JP5726172B2 - アオウキクサ由来の新規アレンオキサイドシンターゼ - Google Patents
アオウキクサ由来の新規アレンオキサイドシンターゼ Download PDFInfo
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- JP5726172B2 JP5726172B2 JP2012509541A JP2012509541A JP5726172B2 JP 5726172 B2 JP5726172 B2 JP 5726172B2 JP 2012509541 A JP2012509541 A JP 2012509541A JP 2012509541 A JP2012509541 A JP 2012509541A JP 5726172 B2 JP5726172 B2 JP 5726172B2
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- oxide synthase
- protein
- dna
- allene oxide
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y402/00—Carbon-oxygen lyases (4.2)
- C12Y402/01—Hydro-lyases (4.2.1)
- C12Y402/01092—Hydroperoxide dehydratase (4.2.1.92)
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
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- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
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- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Description
様々な場所から採取された62種類のアオウキクサを準備し、1/2倍に稀釈したHutnerの培地中において、24〜25℃の昼光色蛍光ライトからの連続的な光照射の下で継代培養した。1/2倍に稀釈したHutnerの培地は、次の成分:
スクロース 10g/l
K2HPO4 200mg/l
NH4NO3 100mg/l
EDTA遊離酸 250mg/l
Ca(NO3)・4H2O 176mg/l
MgSO4・7H2O 250mg/l
FeSO4・7H2O 12.4mg/l
MnCl2・4H2O 8.92mg/l
ZnSO4・7H2O 32.8mg/l
Na2MoO4・2H2O 12.6mg/l
H3BO3 7.1mg/l
Co(NO3)・6H2O 0.1mg/l
CuSO4・5H2O 1.97mg/l
を含み、KOH(50%)を用いてpH6.2〜6.5に調整した。
アオウキクサSH株(Lemna paucicostata, SH)から RNeasy Plant Mini Kit(Qiagen)を用い、全RNAを抽出し、1.8μg の全RNAから、LongRange 2Step RT−PCR Kit(Qiagen)を用いRT−PCR法によりcDNAを合成した。合成したcDNAを、以下に示すシロイヌナズナ(Arabidopsis thaliana)由来プライマーを使用し、PCRのアニーリング温度を45℃と低めに設定し、SH株由来アレンオキサイドシンターゼ(SHLpAOS)の一部配列情報を得た。
AOS-Forward 5’-GGA ACT AAC CGG AGG CTA CCG-3’(配列番号3)
AOS-Reverse 5’-CCG TCT CCG GTC CAT TCG ACC ACA A-3’(配列番号4)
3’-RACE用プライマー
SH-3’-TP:5’-TCTGGGCAGACTCTTCTTTGGAG-3’(配列番号5)
5’-RACE用プライマー
SH-5’-TP:5’-CCGGCTTGCGCTATATATCTGAGAATT-3’(配列番号6)
3’−RACE
上記実験で得られたSH株の全RNA2μgを鋳型として、CapFishingTM target full-length cDNA cloning Kit(Seegene)を用いてcDNAを合成した。このcDNAを鋳型溶液として、キット内アダプタープライマー(3’−RACEプライマー)とSH−3’−TPとそれぞれPCRを行った。
5’−RACE
上記実験で得られたSH株の全RNA2μgを鋳型として、CapFishingTM target full-length cDNA cloning Kit(Seegene)を用いてcDNAを合成した。このcDNAを鋳型溶液として、キット内アダプタープライマー(5’−RACEプライマー)とSH−5’−TPとそれぞれPCRを行った。
SHAOS-F: 5’-CCGCGCCATGGAGAAGAGAATGTCTGTCTCGC-3’(配列番号7)
SHAOS-R: 5’-CCGCGCTCGAGGTTGAATAAGCACGAGTGT-3’(配列番号8)
この結果、SH株より1配列の新規AOSホモログ(核酸配列1443bp、アミノ酸配列480aa、推定分子量53.3KDa)が得られた(図2A−C)。このアオウキクサSH株より得られたアレンオキサイドシンターゼをSHLpAOSと名付けた。
Claims (5)
- 以下の(a)〜(d):
(a) 配列番号1で表される塩基配列からなるDNA;
(b) 配列番号2で表されるアミノ酸配列からなるタンパク質をコードするDNA;
(c) 配列番号2で表わされるアミノ酸配列において1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつアレンオキサイドシンターゼ活性を有するタンパク質をコードするDNA;及び
(d) 配列番号1で表される塩基配列からなるDNAと少なくとも90%の同一性を有し、かつアレンオキサイドシンターゼ活性を有するタンパク質をコードするDNA
からなる群から選ばれるDNAからなる遺伝子。 - 請求項1に記載のDNAとベクター断片とを結合させてなる発現ベクター。
- 請求項2に記載の発現ベクターを、宿主となる微生物、動物細胞又は植物細胞に導入してなる形質転換体。
- 請求項3に記載の形質転換体を培養することを特徴とする、前記アレンオキサイドシンターゼの製造方法。
- 以下の(a)〜(c):
(a) 配列番号2で表わされるアミノ酸配列からなるタンパク質;
(b) 配列番号2で表わされるアミノ酸配列において1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつアレンオキサイドシンターゼ活性を有するタンパク質;及び
(c) 配列番号1で表される塩基配列からなるDNAと少なくとも90%の同一性を有するDNAによりコードされ、かつアレンオキサイドシンターゼ活性を有するタンパク質
からなる群から選ばれるタンパク質。
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JPN6011019822; 横山 峰幸: '9位型オキシリピン,9,10-alphaケトールリノレン酸の植物生長調節における役割' 植物の生長調節,2005,40(1),p.90-100 * |
JPN6011019825; 綾野 まどか: 'シロイヌナズナAOSの9位触媒作用と酵素機能の解析' 日本植物生理学会年会要旨集,2010 Mar 12,51,p.255,P1C023(611) * |
JPN6011019827; 村上 真梨恵: 'アオウキクサ151株のアレンオキシド合成酵素の解析' 日本農芸化学会中四国支部講演会講演要旨集,2010 Jun 5,27,p.29,D-4 * |
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