KR100873353B1 - Novel Burkholderia cepacia SE-4 and Method for Preparing a Gibberellin Using the Same - Google Patents

Novel Burkholderia cepacia SE-4 and Method for Preparing a Gibberellin Using the Same Download PDF

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KR100873353B1
KR100873353B1 KR1020070034118A KR20070034118A KR100873353B1 KR 100873353 B1 KR100873353 B1 KR 100873353B1 KR 1020070034118 A KR1020070034118 A KR 1020070034118A KR 20070034118 A KR20070034118 A KR 20070034118A KR 100873353 B1 KR100873353 B1 KR 100873353B1
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주길재
강상모
김상국
이인중
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Abstract

본 발명은 작물생육촉진 호르몬인 지베렐린을 생산하는 신규 미생물인 불크홀데리아 세파시아 SE-4 및 이를 배양하는 것을 특징으로 하는 지베렐린(GA)의 제조방법에 관한 것이다.The present invention relates to a new microorganism that produces gibberellin, a crop growth promoting hormone, bulkholderia sefacia SE-4, and a method for producing gibberellin (GA), which is characterized by culturing the same.

본 발명에 따른 불크홀데리아 세파시아 SE-4 균주는 GA3, GA4, GA9, GA20 및 GA24 등의 지베렐린을 생성할 수 있고, 상기 지베렐린 생성 균주로 작물의 묘를 처리할 경우, 작물의 생장이 현저하게 촉진되는 효과가 있다.The Vulcanholderia Sephacia SE-4 strain according to the present invention can produce gibberellins such as GA 3 , GA 4 , GA 9 , GA 20 and GA 24 , and when treating seedlings of crops with the gibberellin producing strains, Crop growth is markedly promoted.

지베렐린, 작물성장 호르몬, 불크홀데리아 세파시아 Gibberellin, Crop Growth Hormone, Vulkholdereria Sephacia

Description

작물 생육촉진 미생물 불크홀데리아 세파시아 ＳＥ－４ 및 이를 이용한 지베렐린의 제조방법{Novel Burkholderia cepacia SE-4 and Method for Preparing a Gibberellin Using the Same}Novel Burkholderia cepacia SE-4 and Method for Preparing a Gibberellin Using the Same}

본 발명은 작물생육촉진 호르몬인 지베렐린을 생산하는 신규 미생물인 불크홀데리아 세파시아 (Burkholderia cepacia) SE-4 및 이를 배양하는 것을 특징으로 하는 지베렐린의 제조방법에 관한 것이다.The present invention relates to a new microorganism, Burkholderia cepacia SE-4, which produces gibberellins, which are crop growth promoting hormones, and a method for producing gibberellins, comprising culturing them.

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), 스 파세로마 멘테아(Sphaceloma menthea), 스파세로마 퍼세아(Sphaceloma perseae), 스파세로마 로이스(Sphaceloma rhois), 푸사리움 프로리퍼라툼 (Fusarium proliferatum) 등 8종이며, 세균류는 불크홀데리아 세파시아 (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), 바실러스 시리우스(Bacillus cereus), 바실러스 마크로이더스(Bacillus microides) 등 9종이 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 , Fusarium proliferatum , and 8 kinds of bacteria. Acetobacter diazotropicus , Azotospirillum lipoferum , Azotospirillum brasilense , Bacillus lichenifirmis , Bacillus pumilus , Hercules boss Room flute vertical page digital camera (Herbospirillum seropedicae), Away Resorts Pace duck (Rhizobium phaseoli), Sirius Bacillus (Bacillus cereus), Bacillus makeuroyi more (Bacillus microides) have been reported, including nine of paper until now.

지금까지 128종의 고등작물, 8종의 곰팡이 및 9종의 세균으로부터 126종의 지베렐린(GA)들이 발견되었다.To date, 126 gibberellins (GAs) have been found from 128 higher crops, 8 fungi and 9 bacteria.

현재까지 우리나라에서는 PGPR이 생산하는 지베렐린에 의한 작물의 생육촉진에 관한 연구가 조금씩 진행되고 있는 실정이나, 외국에서는 미생물로부터 GA 생산에 관한 연구가 많이 진행되어 왔다. To date, studies on promoting growth of crops by gibberellin produced by PGPR have been progressed little by little in Korea, but many studies on the production of GA from microorganisms have been conducted in foreign countries.

지금까지 곰팡이로부터 분리된 GAs로 GA1-4, GA7, GA9-16, GA20, GA24-25, GA36-37, GA40-42, GA47, GA54-57, GA78, GA82 등 28종과 세균으로부터 분리된 GAs로 GA1, GA3, GA4, GA20 등 4종 등이 보고되어 있다.GAs so far isolated from fungi include 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 , 28 species including GA 82 and 4 species including GA 1 , GA 3 , GA 4 , and GA 20 have been reported as GAs isolated from bacteria.

아조토스피리룸 리포페륨(Azotospirillum lipoferum), 아조토스피리룸 브라시렌스(Azotospirillum brasilense) 등은 GA1 및 GA3을 생산하며, 바실러스 리케니 피미스(Bacillus lichenifirmis), 바실러스 푸미러스(Bacillus pumilus)는 GA1, GA3, GA4 및 GA20을 생산하며, 헤르보스피리룸 세로페디카(Herbospirillum seropedicae)는 GA3을 생산하며, 리조비움 파세오리(Rhizobium phaseoli)는 GA1 및 GA4를 생산하는 것으로 보고되었다. Azotospirillum lipoferum and Azotospirillum brasilense produce GA 1 and GA 3 , Bacillus lichenifirmis , Bacillus pumilus Produces GA 1 , GA 3 , GA 4, and GA 20 , Herbospirillum seropedicae produces GA 3 , and Rhizobium phaseoli produces GA 1 and GA 4 Has been reported.

본 발명자에 의하여 2001년 바실러스 시리우스(Bacillus cereus), 바실러스 마크로이더스(Bacillus microides), 바실러스 푸밀러스(Bacillus pumilus) 등의 고초균에서 GA 1, GA 3, GA 4, GA 5, GA 7, GA 9, GA 12, GA 19, GA 20, GA 24, GA 34, GA 36, GA 44, 및 GA 53 등의 지베렐린을 생산하는 것이 보고되었다.By the present inventors in 2001 Bacillus sirius ( Bacillus cereus ), Bacillus microides ( Bacillus microides ), Bacillus pumilus (Bacillus pumilus) in Bacillus subtilis such as GA 1, GA 3, GA 4, GA 5, GA 7, GA 9, It has been reported to produce gibberellins such as GA 12, GA 19, GA 20, GA 24, GA 34, GA 36, GA 44, and GA 53 .

그러나 아직 불크홀데리아 세파시아 (Burkholderia cepacia)에서 지베렐린을 생산하는 보고는 없는 상황이다. However, no reports have yet been made of gibberellin from Burkholderia cepacia .

이에 본 발명에서는 신규 미생물 불크홀데리아 세파시아 (Burkholderia cepacia) SE-4가 지베렐린을 생산하고, 또한 상기 불크홀데리아 세파시아 SE-4가 오이 플러그묘의 생육을 촉진시키는 것을 확인한 것이다.Therefore, the present invention confirms that the novel microorganism Burkholderia cepacia SE-4 produces gibberellin, and that the Burkholderia cepacia SE-4 promotes the growth of cucumber plug seedlings.

본 발명의 목적은 작물호르몬 지베렐린을 생산하는 불크홀데리아 세파시아 및 상기 균주를 배양하는 것을 특징으로 하는 지베렐린의 제조방법을 제공하는 데 있다.An object of the present invention is to provide a method for producing gibberellin, characterized in that cultivation of the strain Vulkholderia Sephacia and the strain producing crop hormone gibberellin.

상기 목적을 달성하기 위하여, 본 발명은 작물호르몬 지베렐린을 생산하는 불크홀데리아 세파시아 (Burkholderia cepacia) SE-4 (KCTC 11095BP)을 제공한다.In order to achieve the above object, the present invention provides a Burkholderia cepacia SE-4 (KCTC 11095BP) for producing a crop hormone gibberellin.

또한 본 발명은 상기 미생물을 배양하는 것을 특징으로 하는 지베렐린의 제조방법을 제공한다. 본 발명에 있어서, 상기 지베렐린은 GA3, GA4, GA9, GA20 및 GA24 등으로 구성된 군에서 선택된 어느 하나를 특징으로 할 수 있다.In another aspect, the present invention provides a method for producing gibberellin, characterized in that the culture of the microorganism. In the present invention, the gibberellin may be characterized by any one selected from the group consisting of GA 3 , GA 4 , GA 9 , GA 20 and GA 24 .

본 발명은 상기의 미생물로 작물의 묘를 처리하는 것을 특징으로 하는 작물의 생육촉진방법을 제공한다.The present invention 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 불크홀데리아 세파시아(Burkholderia cepacia) SE-4 균주는 2007년 3월 21일자로 부다페스트 조약에 따른 국제기탁기관인 한국과학기술원 생명공학연구소 내 유전자은행 (Korea Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology)에 기탁번호 KCTC 11092BP로 기탁되었다.The Burkholderia cepacia SE-4 strain, which is a crop growth promoting microorganism (PGPR), is a gene bank (Korea Collection for Biotechnology Research Institute of Korea Institute of Science and Technology), an international depository institution under the Budapest Treaty on March 21, 2007. Type Cultures, Korea Research Institute of Bioscience and Biotechnology) was deposited with the accession number KCTC 11092BP.

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: 불크홀데리아 세파시아 (Example 1: Vulkholdereria Sephacia ( Burkholderia cepaciaBurkholderia cepacia ) SE-4 균주의 동정) Identification of SE-4 Strains

불크홀데리아 세파시아 (Burkholderia cepacia) SE-4 균주는 본 발명의 연 구자들이 작물생육 촉진 미생물 선별과정 실험에서 토양으로부터 분리하였다. Burkholderia cepacia SE-4 strain was isolated from the soil by the researchers of the present invention in the crop growth promoting microbial screening experiment.

SE-4 균주는 16S rDNA 염기서열 분석방법(16S rDNA sequence method)으로 SE-370 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.The SE-4 strain was extracted from the chromosomal DNA of the SE-370 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.

SE-4 균주의 16S rDNA(923bp) 염기서열(서열 3)에 대한 ribosomal database에서의 상동성 검색 결과, 표 1에서와 같이 불크홀데리아 세파시아 (Burkholderia cepacia)와 82.9%의 유사도를 나타내었기에 불크홀데리아 세파시아 (Burkholderia cepacia)로 동정하고, 불크홀데리아 세파시아 (Burkholderia cepacia) SE-4이라 명명하였다.Homology search in the ribosomal database for the 16S rDNA (923bp) sequence (SEQ ID NO: 3) of the SE-4 strain showed 82.9% similarity to Burkholderia cepacia as shown in Table 1. holde Ria Sepharose was identified by a cyano (Burkholderia cepacia), and named as bulkeu holde Ria Sepharose cyano (Burkholderia cepacia) SE-4.

SE-4 균주의 16S rDNA 염기서열 결정에 의한 동정 자료Identification Data by 16S rDNA Sequencing of SE-4 Strains SE-4 균주의 16S rDNA의 염기서열 (923 bp); 서열 3Nucleotide sequence of 16S rDNA of SE-4 strain (923 bp); SEQ ID NO: 3 GGTCTACCGTTTGAAATATTTTTGCGGTTCTCGCGCCCAGGTCTGGTC ACCGCCTATTCAATTCGAGGACTCCAGCGTCCATATCCACGAGCGTGGAC GTCTCGGGTAGCGATGNGGATAAGAAATGAATCCCGCACCATTAATTGGA GTCGAATAGGGTTTATTTCNGAGTTTTTATTNGGCTCAGATTGAACGCTG GCGGCATGCCTTACACATGCAAGTCGAACGGCAGCACGGGTGCTTGCACC TGGTGGCGAGTGGCGAACGGGTGAGTAATACATCGGAACATGTCCTGTAG TGGGGGATAGCCCGGCGAAAGCCGGATTAATACCGCATACGATCTACGGA TGAAAGCGGGGGACCTTCGGGCCTCGCGCTATAGGGTTGGCCGATGGCTG ATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATCAGTAGCTG GTCTGAGAGGACGACCAGCCACACTGGGACTGAGACACGGCCCAGACTCC TACGGGAGGCAGCAGTGGGGAATTTTGGACAATGGGCGAAAGCCTGATCC AGCAATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTTGT CCGGAAAGAAATCCTTGGTTCTAATATAGCCGGGGGATGACGGTACCGGA AGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGG TGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGCGGTTTG CTAAGACCGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTGGTGA CTGGCAGGCTAGAGTATGGCAGAGGGGGGTAGAATTCCACGTGTAGCAGT GAAATGCGTAGAGATGTGGAGGAATACCGATGGCGAAGGCAGCCCCCTGG GCCAATACTGACGCTCATGCACGAA GGTCTACCGTTTGAAATATTTTTGCGGTTCTCGCGCCCAGGTCTGGTC ACCGCCTATTCAATTCGAGGACTCCAGCGTCCATATCCACGAGCGTGGAC GTCTCGGGTAGCGATGNGGATAAGAAATGAATCCCGCACCATTAATTGGA GTCGAATAGGGTTTATTTCNGAGTTTTTATTNGGCTCAGATTGAACGCTG GCGGCATGCCTTACACATGCAAGTCGAACGGCAGCACGGGTGCTTGCACC TGGTGGCGAGTGGCGAACGGGTGAGTAATACATCGGAACATGTCCTGTAG TGGGGGATAGCCCGGCGAAAGCCGGATTAATACCGCATACGATCTACGGA TGAAAGCGGGGGACCTTCGGGCCTCGCGCTATAGGGTTGGCCGATGGCTG ATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATCAGTAGCTG GTCTGAGAGGACGACCAGCCACACTGGGACTGAGACACGGCCCAGACTCC TACGGGAGGCAGCAGTGGGGAATTTTGGACAATGGGCGAAAGCCTGATCC AGCAATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTTGT CCGGAAAGAAATCCTTGGTTCTAATATAGCCGGGGGATGACGGTACCGGA AGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGG TGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGCGGTTTG CTAAGACCGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTGGTGA CTGGCAGGCTAGAGTATGGCAGAGGGGGGTAGAATTCCACGTGTAGCAGT GAAATGCGTAGAGATGTGGAGGAATACCGATGGCGAAGGCAGCCCCCTGG GCCAATACTGACGCTCATGCACGAA 동정된 균주명Identified strain name 유사도Similarity Burkholderia cepacia Burkholderia cepacia 82.9%82.9%

실시예 2: 불크홀데리아 세파시아 SE-4 균주의 배양조건Example 2 Culture Conditions of Vulcanholderia Sephacia SE-4 Strain

불크홀데리아 세파시아 SE-4 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, 오이 생육촉진효과 검정에는 배양한 균체를 원심분리하여 집균하고, 이를 0.1M 인산완충액(pH 7.0)으로 현탁하여 균체수가 109 CFU/ml되게 하여 상토에 처리하였다.Vulcanholderia Sephacia SE-4 strain was incubated at 35-37 ° C. for 3 days 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 cucumber 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: 오이 플러그묘의 생육 촉진 효과Example 3: Growth promoting effect of cucumber plug seedlings

공시작물로는 오이 (Cucumber Cucumis sativus L., Seminis Korea Co., 조은백다다기)를 사용하였으며, 종자를 17~20℃ 물에 12시간 침종한 후 젖은 수건을 이용 28~30℃ 항온실에서 2일간 발아하여 스티로폼 상자 (20 x 30 x 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 36구 tray에 이식 (흥농종묘(주) 바이오 상토 이용)하고 충분히 관수하고 밀폐된 육묘장에서 보온 재배하였다. Cucumber (Cucumber Cucumis sativus L. , Seminis Korea Co., Choeunbaek teapot) was used as a starting material, and the seeds were soaked in water at 17 ~ 20 ℃ for 12 hours and then wet at 28 ~ 30 ℃ in a constant temperature room for 2 days Germinate and add 2/3 of top soil (50% of clay, 30% of crushed soil, 20% of river sand) to a styrofoam box (20 x 30 x 15 cm). It was cultivated at ~ 27 ℃ constant temperature room. After 30 days of sowing, when the main leaves were 3-4 sheets, they were transplanted into a 36-neck tray (using Heung Jongmyo Co., Ltd. Biotope), and then sufficiently irrigated and kept warm in a closed nursery.

불크홀데리아 세파시아 SE-4 균주 배양액 (미생물수 2.0 x 108 cfu/㎖ 이상)은 원심분리하고 균체를 동일량의 살균수에 현탁하여 36구 tray 당 각각의 미생물 배양액 36㎖를 이식한 오이 유묘에 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.Vulcan Holderia Sephacia SE-4 strain culture medium (microbial water 2.0 x 10 8 cfu / ml or more) was centrifuged and the cells were suspended in the same amount of sterilized water and cucumbers transplanted with 36 ml of each microbial culture per 36-neck tray The seedlings were treated. 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.

불크홀데리아 세파시아 SE-4 균주는 표 2에서와 같이 10일 재배후 전체 식물의 길이가 미생물 무처리구에 비해 높게 나타났으며, 뿌리의 길이는 197.7% 이상 증가하는 경향을 나타내었다. 그러나 엽수나 경경은 차이가 거의 없었다. 불크홀데리아 세파시아 SE-4를 처리하여 생산한 오이 플러그묘는 SE-4를 처리하지 않은 대조구에 비해 전체길이는 65.2%, 지상부는 32.4%, 뿌리길이는 197.7%, 생체중은 50.6%, 건물중은 25.0% 증가하였다. As shown in Table 2, the Vulcanholderia Sephacia SE-4 strain showed a higher total length of the plants after 10 days of cultivation than the non-microbial treatment, and the length of the roots increased by more than 197.7%. However, there was little difference between leaves and tillage. Cucumber plug seedlings produced by treatment with Vulcanholderia Sephacia SE-4 had a total length of 65.2%, ground level of 32.4%, root length of 197.7%, and live weight of 50.6%, compared to the control without SE-4. Medium increased by 25.0%.

작물생육 촉진 미생물 불크홀데리아 세파시아 SE-4 균주에 의한 오이 플러그묘의 생육촉진 효과Promoting Growth of Cucumber Plug Seedling by Microbial Vulcanholderia Sephacia SE-4 Strains 미생물 처리Microbial treatment 식물전체 길이(㎝)Plant full length (cm) 지상부 길이(cm)Ground length (cm) 뿌리길이 (㎝)Root Length (cm) 건물중 (g/plant)Building (g / plant) 생체중 (g/plant)Live weight (g / plant) 불크홀데리아 세파시아 SE-4 처리구 Vulkholderia Sephacia SE-4 Treated Area 43.54* 43.54 * 21.2921.29 20.2920.29 0.300.30 4.944.94 무처리구No treatment 26.3426.34 16.0816.08 10.2610.26 0.240.24 2.282.28

* : 36구 오이 개체 평균값, 10일 재배 * : 36 cucumber cucumbers average value, 10 days cultivation

실시예 4: 불크홀데리아 세파시아 SE-4 균주가 생산하는 지베렐린의 분석Example 4 Analysis of Gibberellin Produced by Vulkholderia Sephacia SE-4 Strain

불크홀데리아 세파시아 SE-4 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.The extraction of gibberellins produced by the strains of Vulcanholderia Sephacia SE-4 was performed according to the methods of Foster and Morgan (Plant Physiol. 108: 337-343, 1995) and Lee (Plant Growth Regul. 17: 185-195, 1988). Fractionation by HPLC and analysis by GC / MS SIM.

내부 표준물질로는 [17.17-2H2]로 라벨된 지베렐린 [17.17-2H2] GA1, GA3, GA4, GA5, GA7, GA8, GA9, GA12, GA15, GA19, GA20, GA24, GA34, GA36, GA44 및 GA53을 각각 20ng씩 첨가하였다. 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 불크홀데리아 세파시아 SE-4 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.Table 3 shows the HPLC fractions of the gibberellin-containing sections using internal standards to analyze the gibberellin produced by the Vulcanholderia Sephacia SE-4 strain.The GC / MS KRI values of each gibberellin (Kovats retention index) ) And mass spectrum ( m / z ).

표 4는 불크홀데리아 세파시아 SE-4 균주 배양액 200㎖ 내에 존재하는 GA의 종류를 조사한 결과, GA3, GA4, GA9, GA20 및 GA24 등이 존재하였으며, 각각의 지베렐린 함량은 GA3이 8.63ng,, GA4가 9.31ng, GA9가 0.64g, GA20이 1.08ng 및 GA24가 2.27ng 존재하였다. Table 4 shows the types of GA present in 200 ml of the culture medium of Bulchholderia Sephacia SE-4 strain. As a result, GA 3 , GA 4 , GA 9 , GA 20, and GA 24 were present. 3 is 8.63ng, GA 4 is 9.31ng, GA 9 is 0.64g, GA 20 is 1.08ng and GA 24 is 2.27ng.

지금까지 곰팡이로부터 분리된 GAs는 GA1-4, GA7, GA9-16, GA20, GA24-25, GA36-37, GA40-42, GA47, GA54-57, GA78, GA82 등 28종이며, 세균으로부터 분리된 GAs는 GA1, GA3, GA4, GA20 등 4종으로 보고되어 있다. 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 has been reported as 4 species including GA 1 , GA 3 , GA 4 , and GA 20 .

또한 GA를 생산하는 세균으로 아세토박터 디아조트로피쿠스 (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), 바실러스 시리우스(Bacillus cereus), 바실러스 마크로이더스(Bacillus microides) 등 9종이 보고되었으나, 본 발명에서 신규 미생물 불크홀데리아 세파시아 (Acenetobacter calcoaceticus)가 지베렐린을 생산한다는 것을 확인하였다. In addition, GA-producing bacteria include Acetobacter diazotropicus , Azotospirillum lipoferum , Azotospirillum brasilense , Bacillus lichenifirmis , Although Bacillus Fu mirror's (Bacillus pumilus), Herr boss flutes room vertical page digital camera (Herbospirillum seropedicae), Rizzo Away Pace duck (Rhizobium phaseoli), Bacillus Sirius (Bacillus cereus), Bacillus makeuroyi Traders (Bacillus microides), including nine paper reported, In the present invention, it was confirmed that the novel microorganism Vulcanholderia Sephacia ( Acenetobacter calcoaceticus ) produces gibberellin.

불크홀데리아 세파시아 SE-4 균주 배양액의 지베렐린 분석을 위한 HPLC 분획 및 GC/MS 분석에서의 KRI 값과 mass sperum 비교Comparison of KRI and mass sperum in HPLC Fraction and GC / MS Analysis for Gibberellin Analysis of Vulcanholderia Sephacia SE-4 Strain Cultures 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).

불크홀데리아 세파시아 SE-4 균주가 생산하는 지베렐린의 종류 및 생산량Types and Yields of Gibberellins Produced by Vulcanholderia Sephacia SE-4 Strains GA의 종류Type of GA 생산량 (ng/배양액 200ml)Production amount (ng / culture solution 200ml) GA1 GA 1 NDND GA3 GA 3 8.638.63 GA4 GA 4 9.319.31 GA7 GA 7 NDND GA9 GA 9 0.640.64 GA20 GA 20 1.081.08 GA24 GA 24 2.272.27 TotalTotal 21.9321.93

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 불크홀데리아 세파시아 SE-4 균주 및 이를 배양하는 것을 특징으로 하는 지베렐린의 제조방법을 제공하는 효과가 있다.The present invention has the effect of providing a production method of Gibberellin, characterized in that the cultivation of bulkholderia Sephacia SE-4 strain producing crop hormone gibberellin.

본 발명에 따른 불크홀데리아 세파시아 SE-4 균주는 GA3, GA4, GA9, GA20 및 GA24 등의 지베렐린을 생성할 수 있고, 상기 지베렐린 생성 균주로 오이의 유묘를 처리할 경우, 작물의 생장이 현저하게 촉진되는 효과가 있다.The Vulcanholderia Sephacia SE-4 strain according to the present invention may produce gibberellins such as GA 3 , GA 4 , GA 9 , GA 20, and GA 24 , and when treating seedlings of cucumbers with the gibberellin producing strains, There is an effect that the growth of the crop is significantly promoted.

<110> Kyungpook National University Industry-Academic Cooperation Foundation <120> Novel Burkholderia cepacia SE-4 and Method for Preparing a Gibberellin Using the Same <130> KCTC 11092BP <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> 923 <212> DNA <213> Burkholderia cepacia <400> 3 ggtctaccgt ttgaaatatt tttgcggttc tcgcgcccag gtctggtcac cgcctattca 60 attcgaggac tccagcgtcc atatccacga gcgtggacgt ctcgggtagc gatgnggata 120 agaaatgaat cccgcaccat taattggagt cgaatagggt ttatttcnga gtttttattn 180 ggctcagatt gaacgctggc ggcatgcctt acacatgcaa gtcgaacggc agcacgggtg 240 cttgcacctg gtggcgagtg gcgaacgggt gagtaataca tcggaacatg tcctgtagtg 300 ggggatagcc cggcgaaagc cggattaata ccgcatacga tctacggatg aaagcggggg 360 accttcgggc ctcgcgctat agggttggcc gatggctgat tagctagttg gtggggtaaa 420 ggcctaccaa ggcgacgatc agtagctggt ctgagaggac gaccagccac actgggactg 480 agacacggcc cagactccta cgggaggcag cagtggggaa ttttggacaa tgggcgaaag 540 cctgatccag caatgccgcg tgtgtgaaga aggccttcgg gttgtaaagc acttttgtcc 600 ggaaagaaat ccttggttct aatatagccg ggggatgacg gtaccggaag aataagcacc 660 ggctaactac gtgccagcag ccgcggtaat acgtagggtg cgagcgttaa tcggaattac 720 tgggcgtaaa gcgtgcgcag gcggtttgct aagaccgatg tgaaatcccc gggctcaacc 780 tgggaactgc attggtgact ggcaggctag agtatggcag aggggggtag aattccacgt 840 gtagcagtga aatgcgtaga gatgtggagg aataccgatg gcgaaggcag ccccctgggc 900 caatactgac gctcatgcac gaa 923 <110> Kyungpook National University Industry-Academic Cooperation Foundation <120> Novel Burkholderia cepacia SE-4 and Method for Preparing a          Gibberellin Using the Same <130> KCTC 11092BP <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> 923 <212> DNA <213> Burkholderia cepacia <400> 3 ggtctaccgt ttgaaatatt tttgcggttc tcgcgcccag gtctggtcac cgcctattca 60 attcgaggac tccagcgtcc atatccacga gcgtggacgt ctcgggtagc gatgnggata 120 agaaatgaat cccgcaccat taattggagt cgaatagggt ttatttcnga gtttttattn 180 ggctcagatt gaacgctggc ggcatgcctt acacatgcaa gtcgaacggc agcacgggtg 240 cttgcacctg gtggcgagtg gcgaacgggt gagtaataca tcggaacatg tcctgtagtg 300 ggggatagcc cggcgaaagc cggattaata ccgcatacga tctacggatg aaagcggggg 360 accttcgggc ctcgcgctat agggttggcc gatggctgat tagctagttg gtggggtaaa 420 ggcctaccaa ggcgacgatc agtagctggt ctgagaggac gaccagccac actgggactg 480 agacacggcc cagactccta cgggaggcag cagtggggaa ttttggacaa tgggcgaaag 540 cctgatccag caatgccgcg tgtgtgaaga aggccttcgg gttgtaaagc acttttgtcc 600 ggaaagaaat ccttggttct aatatagccg ggggatgacg gtaccggaag aataagcacc 660 ggctaactac gtgccagcag ccgcggtaat acgtagggtg cgagcgttaa tcggaattac 720 tgggcgtaaa gcgtgcgcag gcggtttgct aagaccgatg tgaaatcccc gggctcaacc 780 tgggaactgc attggtgact ggcaggctag agtatggcag aggggggtag aattccacgt 840 gtagcagtga aatgcgtaga gatgtggagg aataccgatg gcgaaggcag ccccctgggc 900 caatactgac gctcatgcac gaa 923  

Claims (3)

작물호르몬 지베렐린을 생산하는 작물 생육촉진 미생물 불크홀데리아 세파시아 (Burkholderia cepacia) SE-4 (KCTC 11092BP).Crop-promoting microorganisms Burkholderia cepacia SE-4 (KCTC 11092BP) to produce crop hormone gibberellin. 불크홀데리아 세파시아 (Burkholderia cepacia) SE-4 균주를 배양하는 것을 특징으로 하는 불크홀데리아 세파시아 SE-4를 이용한 지베렐린의 제조방법.A method for preparing gibberellins using bulkholderia cepacia SE-4, comprising culturing Burkholderia cepacia SE-4 strain. 제2항에 있어서,The method of claim 2, 지베렐린은 GA3, GA4, GA9, GA20 및 GA24으로 구성된 군에서 선택된 어느 하나 이상인 것을 특징으로 하는 불크홀데리아 세파시아 SE-4를 이용한 지베렐린의 제조방법.Gibberellin is a method for producing gibberellin using bulkholderia Sephacia SE-4, characterized in that any one or more selected from the group consisting of GA 3 , GA 4 , GA 9 , GA 20 and GA 24 .
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KR100690816B1 (en) 2005-03-03 2007-03-09 경상북도농업기술원생물자원연구소 -8 Novel Microbacterium aurum MA-8 and Method for Producing Seed Tuber in Yam Using the Same

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