%, 및 90%가 오옥신, 지베렐린, 사이토키닌을 생성한다고 보고하였다.Crop hormones produced by microorganisms include auxin (Indoleacetic acid (IAA)), gibberellin (GA), cytokinin (cytokinin), ethylene (ethylene), etc. Barea et al. (J. Appl Bacteriol. 40: 129-34. 1976) reported 86%, 58%, and 90%, respectively, in 50 microorganisms isolated from the root zones of various crops. Has been reported to produce oxins, gibberellins, and cytokinins.

특히 지베렐린(GA)을 생산하는 곰팡이류는 지베렐라 푸지쿠로이(Gibbrella fugikuroi), 패오스페리아 속(Phaeospheria sp. L489), 스파세로마 비덴티스(Sphaceloma bidentis), 스파세로마 마니히티코라(Sphaceloma manihiticola), 스파세로마 멘테아(Sphaceloma menthea), 스파세로마 퍼세아(Sphaceloma perseae), 스파세로마 로이스(Sphaceloma rhois) 등 7종이며, 세균류는 아세토박터 디아조크로피쿠스(Acetobacter diazotropicus), 아조토스피리룸 리포페륨(Azotospirillum lipoferum), 아조토스피리룸 브라시렌스(Azotospirillum brasilense), 바실러스 리케니피미스(Bacillus lichenifirmis), 바실러스 푸미러스(Bacillus pumilus), 헤르보스피리룸 세로페디카(Herbospirillum seropedicae), 리조비움 파세오리(Rhizobium phaseoli) 등 7종이 지금까지 보고되었다.In particular fungus that produces gibberellin (GA) is a jibe Relais Fu Chikugo Roy (Gibbrella fugikuroi), L agarose Feria in (Phaeospheria sp. L489), Spa-year-old Roman Providenciales teeth (Sphaceloma bidentis), Spa-year-old Roman Manihi Portico d (Sphaceloma manihiticola ), Sphaceloma menthea , Sphaceloma perseae , Sphaceloma rhois , and 7 species, and bacteria are Acetobacter diazotropicus , Azotospirillum lipoferum , Azotospirillum brasilense , Bacillus lichenifirmis , Bacillus pumilus , Herbospirilum serpedica seropedicae ) and Rhizobium phaseoli have been reported so far.

지베렐린은 작물생장호르몬 중에서 가장 잘 알려진 중요한 그룹으로서, 세포분화와 신장을 자극하여 줄기의 신장을 자극하거나, 장일에 반응하여 개화나 추대를 자극하거나, 발아유도를 위한 빛이나 층형성을 요구하는 종자휴면타파, 곡류 발아에서 가수분해효소(α-아밀라제) 생산을 자극하거나, 자웅이주 꽃(성표현)의 웅성 유도, 과일 성숙에 단위결과(씨 없는)를 유도하거나, 감귤류와 잎의 노화를 연장하는 등 다양한 이화학적 과정을 조절하는 역할을 한다. 지금까지 128종의 고등작물, 7종의 곰팡이 및 7종의 세균으로부터 126종의 지베렐린(GA)들이 발견되었다.Gibberellin is the best known and important group of plant growth hormones, which stimulates cell differentiation and kidneys to stimulate the kidneys of stems, stimulates flowering or sticking in response to intestines, or requires light or layer formation to induce germination. Dormancy breaks, stimulates the production of hydrolase (α-amylase) in grain germination, induces male male flower (sex expression), induces unit results (seedless) in fruit maturation, or prolongs aging of citrus fruits and leaves Regulating various physicochemical processes. To date, 126 gibberellins (GAs) have been found from 128 higher crops, 7 fungi and 7 bacteria.

현재까지 우리나라에서는 PGPR이 생산하는 지베렐린에 의한 작물의 생육촉진에 관한 연구보고가 전혀 없는 실정이다. 그러나 지베렐린 이외 다른 생육촉진물질에 의한 연구로는 문 등(Phytochemistry, 42: 365-368, 1996)이 슈도모나스 세파시아(Pseudomonas cepacia)에 의해 생산되는 4-퀴노리논(4-quinolinone) 대사물질에 의해 고추 생육이 현저하게 촉진된다는 내용을 비롯하여 몇 편이 보고된 바 있다.To date, there have been no reports on promoting growth of crops by gibberellin produced by PGPR. However, studies by other promoters other than gibberellin have shown that Phytochemistry (42: 365-368, 1996) is responsible for the 4-quinolinone metabolite produced by Pseudomonas cepacia . Several reports have been reported, including the fact that the growth of pepper is significantly promoted.

외국에서는 미생물로부터 GA 생산에 관한 연구가 많이 진행되어, 지금까지 곰팡이로부터 분리된 GAs로 GA1-4, GA7, GA9-16, GA20, GA 24-25, GA36-37, GA40-42, GA47, GA54-57, GA78, GA82 등 28종과 세균으로부터 분리된 GAs로 GA1, GA3, GA4, GA20 등 4종 등이 보고되어 있다.In foreign countries, there have been many studies on the production of GA from microorganisms. So far, GAs isolated from fungi are GA 1-4 , GA 7 , GA 9-16 , GA 20 , GA 24-25 , GA 36-37 , GA 40 GAs isolated from 28 species including -42 , GA 47 , GA 54-57 , GA 78 , and GA 82 and 4 species including GA 1 , GA 3 , GA 4 , and GA 20 have been reported.

아세토박터 디아조크로피쿠스(Acetobacter diazotropicus), 아조토스피리룸 리포페륨(Azotospirillum lipoferum), 아조토스피리룸 브라시렌스(Azotospirillum brasilense) 등은 GA1 및 GA3을 생산하며, 바실러스 리케니피미스(Bacillus lichenifirmis), 바실러스 푸미러스(Bacillus pumilus)는 GA1, GA3, GA 4 및 GA20을 생산하며, 헤르보스피리룸 세로페디카(Herbospirillum seropedicae)는 GA3을 생산하며, 리조비움 파세오리(Rhizobium phaseoli)는 GA1 및 GA4를 생산하는 것으로 보고되었으나, 바실러스 시리우스 (Bacillus cereus)에서 지베렐린을 생산하는 보고는 없는 상황이다. Acetobacter diazotropicus , Azotospirillum lipoferum , Azotospirillum brasilense , etc., produce GA 1 and GA 3 , and Bacillus liquenipimis ( Bacillus lichenifirmis ), Bacillus pumilus , produces GA 1 , GA 3 , GA 4, and GA 20 , Herbospirillum seropedicae produces GA 3 , and Rizovium Paseori ( Rhizobium phaseoli ) has been reported to produce GA 1 and GA 4 , but there are no reports of gibberellin production in Bacillus cereus .

이에, 본 발명자들은 신규 미생물 바실러스 시리우스 MJ-1이 지베렐린을 생산하고, 또한 상기 바실러스 시리우스 MJ-1이 고추 플러그묘의 생육을 촉진시키는 것을 확인하고 본 발명을 완성하였다.Accordingly, the present inventors have confirmed that the novel microbial Bacillus sirius MJ-1 produces gibberellin, and also the Bacillus sirius MJ-1 promotes the growth of pepper plug seedlings and completed the present invention.

본 발명의 목적은 작물호르몬 지베렐린을 생산하는 바실러스 시리우스 및 상기 균주를 배양하는 것을 특징으로 하는 지베렐린의 제조방법을 제공하는데 있다.
An object of the present invention is to provide a method for producing gibberellin, characterized in that the cultivation of Bacillus sirius and the strain to produce crop hormone gibberellin.

상기 목적을 달성하기 위하여, 본 발명은 작물호르몬 지베렐린을 생산하는 바실러스 시리우스(Bacillus cereus) MJ-1 (KCTC 10541BP)을 제공한다. In order to achieve the above object, the present invention provides Bacillus cereus MJ-1 (KCTC 10541BP) for producing a crop hormone gibberellin.

본 발명은 또한, 상기 미생물을 배양하는 것을 특징으로 하는 지베렐린의 제조방법을 제공한다. 본 발명에 있어서, 상기 지베렐린은 GA1, GA3, GA4, GA7, GA9, GA12, GA19, GA20, GA24, GA34, GA36, GA44 및 GA53으로 구성된 군에서 선택된 어느 하나 이상인 것을 특징으로 할 수 있다.The present invention also provides a method for producing gibberellin, characterized in that the culture of the microorganism. In the present invention, the gibberellin is from the group consisting of GA 1 , GA 3 , GA 4 , GA 7 , GA 9 , GA 12 , GA 19 , GA 20 , GA 24 , GA 34 , GA 36 , GA 44 and GA 53 It may be characterized in that any one or more selected.

본 발명은 또한, 상기의 미생물로 작물의 묘를 처리하는 것을 특징으로 하는 작물의 생육촉진방법을 제공한다.The present invention also provides a method for promoting growth of crops, wherein the seedlings of the crops are treated with the above microorganisms.

상기 작물생육 촉진 미생물(PGPR)인 바실러스 시리우스 (Bacillus cereus) MJ-1 균주는 2003년 11월 11일자로 부다페스트 조약에 따른 국제기탁기관인 한국과학기술원 생명공학연구소 내 유전자은행 (Korea Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology)에 기탁번호 KCTC 10541BP로 기탁되었다.The Bacillus cereus MJ-1 strain, which is a crop growth promoting microorganism (PGPR), is a gene bank (Korea Collection for Type Cultures, Korea Institute of Science and Technology), which is an international depository institution under the Budapest Treaty on November 11, 2003. Deposited KCTC 10541BP with the Korea Research Institute of Bioscience and Biotechnology.

이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

실시예 1: 바실러스 시리우스 (Example 1: Bacillus sirius ( Bacillus cereusBacillus cereus ) MJ-1 균주의 동정) Identification of MJ-1 Strain

바실러스 시리우스 (Bacillus cereus) MJ-1 균주는 본 발명의 연구자들이 작물생육 촉진 미생물 선별과정 실험에서 토양으로부터 분리하였다. Bacillus cereus MJ-1 strain was isolated from the soil by the researchers of the present invention in the crop growth promoting microbial selection experiment.

MJ-1 균주는 16S rDNA 염기서열 분석방법(16S rDNA sequence method)으로 MJ-1 균주의 염색체 DNA를 추출한 후, 27f 프라이머(5'- AGA GTT TGA TCC TGG CTC AG -3'; 서열 1)와 1492r 프라이머(5'- TAC CTT GTT ACG ACT T -3'; 서열 2)를 이용하여 PCR 산물을 얻고, 상기 PCR 산물의 염기서열의 상동성을 ribosomal database에서 검색하여 동정하였다 (http://rdp.cme.msu.edu/html/analyses.html).The MJ-1 strain was extracted with chromosomal DNA of the MJ-1 strain by the 16S rDNA sequence method, followed by 27f primer (5'- AGA GTT TGA TCC TGG CTC AG -3 '; SEQ ID NO: 1). PCR products were obtained using 1492r primers (5'- TAC CTT GTT ACG ACT T-3 '; SEQ ID NO: 2), and homology of the nucleotide sequences of the PCR products was identified by searching the ribosomal database (http: // rdp). .cme.msu.edu / html / analyses.html).

MJ-1 균주의 16S rDNA(1188bp) 염기서열(서열 3)에 대한 ribosomal database에서의 상동성 검색 결과, 표 1에서와 같이 바실러스 시리우스(Bacillus cereus)와 100%의 유사도를 나타내었기에 바실러스 시리우스(Bacillus cereus)로 동정하고, 바실러스 시리우스(Bacillus cereus) MJ-1이라 명명하였다.Results of homology of the ribosomal database for 16S rDNA (1188bp) nucleotide sequence (SEQ ID NO: 3) of the MJ-1 strain showed 100% similarity to Bacillus cereus as shown in Table 1, and thus Bacillus sirius cereus) and named Bacillus cereus MJ-1.

MJ-1 균주의 16S rDNA 염기서열 결정에 의한 동정 자료Identification Data by 16S rDNA Sequence Determination of MJ-1 Strain MJ-1 균주의 16S rDNA의 염기서열 (1188 bp); 서열 3Nucleotide sequence of 16S rDNA of MJ-1 strain (1188 bp); SEQ ID NO: 3 GCGGCGTGCC TTAATCATGC AAGTCGAGCG AATGGATTAA GAGCTTGCTC TTATGAAGTT AGCGGCGGAC GGGTGAGTAA CACGTGGGTA ACCTGCCCAT AAGACTGGGA TAACTCCGGG AAACCGGGGC TAATACCGGA TAACATTTTG AACCGCATGG TTCGAAATTG AAAGGCGGCT TCGGCTGTCA CTTATGGATG GACCCGCGTC GCATTAGCTA GTTGGTGAGG TAACGGCTCA CCAAGGCAAC GATGCGTAGC CGACCTGAGA GGGTGATCGG CCACACTGGG ACTGAGACAC GGCCCAGACT CCTACGGGAG GCAGCAGTAG GGAATCTTCC GCAATGGACG AAAGTCTGAC GGAGCAACGC CGCGTGAGTG ATGAAGGCTT TCGGGTCGTA AAACTCTGTT GTTAGGGAAG AACAAGTGCT AGTTGAATAA GCTGGCACCT TGACGGTACC TAACCAGAAA GCCACGGCTA ACTACGTGCC AGCAGCCGCG GTAATACGTA GGTGGCAAGC GTTATCCGGA ATTATTGGGC GTAAAGCGCG CGCAGGTGGT TTCTTAAGTC TGATGTGAAA GCCCACGGCT CAACCGTGGA GGGTCATTGG AAACTGGGAG ACTTGAGTGC AGAAGAGGAA AGTGGAATTC CATGTGTAGC GGTGAAATGC GAAGCAACGC GAAGAACCTT ACCAGGTCTT GACATCTTCT GACAACCCTA GAGATAGGGC TTCTCCTTCG GGAGCAGAGT GACAGGTGGT GCATGGTTGT CGTCAGCTCG TGTCGTGAGA TGTTGGGTTA AGTCCCGCAA CGAGCGCAAC CCTTGATCTT AGTTGCCATC ATTAAGTTGG GCACTCTAAG GTGACTGCCG GTGACAAACC GGAGGAAGGT GGGGATGACG TCAAATCATC ATGCCCCTTA TGACCTGGGC TACACACGTG NTACAATGGA CGGTACAAAG AGCTGCAAGA CCGCGAGGTG GAGCTAATCT CATAAAACCG TTCTCAGTTC GGATTGTAGG CTGCAANTCG CNTACATGAA GCTGGAATCG NTAGTAATCG CGGATCAGCA TGCCGCGGTG AATACGTTCC CGGGCCTTGT ACACACCGCC NGTCACACCA CGAGAGTTTG TAACACCCGA AGTCGGTGGG GTAACCTTTT GGAGCCAGCC GCCTAAGGTG GACAGATG　 　 　 　GCGGCGTGCC TTAATCATGC AAGTCGAGCG AATGGATTAA GAGCTTGCTC TTATGAAGTT AGCGGCGGAC GGGTGAGTAA CACGTGGGTA ACCTGCCCAT AAGACTGGGA TAACTCCGGG AAACCGGGGC TAATACCGGA TAACATTTTG AACCGCATGG TTCGAAATTG AAAGGCGGCT TCGGCTGTCA CTTATGGATG GACCCGCGTC GCATTAGCTA GTTGGTGAGG TAACGGCTCA CCAAGGCAAC GATGCGTAGC CGACCTGAGA GGGTGATCGG CCACACTGGG ACTGAGACAC GGCCCAGACT CCTACGGGAG GCAGCAGTAG GGAATCTTCC GCAATGGACG AAAGTCTGAC GGAGCAACGC CGCGTGAGTG ATGAAGGCTT TCGGGTCGTA AAACTCTGTT GTTAGGGAAG AACAAGTGCT AGTTGAATAA GCTGGCACCT TGACGGTACC TAACCAGAAA GCCACGGCTA ACTACGTGCC AGCAGCCGCG GTAATACGTA GGTGGCAAGC GTTATCCGGA ATTATTGGGC GTAAAGCGCG CGCAGGTGGT TTCTTAAGTC TGATGTGAAA GCCCACGGCT CAACCGTGGA GGGTCATTGG AAACTGGGAG ACTTGAGTGC AGAAGAGGAA AGTGGAATTC CATGTGTAGC GGTGAAATGC GAAGCAACGC GAAGAACCTT ACCAGGTCTT GACATCTTCT GACAACCCTA GAGATAGGGC TTCTCCTTCG GGAGCAGAGT GACAGGTGGT GCATGGTTGT CGTCAGCTCG TGTCGTGAGA TGTTGGGTTA AGTCCCGCAA CGAGCGCAAC CCTTGATCTT AGTTGCCATC ATTAAGTTGG GCACTCTAAG GTGACTGCCG GTGACAAACC GGAGGAAGGT GGGGATGACG TCAAATCATC ATGCCCCTTA TGACCTGGGC TACACACGTG NTACAATGGA CGGTACAAAG AGCTGCAAGA CCGCGAGGTG GAGCTAATCT CATAAAACCG TTCTCAGTTC GGATTGTAGG CTGCAANTCG CNTACATGAA GCTGGAATCG NTAGTAATCG CGGATCAGCA TGCCGCGGTG AATACGTTCC CGGGCCTTGT ACACACCGCC NGTCACACCA CGAGAGTTTG TAACACCCGA AGTCGGTGGG GTAACCTTTT GGAGCCAGCC GCCTAAGGTG GACAGATG 동정된 균주명Identified strain name 유사도Similarity Bacillus cereus str. RC607Bacillus cereus str. RC607 100%100% Bacillus cereus str. TA32-5Bacillus cereus str. TA32-5 100%100% Bacillus cereus VKM684Bacillus cereus VKM684 100%100%

실시예 2: 바실러스 시리우스 MJ-1 균주의 배양조건Example 2: Culture conditions of Bacillus sirius MJ-1 strain

바실러스 시리우스 MJ-1 균주는 GY배지(glucose 1g, yeast extract 2.5g, peptone 5g, agar 15g, NaCl 0.5g, 증류수 1ℓ, pH 7.0)에서 35~37℃로 3일간 진탕배양하였다. 배양액은 지베렐린 분석에 사용하였으며, 고추 생육촉진효과 검정에는 배양한 균체를 원심분리하여 집균하고, 이를 0.1M 인산완충액(pH 7.0)으로 현탁하여 균체수가 109 CFU/ml되게 하여 상토에 처리하였다.Bacillus sirius MJ-1 strain was incubated for 3 days at 35 ~ 37 ℃ in GY medium (glucose 1g, yeast extract 2.5g, peptone 5g, agar 15g, NaCl 0.5g, distilled water 1L, pH 7.0). The culture solution was used for Gibberellin analysis, and in the pepper growth promoting effect assay, the cultured cells were collected by centrifugation, suspended in 0.1M phosphate buffer (pH 7.0), and the number of cells was treated at 10 9 CFU / ml.

실시예 3: 고추 플러그묘의 생육 촉진 효과Example 3: Growth promoting effect of red pepper seedlings

공시작물로는 고추 (Red Pepper Capsicum annuum L., Seminis Korea Co., 부 촌)를 사용하였으며, 종자를 17~20℃ 물에 12시간 침종한 후 젖은 수건을 이용 28~30℃ 항온실에서 2일간 발아하여 스티로폼 상자 (20 × 30 ×15 cm)에 상토(참흙 50%, 부엽토 30%, 강모래 20%)를 2/3 가량 넣고 수분을 충분히 공급한 후 파종하고 모래로 복토 후 비닐을 덮고 23~27℃ 항온실에서 재배하였다. 파종 30일 후 본엽이 3~4매 때 105구 tray에 이식 (흥농종묘(주) 바이오 상토 이용)하고 충분히 관수하고 밀폐된 육묘장에서 보온 재배하였다. Red pepper capsicum annuum L. (Seminis Korea Co., Buchon) was used as a starting material, and the seeds were soaked in water at 17 ~ 20 ℃ for 12 hours, and then wetted in a 28 ~ 30 ℃ constant temperature room using a wet towel. Germinate for a day, add 2/3 of top soil (50% of clay, 30% of crushed soil, 20% of river sand) to a styrofoam box (20 × 30 × 15 cm), supply sufficient moisture, sowing, cover with vinyl, cover with vinyl It was cultivated in a constant temperature room at 23 ~ 27 ℃. After 30 days of sowing, when the main leaves were 3 ~ 4 sheets, they were transplanted into 105-hole trays (using Heung Jongmyo Co., Ltd. Biotope), and then sufficiently irrigated and kept warm in a closed nursery.

바실러스 시리우스 MJ-1 균주 배양액 (미생물수 2.0 × 108 cfu/㎖ 이상)은 원심분리하고 균체를 동일량의 살균수에 현탁하여 105구 tray 당 각각의 미생물 배양액 105㎖를 이식한 고추묘에 처리하였다. 육묘관리는 가온 비닐하우스 플러그묘 육묘장(구미시 소재)에서 낮온도 22~25℃, 밤온도 16~22℃로 67일간 재배한 후 플러그묘 초장, 엽수, 경경, 뿌리길이, 생체중 등을 조사하였다.Bacillus sirius MJ-1 strain culture medium (microbial water 2.0 × 10 8 cfu / ㎖ or more) was centrifuged and the cells were suspended in the same amount of sterilized water and treated in pepper seedlings in which 105 ml of each microbial culture solution per 105-neck tray was transplanted. . Nursery management was conducted at Gaon Vinyl House Plug Seedling Nursery (Gumi-si) for 67 days at 22 ~ 25 ℃ and 16 ~ 22 ℃ at night, and then the plug seedlings, leaves, tillage, root length, and live weight were examined.

바실러스 시리우스 MJ-1 균주는 표 2에서와 같이 67일 재배후 초장과 근중은 미생물 처리구에서 높게 나타났으나 엽수나 경경은 차이가 거의 없었다. 바실러스 시리우스 MJ-1 균주를 처리하여 생산한 고추 플러그묘는 MJ-1을 처리하지 않은 대조구에 비해 초장은 13.5%, 뿌리무게는 33.3%가 증가하였다. As shown in Table 2, the Bacillus sirius MJ-1 strains showed high height and weight in the microorganism treatment group after 67 days of cultivation, but there were almost no differences in the number of leaves and the cultivation. Pepper plug seedlings produced by treatment with Bacillus sirius MJ-1 strain increased 13.5% in height and 33.3% in root weight compared to the control without MJ-1.

작물생육 촉진 미생물 바실러스 시리우스 MJ-1 균주에 의한 고추 플러그묘의 생육촉진 효과Growth Promotion Effect of Red Pepper Plug Seedling by Bacterial Bacillus Sirius MJ-1 Strain 미생물 처리Microbial treatment 초장 (㎝)Extra long (cm) 엽수 (개)Conifers 경경 (㎝)Diameter (cm) 근장 (㎝)Root length (cm) 생체중 (g/plant)Live weight (g / plant) 지상부Ground 뿌리Root 전체all 바실러스 시리우스 MJ-1 처리구Bacillus sirius MJ-1 treatment zone 24.34* 24.34 * 12.812.8 2.652.65 1.181.18 3.173.17 1.061.06 4.234.23 무처리구No treatment 21.4421.44 12.412.4 2.612.61 0.830.83 2.462.46 0.810.81 3.273.27

*: 105개 고추 개체 평균값, 67일 재배 * : Average of 105 peppers grown, 67 days

실시예 4: 바실러스 시리우스 MJ-1 균주가 생산하는 지베렐린의 분석Example 4 Analysis of Gibberellin Produced by Bacillus Sirius MJ-1 Strain

바실러스 시리우스 MJ-1 균주가 생산하는 지베렐린 추출은 Foster와 Morgan(Plant Physiol. 108: 337-343, 1995) 및 Lee(Plant Growth Regul. 17:185-195, 1988) 등의 방법에 따라 HPLC로 분획하여 GC/MS SIM으로 분석하였다. 내부 표준물질로는 [17.17-2H2]로 라벨된 지베렐린 [17.17-2H2] GA 1, GA3, GA4, GA5, GA7, GA8, GA9, GA12, GA15, GA19, GA20, GA24, GA34, GA36, GA44 및 GA53을 각각 20ng씩 첨가하였다. Gibberellin extract produced by the Bacillus sirius MJ-1 strain was fractionated by HPLC according to the methods of Foster and Morgan (Plant Physiol. 108: 337-343, 1995) and Lee (Plant Growth Regul. 17: 185-195, 1988). By GC / MS SIM. As the internal standard is labeled with [17.17- 2 H 2] GA [17.17- 2 H 2] GA 1 , GA 3, GA 4, GA 5, GA 7, GA 8, GA 9, GA 12, GA 15, 20 ng of GA 19 , GA 20 , GA 24 , GA 34 , GA 36 , GA 44 and GA 53 were added.

표 3은 바실러스 시리우스 MJ-1 균주가 생산하는 지베렐린을 분석하기 위하여, 먼저 내부표준물질을 이용하여 지베렐린 함유 구간의 HPLC 분획을 분류하고, 지베렐린 각각의 GC/MS KRI값(Kovats retention index)과 mass spectrum(m/z)을 조사하였다. 표 4는 바실러스 시리우스 MJ-1 균주 배양액 300㎖ 내에 존재하는 GA의 종류를 조사한 결과, GA1, GA3, GA4, GA7, GA9, GA12, GA19, GA20, GA24, GA34, GA36, GA44, GA53 등이 존재하였으며, 각각의 지베렐린 함량은 GA44가 46.5ng, GA4가 23.5ng, GA7이 16.8ng 순으로 많이 존재하는 결과를 나타내었다. Table 3 shows how to analyze the Gibberellin produced by the Bacillus sirius MJ-1 strain, first classify the HPLC fraction of the Gibberellin containing section using internal standards, and the GC / MS KRI (Kovats retention index) and mass of each of the Gibberellin The spectrum ( m / z ) was investigated. Table 4 shows the types of GA present in 300 ml of Bacillus Sirius MJ-1 strain culture medium. As a result, GA 1 , GA 3 , GA 4 , GA 7 , GA 9 , GA 12 , GA 19 , GA 20 , GA 24 , GA 34 , GA 36 , GA 44 , GA 53 and the like, each of the gibberellin content of the GA 44 was 46.5ng, GA 4 23.5ng, GA 7 was found in the order of 16.8ng.

지금까지 곰팡이로부터 분리된 GAs는 GA1-4, GA7, GA9-16, GA20, GA24-25, GA36-37, GA40-42, GA47, GA54-57, GA78, GA82 등 28종이며, 세균으로부터 분리한된 GAs는 GA1, GA3, GA4, GA20 등 4종으로 보고되었으나, 본 발명에서는 GA1, GA3, GA4, GA7, GA9, GA12, GA19, GA20, GA24, GA34, GA36, GA44, GA53 등이 존재하여 지금까지 보고된 기존 곰팡이나 세균이 생산하는 GA와는 달리 GA34, GA44, GA53 등이 처음으로 존재하는 것을 확인하였다. So far, GAs isolated from fungi are GA 1-4 , GA 7 , GA 9-16 , GA 20 , GA 24-25 , GA 36-37 , GA 40-42 , GA 47 , GA 54-57 , GA 78 , There are 28 species including GA 82 , and GAs isolated from bacteria have been reported as 4 species including GA 1 , GA 3 , GA 4 , GA 20 , but in the present invention, GA 1 , GA 3 , GA 4 , GA 7 , GA 9 , GA 12 , GA 19 , GA 20 , GA 24 , GA 34 , GA 36 , GA 44 , GA 53 , and so on, unlike GA produced by conventional molds or bacteria reported so far, GA 34 , GA 44 , GA 53, etc. It confirmed that this existed for the first time.

또한 GA를 생산하는 세균으로 아세토박터 디아조크로피쿠스(Acetobacter diazotropicus), 아조토스피리룸 리포페륨(Azotospirillum lipoferum), 아조토스피리룸 브라시렌스(Azotospirillum brasilense), 바실러스 리케니피미스(Bacillus lichenifirmis), 바실러스 푸미러스(Bacillus pumilus), 헤르보스피리룸 세로페디카(Herbospirillum seropedicae), 리조비움 파세오리(Rhizobium phaseoli)등 7종으로 보고되었으나, 본 발명에서 신규 미생물 바실러스 시리우스(Bacillus cereus)가 지베렐린을 생산한다는 것을 확인하였다. In addition, GA-producing bacteria include Acetobacter diazotropicus , Azotospirillum lipoferum , Azotospirillum brasilense , and Bacillus lichenifirmis . Bacillus Fu mirror's (Bacillus pumilus), Herr boss flutes room vertical page digital camera (Herbospirillum seropedicae), Rizzo Away Pace duck (Rhizobium phaseoli), etc. was reported in seven species, Bacillus novel microorganism in the present invention Sirius (Bacillus cereus) the GA It was confirmed that it produces.

바실러스 시리우스 MJ-1 균주 배양액의 지베렐린 분석을 위한 HPLC 분획 및 GC/MS 분석에서의 KRI 값과 mass sperum 비교Comparison of KRI values and mass sperum in HPLC Fraction and GC / MS Analysis for Gibberellin Analysis of Bacillus Sirius MJ-1 Strain Culture Fraction no. in HPLCFraction no. in HPLC GAs identifiedGAs identified KRIa KRI a m/z(%, relative intensity of base peak)b m / z (%, relative intensity of base peak) b 13∼1513-15 GA1 GA 1 26742674 Sample StandardSample Standard 506(100) 508(100)506 (100) 508 (100) 448(20) 450(17)448 (20) 450 (17) 313(17) 315(15)313 (17) 315 (15) 13∼1513-15 GA3 GA 3 26922692 Sample StandardSample Standard 504(100) 506(100)504 (100) 506 (100) 208(45) 210(42)208 (45) 210 (42) 347(10) 349(8)347 (10) 349 (8) 34∼3534-35 GA4 GA 4 25062506 Sample StandardSample Standard 284(100) 286(100)284 (100) 286 (100) 225(80) 227(78)225 (80) 227 (78) 289(70) 291(66)289 (70) 291 (66) 21∼2321-23 GA5 GA 5 24772477 Sample StandardSample Standard 416(100) 418(100)416 (100) 418 (100) 299(25) 301(22)299 (25) 301 (22) 401(18) 403(16)401 (18) 403 (16) 34∼3534-35 GA7 GA 7 25252525 Sample StandardSample Standard 222(100) 224(100)222 (100) 224 (100) 223(93) 225(80)223 (93) 225 (80) 356(66) 356(54)356 (66) 356 (54) 7∼87-8 GA8 GA 8 28182818 Sample StandardSample Standard 594(100) 596(100)594 (100) 596 (100) 238(28) 240(28)238 (28) 240 (28) 448(25) 450(24)448 (25) 450 (24) 37∼3837-38 GA9 GA 9 23052305 Sample StandardSample Standard 298(100) 300(100)298 (100) 300 (100) 270(78) 272(77)270 (78) 272 (77) 227(48) 229(50)227 (48) 229 (50) 42∼4442-44 GA12 GA 12 23352335 Sample StandardSample Standard 300(100) 302(100)300 (100) 302 (100) 240(31) 242(28)240 (31) 242 (28) 328(21) 330(20)328 (21) 330 (20) 39∼4039-40 GA15 GA 15 26082608 Sample StandardSample Standard 239(100) 241(100)239 (100) 241 (100) 284(50) 286(45)284 (50) 286 (45) 344(23) 346(21)344 (23) 346 (21) 30∼3130-31 GA19 GA 19 26002600 Sample StandardSample Standard 434(100) 436(100)434 (100) 436 (100) 374(59) 376(57)374 (59) 376 (57) 402(41) 404(40)402 (41) 404 (40) 25∼2625-26 GA20 GA 20 24852485 Sample StandardSample Standard 418(100) 420(100)418 (100) 420 (100) 375(45) 377(40)375 (45) 377 (40) 403(14) 405(13)403 (14) 405 (13) 36∼3736 to 37 GA24 GA 24 24442444 Sample StandardSample Standard 314(100) 316(100)314 (100) 316 (100) 226(89) 228(85)226 (89) 228 (85) 286(77) 288(76)286 (77) 288 (76) 32∼3332 to 33 GA34 GA 34 26652665 Sample StandardSample Standard 506(100) 508(100)506 (100) 508 (100) 217(22) 219(21)217 (22) 219 (21) 229(12) 231(10)229 (12) 231 (10) 28∼2928-29 GA36 GA 36 26002600 Sample StandardSample Standard 284(100) 286(100)284 (100) 286 (100) 430(58) 432(56)430 (58) 432 (56) 312(47) 314(45)312 (47) 314 (45) 30∼3130-31 GA44 GA 44 27892789 Sample StandardSample Standard 207(100) 209(100)207 (100) 209 (100) 432(63) 434(61)432 (63) 434 (61) 238(41) 240(40)238 (41) 240 (40) 36∼3736 to 37 GA53 GA 53 24502450 Sample StandardSample Standard 448(47) 450(47)448 (47) 450 (47) 251(30) 253(26)251 (30) 253 (26) 235(30) 237(26)235 (30) 237 (26)

aKRI, Kovats retention index. bIdentified as methyl ester trimethylsilyl ether derivatives by comparison with reference spectra and KRI data (Gaskin and MacMillan, 1991). a KRI, Kovats retention index. b Identified as methyl ester trimethylsilyl ether derivatives by comparison with reference spectra and KRI data (Gaskin and MacMillan, 1991).

바실러스 시리우스 MJ-1 균주가 생산하는 지베렐린의 종류 및 생산량Types and Yields of Gibberellin Produced by Bacillus Sirius MJ-1 Strain GA의 종류Type of GA 생산량 (ng/배양액 300ml)Production (ng / culture solution 300ml) GA1 GA 1 6.4996.499 GA3 GA 3 12.60212.602 GA4 GA 4 23.50023.500 GA5 GA 5 NDND GA7 GA 7 16.85516.855 GA8 GA 8 NDND GA9 GA 9 1.4341.434 GA12 GA 12 0.2320.232 GA15 GA 15 NDND GA19 GA 19 4.6724.672 GA20 GA 20 0.9420.942 GA24 GA 24 3.7883.788 GA34 GA 34 4.5454.545 GA36 GA 36 11.68811.688 GA44 GA 44 46.47646.476 GA53 GA 53 0.3500.350 TotalTotal 133.583133.583

이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.Having described the specific part of the present invention in detail, it is obvious to those skilled in the art that such a specific description is only a preferred embodiment, thereby not limiting the scope of the present invention. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

본 발명은 작물호르몬 지베렐린을 생산하는 바실러스 시리우스 MJ-1균주 및 이를 배양하는 것을 특징으로 하는 지베렐린의 제조방법을 제공하는 효과가 있다.The present invention is effective to provide a Bacillus sirius MJ-1 strain producing crop hormone gibberellin, and a method for producing gibberellin, characterized in that it is cultured.

본 발명에 따른, 바실러스 시리우스 MJ-1균주는 GA1, GA3, GA4, GA7 , GA9, GA12, GA19, GA20, GA24, GA34, GA36, GA44, GA53 등 다양한 지베렐린을 생성할 수 있고, 상기 지베렐린 생성 균주로 작물의 묘를 처리할 경우, 작물의 생장이 현저하게 촉진되는 효과가 있다.According to the present invention, Bacillus sirius MJ-1 strain GA 1 , GA 3 , GA 4 , GA 7 , GA 9 , GA 12 , GA 19 , GA 20 , GA 24 , GA 34 , GA 36 , GA 44 , GA 53 It is possible to produce a variety of gibberellin, and when the seedling of the crop is treated with the gibberellin producing strain, there is an effect that the growth of the crop is significantly promoted.

<110> Kyungpook University <120> Novel Bacillus cereus MJ-1 and Method for Preparing a Gibberellin Using the Same <130> P03-307 <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PCR Primer <400> 1 agagtttgat cctggctcag 20 <210> 2 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> PCR Primer <400> 2 taccttgtta cgactt 16 <210> 3 <211> 1188 <212> DNA <213> Bacillus cereus <400> 3 gcggcgtgcc ttaatcatgc aagtcgagcg aatggattaa gagcttgctc ttatgaagtt 60 agcggcggac gggtgagtaa cacgtgggta acctgcccat aagactggga taactccggg 120 aaaccggggc taataccgga taacattttg aaccgcatgg ttcgaaattg aaaggcggct 180 tcggctgtca cttatggatg gacccgcgtc gcattagcta gttggtgagg taacggctca 240 ccaaggcaac gatgcgtagc cgacctgaga gggtgatcgg ccacactggg actgagacac 300 ggcccagact cctacgggag gcagcagtag ggaatcttcc gcaatggacg aaagtctgac 360 ggagcaacgc cgcgtgagtg atgaaggctt tcgggtcgta aaactctgtt gttagggaag 420 aacaagtgct agttgaataa gctggcacct tgacggtacc taaccagaaa gccacggcta 480 actacgtgcc agcagccgcg gtaatacgta ggtggcaagc gttatccgga attattgggc 540 gtaaagcgcg cgcaggtggt ttcttaagtc tgatgtgaaa gcccacggct caaccgtgga 600 gggtcattgg aaactgggag acttgagtgc agaagaggaa agtggaattc catgtgtagc 660 ggtgaaatgc gaagcaacgc gaagaacctt accaggtctt gacatcttct gacaacccta 720 gagatagggc ttctccttcg ggagcagagt gacaggtggt gcatggttgt cgtcagctcg 780 tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac ccttgatctt agttgccatc 840 attaagttgg gcactctaag gtgactgccg gtgacaaacc ggaggaaggt ggggatgacg 900 tcaaatcatc atgcccctta tgacctgggc tacacacgtg ntacaatgga cggtacaaag 960 agctgcaaga ccgcgaggtg gagctaatct cataaaaccg ttctcagttc ggattgtagg 1020 ctgcaantcg cntacatgaa gctggaatcg ntagtaatcg cggatcagca tgccgcggtg 1080 aatacgttcc cgggccttgt acacaccgcc ngtcacacca cgagagtttg taacacccga 1140 agtcggtggg gtaacctttt ggagccagcc gcctaaggtg gacagatg 1188 <110> Kyungpook University <120> Novel Bacillus cereus MJ-1 and Method for Preparing a Gibberellin          Using the same <130> P03-307 <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PCR Primer <400> 1 agagtttgat cctggctcag 20 <210> 2 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> PCR Primer <400> 2 taccttgtta cgactt 16 <210> 3 <211> 1188 <212> DNA <213> Bacillus cereus <400> 3 gcggcgtgcc ttaatcatgc aagtcgagcg aatggattaa gagcttgctc ttatgaagtt 60 agcggcggac gggtgagtaa cacgtgggta acctgcccat aagactggga taactccggg 120 aaaccggggc taataccgga taacattttg aaccgcatgg ttcgaaattg aaaggcggct 180 tcggctgtca cttatggatg gacccgcgtc gcattagcta gttggtgagg taacggctca 240 ccaaggcaac gatgcgtagc cgacctgaga gggtgatcgg ccacactggg actgagacac 300 ggcccagact cctacgggag gcagcagtag ggaatcttcc gcaatggacg aaagtctgac 360 ggagcaacgc cgcgtgagtg atgaaggctt tcgggtcgta aaactctgtt gttagggaag 420 aacaagtgct agttgaataa gctggcacct tgacggtacc taaccagaaa gccacggcta 480 actacgtgcc agcagccgcg gtaatacgta ggtggcaagc gttatccgga attattgggc 540 gtaaagcgcg cgcaggtggt ttcttaagtc tgatgtgaaa gcccacggct caaccgtgga 600 gggtcattgg aaactgggag acttgagtgc agaagaggaa agtggaattc catgtgtagc 660 ggtgaaatgc gaagcaacgc gaagaacctt accaggtctt gacatcttct gacaacccta 720 gagatagggc ttctccttcg ggagcagagt gacaggtggt gcatggttgt cgtcagctcg 780 tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac ccttgatctt agttgccatc 840 attaagttgg gcactctaag gtgactgccg gtgacaaacc ggaggaaggt ggggatgacg 900 tcaaatcatc atgcccctta tgacctgggc tacacacgtg ntacaatgga cggtacaaag 960 agctgcaaga ccgcgaggtg gagctaatct cataaaaccg ttctcagttc ggattgtagg 1020 ctgcaantcg cntacatgaa gctggaatcg ntagtaatcg cggatcagca tgccgcggtg 1080 aatacgttcc cgggccttgt acacaccgcc ngtcacacca cgagagtttg taacacccga 1140 agtcggtggg gtaacctttt ggagccagcc gcctaaggtg gacagatg 1188

Claims (5)

삭제delete 작물호르몬 지베렐린을 생산하는 바실러스 시리우스(Bacillus cereus) MJ-1 (KCTC 10541BP). Bacillus cereus MJ-1 (KCTC 10541BP), which produces crop hormone gibberellin. 바실러스 시리우스(Bacillus cereus) MJ-1을 배양하는 것을 특징으로 하는 지베렐린의 제조방법.A method for producing gibberellin, comprising culturing Bacillus cereus MJ-1. 제3항에 있어서, 지베렐린은 GA1, GA3, GA4, GA7, GA9, GA12, GA19, GA20, GA24, GA34, GA36, GA44 및 GA53으로 구성된 군에서 선택된 어느 하나 이상인 것을 특징으로 하는 방법.The method of claim 3, wherein gibberellin is in the group consisting of GA 1 , GA 3 , GA 4 , GA 7 , GA 9 , GA 12 , GA 19 , GA 20 , GA 24 , GA 34 , GA 36 , GA 44 and GA 53 At least one selected. 바실러스 시리우스(Bacillus cereus) MJ-1으로 작물의 묘를 처리하는 것을 특징으로 하는 작물의 생육촉진방법. Bacillus cereus ( Bacillus cereus ) A method for promoting the growth of crops, characterized by treating the crop seedlings with MJ-1.
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