KR100982824B1 - Phenylalanine ammonia lyase gene and cinnamoyl-CoA reductase gene isolated from Tricholoma matsutake - Google Patents
Phenylalanine ammonia lyase gene and cinnamoyl-CoA reductase gene isolated from Tricholoma matsutake Download PDFInfo
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Abstract
본 발명은 송이버섯의 페닐알라닌 암모니아리아제 유전자 및 신나모일-코에이 리덕타제 유전자에 관한 것이다. 상기의 유전자는 송이버섯(Tricholoma matsutake)의 향기의 주성분으로 알려진 신남산메틸(methylcinnamate)생성에 관여하는 것이다.The present invention relates to the phenylalanine ammoniaase gene and cinnamoyl-CoA reductase gene of Matsutake mushroom. The gene is a tricholoma It is involved in the production of methylcinnamate (methylcinnamate), which is known as the main component of the fragrance of matsutake .
신남산메틸, 페닐알라닌 암모니아 리아제, 신나모일-코에이 리덕타제, 송이버섯 Methyl Cinnamate, Phenylalanine Ammonia Lyase, Cinnamoyl-Coy Reductase, Matsutake Mushroom
Description
본 발명은 송이버섯의 페닐알라닌 암모니아리아제 유전자 및 신나모일-코에이 리덕타제 유전자에 관한 것이다.The present invention relates to the phenylalanine ammoniaase gene and cinnamoyl-CoA reductase gene of Matsutake mushroom.
송이버섯의 향기성분에 대한 연구는 특허 10-0769652에 의하면 성분으로는 유리아미노산 27종, 에르고스테롤(ergosterol), 미량 금속 원소 13종, 비타민 B1, B2, C, D 및 니아신(niacin), 글리세롤(glycerol), 만니톨(mannitol), 트레할로즈(trehalose), 포도당 셀룰로오스(glucose cellulose), 헤미셀룰로오스(hemicellulose), 키틴(chitin), 펙틴 리그닌(pectin lignin), 5´-AMP, 5´-CMP, 5´-GMP, 5´-UMP, 1-옥텐-3-올(1-octene-3-ol), 메틸 시나메이트(methyl cinnamate), 에미타민(emitamin), 산성 포스포디에스테라아제(phosphodiesterase)등이 알려져 있다. 이 중 메틸신나메이트는 신남산에 메틸기가 결합된 것으로서 이와 유사한 형태의 신남산 알데히드가 송이버섯의 주요 향기 성분으로 인식되어 있다.According to patent 10-0769652, the odor component of pine mushrooms includes 27 free amino acids, ergosterol, 13 trace metal elements, vitamins B1, B2, C, D and niacin and glycerol. (glycerol), mannitol, trehalose, glucose cellulose, hemicellulose, chitin, pectin lignin, 5´-AMP, 5´-CMP, 5´-GMP, 5´-UMP, 1-octene-3-ol, methyl cinnamate, emitamin, acid phosphodiesterase Known. Among these, methyl cinnamate is a methyl group bonded to cinnamic acid, and a similar form of cinnamic acid aldehyde is recognized as a main scent component of pine mushroom.
한편 페닐알라닌에서 트랜스신남산이 되는데 이때에 관여하는 효소가 페닐알라닌 암모니아 리아제이다. 트랜스신남산에서 분지되어 파라큐마린산이 되어 플라보노이드, 스코포린, 리그닌, 시린진 그리고 레스베라트롤 등이 생합성 되는 것으로 알려져 있다 (ref). 트랜스신남산의 한 분지는 신나모일-코에이로 되며 여기서 다시 피노실빈이나 신남알데하이드가 되는데 신남알데하이드로 되는 과정에 관여하는 효소가 신나모일-코에이 리덕타제이다. 따라서 페닐알라닌 암모니아리아제와 신나모일-코에이 리덕타제는 송이버섯의 향기에 주요성분인 신남알데하이드의 생성에 주요한 두 가지의 효소이다.On the other hand, from phenylalanine to transcinnamic acid, the enzyme involved in this is phenylalanine ammonia lyase. It is known to be branched from transcinnamic acid to paracumarin acid to biosynthesize flavonoids, scoporin, lignin, sirin and resveratrol (ref). One branch of transcinnamic acid is cinnamoyl-coei, where it becomes pinocylbin or cinnamic aldehyde, and the enzyme involved in the process of cinnamic aldehyde is cinnamoyl-coei reductase. Thus, phenylalanine ammonia lyase and cinnamoyl-CoA reductase are two major enzymes in the production of cinnamic aldehyde, which is a major component of the scent of pine mushroom.
그러나, 상기한 연구 보고들에서 송이버섯의 향기에 대한 물질이나 이의 복합물 또는 유전자 수준에 대한 연구는 전혀 보고된 바 없다.However, there have been no reports on the substance, complex or gene level of pine mushroom scent in the above mentioned studies.
본 발명은 상기와 같은 문제점을 해결하기 위해서 안출된 것으로서, 공지된 페닐알라닌 암모니아리아제 유전자 및 신나모일-코에이 리덕타제 유전자와 구별되는 송이버섯의 페닐알라닌 암모니아리아제 유전자 및 신나모일-코에이 리덕타제 유전자를 제공하는 것을 목적으로 한다.The present invention has been made to solve the above problems, and the phenylalanine ammoniaase gene and cinnamoyl-coei reductase gene of Matsutake mushroom distinguished from known phenylalanine ammoniaase gene and cinnamoyl-coei reductase gene. It aims to provide.
상기와 같은 문제점을 해결하기 위해서 안출된 본 발명은 서열번호 1로 표시되는 DNA 염기서열을 제공하는 것을 특징으로 한다.The present invention devised to solve the above problems is characterized by providing a DNA base sequence represented by SEQ ID NO: 1.
또한, 본 발명은 서열번호 2로 표시되는 DNA 염기서열을 제공하는 것을 특징으로 한다.In addition, the present invention is characterized by providing a DNA base sequence represented by SEQ ID NO: 2.
이상에서 상술한 바와 같이, 본 발명은 송이버섯의 페닐알라닌 암모니아리아제(phenylalanine ammonia lyase) 유전자 및 신나모일-코에이 리덕타제(cinnamoyl-CoA reductase) 유전자를 제공하는 효과가 있다.As described above, the present invention has an effect of providing a phenylalanine ammonia lyase gene and a cinnamoyl-CoA reductase gene of Matsutake mushroom.
본 발명은 송이버섯으로부터 페닐알라닌 암모니아리아제(phenylalanine ammonia lyase) 및 신나모일-코에이 리덕타제(cinnamoyl-CoA reductase)를 클로닝하여 확인하고, 이들의 유전자의 염기서열을 해명하였다. The present invention was identified by cloning phenylalanine ammonia lyase and cinnamoyl-CoA reductase from pine mushrooms and elucidated the nucleotide sequences of these genes.
따라서, 본 발명은 서열번호 1로 표시되는 페닐알라닌 암모니아리아제 유전 자와 서열번호 2로 표시되는 신나모일-코에이 리덕타제 유전자를 제공한다.Accordingly, the present invention provides a phenylalanine ammoniaase gene represented by SEQ ID NO: 1 and a cinnamoyl-coei reductase gene represented by SEQ ID NO: 2.
이하, 본 발명을 좀더 구체적으로 살펴보면 다음과 같다.Hereinafter, the present invention will be described in more detail.
본 발명은 하기의 실험예에 의하여 보다 명확히 이해될 수 있으며, 하기의 실험 예는 본 발명의 예시 목적에 불과하며 발명의 영역을 제한하거나, 본 발명의 내용이 여기에 한정되는 것은 아니다. The present invention can be more clearly understood by the following experimental examples, the following experimental examples are only for the purpose of illustrating the invention and limit the scope of the invention, or the content of the present invention is not limited thereto.
실시예1Example 1 : 송이버섯으로 제작한 : Made with Matsutake Mushroom cDNAcDNA 라이브러리( library( librarylibrary ) ) 로부터from 클로닝한Cloned 페닐알라닌 Phenylalanine 암모니아리아제Ammonia 및 And 신나모일Cinnamo -- 코에이Koei 리덕타제의Reductant 염기서열과 아미노산 서열 Sequence and Amino Acid Sequence
국내 대구 근교의 가창 송림에서 채취한 송이버섯으로부터 QIAGEN의 RNeasykit를 이용하여 토탈(total) RNA를 분리, 정제하고 RT-PCR반응으로 1차 cDNA를 합성하였다. 1차 cDNA를 주형으로 다시 PCR하여 최종 cDNA를 만들고 벡터에 삽입하여 이를 일렉트로포레이션(electroporation) 하여 cDNA library를 제작하였다. 그리고 Seq I 씨퀀싱 프라이머(sequencing primer)를 이용하여 각각의 클론을 씨퀀싱(sequencing)하고 그결과를 NCBI에서 블라스트 써치(blast search)를 해서 phenylalanine ammonia lyase, cinnamoyl-CoA reductase 유전자와 높은 상동성을 나타냄을 확인하였다.Total RNA was isolated and purified from matsutake mushrooms in Songchang, Korea near Daegu using QIAGEN's RNeasykit, and primary cDNA was synthesized by RT-PCR reaction. The primary cDNA was PCR again as a template to make a final cDNA, inserted into a vector, and electroporated the resulting cDNA library. Sequencing each clone using Seq I sequencing primer and blast search the result in NCBI to obtain high homology with phenylalanine ammonia lyase and cinnamoyl-CoA reductase gene. It confirmed that it was shown.
실험예Experimental Example 1: 타 미생물과 송이버섯의 페닐알라닌 1: Phenylalanine from other microorganisms and Matsutake 암모니아리아제Ammonia 및 And 신나모일Cinnamo -- 코에이Koei 리덕타제의 아미노산 서열의 Of the amino acid sequence of the reductase 다상정렬Multiphase alignment ( ( multiplemultiple -- alignmentalignment ))
타 미생물과 송이버섯의 페닐알라닌 암모니아리아제(도 5(a)-(d)) 및 신나모일-코에이 리덕타제(도 6(a)-(b))의 아미노산 서열을 Clustal X 프로그램을 이용하여 다상정렬(multiple-alignment) 하였다.The amino acid sequence of phenylalanine ammonia lyase (Figs. 5 (a)-(d)) and cinnamoyl-Coei reductase (Fig. 6 (a)-(b)) of other microorganisms and pine mushrooms was determined using the Clustal X program. Multiple-alignment.
도 1(a)-(d)는 페닐알라닌 암모니아리아제의 DNA 염기서열을 나타낸 것이다.Figure 1 (a)-(d) shows the DNA sequence of the phenylalanine ammonia.
도 2(a)-(b)는 페닐알라닌 암모니아리아제의 cDNA(tmpal) 염기서열을 나타낸 것이다.Figure 2 (a)-(b) shows the cDNA ( tmpal ) base sequence of phenylalanine ammonia.
도 3(a)-(b)은 신나모일-코에이 리덕타제의 DNA 염기서열을 나타낸 것이다.Figure 3 (a)-(b) shows the DNA sequence of cinnamoyl-CoA reductase.
도 4는 신나모일-코에이 리덕타제의 cDNA(tmcin) 염기서열을 나타낸 것이다.Figure 4 shows the cDNA ( tmcin ) nucleotide sequence of cinnamoyl-CoA reductase.
도 5(a)-(d)는 타 미생물과 송이버섯의 페닐알라닌 암모니아리아제의 아미노산 서열을 다상정렬한 것이다.5 (a)-(d) are polyphase alignments of amino acid sequences of phenylalanine ammonia from other microorganisms and pine mushrooms.
도 6(a)-(b)는 타 미생물과 송이버섯의 신나모일-코에이 리덕타제의 아미노산 서열을 다상정렬한 것이다.6 (a)-(b) are polyphase alignments of amino acid sequences of cinnamoyl-Coei reductases of other microorganisms and pine mushrooms.
<110> KYUNGPOOK NATIONAL UNIVERSITY INDUSTRY-ACADEMIC COOPERATION FOUNDATION
<120> Phenylalanine ammonia lyase gene and cinnamoyl-CoA reductase gene
isolated from Tricholoma matsutake
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<141> 2008-02-12
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ctggatctcc cgcgcaaccc cggtgcactt gagagacgct acatcgtgaa tggtggcaat 900
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attaccgact tgaagttcgg caagttcatt gacgtcaagc aaactatcga tgacgccgtt 1080
gttgcgttgc aggaggttga ggggagctgg tcgtaa 1116
<110> KYUNGPOOK NATIONAL UNIVERSITY INDUSTRY-ACADEMIC COOPERATION FOUNDATION
<120> Phenylalanine ammonia lyase gene and cinnamoyl-CoA reductase gene
isolated from Tricholoma matsutake
<130> 1036315
<140> 10-2008-0012448
<141> 2008-02-12
<160> 2
<170> KopatentIn 1.71
<210> 1
<211> 2160
<212> DNA
<213> Tricholoma matsutake
<400> 1
atgtctttgc ttctcaatgg ttccgtcaat ggtatacctc attcaaaacc ctacaattat 60
tcgactcttc tatctaggtt tattgaatcc tacaatgagc tccagagcta caaaagtgga 120
aaggctgtca aggttgatgg tcgcacactc tctgttgctg cagtgactgc tgctgctcgt 180
taccatgcct ccgtagaatt agacgactgt cctactgcta gagcacgtct agcaaaaagt 240
agaaaggtta tcacggacaa agttgaaagc ggtaccagcg tttacgggtt gtcgaccggt 300
ttcggtggaa gtgctgatac tcgaactgat caacctctgc ttcttggtca tgccctgctt 360
caacaccagc atatgggtgt tcttccttct tccagtcagg ctctcgatgt tttacctttg 420
ctggatccta acagttctac aactatgccc gaatcttggg ttcggggtgc aatgcttatt 480
cggatgaact cgcttattcg tggtcactca ggcgtcagat gggagctcat cgagaagata 540
aatgaagttt tgagtgcaaa tgtcactccg gttgtacctc ttcgaggaag tatctctgcg 600
tctggagacc tgtcacccct atcatatatt gctggcaccc tagtcgctaa cccagctatt 660
cgagtctatc atggccccgc cgcatacggc gcccgccaaa
Claims (2)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020080012448A KR100982824B1 (en) | 2008-02-12 | 2008-02-12 | Phenylalanine ammonia lyase gene and cinnamoyl-CoA reductase gene isolated from Tricholoma matsutake |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020080012448A KR100982824B1 (en) | 2008-02-12 | 2008-02-12 | Phenylalanine ammonia lyase gene and cinnamoyl-CoA reductase gene isolated from Tricholoma matsutake |
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| Publication Number | Publication Date |
|---|---|
| KR20090087167A KR20090087167A (en) | 2009-08-17 |
| KR100982824B1 true KR100982824B1 (en) | 2010-09-16 |
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| KR1020080012448A KR100982824B1 (en) | 2008-02-12 | 2008-02-12 | Phenylalanine ammonia lyase gene and cinnamoyl-CoA reductase gene isolated from Tricholoma matsutake |
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| KR (1) | KR100982824B1 (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1995027790A2 (en) | 1994-04-11 | 1995-10-19 | Centre National De La Recherche Scientifique | DNA SEQUENCES CODING FOR A CYNNAMOYL CoA REDUCTASE AND THEIR USE IN THE REGULATION OF PLANT LIGNIN CONCENTRATIONS |
| KR20070008643A (en) * | 2004-04-20 | 2007-01-17 | 이 아이 듀폰 디 네모아 앤드 캄파니 | Methods for the preparation of para-hydroxycinnamic acid and cinnamic acid at alkaline ph |
-
2008
- 2008-02-12 KR KR1020080012448A patent/KR100982824B1/en active IP Right Grant
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1995027790A2 (en) | 1994-04-11 | 1995-10-19 | Centre National De La Recherche Scientifique | DNA SEQUENCES CODING FOR A CYNNAMOYL CoA REDUCTASE AND THEIR USE IN THE REGULATION OF PLANT LIGNIN CONCENTRATIONS |
| KR20070008643A (en) * | 2004-04-20 | 2007-01-17 | 이 아이 듀폰 디 네모아 앤드 캄파니 | Methods for the preparation of para-hydroxycinnamic acid and cinnamic acid at alkaline ph |
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| Publication number | Publication date |
|---|---|
| KR20090087167A (en) | 2009-08-17 |
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